UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

The idiotypic response to ferredoxin in mice Demetrick, Douglas James 1984

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1984_A1 D44.pdf [ 8.96MB ]
Metadata
JSON: 831-1.0096398.json
JSON-LD: 831-1.0096398-ld.json
RDF/XML (Pretty): 831-1.0096398-rdf.xml
RDF/JSON: 831-1.0096398-rdf.json
Turtle: 831-1.0096398-turtle.txt
N-Triples: 831-1.0096398-rdf-ntriples.txt
Original Record: 831-1.0096398-source.json
Full Text
831-1.0096398-fulltext.txt
Citation
831-1.0096398.ris

Full Text

THE IDIOTYPIC RESPONSE TO FERREDOXIN IN MICE. By DOUGLAS JAMES DEMETRICK B . S c , The Un ive rs i t y of B r i t i s h Columbia, 1980 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF MICROBIOLOGY We accept t h i s thes i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA July 1984 (c) Douglas James Demetrick, 1984 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r equ i r ements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Co lumb i a , I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s tudy . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copy ing o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g ran ted by the head o f my department o r by h i s o r her r e p r e s e n t a t i v e s . I t i s unde r s tood t h a t copy ing o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l no t be a l l owed w i t hou t my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y o f B r i t i s h Co lumbia 1956 Main Mall Vancouve r , Canada V6T 1Y3 Date T w \ v * , \<\QVr i i ABSTRACT These inves t iga t ions def ine a new prote in i d i o t y p i c system-:- that of C los t r id ium pasteurianum fer redoxin (Fd). Previous studies from our laboratory have shown that the response to Fd , a sma l l , non-mammalian e lec t ron t ransport prote in i s con t ro l l ed by H-2K/I-A l inked l o c i in mice and i s almost a unideterminant response in H-2 mice. S t ra ins of mice vary ing in Igh, I g l , and H-2 l o c i were immunized with Fd. The i r Fd-immune sera were tested by i n h i b i t i o n in the ELISA with four s p e c i f i c ant i- (ant i-Fd)- id iotypes produced in rabbi ts to the p u r i f i e d , pooled anti-Fd sera from four d i f f e r e n t s t ra ins of mice. Major id iotypes were observed reproducib ly in the anti-Fd responses of AKR/J, RF/J, and B10.BR/SnJ mice, while the anti-A/J id io type d id not reproducib ly i n h i b i t a high frequency of A/J ant i-Fd an t i s e r a . S i g n i f i c a n t d i f fe rences were observed between the l eve l s of i n h i b i t i o n of pooled or i n d i v i d u a l l y analyzed anti-Fd ant i se ra from the same mice. Further experiments showed that ant i-Fd ant i sera from B10.BR mice have a high frequency of c ross- reac t i v i t y with AKR/J ant i-Fd id iotypes as do RF/J, C58/J, C3H/HeJ and CBA/J an t i s e r a . C58/J, C57BR/cdJ, and RF/J an t i se ra a lso cross-react s i g n i f i c a n t l y with A/J ant i-Fd id io t ypes . Cu r ious l y , A/J and AKR/J anti-Fd ant i se ra are not i nh ib i t ed with anti-RF i d i o t y p e , and AKR anti-Fd ant i sera are not i nh ib i t ed with anti-B10.BR id io t ype . i i i The idiotype profile of monoclonal anti-Fd antibodies was studied. The results demonstrated interstrain idiotypic cross-reactivity that paralled the results obtained with the serum antibodies of the appropriate mouse strain. These data, with the data from experiments u t i l i z i n g serum antibodies do not demonstrate a correlation between idiotypic cross-reactivity and Igh or Igl allotypes. H-2 genes were observed to vary the pattern of idiotype expression in BIO recombinant mice, but no correlation between H-2 genes and idiotype expression could be observed. These results demonstrate that the anti-Fd response in H-2 mice is very complex with many intra and interstrain cross-reactive idiotypic families. Confirmatory results from 2-D electrophoretic analyses show-the presence- of many light chains in the anti-Fd response: These data are discussed in the context of existing hapten, carbohydrate, and protein antigenic systems, as well as in the context of current theories of idiotypic structure and antibody cross-reactivity. i v TABLE OF CONTENTS Page Abstract i i L i s t of Tables ix L i s t of F igures x L i s t of Abbreviat ions x i i Dedicat ion xv Acknowledgements xvi Chapter I. Introduct ion 1 II. Review of Hapten Systems 2 A) p-Azophenylarsonate 2 B) Phosphorylchol ine 7 C) (4-Hydroxy-3-nitrophenyl) acety l 12 III. Carbohydrate Antigen Systems 15 A) Dextran 15 B) Streptococcal group A antigen 17 C) L ipopolysacchar ide antigens 20 IV. Polypeptide and Prote in Antigen Systems 22 A) (Glutamic a c i d 6 0 , a l a n i n e 3 0 , t y r o s i n e 1 0 ) random terpolymer 22 B) Myoglobins 26 C) Lysozymes 28 D) Staphylococcal nuclease 30 V. Introduct ion Summary 32 A) Hapten Systems 32 B) Carbohydrate antigen systems 34 C) Polypeptide and prote in antigen systems 36 V Page VI. Review of the Ferredoxin system 39 Chapter 2. Mater ia ls and Methods 43 I. Immunological Methods 43 A) Ferredoxin and Keyhole Limpet Hemocyanin antibody EL ISA 43 1) EL ISA 43 2) I nh ib i t i on assays f o r id iotype exp ress ion . . 44 a) Pooled sera 44 b) Indiv idual sera 44 B) Preparat ion of immunoadsorbents 45 1) Ferredoxin-Sepharose 45 2) Immunoglobulin-Sepharose 45 a) Preparat ion of immunoglobulin 45 b) Ac t i va t ion and coupl ing to Sepharose. . . 46 C) Animals 47 1) Mice 47 2) Rabbits 47 D) Production of immunological reagents 47 1) Anti-Fd ant i se ra 47 2) Anti-KLH ant i se ra 48 3) Monoclonal ant ibodies to Fd 48 a) Production of hybridomas 48 b) Se lec t ion and c lon ing of hybridomas 51 c) Asc i tes production 51 4) Production of a n t i - i d i o t y p i c reagents 52 a) P u r i f i c a t i o n of ant i-Fd ant ibodies 52 b) Immunization and bleeding of r a b b i t s . . . 52 vi Page c) P u r i f i c a t i o n of a n t i - i d i o t y p i c an t i se rum. . . 52 d) Concentrat ion of a n t i - i d i o t y p i c and contro l normal rabb i t sera 53 II. Biochemical Methods 53 A) Reagent P u r i f i c a t i o n 53 B) Mini column immunoadsorbents 55 1) Preparat ion 55 2) Use of columns 55 C) SDS polyacrylamide gel e lec t rophores i s 56 D) Two-dimensional e l e c t r o p h o r e s i s . . . . . 56 E) S i l v e r s ta in ing 57 Chapter 3. Major Idiotypes in the Murine Ant i-Ferredoxin Response 58 I. Results 58 A) S p e c i f i c i t y of Ant i- id io types 58 B) I nh ib i t ion of Anti-KLH A c t i v i t y of Indiv idual Sera by Anti-Fd Idiotypes 64 C) I nh ib i t i on of Anti-Fd Indiv idual sera 69 D) K ine t i cs of Idiotype Expression 74 II. D iscuss ion 77 Chapter 4. I n te rs t ra in Cross-react ive Idiotypes in the An t i -Ferredoxin Response 80 I. Results 80 A) In te rs t ra in Cross-react ive Idiotypes in the Pooled Anti-Fd Response.. 80 B) In te rs t ra in Cross-react ive Idiotypes in Indiv idual Anti-Fd Sera 87 C) K ine t i cs of Indiv idual In te rs t ra in Cross-react ive Idiotype Expression 109 v i i Page D) E f f e c t of H-2 Genes on Idiotype Expression 121 II. Discuss ion 124 Chapter 5. Id io typ ic Charac te r iza t ion of Monoclonal An t i -Ferredoxin Ant ibodies 135 I. Results 135 A) S p e c i f i c i t y of Monoclonal Anti-Fd Ant ibodies 135 B) Determinant Charac te r iza t ion of Monoclonal Anti-Fd Ant ibodies 135 C) Id io typ ic Charac te r iza t ion of Monoclonal Anti-Fd Ant ibodies 138 II. D iscuss ion 143 Chapter 6. Two Dimensional E l ec t rophore t i c Ana lys is of An t i -Ferredoxin Ant ibodies 145 I. Results 145 II. D iscuss ion 159 Chapter 7. Impl icat ions of Results and Further Experiments 162 I. Thesis Discuss ion 162 A) The T Ce l l Receptor 162 B) What Const i tutes Idiotype? 163 C) What Const i tutes C ross- reac t i v i t y and S p e c i f i c i t y ? 165 D) Id io typ ic Regulation 171 II. Further Experiments 172 III. Summary 174 References 175 v i i i Page Appendix 1 201 1) S t a t i s t i c a l Methods 201 2) Threshold of Response 201 3) S t a t i s t i c a l Comparisons 202 Appendix 2. A Summary of Id io typ ic Systems 203 Appendix 3. The Primary Structure of Ferredoxin 204 ix LIST OF TABLES Page I. Cha rac t e r i s t i c s of Mouse St ra ins U t i l i z e d 60 II. Summary of Pooled Versus Indiv idual Anti-Fd I n h i b i t i o n s . . . 73 III. Summary of S t a t i s t i c a l Comparisons f o r Chapters 3 and 4 . . . 106 IV. Summary of K ine t i cs of Idiotype Expression 115 V. A l lo types of Mice at Loci of Immunological Interest 127 X LIST OF FIGURES Page 1. S p e c i f i c i t y of A n t i - i d i o t y p i c Sera on Pooled Anti-Fd or Anti-KLH Sera 62 2. S p e c i f i c i t y of A n t i - i d i o t y p i c Sera on Indiv idual Anti-KLH Sera 66 3. I nh ib i t ion of Anti-KLH Sera from Ind iv idua l l y Marked BIO.BR Mice 68 4. I nh ib i t i on of Indiv idual Anti-Fd Sera with Appropriate Ant i- id io type 71 5. K ine t i cs of Inh ib i t ion of Anti-Fd Sera from Ind i v idua l l y Marked Mice with Appropriate Ant i- id io type 76 6. I nh ib i t i on of Pooled Te r t i a r y Anti-Fd Sera from AKR/J, RF/J, A/J and B10.BR/SnJ Mice 82 7. I nh ib i t ion of Pooled Te r t i a r y Anti-Fd Sera from C3H/HeJ, ST/bJ, C58/J and CE/J Mice 84 8. I nh ib i t ion of Pooled Te r t i a r y Anti-Fd Sera from C57BR/cdJ Mice 86 9. Inh ib i t ion of Indiv idual Anti-Fd Sera from AKR/J Mice 89 10. I nh ib i t ion of Indiv idual Anti-Fd Sera from RF/J Mice 91 11. I nh ib i t i on of Indiv idual Anti-Fd Sera from A/J Mice 93 12. I nh ib i t i on of Indiv idual Anti-Fd Sera from B10.BR/SnJ Mice 95 13. I nh ib i t i on of Indiv idual Anti-Fd Sera from C58/J Mice 97 14. I nh ib i t i on of Indiv idual Anti-Fd Sera from C57BR/cdJ Mice 99 15. I nh ib i t i on of Indiv idual Anti-Fd Sera from C3H/HeJ Mice 101 16. I nh ib i t i on of Indiv idual Anti-Fd Sera from CBA/J Mice 103 17. I nh ib i t i on of Anti-Fd Sera from Ind iv idua l l y Marked AKR/J Mice: K ine t i c s of the Response I l l 18. I nh ib i t i on of Anti-Fd Sera from Ind i v idua l l y Marked RF/J Mice: K ine t i cs of the Response 113 xi Page 19. I nh ib i t ion of Anti-Fd Sera from Ind iv idua l l y Marked A/J Mice: K ine t i cs of the Response 117 20. I nh ib i t ion of Anti-Fd Sera from Ind iv idua l l y Marked B10.BR/SnJ Mice: K ine t i cs of the Response 120 21. I nh ib i t ion of Secondary Anti-Fd Sera from Various S t ra ins of Mice D i f f e r i n g at H-2 l o c i 124 22. Simple Relatedness Tree of Heavy Chain A l lo types in Mice 129 23. Re lat ionship of Various Anti-Fd Id io typ ic Fami l ies 131 24. S p e c i f i c i t y of C57BR Anti-Fd Monoclonal Ant ibodies 137 25. Determinant S p e c i f i c i t y of C57BR Anti-Fd Monoclonal Ant ibodies 140 26. I nh ib i t i on of Monoclonal Anti-Fd Ant ibodies with Various Ant i- Id iotypes 142 27. Two Dimensional Gel E lec t rophores is of Normal Mouse Serum 148 28. Two Dimensional Gel E lec t rophores is of Monoclonal Anti-Fd Antibody (Fd-1) 150 29. Two Dimensional Gel E lec t rophores is of Pooled Anti-Fd Ant ibodies from AKR/J Mice 152 30. Two Dimensional Gel E lec t rophores is of Pooled Anti-Fd Ant ibodies from A/J Mice 154 31. Two Dimensional Gel E lec t rophores is of Pooled Anti-Fd Ant ibodies from B10.BR/SnJ Mice 156 32. Representative Two Dimensional Gel E lec t rophores is of an Indiv idual B10.BR/SnJ Anti-Fd Serum 158 x i i LIST OF ABBREVIATIONS A5A GAC id iotype ARS p-Azobenzenearsonate hapten ARS-Br ARS conjugated to B ruce l l a abortus Bl-8 Monoclonal anti-NP antibody BI Bovine i n s u l i n BSA Bovine serum albumin CGAT GAT id iotype CFA Complete Freund's Adjuvant C Complement CRI Cross-react ive id io type in ARS system DNP Dini t rophenyl hapten DTH Delayed type hype rsens i t i v i t y FCS Fetal c a l f serum Fd C los t r id ium pasteurianum fer redoxin GA Glutamic a c i d , a lanine polypept ide GA-1 GA id io type GAC Group A carbohydrate GAT G l u , a l a , t y r random tercopolymer GL G lu , l ys polypeptide GT G l u , t y r polypeptide Gte GAT id iotype HEL Hen egg-white lysozyme Id I Indiv idual id io type (various systems) Id X Cross-react ive id iotype (various systems) IEF I soe l e c t r i c focuss ing I FA Incomplete Freund's Adjuvant igh Immunoglobulin heavy chain gene igk Immunoglobulin kappa chain gene i g X Immunoglobulin lambda chain gene i g i Immunoglobulin l i g h t chain gene i/p Intraper i toneal i/v Intravenous J558 Dextran binding myeloma prote in KLH Keyhole l impet hemocyanin LPS L ipopolysacchar ide MAb Monoclonal antibody MHC Major h is tocompatab i l i t y l o c i MIg Mouse immunoglobulin M104E Dextran binding myeloma prote in M460 DNP binding myeloma prote in Nase Staphyloccal nuclease NIP (4-Hydroxy-5-iodo-3-nitrophenyl) acety l hapten NMS Normal mouse serum NNP (4-Hydroxy-3,5-dinitrophenyl) acety l hapten NP (4-Hydroxy-3-nitrophenyl) acety l hapten NRS Normal rabb i t serum Ox-1 phOx id iotype PAGE Polyacrylamide gel e lec t rophores i s PC Phosphorylchol ine hapten PFC Plaque forming co lon ies pGAT GAT id io type xiv PhOx 2-Phenyloxazolone hapten RaMIg Rabbit anti-mouse immunoglobulin SRBC Sheep red blood c e l l s SWM Sperm whale myoglbin S117 GAC id io type TGAL. Poly-L-(Tyr, Glu)-poly-D,L-(Ala)-poly-L-(Lys) polypept ide T s C T suppressor c e l l T s F T suppressor f a c to r T15 PC binding myeloma prote in (major PC id io type) V H Heavy chain va r i ab le region V L L ight chain va r i ab le region 2-D Two dimensional gel e lec t rophores i s 6-90 Ident ica l to Fd-1 Fd binding mAb XV DEDICATION This thes is i s dedicated and my parents Vic and L o i s . to my wife L yd i a , xvi ACKNOWLEDGEMENTS It would have been impossible to do many of the experiments, only some of which are presented here , that I d id as a graduate and fourth year student without the help and moral support so read i l y ava i l ab le from the members of UBC Department of Microb io logy . As w e l l , I would l i k e to acknowledge the support of Dr. P. Candido and the exce l l en t ass is tance of Mr. J . Durgo in the modi f i ca t ion of the Beckman 890 B sequencer to micro-sequencing c a p a b i l i t y -- the use of which, very sad l y , could not be appropr ia te ly taken advantage of in the f i n a l form of my p ro jec t . I wish to acknowledge my debt to Drs. J . J . R. Campbell and J . Levy f o r i n i t i a t i n g and encouraging my in te res t s in microbiology and immunology r e spec t i v e l y , and to Dr. J u l i a Levy and the members of her lab who made my stay so academical ly and s o c i a l l y enjoyable . In a d d i t i o n , I would l i k e to thank a l l of the members of my thes is committee f o r t h e i r carefu l reading of t h i s manuscript. F i n a l l y I would l i k e to thank Dr. Lydia S ikora f o r paving the way f o r my work, f o r her exce l l en t help in the preparat ion of my t h e s i s , and f o r her encouragement and company during my years in the Department of Microbio logy at UBC. 1 Chapter 1. Introduct ion 1. Introduct ion An important cons iderat ion to address p r i o r to any s c i e n t i f i c pursu i t i s the reason f o r pursu i t at a l l . The continued study of immune responsiveness has the potent ia l of y i e l d i n g benef i ts in c l i n i c a l f i e l d s such as t r ansp l an t a t i on , i n f e c t i o n , cancer therapy and preventat ive medicine. As w e l l , i ns igh ts in to f i e l d s such as b iochemist ry , molecular gene t i c s , c e l l b io logy and pharmacology have been made. Indeed, the study of the regulat ion of the immune response has become a very large f i e l d of study in i t s e l f . The concept of id io type was introduced by Oudin (1) and Kunkel (2) and i t s poss ib le ro le in immune regulat ion was postulated by Jerne (3). Idiotype has been def ined as the set of ant igen ic s p e c i f i t i e s d isp layed by the va r i ab le regions of a set of antibody molecules produced by an ind iv idua l or group of i nd i v idua l s in response to a given antigen (3). Idiotypes can be composed of two kinds of determinants. A paratope i s a determinant at the s i t e of actual antigen b ind ing , and an id iotope i s an i d i o t y p i c determinant that i s not assoc iated with antigen binding (3). Aside from much data showing the importance of id iotypes in the regulat ion of both the humoral (4) and c e l l mediated immune responses (5), id iotypes have been used as genet ic markers in the study of immunoglobulins at the molecular leve l (6) and have been l inked to ce r ta in c l i n i c a l d isorders such as myasthenia grav is (7), Hashimoto's disease (8), rheumatoid a r t h r i t i s (9) and to the loss of immune responsiveness, due to aging (10). Current ly the study of the ro le of the id io type in immune regulat ion i s a top i c of great i n t e r e s t . In order to demonstrate where the inves t iga t ions comprising th i s 2 thes is f i t into the f i e l d , a review of the major systems involved in the study of i d i o t y p i c s t ructure and regu la t ion of the immune response i s needed. Although a number of model systems have been i nves t i ga t ed , ce r t a in systems have been extremely well cha rac te r i zed , sometimes to the molecular leve l of id io type s t ruc tu re . These studies w i l l be reviewed in depth. II. Review Of Hapten Systems A) p-Azophenylarsonate The immune response to p-Azophenylarsonate (ARS) or p-Azobenzenear-sonate hapten i s one of the e a r l i e s t and best character ized i d i o t y p i c systems a v a i l a b l e . When A/J mice are immunized with the ARS hapten coupled to the prote in c a r r i e r keyhole l impet hemocyanin (KLH), they produce in t he i r asc i t es f l u i d or serum, large amounts of anti-ARS an t ibod ies . Of these , 20 - 70% bear a major c ross-react i ve id io type (CRI). These id iotypes are detectable with a rabbi t an t i- id io type (6 ) , and have an i s o - e l e c t r i c point in the range of 6.65-6.90 pH uni ts (11). Ant ibodies bearing CRI were found to be of IgG^ isotype (12) , and studies l inked the expression of the A/J CRI to the immunoglobulin heavy chain locus (Igh) (6) . Thus, the CRI was proposed as a marker f o r heavy chain va r i ab le region genes (V^) in A/J mice. Pre l iminary amino ac id sequence studies (12, 13) showed that the heavy chain va r i ab le regions and the l i g h t chain hypervar iable regions (14) of induced polyc lona l anti-ARS ant ibodies in A/J mice were homogeneous in t h e i r respect ive primary s t ruc tu res . Further i nves t iga t ion using CRI pos i t i v e monoclonal ant ibodies (mAb) d id not corroborate these o r i g i n a l observat ions . It was found that MAb exh ib i ted a wide array of b inding curves when assayed with ant i-po lyc lona l CRI (15) , implying s t ruc tu ra l heterogeneity of the o r i g i na l antibody popula t ion . 3 They were not usua l l y of IgG^ isotype (15,16) and d i f f e r ed extens ive ly (16) from the e a r l i e r reported (13) heavy chain V region sequence. When ind i v idua l an t i- id io types were made by rabbi ts to each monoclonal , i n h i b i t i o n studies e legant ly i l l u s t r a t e d the concepts of " Pub l i c " or shared and " P r i va te " or ind iv idua l id iotypes (15,17). Prote in sequence ana lys is corroborated the se ro log i ca l f ind ings of heterogeneity and showed that there was microheterogeneity assoc iated with both the heavy and l i g h t chain V reg ions . However, as might be a n t i c i p a t e d , fu r the r work showed that even though CRI pos i t i v e monoclonal heavy chain V regions exh ib i ted some primary s t ructure heterogenei ty , the CRI negative heavy chains were much more d iverse (19). Inspection of more prote in sequence data revealed that the anti-ARS heavy chains could be c l a s s i f i e d into three f am i l i e s based on homology to a prototype monoclonal (20). One of these f a m i l i e s , 91A3, possesses i d i o t y p i c determinants that are assoc iated with a l l CRI pos i t i ve anti-ARS ant ibodies (20). The 91A3 determinants were a lso found in some CRI negative anti-ARS monoclonals as well as polyc lona l non-ARS binding ant ibodies (20). It was thus postulated that 91A3 encodes a s ing le gene respons ib le f o r the CRI i d io t ype . This observat ion i s i n t e r e s t i n g , s ince genet ic s tudies had shown that the expression of the CRI id iotype was a lso l i nked to the K l i g h t chain l o c i (21). The s i t ua t i on was c l a r i f i e d when i t was shown that indeed a s p e c i f i c 91A3 fami ly l i g h t chain from e i the r CRI pos i t i v e or negative ant ibodies was required to form a CRI pos i t i v e heavy chain (22). Thus, the expression of the CRI required both s p e c i f i c heavy and l i g h t cha ins , but only heavy chains from a CRI pos i t i v e antibody could recons t i tu te the CRI (22). It i s worthy to note, however, that the l i g h t chain had to be from the 91A3 fami ly - none of the other prototype f ami l i e s could provide the necessary l i g h t cha in . 4 Recent studies have analyzed the gene DNA at the germ l i n e l e v e l . The postu late that members of the 91A3 prote in fami ly are somatic descendants of a s ing le germ l i n e gene (23) has been shown to be cor rec t (24). It was concluded that the d i v e r s i t y seen among CRI pos i t i v e ant ibodies was a r e su l t of somatic mutation processes. It i s expected that only one or two genes w i l l code f o r the necessary K l i g h t chain gene (24) but fu r the r work i s in progress. The exact s t ruc tu ra l basis of the CRI id io type i s s t i l l unknown, although recent work u t i l i z i n g monoclonal a n t i - i d i o t y p i c probes and i so l a t ed H and L chains (25) may eventual ly c l a r i f y id iotype primary s t ructure in the ARS system. In p a r a l l e l with studies to determine the s t ructure o f the CRI i d i o t y p e , inves t iga t ions of i t s ro le in immune regu la t ion of the anti-ARS response was undertaken. It was found in A/J mice that T c e l l s capable of suppressing the en t i r e delayed type hype r sens i t i v i t y (DTH) response to ARS could be induced by intravenous (i/v) immunization with rabb i t anti-CRI antibody (5) . Th is r esu l t was assumed to show that T suppressor c e l l s had a n t i - i d i o t y p i c receptors , and that the T c e l l r eper to i re makes great use of id io types c ross-reac t i ve with B c e l l s . It was a lso shown that the anti-CRI induced suppression was gene t i c a l l y r e s t r i c t e d and l inked with the same Igh a l lo types as the humoral CRI anti-ARS response (5) . I nves t iga t ions , at t h i s t ime, of the humoral response showed that A/J mice immunized i/v with syngeneic thymocytes conjugated with s p e c i f i c a l l y p u r i f i e d A/J anti-ARS ant ibodies had a suppressed CRI pos i t i v e response to KLH-ARS, although the to ta l anti-ARS response was normal (26). Subsequent work showed that suppressor T c e l l s were •induced, which s p e c i f i c a l l y suppressed the CRI response, and that these T c e l l s could a lso suppress the en t i r e DTH response to ARS (27). A f l u r r y of i nves t iga t i ve a c t i v i t y fo l lowed. Two 5 c lasses of suppressor T c e l l s were found. One c l a s s , designated T 1 was found to bear CRI determinants and was produced by immunization with ARS coupled thymocytes (29). The other c l a s s , T s 2 , was found to be CRI-binding and was produced by immunizing A/J mice with so luble fac tors (T S F1) der ived from T 1 c e l l s (28,29) , or produced, even tua l l y , by immunization with rabb i t anti-CRI (30). Other studies a lso showed that T 1 c e l l s produced T S F1 fac tors that were ARS-binding, CRI pos i t i v e and ca r r i ed major h is tocompatab i l i t y complex (MHC) markers. A l s o , T g F2 c e l l f ac tors d id not bind ARS, but d id bind CRI (28). It was concluded that appropriate a n t i - i d i o t y p i c and i d i o t y p i c in te rac t ions were essent ia l f o r the manifestat ion of suppressor T c e l l funct ion in ARS s p e c i f i c suppressor pathways (32). Recent ly , several T c e l l hybridomas have been produced (33). Suppressor c e l l hybrids producing fac tors binding both antigen and anti-CRI ant ibodies have been character ized (34) , as have T lymphocyte hybrids that bind to CRI pos i t i v e monoclonal ant ibodies (33,35). Unfor tunate ly , an ARS binding CRI pos i t i v e non-immunoglobulin molecule , thought to be a T c e l l f a c to r but s ince found to be ubiquitous to almost a l l c e l l s (and poss ib l y a phosphatase), has complicated matters (36,37). Presumably, work on more c a r e f u l l y character ized T c e l l f ac tors from hybridomas or c e l l l i nes w i l l provide much more s t ruc tu ra l information on i d i o t y p i c determinants of T c e l l s . Recent experiments have complicated some of the e a r l i e r apparent s i m p l i c i t i e s of the anti-ARS CRI systems. Although the CRI produced in the anti-ARS-KLH response very c l e a r l y cor re la ted to the A/J Igh a l lo type (6 ) , CRI i s a lso produced by other mouse s t ra ins in IgM anti-ARS plaque forming c e l l (PFC) assays (38), when the mice were immunized once with ARS conjugated to B ruce l l a abortus (ARS-Br) (a T-independent c a r r i e r ) . These 6 resu l t s c l e a r l y show that the hyperimmune response to a hapten l inked to a T-dependent c a r r i e r does not represent the f u l l B c e l l reper to i re capable of hapten binding (38). Obv ious ly , some regulatory phenomenon i s occurr ing that has very s t r i k i n g and reproduceable e f f e c t s . When mice are primed with ARS-KLH and boosted with ARS-Br, the resu l t s are s im i l a r to those seen when only ARS-KLH i s used as an antigen (39). Thus, whatever phenomenon occurs , i t s e f f e c t s d i r e c t antibody production on subsequent immunization. Other work has v e r i f i e d that d i f f e r e n t immunization techniques can r e su l t in CRI expression in supposedly CRI-negative s t ra ins (40). A l s o , in A/J animals the CRI id iotype i s ra re l y seen at the B c e l l precursor l eve l (41) and i s magnified one hundred f o l d upon immunization with ARS coupled prote in conjugates. This i s probably not due so l e l y to c lona l expansion dr iven by antibody a f f i n i t y , as CRI pos i t i v e antibody does not have a p a r t i c u l a r l y high a f f i n i t y f o r ARS (42,43). Obv ious ly , regu la t ion of CRI production i s very complex in mice (39,44) and depends heav i ly on the immunization p ro toco l . To fur ther complicate matters , i t seems that some members of the CRI pos i t i v e antibody fami ly have no r e a c t i v i t y towards ARS (45). Thus, inves t iga t ions of anti-ARS id iotypes have y i e lded a l o t of de ta i l at the molecular leve l about CRI express ion. In A/J mice, CRI pos i t i v e ant ibodies are descendants of a s ing le gene (46); t h i s gene i s not found in inbred s t ra ins that do not express the CRI id iotype (24). T c e l l s express CRI determinants, and a n t i - i d i o t y p i c modulation can produce large changes in the expression of CRI. Thus both genet ics and regulat ion play a ro le in CRI expression in the A/J anti-ARS response. However, in sp i te of a l l o f the i n v e s t i g a t i o n , the s t ruc tura l bas is of the CRI and the mechanism of i t s regulat ion are l a rge l y unknown. 7 B) PhosphoryTcholine The study of the ant i-phosphory lchol ine (PC) response in mice began with the i s o l a t i o n of immunoglobulins produced by mineral o i l induced myelomas in BALB/c mice. Some IgA mouse plasmacytomas were found to produce antibody which bound to bac te r i a l polysacchar ides (47). Character -i z a t i on of one of the ant igen ic determinants revealed i t to be phosphoryl-chol ine (48). E ight of these monoclonal ant ibodies were character ized and in jec ted in to s t r a i n A mice (eg. A/J) to produce homologous a n t i - i d i o t y p i c se ra . F ive of these monoclonals (T15, H8, S107, M299, and S63) were found to cross-react extens ive ly with one an t i- id io type (49). When BALB/c mice were in jec ted with rough s t r a i n pneumococcus (R36A) c e l l s , they produce a strong anti-PC response. Up to 98% of the anti-PC B c e l l s reacted with anti-T15 id io type as measured by a plaque assay (50). The serum antibody response to PC was studied in other s t r a i n s , and large amounts of T15 id io type were found to be present in BALB/c, C57L, C58, 129 and ST mouse s t r a i n s . Other s t r a ins d id not appear to express the T15 id iotype (51). Genetic studies es tab l i shed the l inkage of the T15 id io type to Igh l o c i (51). S t ructura l i nves t iga t ions of the murine anti-PC response showed that probably only 3 K l i g h t chains were involved in the en t i r e anti-PC response. IEF inves t iga t ions of i so l a t ed heavy chains could not be ca r r i ed ou t , however, due to s i a l i c ac id charge heterogeneity (52). Other ear ly work showed the anti-PC response to be qui te homogeneous wi th in some s t ra ins (51,53) . This work a lso showed that some s t ra ins of mice which d id not express the T15 id iotype (CBA, A , CE) produced antibody having s im i l a r b inding s p e c i f i c i t i e s and L chain patterns as T15 pos i t i v e antibody (52,53). These data s t rongly suggested the presence of a T15-l ike id iotype 8 in some T15-negative s t ra ins of mice. A binding myeloma prote in from C57BL/6 mice (C3) was sequenced and found to have 96% homology to T15 (54). Homologous an t i - id io type to C3 very s t rongly i nh ib i t ed T15 pos i t i v e ant ibodies from binding to PC (55). It was a lso noted that anti-T15 id iotypes were s t rongly c ross-react ive to the C3 id io type (55). The C3 i d i o t ype , l i k e the T15 i d i o t ype , was found to be l inked to Igh a l lo t ype and was seen in the anti-PC response of C57BL/6, C57BL/10, SJL, A , and CBA mice. S u r p r i s i n g l y , C3H mice, which are very s im i l a r to CBA mice (56) d id not produce C3 id io type in response to PC (55). Since C3 and T15 id io types are s t rongly c ros s r eac t i v e , one cannot help but wonder why C3 pos i t i v e s t ra ins were not found to be T15 pos i t i v e in e a r l i e r experiments (51). I n t e r e s t i ng l y , ant ibodies from BAB.14 mice exh ib i ted both C3 and T15 id iotypes (55). This might be due to unequal c ross ing over of mendelian (or true) a l l e l e s (55) in the generation of the BAB.14 V-C recombinant. The use of monoclonal ant ibodies made from PC immunized mice has added grea t l y to the knowledge of the T15 i d i o t y p i c system. Though previous work (51,53) had ind ica ted a homogeneous T15 response, c l ose r sc ru t iny showed that the response was heterogeneous at the c e l l u l a r (57) as well as serum antibody l eve l s (58,59) . Sequence ana lys is of monoclonal T15 pos i t i v e ant ibodies confirmed th i s observat ion (60). Indeed, the PC response was theor ized to comprise 3 d i s t i n c t i d i o t y p i c f ami l i e s (61) , a l l of which could have been somat ica l ly der ived from T15 (60). The expansion of mutant clones was probably dr iven by antigen a f f i n i t y , as more "mature" id iotypes have greater a f f i n i t y f o r PC than T15 (62). The p re fe ren t i a l expression of the T15 id io type may be due to i t s assoc i a t ion with a f f i n i t y f o r PC (63) , probably co r r e l a t i ng with the presence of a glutamic ac id residue at the 35 pos i t i on of the T15 heavy chain in V H (64). This residue has been 9 demonstrated to s t a b i l i z e the hapten binding pocket (64) and i t s mutation to a lanine destroys the PC binding c a p a b i l i t y , although T15 determinants are l e f t i n t ac t (63). Molecular genet ic s tudies showed that a T15 gene probe hybr id ized to 4 genes in germ l i n e BALB/c DNA under s t r ingent condi t ions (65). Sequence ana lys is of these genes revealed that 3 of them could be derived from the f o u r t h , the T15 germ l i n e gene (66). Studies of monoclonal ant ibodies showed that a l l known H chains sequences, but one could be der ived from the T15 germ l i n e gene. The only exception was encoded by one of the other 3 germ l i n e genes (67). Thus, the en t i r e immune response to PC i n , at l e a s t , BALB/c mice i s der ived from one germ l i n e gene (66). It was a lso shown that somatic mutations of t h i s gene were cor re la ted with IgA or IgG i so t ypes , and not IgM which expressed the germ l i n e T15 sequence (66). This corroborated e a r l i e r prote in sequence s tud i e s , implying that somatic mutation i s an event occurr ing a f t e r , or du r ing , the c lass switch events and thus appears to be a regulated phenomenon (60). Genetic ana lys is of PC binding monoclonal ant ibodies showed that most u t i l i z e d the J^l gene segment, exh ib i ted D^ d i v e r s i t y , and u t i l i z e d one of the 3 K l i g h t chain f am i l i e s (63). Unfor tunate ly , though the T15 id iotype system represents one of the best s t r u c t u r a l l y character ized systems, the chemical cor re la tes of id io typy remain undef ined. Regulatory studies of the T15 id io types have been very popular . As prev ious ly mentioned, when BALB/c mice are immunized with R36A vacc ine , the anti-PC response i s almost e n t i r e l y T15 pos i t i v e (51). When adult BALB/c mice are pre-treated with ant i-T15, the subsequent anti-PC response exh ib i ted a t r ans ien t absence of T15 i d i o t y p e , which reaches normal l e ve l s wi th in 2-3 weeks (68). When neonates, however, were t reated with ant i-T15, 10 the subsequent lack of T15 ant ibodies pers i s ted f o r the l i f e of the mouse (69) though the anti-PC response recovered a f t e r 15 weeks. In ea r l y s tud i e s , t h i s chronic suppression could not be adopt ive ly t rans fe r red to young BALB/c mice (70) although l a t e r i t was shown that immunization with anti-T15 induced Lyt-2 pos i t i v e T suppressor c e l l s (T S C) which could t rans fe r suppression (71). When neonates were t reated with monoclonal a n t i - i d i o t y p e , d i rec ted to var ious T15 i d i o topes , a suppression of the T15 response was seen, although expression of the p a r t i c u l a r id io tope may s t i l l be qui te detectable (72). Hyperimmunization of BALB/c mice with R36A vaccine profoundly lowers the T15 expression (73,74). This "suppress ion" cor re la tes with the appearance of anti-T15 ant ibodies in the sera of these mice (73,74) . It should be noted that treatment of mice with s t r a i n A anti-T15 antibody suppressed the anti-PC plaque response in both C57BL/6 and BALB/c mice (72) even though C57BL/6 mice do not express the T15 id iotype (per se) in the anti-PC response. This r e su l t may be due to T15 c ross- reac t i v i t y with C3. D i rec t studies of T c e l l s involved in regu la t ing the T15 pos i t i v e anti-PC response have been undertaken. At l eas t two helper c e l l s have been cha rac te r i zed . One i s involved in MHC product recogni t ion (75) and the other i s involved in T15 recogni t ion (76). Both are required to s y n e r g i s t i c a l l y i n s t i ga t e a T15 pos i t i v e anti-PC response (77). T suppressor c e l l s have a lso been inves t iga ted . Immunization of BALB/c mice with PC-coupled syngeneic splenocytes caused the production of PC s p e c i f i c suppressor c e l l s that could be neut ra l i zed with anti-T15 id io type (78). A T c e l l hybridoma that s ta ins with f luorescent anti-T15 id iotype and binds PC (79) , produces a f a c to r that has a PC binding s i t e with T15 determinants, but does not express the T15 germline gene segment (80). 11 Recent work has analyzed T helper c e l l s induced by PC-KLH pr iming, or monoclonal anti-T15 i d i o t y p e , and has shown that these c e l l s cannot d i s t i ngu i sh between T15 and M167 id iotypes (81). Such f ind ings ind i ca te that id iotype s p e c i f i c priming can induce non-idiotype s p e c i f i c T c e l l s , implying that the i d i o t y p i c T c e l l network i s based on a d i f f e r e n t se l ec t i on of i d i o t y p i c determinants than the se l ec t ion of the B c e l l id io type network (81). As with the anti-ARS CRI system, the hapten c a r r i e r may dramat ica l l y change the anti-hapten i d i o t y p i c response to PC. BALB/c mice immunized with Proteus morganii (Potter) produce an anti-PC response cons i s t ing of M603 id io type (82) and not T15. This r esu l t may be due to the higher a f f i n i t y M603 has f o r P. morganii (Potter) PC (82). Cu r i ous l y , CBA/N mice bear an X-l inked immune d e f i c i e n c y , such that females, car ry ing one copy of the gene, w i l l respond normal ly , but F^  males w i l l not produce an anti-PC response (83). It has been shown that the F^  males possess a T15 (or C3) s p e c i f i c helper T c e l l , but lack an Lyb-5 pos i t i v e B c e l l populat ion respons ib le f o r the product ion of anti-PC IgM (84). Another c u r i o s i t y was observed when neonatal BALB/c mice were immunized with PC l inked to a prote in c a r r i e r . When the mice were boosted 30-40 days l a t e r , the anti-PC response was less in both amount and a f f i n i t y than the placebo-immunized c o n t r o l s , and a lower r a t i o of T15 pos i t i v e ant ibodies i s observed. Apparent ly , immunization of neonates with PC coupled to prote in c a r r i e r s i s assoc iated with the production of low a v i d i t y , T15 negative ant ibodies (85). This phenomenon i s not mediated by a T c e l l or auto an t i - id io type (85). Another compl icat ion i s the f a c t that a monoclonal antibody to a T15 determinant cross-reacts with a Thy-1 determinant (86). 12 Thus, the molecular genet ics of the B c e l l T15 pos i t i v e anti-PC response are well cha rac te r i zed . The T15 id iotype seems to be r e s t r i c t e d to the Igh a a l l o t ype f am i l y , but a very c l o se l y re la ted and poss ib l y a l l e l i c gene i s expressed by mice of d i f f e r e n t Igh a l l o t ypes . Various studies have evaluated the ro le and the cha r a c t e r i s t i c s of the T c e l l T15 response, and several s tudies have examined the regu la t ion of the T15 id io t ype . In sp i te of these inves t iga t ions the s t ruc tu ra l character of the T15 id io type i s unknown, and the i d i o t y p i c regulat ion seems very complex and l a rge l y undef ined. C) 4-Hydroxy-3-nitropheny) acety l (NP) Another hapten system in which the i d i o t y p i c response has been extens ive ly studied i s that of the (4-hydroxy-3-nitropheny) acety l (NP) hapten. When C57BL, 101 or LP mice are immunized with NP on a prote in c a r r i e r , such as bovine serum albumin (BSA), or chicken serum g lobu l i n (CSG), the ant ibodies produced bear a major cross react ive id io type (NP-b) in the primary response (87,88) . These ant ibodies a lso have a pecu l i a r binding s p e c i f i c i t y ( h e t e r o c l i t i c i t y ) by which anti-NP ant ibodies a c tua l l y bind to re la ted haptens such as (4-hydroxy-3,5-dinitrophenyl) acety l (NNP) or (4-hydroxy-5-iodo-3-nitrophenyl) acetyl (NIP) bet te r than they bind to NP i t s e l f (89). Another id io type (NP-a) has been l inked to the Igha a l lo type (90). It was suggested (90) that the Np-a and NP-b id iotypes were a l l e l i c s ince both id io types are associated with M i g h t chain bearing an t ibod i es , which are detected most e a s i l y in the primary response, are h e t e r o c l i t i c (91) and are mutually exc lus ive in a l l conventional and recombinant s t r a ins studied (90). Recent inves t iga t ions have u t i l i z e d monoclonal ant ibodies produced from the NP-a pos i t i v e anti-NP response in BALB/c mice. These studies show that 50% of the monoclonal ant ibodies 13 express the K l i g h t chain -- the res t express \ , as seen in the primary serum response (92). A f t e r 3 weeks post immunization, or in the secondary response, the BALB/c ant ibodies express the K chain predominantly (92). Both X and K bearing monoclonals were h e t e r o c l i t i c , but only X bearing monoclonals expressed the NP-a id iotype (92). When NP-a and NP-b bearing monoclonals were compared, a subset of NP-b shared i d i o t y p i c determinants with NP-a (93). This c ross- reac t i v i t y was a lso observed with immune sera of ind iv idua l NP-b pos i t i v e anti-NP responding mice (93). One monoclonal antibody bearing NP-b i d i o t y p i c determinants has been very well s tud ied . Bl-8 i s an IgM antibody that ca r r i e s expressed germ l i n e V^ and genes (94,95) and possesses at l eas t two i d i o topes , corresponding to the s p e c i f i c i t i e s of two monoclonal an t i - i d i o t ypes , Ac38 and Acl46 (96). The Bl-8 id iotopes are regu la r l y expressed in the NP-b pos i t i v e anti-NP response (97). S t ructura l s tudies of Bl-8 id iotopes have shown that both and regions are important in id io type expression (98). Very small changes in primary s t ructure can d r a s t i c a l l y a f f e c t id io type s p e c i f i c i t y , such as the case of a mutant of B l-8 . There , a s ing le amino ac id i s a l te red in causing the loss of seven parental id iotopes and changes in an e i gh th , but re tent ion of NP binding (98). When Igh^ mice are immunized with monoclonal ant i -B l-8 i d i o t y p e s , they produce Bl-8 id io type pos i t i v e an t ibod ies , some of which do not bind NP (99). The pattern of s p e c i f i t i e s produced i s s t r a i n s p e c i f i c and i t i s thought that these observat ions provide evidence that the i d i o t y p i c reper to i re i s a r e su l t of V^ region polymorphisms (99) and that i d i o t y p i c and antigen binding residues are s t r u c t u r a l l y separate. This proposal i s re in forced by f i nd ing that an NP-b id io type p o s i t i v e , non-NP binding antibody can be 14 generated by rep lac ing most of Bl-8 gene with a neighboring gene (100). Recent studies have compared the genes coding f o r the NP-a and NP-b id iotypes in mice (101). A cDNA probe f o r Bl-8 was used to screen C57BL/6 germ l i n e DNA, revea l ing 7 bands on Southern b l o t t i n g and hybr id i za t ion under f a i r l y s t r ingent condi t ions (94). This same probe was used to screen BALB/c germ l i n e DNA, and 5 genes were subsequently sequenced. The two that were not pseudogenes shared homology with the NP-b pos i t i v e cDNA probe, but could not code f o r a prote in binding NP (101). When the NP-b probe was compared to NP-a cDNA, 28 (of 98) d i f fe rences were noted (101). Thus the NP-b and NP-a genes are qui te d i f f e r e n t . T suppressor c e l l s that suppress the DTH response in Igh*3 mice can be induced by immunizing C57BL/6 mice with NP-coupled syngenic splenocytes (102). These suppressor T c e l l s are Ig and MHC r e s t r i c t e d (103). It was subsequently found that two populat ions of T c e l l s were involved (104). One populat ion was NP-b id iotype bearing and bound NP (104) but was probably not h e t e r o c l i t i c (102). The other populat ion possessed a n t i - i d i o t y p i c receptors (104). Ear ly r esu l t s showed that NP b ind ing , Ig negative molecules could be p u r i f i e d from sens i t i zed T c e l l s by antigen b ind ing . These molecules were shown to bear the NP-b id io type (105). More recent work has corroborated these f i n d i n g s . A T c e l l hybridoma from B10.BR has been i so l a t ed that binds to NP and possesses NP-b and framework Ig determinants. This c e l l produces a T suppressor f a c to r s p e c i f i c f o r the anti-NP response that i s NP-b p o s i t i v e , I-J pos i t i v e and i s d i s t i n c t from the c e l l surface receptor (106). In v ivo studies with an t i- id io types have shown that monoclonal anti-NP-b id io types can suppress the response to NP-coupled sheep red blood 15 c e l l s (SRBC) (107). This suppression could be mediated by IgG but not IgM or IgD monoclonals, or even F (ab ) 2 fragments (107). In ject ions of small amounts of NP-b pos i t i v e monoclonal antibody ( B l - 8 ) led to an increase in B l - 8 id iotype expression in the anti-NP response (98). Recent work, however, has shown that v i r t u a l l y a l l of the NP-b id io type of T c e l l preparat ions i s due to conventional NP-b pos i t i v e anti-NP antibody t i g h t l y bound to T c e l l s (108). Such resu l t s make fu r the r i n t e rp re ta t i on of the T c e l l i d i o t y p i c response and control of id io type expression d i f f i c u l t . In summary, two major id iotypes expressed in the anti-NP response in mice have been charac te r i zed . These id io types are l inked to d i f f e r e n t Igh a l l e l e s and are probably not a l l e l i c . Some s t ruc tura l information of the NP-b id io type has been prov ided, both at the prote in and gene l e v e l s . The regu la t ion of the anti-NP response by i d i o t y p i c and T c e l l involvement has been i nves t i ga ted , but , unfor tunate ly , some controversy i s encountered in i n te rp re t i ng the r e s u l t s . III. Carbohydrate Antigen Systems A) Dextran This T-independent system has been extens ive ly studied and has revealed the most d e f i n i t i v e information on s t ruc tura l co r re l a tes of i d io t ypy . Two i d i o t y p i c f ami l i e s of dextran binding myeloma ant ibodies have provided much of the informat ion. M104E (IgM,A) has optimal s p e c i f i c i t y f o r the «*(l-3) l i nked dextran t r i s a ccha r ide (109), whi le J558 ( I g A , X ) has s p e c i f i c i t y f o r the pentasaccharide (110). The l i g h t chains of the two myeloma prote ins are iden t i ca l (111) and t h e i r regions d i f f e r only at residues 100 and 101 (112). When mice of Igh a a l l o t ype are immunized with B1355 dextran, they produce ant ibodies bearing 1ight l* ) chain and sharing i d i o t y p i c determinants with J558 (113). This response i s l i nked to 16 the Igh a a l l o t ype (113). Further studies showed that over 50% of these induced ant ibodies share i d i o t y p i c determinants with both J558 and M104E (IdX) (114) and a l l of the serum ant ibodies have homologous l i g h t cha ins , as determined by IEF (115), to the myeloma ant ibod ies . Thus most, i f not a l l , of the information determining i d i o t y p i c s p e c i f i c i t y must l i e in the reg ion . S t ructura l s tudies of many monoclonal ant i-dextran ant ibodies have been undertaken. The presence of arg in ine and ty ros ine at pos i t ions 100 and 101 r e spec t i v e l y , co r re l a te with the ind i v idua l id io type ( Idl ) and binding s p e c i f i c i t y of J558 (112,116), while the presence of ty ros ine and aspar t i c ac id co r re l a te with the Idl and binding s p e c i f i c i t y of M104E (112,116). The e l im ina t ion of pos i t i on 101 destroys both the J558 and M104E Idl determinants (112). IdX determinants co r re l a te with residues at pos i t ions 54 and 55 in the reg ion . Since a carbohydrate moiety i s attached to the asparagine at pos i t i on 55, i t i s tempting to postu late that carbohydrate may have a ro le in de f in ing IdX determinants (112). and sequences have been found to vary a l o t without destroy ing e i t he r dextran binding or IdX s p e c i f i c i t y (112,116). The dextran id iotype resu l t s are s im i l a r to those seen with a n t i - i n u l i n i d i o t ypes . Studies of t h i s T-independent carbohydrate system show that ind i v idua l id iotypes ( Idl ) are probably determined by two amino a c i d s , though IdX determinants are more complex and involve residues ins ide and outs ide the hypervar iable reg ion . Some pa i rs of monoclonal ant ibodies d i f f e r e d by more i d i o t y p i c determinants than amino ac id s u b s t i t u t i o n s , implying conformational e f f e c t s ( i n u l i n reviewed in 64) . Though the s t ruc tura l studies of ant i-dextran id iotypes have u t i l i z e d the T-independent response, a T-dependent response can be studied by using dextran attached to a prote in c a r r i e r (117). When the T-independent and 17 T-dependent responses are observed, one f inds IdX and Idl expression assoc iated f a r more with the T-independent response (118). Thus, the two forms of the dextran antigen t r i gge r d i f f e r e n t B c e l l precursor subpopulations (118). In te res t ing ly enough, the frequency of T-dependent precursors i s almost 3 times greater than the frequency of T-independent precursors (117). In summary, the study of ant i-dextran id io types has been very valuable in de f in ing the ro le of primary s t ructure in r e l a t i on to i d i o t y p i c determinants, though the conformational aspects are s t i l l unknown. Unfor tunate ly , the T-dependent ant i-dextran response i s r a re l y assoc iated with T-independent i d i o t ypes , making studies of regu la t ion dependent on redef in ing ant i-dextran id io types f o r the T-dependent response. B) Streptococcal Group A Antigen When A/J mice are immunized with group A st reptococca l vacc ine , they respond by producing ant ibodies of l im i t ed heterogeneity and bearing a major id io type (119). This i d i o t y p e , designated A5A, was found to be l inked to the Igh a l lo type (120). A/J mice were reported to make from 0 to 60% of a l l ant i-s t reptococca l A (group A carbohydrate = GAC) antibody with A5A determinants (121), and these ant ibodies have l i m i t e d , c h a r a c t e r i s t i c pl values (122). Such serum ant ibodies were i n i t i a l l y found to be predominantly of IgG^g isotype (121), though now i t i s evident that they are usua l l y of IgG^ (123) or IgM (124) isotypes and express l i g h t chains that share p l valves by IEF(125). Other anti-GAC serum cross-react i ve id iotypes have been cha rac te r i zed . The S117 id io type i s seen in the BALB/c anti-GAC response, l inked to the Igh a a l l o t y p e , and appears not to cross-react with the A5A id io type (126). A l s o , in s tudies of .hyperimmune A/J mice, i t was found that monoclonal 18 anti-GAC ant ibodies share the common l i g h t chain spectrotype prev ious ly seen in serum (127). Ant i- id io type made to a monoclonal antibody possessing th i s l i g h t chain (ant i-V K GAC id iotype) reacted with a l l prote ins possessing the V K GAC l i g h t chain (127), even from mice outs ide the Igh e a l l o t ype group (127). Some inves t iga tors are very unhappy with the way in which ant ibodies bearing A5A id io type have been character ized (127). It i s thought that there i s no dominant clone with A5A id io type in the A/J anti-GAC response, because i t i s not a f requent ly occurr ing spectrotype (128). Though d i f f e r e n t anti-A5A reagents detect d i f f e r e n t determinants (125) i t i s not c l e a r that they are on the same molecule (127). Since GAC id io type i s present on 50% of A/J anti-GAC ant ibodies and reacts across Igh b a r r i e r s , i t i s strange that A5A i s only found in the A/J s t r a i n (121). The Igh a l lo t ype l inkage of A5A may be a r e su l t of such l o c i a f f e c t i ng l i g h t chain express ion , thus the A5A id io type may not be a heavy chain marker (129). The anti-A5A reagents are known to react with some determinants (125). Thus i t was thought that in the anti-GAC response, d iverse regions are assoc iated with a r e s t r i c t e d number of regions (127). Some i n t e r es t i ng new experiments have shed more l i g h t on anti-GAC immunoglobulin s t ruc tu re . M u l t i p l e , c l o se l y homologous gene segments contr ibute to the generation of anti-GAC ant ibodies (130). A common framework sequence, re la ted to the V K27 subgroup probably def ines V K GAC (130). As w e l l , the A/J anti-GAC and BALB/c a n t i - i n u l i n regions are 95% homologous at the prote in leve l and are l i k e l y encoded by overlapping gene f ami l i e s (130). Furthermore, in the anti-GAC response, as in other antibody responses (24,66) somatic mutation events amplify the d i v e r s i t y of the mul t ip le heavy chain genes 19 (130) . At present , more experiments are in progress to determine the bas is of the V K GAC id io t ype . Results of experiments done to evaluate the regu la t ion of the A5A i d i o t y p e , may be complicated by work imp l i ca t ing the anti-GAC response to be T-independent (123,124), work showing the response to be T-dependent (131) , and work showing i t to be both (132). The most convincing data ind ica ted thymus dependence (132). Guinea pig ant i se ra were ra ised to p u r i f i e d A5A pos i t i v e A/J anti-GAC ant ibod ies . Mice rece iv ing anti-A5A of IgG2 isotype were suppressed f o r the production of the A5A id io type without concurrent loss of anti-GAC a c t i v i t y (122). Mice rece iv ing anti-A5A of IgG^ isotype had s l i g h t l y enhanced expression of A5A i d i o t y p e , again without a f f e c t i ng the to ta l anti-GAC response (122). If A/J mice were immunized with the guinea pig IgGj anti-A5A i d i o t y p e , the mice could be shown to be sens i t i zed to GAC without antigen (133). Strangely enough, t h i s phenomenon could be observed in C57BL mice, which do not express the A5A id io type in t h e i r anti-GAC response (121). Guinea pig IgG 2 anti-A5A id iotype was observed to induce id iotype s p e c i f i c suppressor T c e l l s (134), probably bearing Lyt-2,3 antigens and suscept ib le to k i l l i n g with anti-A5A and complement ( C ) (135). High doses of the IgG 2 anti-A5A id iotype were assoc iated with short l i v ed suppression (134) whereas lower doses were assoc iated with a much longer l a s t i n g suppression (134) that could be adopt ive ly t rans fe r red with as few as 10^ c e l l s (134). IgG^ anti-A5A was assoc iated with the production of id io type s p e c i f i c T helper c e l l s (133) that were found to respond only to an t i- id io types prepared to id iotypes expressed by the donor s t r a i n (136). This phenomenon was i l l u s t r a t e d by observing the A/J or BALB/c anti-GAC responses. Anti-A5A primed only A/J mice to GAC, whereas 20 anti-S117 primed only BALB/c mice (137), seemingly in con t rad i c t ion to e a r l i e r r e su l t s (121). The A5A helper T c e l l s were only recognized by an t i - id io type having s p e c i f i c i t y f o r region determinants to A5A (125). These experiments suggested that T c e l l s c a r r i ed B c e l l i d i o t y p i c determinants. Since there appears to be some controversy about the s t ructure of B c e l l anti-GAC i d i o t y p e s , perhaps i t would be bet te r to wait f o r c l a r i f i c a t i o n before i n te rp re t ing studies with T c e l l s done with anti-B c e l l i d i o t y p i c reagents. In summary, several anti-GAC id iotypes have been cha rac te r i zed , of which A5A has been the most important. Recent work has descr ibed another major A/J anti-GAC id iotype (V^GAC) which i s assoc iated with a p a r t i c u l a r l i g h t chain fami l y . Further work has cor re la ted the V^GAC id io type with a subgroup s i m i l a r to 27. Molecular genet ic s tudies have shown the presence of mul t ip le heavy chain genes in the A/J mouse genome that can provide the anti-GAC response. These gene products combine with a small number of l i g h t cha ins . These molecular gene t i c i s t s in p a r t i c u l a r have been c r i t i c a l of the e a r l i e r d e f i n i t i o n of the A5A id io t ype . Regulatory studies have been concerned with the A5A system in A/J mice and the S117 system in BALB/c mice. Helper and suppressor T c e l l s have been charac ter ized and some elegant in v ivo experiments have provided data useful to theore t i ca l immunologists (138,139). However, more inves t iga t ions are needed to proper ly def ine the A5A id io t ype . In th i s system, only the s t ruc tura l co r re la tes with id iotype have been d iscovered . C) L ipopolysacchar id Antigens L ipopolysacchar ide (LPS) i s a prote in-free bac te r i a l endotoxin composed of a l i p i d moiety ( L i p id A) and a polysacchar ide moiety, 21 determining the se ro log i ca l c l a s s i f i c a t i o n of the organism (reviewed in 140). Aside from i t s b i o l o g i c a l importance in i n s t i g a t i ng a strong an t i -bac te r i a l response, LPS i s best known to immunologists as a potent po lyc lona l B c e l l a c t i va to r (141); yet the s p e c i f i c immunogenicity of LPS i s very strong (142). Recent work has shown that the immune response to LPS i s gene t i c a l l y con t ro l l ed by genes l inked to the H-2 and to the X-chromosome (143). The memory response character ized by production of IgG ant ibodies to LPS has been mapped to genes l inked to the Igh l o c i in mice (144). Invest igat ions of the i d i o t y p i c responses to LPS have commenced with the product ion of monoclonal anti-LPS ant ibodies from BALB/c mice responding to d i f f e r e n t carbohydrate residues and d i f f e r e n t bac te r i a l LPS ant igens , which share common id io types (IdX) (145). When ant i se ra from mice immunized with LPS from E. c o l i 0113 were s tud i ed , the IdX was seen in ind iv idua l immune sera from a l l s t r a ins of mice tested (146). These resu l t s demonstrate that no a l lo t ype l inkage of LPS id iotypes i s observed in the PFC anti-LPS response. It was a lso found that a minor id io type ( IdZ) , not seen in the primary CE/J or C57BL/6 response, was seen in hyperimmune s e r a , demonstrating some kind of i d i o t y p i c maturation (146). It was thought by these inves t iga tors that shared id io types are common f o r ant ibodies s p e c i f i c f o r an ant igen ic fami ly (145). Unfor tunate ly , these LPS r e su l t s are complicated because i t i s unclear whether IgG or IgM PFC are detected in the hyperimmune anti-LPS responses. I f IgG plaques are being produced by a l l s t r a i n s , e a r l i e r f ind ings of memory l inkage to a l l o t ype are contrad ic ted and i f IgM i s found to undergo i d i o t y p i c maturat ion, then qu i te a l o t of other e a r l i e r work i s contrad ic ted 22 (60,62,66) . Obv ious ly , more experiments have to be done to b iochemica l ly def ine LPS id io type i n t e r s t r a i n c r o s s - r e a c t i v i t y . IV. Polypeptide and prote in Antigen Systems A) (Glutamic ac id 6 0 a lanine 3 0 t y ros ine 1 0 ) Random Terpolymer (GAT) The examination of the immune response to GAT may be thought of as the missing l i n k between studies of hapten and prote in systems. The response to GAT and re la ted random copolymers has been extens ive ly reviewed elsewhere (147), but some key points w i l l be b r i e f l y d i scussed . GAT i s a random copolymer of var ious proport ions of glutamic a c i d , a lanine and t y ros ine . The response to GAT has been l inked with H-2 l o c i (148), but non-H-2 l inked gene in f luence was a lso observed (149). Idiotype studies have been undertaken with GAT even though, i f one assumes a pentapeptide-size determinant as seen with other studies (150,151,152) there could be at l eas t (3)^ or 243 theore t i ca l determinants, ignor ing epitope dominance or pH e f f e c t s . In var ious inbred s t ra ins of mice, GAT immunization caused the production of ant ibodies bearing an i n t e r s t r a i n c ross-react i ve i d io t ype . Those def ined using a guinea pig an t i- id io type are re fe r red to as CGAT (153) whereas those def ined with rabb i t reagents are re fe r red to as pGAT (154). They may be assumed to be de f in ing s i m i l a r i f not i den t i ca l id io types (155). Both CGAT and pGAT can be found in ra t an t i se ra (153,156) and pGAT has been observed in guinea pig anti-GAT sera (156). CGAT can be observed upon immunization of mice with poly (glutamic a c i d , t y ros ine ) (GT) (157), but not with poly (glutamic a c i d , a lanine) (GA) (158). Studies of antibody production (153,154) as well as IEF ana lys is (159) ind ica te that a l l inbred s t r a ins of mice produce CGAT/pGAT (153,154) and th i s may be due to conservat ion of common anti-GAT V„ and V„ germline genes, s ince "responder" and "non-responder" mice made 23 pGAT/CGAT pos i t i v e an t ibod ies . Monoclonal anti-GAT ant ibodies possessing CGAT i d i o t y p i c determinants were character ized (160) as well as some that share i d i o t y p i c s p e c i f i c i t i e s with anti-GA ant ibodies (GA-1 id io type) (161). In a d d i t i o n , mice of I g h - l a , I g h - l c , and I gh- l e a l lo types share a common id io type (srGAT-1) (162) as do mice of Igh b a l lo type (Gte) (163). The Gte id iotype has been mapped to the Igh-V l o c i near Igh-NP, Igh-Nbp and Igh-Bgl markers, and i t s expression i s not l inked to Lyt-3 (Igk) l o c i (164) . As w e l l , ant ibodies uniquely s p e c i f i c to GAT share a c ross-react i ve id io type with ant ibodies to poly (GT) or poly (glutamic a c i d , l y s ine ) (GL) (165) . As can be seen, many types of c ross-reac t i ve id iotypes are assoc iated with the anti-GAT response. The best s t ruc tura l charac te r iza t ions have u t i l i z e d the pGAT/CGAT i d i o t ypes . These s p e c i f i c i t i e s require the i n t e rac t i on of both heavy and l i g h t chains to be expressed (166). The pGAT/CGAT determinants were a lso found to be assoc iated with the antibody combining s i t e , as id io type-ant i- id io type in te rac t ions were i nh ib i t ed with antigen (166). Amino ac id sequence data from monoclonal anti-GAT immunoglobulins have shown that the kappa l i g h t chains from CGAT pos i t i v e ant ibodies are i den t i c a l and def ine a new V K subgroup (167). Analyses of CGAT pos i t i v e heavy chains ind ica te that hybridoma sequences are present in polyc lona l anti-GAT immunoglobulins, pGAT/CGAT s p e c i f i t i e s are assoc iated with heavy chains of l im i t ed heterogeneity and CGAT negative GA-1 pos i t i v e heavy chains are s i m i l a r to CGAT pos i t i v e heavy cha ins , but are assoc iated with a d i f f e r e n t l i g h t chain (155). It i s thought that CGAT and GA-1 i d i o t y p i c determinants are germline markers f o r both and genes (155). 24 Recent data have been obtained at the leve l of the anti-GAT genes. The mRNA encoding the regions (V-D-J genes) of 4 BALB/c pGAT pos i t i v e monoclonal anti-GAT ant ibodies was sequenced, showing that 2 of the monoclonals were iden t i ca l and the other two d i f f e r e n t in 3 and 8 pos i t ions respec t i ve l y (168). When a cDNA region probe from one of the homologous monoclonals was used to inves t igate germ!ine DNA from 3 d i f f e r e n t s t ra ins d i f f e r i n g in Igh a l l o t y p e , h y b r i d i z a t i o n , under s t r ingent cond i t i ons , of Southern b lo t patterns made with var ious r e s t r i c t i o n endonucleases, showed 3-7 non-ident ica l gene fragments (169). These resu l t s ind ica te that a small number of non-ident ica l germ l i n e genes (3 to 7) hybr id ize with the pGAT cDNA probe, suggesting that the anti-GAT reper to i re d i f f e r s between at l eas t 3 s t r a i n s , even though t h e i r anti-GAT ant ibodies bear the pGAT id iotype (169). Recent work, comparing the gene sequences of anti-GAT and anti-NP an t ibod ies , shows that some heavy chains are der ived from the same germ!ine gene - but are assoc iated with d i f f e r e n t l i g h t chain isotypes (170), prov id ing an elegant example of heavy and l i g h t chain combinational assoc ia t ion determining immunoglobulin s p e c i f i t y . Subsequently, the genes were i nves t i ga ted , using the pGAT bearing monoclonals descr ibed prev ious ly (168,169). BALB/c, DBA/2 and C57BL/6 mice were shown to possess 3-5 iden t i ca l Bam HI or Eco Rl Southern b lo t patterns of germline DNA, hybr id ized under s t r ingent condi t ion to a pGAT pos i t i v e DNA probe (171). Further study of V^ , regions (V-J genes) revealed that 3 germline genes are present in a l l s t r a i n s , but that a given s t r a i n may not use a l l of them fo r the anti-GAT response. A l l of these regions are very s i m i l a r in primary s t ructure (172). The ro le of the CGAT/pGAT id iotypes in the T c e l l response has been s tud ied . When mice of genet ic "non-responder" status are immunized with 25 GAT, they produce T suppressor c e l l s from which f a c to r may be i so l a t ed (173). Th is f a c to r binds to GAT, bears I-J but not Igh or Igl constant region determinants (174) and can st imulate the in v i t r o generation of T g 2 c e l l s in "responder" s t r a ins (175). This T $ F has been shown to express the CGAT id io type (176) and i s now being produced by T c e l l hybridomas (177). Another T s F , produced by a "responder" s t r a i n has been immortalized in a T c e l l hybridoma and possesses propert ies s im i l a r to T g F produced by "non-responder" s t ra ins (178). The i d i o t y p i c p r o f i l e of these e legant ly p u r i f i e d monoclonal T suppressor fac tors (178,179) has not ye t been publ ished. The presence of CGAT on helper T c e l l l i nes has recent ly been es t ab l i shed . Syngeneic an t i- id io type that detects CGAT determinants produced by an anti-GAT monoclonal antibody w i l l s p e c i f i c a l l y i n h i b i t help to an in v i t r o GAT-DNP (d in i t rophenol ) response by two T helper c e l l l i nes (179). Molecular genet ic • ana lys i s of 10 GAT-speci f ic suppressor T c e l l hybridomas (6 of which secrete fac tors with CGAT s p e c i f i c i t i e s ) and 3 GAT-spec i f ic helper T c e l l l i nes or hybridomas, has shown that T and B c e l l s that recognize the same antigen do not t ransc r ibe s im i l a r heavy chain va r i ab le region gene segments, with a s e n s i t i v i t y of 1-2 mRNA copies per hybridoma c e l l and 5-10 mRNA copies per T c e l l l i n e c e l l (180). In f a c t , one suppressor T c e l l hybrid producing antigen s p e c i f i c f a c to r was found to produce below the minimum detectable l i m i t s (10 cop i e s/ce l l ) of any IgV^ gene, with a p r o b a b i l i t y of detect ion greater than 99% (181). In summary, the immune response to GAT i s under the contro l of several genes, though a l l mice can produce anti-GAT ant ibod ies . These ant ibodies exh ib i t i d i o t y p i c determinants, the best character ized of which are 26 CGAT/pGAT. Both B and T c e l l s may express CGAT determinants. Molecular genet ic ana lys i s of B c e l l s has shown that there are 3-7 anti-GAT-1ike germline genes, that d i f f e r between s t r a i n s . One of these may code f o r both NP or GAT b ind ing , depending on i t s products assoc ia t ion with a d i f f e r e n t l i g h t cha in . There are probably only 3 anti-GAT l i g h t chain genes conserved between s t r a i n s . T c e l l s expressing CGAT/pGAT i d i o t y p i c determinants do not express genes. As of y e t , no s t ruc tu ra l co r r e l a t i on between anti-GAT id iotypes and amino ac id sequence of e i t he r anti-GAT H or L chains has been shown. B) Myoglobins The mammalian myoglobins have been of immunological i n t e r es t f o r the same reasons as the lysozymes. Myoglobins are sma l l , easy to obtain and pur i f y and nature has k ind ly provided many chemical var iants whose st ructures have, in many cases , been determined (reviewed in 182). Studies with rabb i t and goat an t i se ra showed that there are 5 major ant igen ic regions and that ant ibodies are d i rec ted to conformational determinants (150,182). These studies were confirmed in mice at both B and T c e l l l e ve l s (183). Ear ly work a lso showed that the an t i gen i c i t y of a s t ruc tu ra l region of sperm whale myoglobin (SWM) i s re la ted only to the s t ruc tura l l oca t ion and not the d i f f e rence between SWM and other myoglobins (183). Further work showed that the response to d i f f e r e n t determinants of SWM does not change over the time course of the response (184). Thus, one would not expect to see a d i f f e rence between epitope recogni t ion by ear l y ant i sera (such as used by Atass i to def ine SWM) (182) and ant ibodies produced l a t e r in the response. S u r p r i s i n g l y , when monoclonal ant ibodies were used, (185,186), the ant igen ic determinants recognized were vas t l y d i f f e r e n t from those observed e a r l i e r (150). These resu l t s were confirmed independently 27 with human myoglobin (187). A l l of the "new" B c e l l epitopes were topograph ica l . Experiments to charac ter ize the T c e l l response i n i t i a l l y revealed that T c e l l s recognized the same s t ruc tura l regions of SWM as B c e l l s , but that T c e l l s recognized l i n e a r instead of conformational determinants (183). Later i t was suggested, based on work with T c e l l c l ones , that T c e l l s were incapable of binding conformational determinants (188), although th i s hypothesis was shown to be wrong, as some T c e l l s were found to require a conformational determinant both in v i t r o (189) and in v ivo (190). Thus, there may not be a separat ion based on epitope recogn i t ion of T c e l l s and B c e l l s in the SWM response. The genet ic response to SWM has been c a r e f u l l y reviewed elsewhere (147). Ear ly s tudies showed that both H-2 and non-H-2 genes con t ro l l ed responsiveness to SWM in mice (191), but the H-2 l inked regu la t ion could be overcome with high doses of antigen (192). It a l so appears that the chemical proper t ies of the ant igen ic s i t e can determine the genet ic control of the response to a p a r t i c u l a r myoglobin (193). Idiotype has only recent ly been studied in the myoglobin system. Monoclonal ant ibodies were used to induce the formation of a n t i - i d i o t y p i c an t i se ra to each monoclonal in guinea p igs . The resu l t s showed that high a f f i n i t y monoclonal ant ibodies to d i f f e r e n t determinants, cross-reacted with respect to id io type (194). Further data eva luat ing the i d i o t y p i c response in the myoglobin system are expected soon. The myoglobin system (notably SWM) has been extremely well charac te r ized with regard to epitope s t ruc tu r e , genet ics of responsiveness, T and B c e l l i n te rac t ions and recent work has involved studies of the presence of c ross-react ive id io types on anti-SWM ant ibod ies . 28 Unfor tunate ly , the determinants assoc iated with myoglobin are almost a l l conformational and many e x i s t . The genet ics of responsiveness are complex, and the id iotypes of ant ibodies to d i f f e r e n t determinants c ross-react . Thus, i t may be d i f f i c u l t to obtain d e f i n i t i v e data on the ro le of id io types in the myoglobin system. C) Lysozymes The lysozymes ex i s t in nature as a number of r e l a t e d , but d i s t i n c t molecules. Such a s i t ua t i on i s very useful immunological ly , s ince response to molecules d i f f e r i n g only in a few chemical ly (and sometimes conformat ional ly ) def ined loca t ions can be s tud ied . Studies of the epitopes of hen egg-white lysozyme (HEL) o r i g i n a l l y revealed 3 ant igen ic conformational s i t e s , as def ined by rabbi t and goat primary ant ibodies (although e s s e n t i a l l y a l l of them were IgG) (195). Subsequent inves t iga t ions have discovered many other determinants assoc iated with HEL as def ined by heterologous (196,197) or monoclonal ant ibodies (198). These determinants seem to be predominantly conformat ional . Genetic s tudies of the response to the ga l l inaceous egg-white lysozymes showed that H-2 l inked genes control the response s t a tus , while non-H-2 l inked genes control the magnitude of the hyperimmune response (199). T and B c e l l s recognize several epitopes and the HEL response i s regulated through an antigen br idg ing mechanism (200). Some T and B determinants are located on the same fragments of HEL (200,202,203,204) and helper and suppressor T c e l l s were thought to recognize d i f f e r e n t determinants in non-responder mice (202). The model of antigen br idg ing between T and B c e l l s recogniz ing d i f f e r e n t determinants has recent ly been independently confirmed (205). 29 Experiments with responder sera show that there i s a major i n t e r s t r a i n c ross-reac t i ve i d i o t y p e , whose expression does not co r re l a te with H-2 or Igh a l l o t ype (206). Only serum ant ibodies s p e c i f i c f o r the NC-HEL fragment express th i s id iotype (IdX) (206), but monoclonal anti-HEL an t ibod i es , s p e c i f i c f o r d i f f e r e n t ep i topes , a lso express IdX determinants (207,208). Further studies showed that IdX determinant-bearing ant ibodies replace an IdX negative populat ion during maturation of the anti-HEL response, implying a se l e c t i v e mechanism fo r id iotype expression (208). This observat ion was confirmed u t i l i z i n g hybridomas from fus ions ca r r i ed out on HEL immune splenocytes at var ious times during the anti-HEL response (208,209). Studies of IdX expression at the T c e l l l eve l have provided several i n t e r es t i ng observat ions . U t i l i z i n g several B10 recombinant s t r a i n s , T suppressor c e l l s in nonresponders s p e c i f i c f o r HEL may be induced by immunizing with an t i - id io t ype . Conversely , anti-HEL T suppressor c e l l s may be k i l l e d with anti-IdX and complement (203). Thus, T suppressor c e l l s and B c e l l s can share IdX, and a T c e l l l i n e producing a f a c to r suppressing the primary and secondary anti-HEL responses vn v ivo has been i s o l a t e d . The f a c to r w i l l not suppress the response to a very c l o se l y re la ted lysozyme (ringneck pheasant lysozyme) nor the LJJ fragment of HEL, thus demonstrating f i ne epitope s p e c i f i c i t y (210). Subsequently, the epitope s p e c i f i c i t y was narrowed to the phenylalanine residue at pos i t i on 3 of HEL and the f a c to r was shown to be both Igh and H-2 r e s t r i c t e d , although the B c e l l response i s not so r e s t r i c t e d (206,211). Studies of the i d i o t y p i c nature o f t h i s f a c to r are expected soon. Thus, the lysozymes, most notably HEL, are sma l l , re la ted prote ins that provide many conformational determinants f o r the B c e l l response, 30 although the T c e l l s may recognize l i n e a r determinants (204). Careful studies have determined the genet ic response and the ro le of T and B c e l l s in the anti-HEL response. A major c rossreac t i ve id iotype that i s seen in the mature anti-HEL response of many diverse s t r a i n s , and i s expressed by both B c e l l s recogniz ing d i f f e r e n t HEL ep i topes , and T c e l l s has been cha rac te r i zed . Recent work, not yet ava i l ab le in the publ ished l i t e r a t u r e , may shed more l i g h t on the anti-HEL IdX system (212). D) Staphylococcal Nuclease Staphylococcal nuclease (Nase) i s an enzyme i so l a ted from cu l tures of Staphylococcus aureus, whose s t ructure has been well character ized (213). Since Nase has an enzymatic a c t i v i t y , ea r l y studies used to assay antibody binding by the drop in Nase a c t i v i t y (214). Studies of Nase B c e l l epitopes showed that most were assoc iated with the conformation of the antigen (215), making i t very d i f f i c u l t to def ine the s t ructure of determinants. T c e l l s appear to be less r e s t r i c t e d , and respond well to Nase fragments a f t e r Nase priming (214). The response to Nase in mice has been shown to be gene t i c a l l y c o n t r o l l e d , at the leve l of the primary response, by H-2 l inked genes (216), although the hyperimmune l eve l s d id not co r re l a te with e i t he r the H-2 or Igh l o c i (217). Later work showed that the Ir genes were a f f e c t i ng the determinants s p e c i f i c i t y of the anti-Nase response (218) and non-H-2 l inked genes con t ro l l ed the magnitude of the response (217). Further experiments character ized the genet ics of anti-Nase id iotype express ion . The presence of A/J H-2 l inked genes was not necessary f o r the expression of id iotypes c ross-react ive with A/J anti-Nase i d i o t ypes , in s tudies with congenic recombinants (219); the H-2 genes were necessary f o r the determinant response to Nase (218). The A/J and SJL anti-Nase 31 responses both exh ib i ted major i n t r a s t r a i n c ross-react ive i d i o t ypes , but the two id iotypes d id not share s p e c i f i c i t i e s and were subsequently found to be l inked to t h e i r respect ive Igh l o c i (219). BALB/c mice expressed both id iotypes in t he i r anti-Nase responses (219). Further i nves t iga t ion charac ter ized 5 separate id iotypes in the Nase response (220). These markers were shown to map to d i f f e r e n t genes and were useful in detect ing the V^-C^ recombination event observed in the BAB.14 s t r a i n (220). Various studies of the regulatory proper t ies of anti-Nase ant ibodies have been undertaken. BALB/c mice t reated with pig anti-BALB/c id io type w i l l produce id iotype pos i t i v e antibody that w i l l not bind Nase (221). These mice were found to contain an t igen-spec i f i c helper T c e l l s that were suscept ib le to an t i- id io type and complement, and were s i m i l a r to those induced by priming with Nase (221, 222). A s i m i l a r s i t ua t i on occurred in the A/J anti-Nase i d i o t y p i c system (223). In terest ing experiments in the BALB/c system u t i l i z e d recombinant s t r a i n s . Pig (anti-BALB/c id io type) an t i se ra and complement k i l l e d only T helper c e l l s from BALB/c and not B10.D2 mice (224). As w e l l , B10.D2 animals d id not produce anti-Nase ant ibodies of BALB/c i d io t ype . Yet B10.D2 mice given pig anti-BALB/c id iotype produced non-Nase b ind ing , BALB/c id iotype pos i t i v e an t ibod ies , and id io type p o s i t i v e , antigen s p e c i f i c T helper c e l l s (224). Thus, id io types and binding s i t e s could be d i s t ingu ished at the B c e l l , but not at the T c e l l l e v e l . In summary, the Staphylococcal nuclease system exh ib i t s qu i te complex genet ic mechanisms of response. T and B c e l l determinant recogn i t ion seem to be d i f f e r e n t , with B c e l l s responding to a va r i e t y of conformational determinants. At l eas t 5 d i f f e r e n t id iotypes are seen in the murine 32 anti-Nase response, which a l l map to regions l inked to Igh l o c i . T helper c e l l s , bearing some of these id iotypes have been character ized and some elegant experiments have shown d i f f e r e n t modes of id iotype expression between B and T c e l l s . Unfor tunate ly , the s t ructures of B and T c e l l id io types in the Nase system are unknown, and id io type r egu l a t i on , which has not ye t been studied to any great extent , appears to be extremely complex. V. Introduct ion Summary A) Hapten Systems Idiotype analyses invo lv ing responses to haptens probably represent the s implest ava i l ab le systems. Haptens are small antigens and w i l l have only one ep i tope . A l s o , the immune response to haptens i s not usua l l y con t ro l l ed by H-2 l inked genes. One may use haptens with T-dependent (11) and T-independent (38) c a r r i e r s to control f o r c a r r i e r e f f e c t s . Many important observat ions have been made about i d i o t y p i c s t ructure and r egu l a t i on , u t i l i z i n g hapten systems. The best character ized of these are the ARS, PC and NP systems, and these have already been d i scussed . Pert inent work has been done with other haptens. 2-Phenyloxazolone (phOx) on a prote in c a r r i e r causes the production of large amounts of ant ibodies bearing s p e c i f i c id io type in BALB/c and DBA/2 mice (225) as def ined by rabb i t an t i - id io type and IEF. This marker (Ox-1) was l inked to the BALB/c Igh a l lo t ype in studies with recombinant mice (225), though Ox-l was expressed in the anti-phOx response of 5 s t ra ins of varying a l lo type (BALB/c, DBA, A/J , SM, RIII) (225). Monoclonal anti-phOx ant ibodies made at d i f f e r e n t times during the primary response were tested f o r i d i o t y p i c determinants recognized by conventional an t i- id io type (225) or sera recogniz ing anti-phOx heavy, and normal cha in , recombinant an t ibod ies . 33 Strangely enough, no monoclonal was recognized by the conventional a n t i - i d i o t y p e , whereas almost a l l day 7 but no day 14 monoclonals reacted with the an t i - ( reconst i tu ted antibody) id iotype (226). mRNAs of the and regions of three id io type pos i t i v e and one id io type negative monoclonals have been sequenced, and consensus V^QX J and j germline genes have been def ined (226). More work in th i s system i s forthcoming. Another useful system is the study of the response to d in i t rophenyl hapten (DNP). The presence of i d i o t y p i c determinants of the DNP-binding myeloma prote in M-460 were seen in the sera of immune mice (227). The expression of M-460 id iotype was cor re la ted with the Igh a a l lo type using BALB/c recombinants (227). C loser sc ru t iny revealed that some M-460 determinant expression was l inked to l o c i (228). The expression of M-460 id io type in the anti-DNP response i s t r ans ien t and regulated independently of the anti-DNP response (229). Pre-immunization of BALB/c mice with syngeneic anti-M-460 id io type w i l l enhance the production of M-460 determinants (230). Thus, the expression of the M-460 id io type i s probably assoc iated with genes l inked to both Igh and Igk l o c i . Studies of the T c e l l anti-DNP response w i l l not be reviewed here. In summary, the majority of i d i o t y p i c studies have been done in the ARS, PC, NP, ph(Ox) and DNP hapten systems. In the majority of these cases , i d i o t y p i c determinants appear to be Igh germline markers, although Igk c e r t a i n l y can play a ro le in i d i o t y p i c express ion. Some of these germline genes are shared by mice of d i f f e r i n g Igh a l l o t y p e . Molecular genet ic s tudies have shown that somatic mutation plays a major ro le in de f in ing antigen binding or id io type s p e c i f i c i t y of an expressed germline gene, but the s t ruc tu ra l co r re la tes of id io typy are poor ly def ined by hapten systems as of y e t . Much data concerning immune regu la t ion have been 34 obtained by the study of id io type an t i- id io type in te rac t ions in hapten systems. Despite these inves t iga t ions of i d i o t ypy , even the s implest mechanisms of i d i o t y p i c regu la t ion are l a rge l y undef ined. Some object ions to the use of haptens may be that while being very useful t o o l s , they represent qui te abnormal ant igen ic s t ructures (with the exception of PC), the immune responses to them may be too simple in comparison to the responses to more complex, natural ant igens , and s ince they are small molecules (231), they may be r ead i l y bound by antibody s p e c i f i c f o r other ant igens. Thus, while the information forthcoming from i d i o t y p i c s tudies may be very re levant in de f in ing many aspects of id io type expression and r egu l a t i on , i t should be kept in mind that any comprehensive conclusions regarding th i s element of immune responsiveness must inc lude comparable data der ived from more complex ant igens. B) Carbohydrate Antigen Systems One reason that simple carbohydrate systems such as dextran, i n u l i n and galactan are studied with regard to id io typy i s due to the ear ly i s o l a t i o n of myeloma prote ins with s p e c i f i c i t y f o r simple carbohydrate moie t i es . Carbohydrate determinants in polymers are n a t u r a l , large enough to f i l l the binding s i t e of ant ibodies (109,110), are e a s i l y ava i l ab le (except f o r GAC) independently of major regulatory in f luences from T c e l l s in t he i r nat ive form. The responses to LPS or GAC cons t i tu te a natural and high leve l of response which i s important f o r the host defence against ba c t e r i a . The more simple carbohydrates studied immunologically often are found on some part of the bac te r i a l c e l l w a l l , outer membrane, or capsule . The i d i o t y p i c response to simple carbohydrates i s often very r e s t r i c t e d , hence the i n t e res t in dextrans, i n u l i n s , and ga lactans . As has been shown, the dextran and i n u l i n systems have provided very useful 35 information about the s t ructure of i d i o t y p i c determinants at the primary s t ruc ture or genet ic l e v e l s . The galactan system has been used more to answer questions regarding antigen binding (reviewed extens ive ly in 64) . Very elegant computer s imulat ions of changes in prote in s t ructure assoc iated with antigen b ind ing , and the d i f f e r e n t forms of ant i-galactan ant ibodies have been u t i l i z e d (232). L a t e l y , ant i-galactan hybridoma ant ibodies have been character ized in terms of t h e i r i d i o t y p i c determinants (233). The ant ibodies studied had v i r t u a l l y i den t i ca l V^ reg ions , but d i f f e r e d at with no obvious pa t te rn . Thus, l i g h t chains probably contr ibute l i t t l e to the actual s t ructure of the def ined ant i-galactan i d io topes . Several co r re l a t i ons between id iotopes and primary sequence have been character ized (233) in the complementarity determining regions (CDR) of the H chain V reg ion . The e f f e c t of these residues on the t e r t i a r y s t ructure has not yet been de f ined . Thus, the simple carbohydrates have been very useful in the study of antigen binding and i d i o t y p i c determinants at the s t ruc tu ra l l e v e l . More complicated carbohydrates such as LPS and GAC, as well as DNA have a lso been s tud ied . As prev ious ly d i s cussed , the LPS and GAC antigens st imulate a complex immune response, due to regulatory in f luences which have not been f u l l y de f ined . In these systems s t ruc tura l work i s progress ing . In the GAC system, no concrete evidence de f in ing i d i o t y p i c determinants i s obv ious, though the V^GAC id iotype i s associated with a s p e c i f i c V^ sequence (130). Mu l t ip l e heavy chains encode the anti-GAC response (130). The LPS system has revealed some i n t e r es t i ng data regarding an i n t e r s t r a i n c ross-react i ve i d i o t y p e , but more work i s needed. Anti-DNA id io types have been invest igated in several autoimmune model systems. In at l eas t three independent systems (234,235,236) autoimmune 36 anti-DNA ant ibodies share a dominant i d i o t y p i c marker. These systems may be very useful in studying i d i o t y p i c regulatory phenomena. Thus, in sp i t e of some s t ruc tu ra l in format ion , the complex carbohydrate systems have been more useful in descr ib ing regulatory phenomena. In summary, carbohydrate systems have been provided much s t ruc tura l and regulatory information on id io t ypes . Carbohydrates usua l l y represent natural antigens of r e l a t i v e l y well def ined epitope s t ructure and are f requent ly f ree of T c e l l d i rec ted r egu la t i on . The major disadvantage i s that these systems often cannot be used to inves t iga te T c e l l mediated regulatory phenomena, though one can conjugate a carbohydrate determinant to a c a r r i e r p ro t e i n . In at l eas t one of these systems (117) the i d i o t y p i c reper to i re d i f f e r s between the T-dependent and T-independent responses. C) Polypeptide and Prote in Antigen Systems Proteins have many epitopes and are usua l l y T c e l l dependent. The disadvantage of using a prote in in studies of id iotype expression i s that the immune response to them i s very complex. As a compromise, a r t i f i c i a l polypeptides such as GAT, or poly-L-(Tyr , Glu)-poly-D,L-(Ala)-poly-L-( lys) (TGAL) have been used to t h e o r e t i c a l l y l i m i t the number and conformation of epitopes and to al low easy s t ruc tura l changes in the ant igen. These polypeptides have y i e lded some i n t e r es t i ng data on id io t ypy . The study of GAT, as desc r ibed , has provided s t ruc tura l and genet ic information about ant i-pept ide i d i o t ypes , as well as information on i d i o t y p i c r egu l a t i on . The study of TGAL and other synthet ic polypept ide antigens has y i e lded data concerning immune responsiveness and c e l l u l a r cooperat ion (extens ive ly reviewed in 147 and 237). TGAL has a lso been used in i d i o t y p i c s tud ies . Two s t r a i n s p e c i f i c id iotypes have been found in the anti-TGAL response (BIO and C3H.SW id io types ) (238). The major i ty of these i d i o t y p i c 37 determinants cross-react with those seen in the anti-GAT response (239). This was not observed e a r l i e r due to the poor s e n s i t i v i t y of the assay (238). Some monoclonal ant ibodies recogniz ing only TGAL have been i s o l a t e d . None of these hybridoma ant ibodies (of 22) def ined a l l of the serum i d i o t y p i c determinants, implying that no s ing le s t ruc tu ra l gene i s respons ib le f o r the predominant TGAL i d i o t y p i c fami ly (240). Thus, of the synthet i c polypeptide systems used to study i d i o t y p e s , the anti-GAT response i s the best de f ined . Unfor tunate ly , most of the id iotypes s tud i ed , even to d i f f e r e n t synthet ic po lypept ides , c ross-react ex tens ive l y . Immunologists in te res ted in a cha l lenging occupation have studied the i d i o t y p i c responses to p ro te ins . So many of these systems ex i s t that i t would be impossible to t r y to review them a l l . The most s tud i ed , or the systems with the greatest potent ia l are those invo lv ing the responses to staphylococcal nuc lease , hen egg-white lysozyme, sperm whale myoglobin and C los t r id ium pasteurianum fer redoxin (Fd). Ferredoxin w i l l be discussed in depth l a t e r . The studies of prote in epitopes have been done in excruc ia t ing de ta i l f o r SWM and HEL by Atass i (182,195). Many epitopes e x i s t , even f o r these r e l a t i v e l y low molecular weight p ro te ins . The majority of these determinants appear to be conformat ional . As w e l l , the immune responses to these prote ins appear to be con t ro l l ed by a number of r e l a t i v e l y i l l def ined genes. There seem to be major id iotypes expressed in the immune responses to these p ro t e i n s , but ant ibodies to d i f f e r e n t epitopes have been reported to bear the same i d i o t y p i c determinants. It i s a very complicated business to accurate ly study i d i o t y p i c regu la t ion when many prote in epitopes may be invo lved . The i d i o t y p i c response to Nase i s probably the best def ined of the natural prote in systems. The ant igen ic determinants of 38 Nase and the genet ic control of the anti-Nase response have been s tud i ed , though not in the f i ne de ta i l of the HEL or SWM systems. The i d i o t y p i c response has been r e l a t i v e l y well s tud ied . Major c ross-react i ve id io types have been produced in the response to Nase and f i v e of these have been mapped to separate l o c i . The molecular genet ic studies have not yet been pub l i shed . In a d d i t i o n , the regulatory proper t ies of the anti-Nase id iotypes have been studied and show an i n t e res t i ng dichotomy between id io type bearing T and B c e l l r epe r to i r e s . Recent ly , i nves t iga t ions of id io types produced during the immune responses to other prote ins have added some i n t e r es t i ng data . Such work has character ized id iotypes in the anti-H-2 antigen responses. Monoclonal ant ibodies s p e c i f i c f o r H-2 molecules were produced and used to prepare e i t he r pig or rabb i t an t i- id io types (241). Mice preimmunized with an t i- id io type produced up to 65% id iotype pos i t i v e ant ibodies when immunized with H-2K ant igen , compared to undetectable l eve l s seen in conventional BALB/c a l loant iserum (241). This enhancement of id io type expression i s probably due to the production of id io type s p e c i f i c helper T c e l l s (242) by the in v ivo treatment with an t i - i d i o t ype . As w e l l , the cha rac te r i za t ion of the monoclonal anti-H-2K id io types showed that most were assoc iated with the H chain (243). This system has potent ia l as an important model of idiotype-MHC immune r egu l a t i on , but the potent ia l f o r useful s t ruc tu ra l information on id io types seems l i m i t e d . Another useful system appears to be that of bovine i n s u l i n (BI). The epitope s t ructure and responder studies of BI have been reviewed extens ive ly elsewhere (147). IEF experiments have shown that ind iv idua l mice responding to BI present complex spectrotypes that are not conserved between ind i v idua l s (244). Monoclonal ant ibodies against BI were used to 39 immunize guinea p i g s . Two of these were found to def ine pub l i c id io types (244). One was found in 17% of BALB/c sera and was Igh a -1inked, but could a lso be found in DBA/2 mice. The other id iotype i s found in ( Igh)a, b and c mice (244). Within each group, members could be found that bound to d i f f e r e n t determinants of BI (244). Thus, the i n s u l i n system appears to be qu i te complex with regard to id iotype express ion . In summary, the immune response to many prote ins has been studied ( for a pa r t i a l l i s t see 145), though only a few have been well character ized at the leve l of the i d io t ype . Since prote ins induce complex responses, the best systems are those in which the genet ics of response and epitope s t ructure are well cha rac te r i zed . Such systems include the responses to GAT, Nase, HEL, SWM. and now BI. I dea l l y , the best system should u t i l i z e a prote in that i s : a) fo re ign to mammals so that no a r t i f a c t s due to ubiqui t iousness are observed; b) small in s i z e ; c) e a s i l y obtained or inexpensive, with no tox i c p rope r t i e s ; d) well character ized and simple with regard to immunogenicity. Such a system i s the C. pasteurianum ferredoxin (Fd) system. VI. Review of the Ferredoxin System The fer redoxins are a large group of e lec t ron t ransport prote ins found in bac te r ia and p l an t s . The f i r s t fe r redox in was discovered in C los t r id ium  pasteurianum in 1962 (245). The funct ion of Fd i s that of an e lec t ron c a r r i e r . Fd accepts e lec t rons from an enzyme-catalyzed ox idat ion and i s in turn ox id ized by another enzyme involved in e lec t ron t ranspor t . As such, Fd can pa r t i c i pa t e in many d i f f e r e n t react ions (246). C. pasteurianum Fd has 55 amino a c i d s , inc lud ing 8 cyste ine residues used to chelate i r o n . Although many types of Fd e x i s t , C. pasteurianum Fd i s re la ted to several other bac te r i a l fer redoxins (247). The best studied of these i s the 40 fer redox in of Micrococcus aerogenes. Since i t appears that C. pasteurianum Fd has not been studied at the leve l of the t e r t i a r y s t ru c tu r e , but i s s i m i l a r to the M. aerogenes fer redoxin sequence (247), i t w i l l be assumed that i t s t e r t i a r y s t ructure i s a lso s im i l a r to that of M. aerogenes Fd. S t r u c t u r a l l y , t h i s fer redoxin molecule cons is ts of two s im i l a r Fe^S^ ac t i ve s i t e s which are about 1.2 mm apart . The ac t i ve s i t e s are buried in a hydrophobic environment, and communicate through the ty ros ine residue to the aqueous environment (248). The molecule can be descr ibed as a pro late e l l i p s o i d with r ad i i of 2.2 and 2.7 nm. The major ax is i s p a r a l l e l to the l i n e between the two Fe^S^ centers . In the s t r u c tu r e , an approximate 2-fold axis re la tes the two halves of the molecule, with the amino and carboxy termini in c lose proximity . Needless to say, the charges of the termini should ensure t h e i r pos i t i on on the hydroph i l i c ex t e r i o r of the prote in (248). Due to i t s small s i z e , and r e l a t i v e l y simple s t ruc tu r e , C. pasteurianum Fd has been used as an immunological probe in our laboratory f o r many years . In order to determine the epitopes of Fd , several small peptides were synthesized and tested f o r t h e i r r e l a t i on to epitopes of nat ive Fd in a competit ion assay. These studies showed that the amino terminal heptapeptide and the carboxy terminal pentapeptide of the molecule const i tu ted two major ant igen ic determinants of ox id ized Fd (249). Further 14 s t u d i e s , using C-acetylated peptides in equ i l ib r ium d i a l y s i s es tab l i shed that these two peptides accounted f o r e s s e n t i a l l y a l l antibody synthesized in rabb i ts to ox id ized Fd (151). Subsequently, these resu l t s were confirmed with inbred mice (250). The primary s t ructure of Fd i s shown in Appendix 3. Further work showed that both the magnitude (251) and 41 determinant s e l e c t i v i t y (250) of the anti-Fd response in inbred mice were l inked to the genes of the H-2-K/IA region of the mouse MHC. Idiotypes present in the Fd system have only recent ly been inves t iga ted . Two monoclonal ant ibodies have been produced, and experiments have character ized the expression of these id iotypes in the serum anti-Fd response. One of these an t ibod ies , Fd-1 (amino determinant s p e c i f i c ) was shown to represent i d i o t y p i c determinants shared by the amino determinant s p e c i f i c ant ibodies of about ha l f of the B10.BR mice tested (252). Subsequently, t h i s id iotype was found only in sera produced by mice of Igh*3 a l lo type (253). Ce l l mixing studies a lso showed an inf luence of anti-Fd-1 on T c e l l s (253). Another monoclonal ant ibody, Fd-B2, which i s carboxyl determinant s p e c i f i c has been e spec i a l l y well s tud ied . The id iotype of Fd-B2 i s not represented in the anti-Fd response of B10.BR mice (254). Primed T c e l l s t reated with anti-Fd-B2 and complement were associated with a large increase in ant i-Fd production when t rans fe r red to i r r ad i a t ed B10.BR rec ip i en t s and boosted. There was no increase in Fd-B2 id io t ype . Very i n t e r es t i ng resu l t s were obtained when the same experiments were done with B10.D2 non-responder mice. The r e c ip i en t mice were capable of responding to Fd , but again no Fd-B2 id io type was observed in the response (254). Subsequent study showed that T c e l l id io type an t i- id io type in te rac t ions were invo lved , and that id iotype-bear ing molecules were ne i ther H-2 or Igh l i n k e d . The an t i- id io type was shown to recognize an Lyt-1 pos i t i v e T c e l l populat ion (255). Further work i s in progress . In sp i t e of the extensive study of the anti-Fd response in mice, e s s e n t i a l l y nothing i s known about the serum i d i o t y p i c response. In order to def ine immune regu la t ion in th i s system, such knowledge i s requ i red . 42 The Fd system is admirably su i ted f o r these s tud i e s , as the Fd molecule i s very s imple , possessing the minimum number of determinants necessary f o r an immune response, and the response i s almost unideterminant in high responder s t r a i n s , regardless of t h e i r Igh a l lo type (250). There fore , studies to def ine major id io types in the serum anti-Fd response of several s t ra ins of mice were i n i t i a t e d . 43 Chapter 2. Mater ia ls and Methods I. Immunological Methods A) Ferredoxin and Keyhole Limpet Hemocyanin Antibody EL ISA 1) ELISA The enzyme l inked immunosorbent assay as descr ibed elsewhere (256) was used to detect the antibody responses to Fd and KLH in mice. This assay depends on the i r r e v e r s i b l e binding of the antigen to a polystyrene p l a t e . Antibody i s added and binds to the antigen on the p l a t e . At th i s stage the antibody may be i nh ib i t ed from binding to the p late by an t i- id io type which binds to the antigen binding s i t e . The antibody that attaches to the p late may be detected by the add i t ion of an enzyme l abe l l ed reagent s p e c i f i c f o r the bound ant ibody. When a substrate i s added to the enzyme, the amount of bound antibody may be quant i tated by the amount of enzymatic a c t i v i t y from the detect ing reagent. The technica l de t a i l s of the use of th i s assay in th i s thes i s are as fo l l ows : Polystrene substrate p lates (Dynatech Immulon 1) were coated with 0.1 ml per well of 1.0 mg/ml of antigen in carbonate coat ing bu f f e r . The plates were incubated approximately 12 hours at 37°C in a humidif ied incubator . A f t e r washing at l eas t twice with PBS-Tween, antibody to KLH or Fd , d i l u t ed in PBS-Tween, was added at 0.1 ml/well of s o l u t i o n . The plates were l e f t at room temperature fo r 1-2 hours. 0.1 ml of a d i l u t i o n of rabb i t anti-mouse Ig (RaMIg) conjugated to a l ka l i ne phosphatase was added a f t e r 3 washes with PBS-Tween and the p lates were l e f t again 1-2 hours. A f t e r 3 more washes, the substrate (2 tab le ts of Sigma #104-105 per 10 ml of diethanolamine buf fe r pH9.8) was added and co lo r allowed to develop at room temperature. Color development was quant i tated at 405 mm with a 44 T i t r e t ek Mult iscan (Flow ). Fd was e i t he r a g i f t from Dr. L. S ikora ( pu r i f i ed as descr ibed) (257) or purchased (Sigma F7629) and KLH was purchased from Calbiochem (374805). Sheep an t i- rabb i t immunoglobulin (SaRIg) conjugated to a l ka l i ne phosphatase was a g i f t from Agnes Chan (prepared as described) (256) and was used to quant i fy the amount of RaMIg in rabbi t se ra . The RaMIg - a l ka l i ne phosphatase used was g i f t of Rahesh Singhai (prepared as descr ibed) (256). 2) I nh ib i t ion Assays f o r Idiotype Expression a) Pooled Sera Pooled sera from mice immune to e i the r KLH or Fd were t i t r a t e d in t r i p l i c a t e . Tubes conta in ing e i t he r control or normal rabbi t serum, or d i l u t i o n s of an t i- id io type (see below) in PBS-Tween were mixed with d i l u t i o n s of mouse ant i se ra at a d i l u t i o n which had prev ious ly been determined to give an absorbance of 0.5 at 405 nm a f t e r 1 hour in a standard ELISA. The so lu t ions were incubated overnight at room temperature, and tested f o r antigen binding by the ELISA. The normal rabb i t serum control was used to def ine the 100% react ion in a l l assays. b) Indiv idual Sera 0.2 ml of prev ious ly t i t r a t e d ind iv idua l sera at twice the f i n a l concentrat ion needed were dispersed from a common stock d i l u t i o n in to tubes conta in ing 0.2 ml of an t i- id io type or control normal rabbi t serum, usua l l y at 1:50. The f i n a l concentrat ion of mouse serum was that necessary to give a reading of 0.5 in the ELISA a f t e r 1 hour, and f i n a l concentrat ion of rabb i t reagents was 1:100. The tubes were incubated overnight and assayed f o r antigen binding in t r i p l i c a t e , thus prov id ing a convenient double check that both serum and an t i- id io type had been added to the tube. A pos i t i v e control cons i s t i ng of a d i l u t i o n of poo led , appropriate ant i-Fd serum was 45 used f o r each assay. The control in which normal rabbi t serum was used f o r each ind i v idua l serum def ined the 100% r e a c t i v i t y . B) Preparat ion of Immunoadsorbents 1) Ferredoxin - Sepharose 0.35 g of Thiopropyl Sepharose 6B (Pharmacia 17-0420-01) was reconst i tu ted with d i s t i l l e d water f o r 2 hours at 22 °C , then washed extens ive ly by suct ion f i l t r a t i o n with d i s t i l l e d water fol lowed by PBS. The beads were t rans fe r red to a glass column (Bio Rad 737-2240) and washed with a so lu t ion of degassed 4M p u r i f i e d urea , 5mM sodium EDTA in PBS pH 7.5 (urea b u f f e r ) . The beads were then incubated with degassed urea bu f fe r conta in ing supernatant from a saturated so lu t ion of 2,2 - D ipyr idy l d i su lph ide (Sigma D5767) f o r 15 minutes at room temperature. The column was then washed with 3 volumes of degassed urea bu f f e r . 1.0 mg of Fd that had been l y o p h i l i z e d from d i s t i l l e d water was resuspended with 1.0 ml of degassed urea buf fe r and slowly washed into the column. The tube was r insed with 0.5 ml of urea b u f f e r , which was added to the column. The column was then f lushed with a stream of nitrogen gas, sea led , and incubated at 37°C f o r 24 hours. At t h i s time the Fd so lu t ion had l o s t i t s o r i g i na l brown c o l o r . The column was washed a l t e rna te l y with PBS or 0.1M HC1, and then the column a c t i v i t y was tested by absorpt ion of anti-Fd ant ibody. The capac i ty of the column was estimated by t i t r a t i o n of absorbed antibody by ELISA and by polyacrylamide gel e lec t rophores i s (PAGE). Th is method of column preparat ion was found to be simple and reproduceable, as long as urea was used to denature the Fd. 2) Immunoglobulin Sepharose a) Preparat ion of Immunoglobulin 46 5.0 ml of normal mouse serum was f rac t iona ted with ammonium sulphate at 4 °C to a f i n a l concentrat ion of 45% (257). The p rec ip i t a t e was co l l e c t ed by cen t r i fuga t ion at 10,000 rpm with an SS-34 rotor in an RC5B r e f r i ge ra t ed cent r i fuge (Sorval) and resuspended in 2.0 ml of PBS. D i a l y s i s f o r 24 hours at 4 ° C , with 2 1 of PBS was fol lowed by d i a l y s i s f o r 24 hours with 2 1 of 0.2M N a 2 C 0 3 , 0.5M NaCl pH 8 .0 . The absorbance at 280 nm of the so lu t ion was noted and the immunoglobulin conta in ing so lu t ion was r e f r i ge ra ted at 4 °C immediately before use, or stored at -30°C. b) Ac t i va t i on and Coupling to Sepharose 20.0 g of CL-Sepharose 4B (Pharmacia (7-0150-01) was ac t i va ted with CNBr (Baker F946 only) as a va r i a t i on of a method prev ious ly descr ibed (258). In summary: The f i nes from sepharose 4B were removed by decantat ion and the gel was washed with 100 volumes of d i s t i l l e d water on a suct ion f i l t e r . 20.0 g of the mois t , packed gel was t rans fe r red to a 100 ml beaker conta in ing a magnetic s t i r r i n g bar and 20.0 ml of 0.5 M NaPO^ pH 11.0. The beaker was placed in a 10°C water bath , on top of an external rheostat con t ro l l ed magnetic s t i r r e r in a fume hood. A thermometer and pH e lect rode was added to the beaker. When the so lu t ion temperature had reached 10 °C , 6.0 g of c r y s t a l l i n e CNBr (Baker gave twice the y i e l d of F isher or Eastman CNBr) was added and the rate of s t i r r i n g was increased. The pH was kept to 11.0 +/-0.2 un i ts with ice co ld 5.0 M NaOH, while the react ion temperature was kept below 18°C with i c e . The react ion was allowed to continue f o r 30 minutes (about 19 mis of 5M NaOH). At th i s time the contents were qu ick ly r insed by suct ion f i l t r a t i o n in the fume hood with 50 volumes of i ce co ld d i s t i l l e d water. 5.0 g of packed beads were added to 4.0 ml of immunoglobulin conta in ing so lu t ion in a 15.0 ml disposable p l a s t i c tube 47 (Falcon 2001) and the tube was rotated f o r 24 hours at 4 °C on a Labquake ro ta to r (Western 56264-302). The supernatant was then removed by suct ion f i l t r a t i o n and the gel was washed with PBS and incubated with 5.0 ml of 10% monoethoanolamine, pH 9 .0 , f o r 24 hours at 4 ° as before . The e f f i c i e n c y of binding was ca l cu la ted using before and a f t e r coupl ing A 2 8 0 n m readings. A f t e r b lock ing of react ive groups was completed, the gel was washed with a l t e rna t ing rounds of PBS and 0.1 M HC1, t rans fe r red to a glass column (Bio Rad 737-2240) and stored at 4 °C with PBS conta in ing 0.5% sodium az ide . Columns prepared in th i s way contained approximately 28 mg of prote in per ml of beads, c) Animals 1) Mice Female mice between 4 and 8 weeks of age were obtained from Jackson Laborator ies (Bar Harbor, Maine). S t ra ins used in these studies were CBA/J, C3H/HeJ, C57BR/cdJ, B10.BR/SnJ, RF/J, A/J , AKR/J, ST/J, CE/J, and C58/J. Mice from our animal f a c i l i t y were random bred, aged 1-2 weeks and used as a source of thymocytes or aged 5-8 weeks and used to grow asc i t es f l u i d conta in ing monoclonal an t ibod ies . A l l mice were maintained in our f a c i l i t y . 2) Rabbits Female New Zealand White rabbi ts weighing approximately 2 kg were obtained from the Un ive rs i t y of B.C. Animal Care un i t and were maintained there or in our animal f a c i l i t y . D) Production of immunological Reagents 1) Anti-Fd Ant isera Mice of s t r a ins CBA/J, C3H/HeJ, C58/J, CE/J, C57BR/cdJ, RF/J, A/J , AKR/J, B10.BR/SnJ, and ST/bJ were immunized with Fd (prepared by Dr. 48 S ikora ) . Each mouse was immunized with 0.1 ml to ta l volume of a 50% so lu t ion of 1.0 mg/ml Fd in PBS and complete Freund's adjuvant (CFA) purchased from Di fco (H-37Ra). Mice were immunized with 2 x 50 ul a l iquots in jec ted subcutaneously into each f lank at i n te r va l s of 30 days from primary ( 1 ° ) to secondary ( 2 ° ) and 50 days from secondary to t e r t i a r y ( 3 ° ) immunizations. The sera from these mice y i e lded immunoglobulin used to pur i f y a n t i - i d i o t y p i c reagents. Another group of mice from AKR/J, A/J , RF/J and B10.BR/SnJ s t ra ins were immunized with commercial Fd as above but at 28 day i n t e r v a l s , and each of these mice were earpunched f o r i d e n t i f i c a t i o n . Mice immunized with p u r i f i e d Fd , were e ther ized and bled from the t a i l at approximately 11, 15 and 21 days from 1° st imulus and 7 and 14 days fo l lowing both 2° and 3° s t i m u l i . Mice immunized with commercial Fd were bled at 28 days a f t e r i n i t i a l priming and 15 days a f t e r both 2 ° or 3 ° immunization. 2) Anti-KLH Ant i sera Mice of each s t r a i n were immunized as descr ibed with a 50% so lu t ion of CFA and 1.0 mg/ml of KLH. A separate group of B10.BR/snJ mice was immunized with KLH at 21 day i n te r va l s and i n d i v i d u a l l y marked by earpunching. A l l mice were bled fo r anti-KLH sera as descr ibed f o r Fd-immune animals. Normal mouse serum (NMS) was obtained by bleeding 10-15 unimmunized mice and pool ing t h e i r sera . 3) Monoclonal Ant ibodies to Fd a) Production of Hybridomas Monoclonal ant ibodies RIO and R32 were produced from B10.BR/snJ mice and were a g i f t of Rakesh S ingha i . Cloned c e l l s of Fd-1 from B10.BR/snJ mice were a g i f t of Dr. M. Weaver and were recloned 5 times by l i m i t i n g 49 d i l u t i o n before asc i t es production of antibody. H-l , H-4, H-5, H-16 and H-32 hybrid l i nes were produced from C57BR/cdJ mice. Hybridomas were produced by fus ing hyperimmune murine splenocytes with myeloma c e l l s in the presence of polyethylene g lyco l (PEG) in a manner s i m i l a r to that descr ibed prev ious ly (252), but with s i g n i f i c a n t mod i f i c a t i ons . B r i e f l y : Mice were hyperimmunized with Fd in CFA subcutaneously. At l eas t 30 days a f t e r the l a s t i n j e c t i o n , C57BR mice received 50 ug of Fd i/p in 50% Incomplete Freund's Adjuvant (IFA) as well as 20 ug of Fd in PBS intravenously ( i /v ) . Mice received 20 ug Fd i/v da i l y f o r the next 2 days and t h e i r spleens were fused 3 days a f t e r the l a s t i n j e c t i o n . The parental myeloma l i n e used f o r the production of Fd-1 was SP2/0 (252); a l l other hybridomas were produced with NS-1 (259). NS-1, a g i f t of Dr. F. T ake i , was grown in Dulbecco's Modif ied Eagles Medium (Gibco 430-1600) supplemented with 0.29 g/1 glutamine (Sigma G3126), 0.11 g/1 sodium pyruvate (Gibco 890-1840), 2.38 g/1 HEPES (Sigma H7006), 3.7 g/1 sodium bicarbonate (F isher S-233), 2.0 g/1 D-glucose (Baker 1916) and 10.0 ml/1 of pen i c i l l i n-s t rep tomyc in (Gibco 600-5145) re fe r red to as complete DME (or cDME) with the addi t ion of fus ion screened, heat inac t i va ted f e t a l c a l f serum (FCS) from Gibco (200-G140). Ce l l s were maintained at 37 °C , 10% C0£, and 95% of r e l a t i v e humidity in a Forma incubator . NSI was se lected with 20 ug/ml and 40 ug/ml of 8-azaguaine (Sigma A1007) f o r r e s i s t an t c e l l s and grown f o r only one month a f t e r s e l e c t i o n , before replacement with f r e sh l y se lected c e l l s from frozen stock. NS-1 c e l l s were grown to a 5 densi ty of 1-2 x 10 ce l ls/ml p r i o r to f u s i o n . On the day of f u s i o n , immune mice were k i l l e d by ce rv i ca l d i s l o c a t i o n , and a s ing le c e l l suspension of splenocytes was prepared. The c e l l s were washed 3 times with s t e r i l e , 37°C PBS by 5 minute cent r i fuga t ions at 400 50 x g. NS-1 c e l l s were harvested by cent r i f l iga t ion and washed in the same way. C e l l s were counted by trypan blue exc lus ion and mixed at a r a t i o of 4 splenocytes per NS-1 c e l l in PBS. This mixture was centr i fuged at 400 x g f o r 10 minutes. Fusion was accomplished by decanting res idual PBS, p lac ing the tube in a 37°C water bath and adding 1.0 ml of 3 7 ° , 50% polyethylene g lyco l (PEG) (Baker 1540 or Serva 4000) in PBS prepared that morning. The PEG so lu t ion was added to c e l l s over 1 minute while s t i r r i n g with the p ipet te t i p . The c e l l s were s t i r r e d f o r another minute, then the PEG was d i l u t ed with 2 x 1.0 ml a l iquots of 37 °C cDME (without FCS) with s t i r r i n g over 2 minutes. PEG was fu r ther d i l u t ed with 5.0 mis of warm cDME added over 2 minutes. The suspension was then centr i fuged at 400 x g f o r 10 minutes. The supernatant was decanted, and 10 ml of 37°C cDME was added without d i s tu rb ing the c e l l p e l l e t . The tube was capped and incubated at 37°C f o r 20-40 minutes while thymocytes were prepared from random bred mi ce. Fol lowing incubat ion , thymocytes and fus ion products were combined at a r a t i o of 2 thymocytes per o r i g i na l splenocyte in warm cDME conta in ing 20% fus ion se lected FCS. To th i s was added s t e r i l e PBS conta in ing 1.3 mg/ml hypoxanthine (Sigma H9377) and 0.4 mg/ml thymidine (Sigma T9250) termed HT s tock , to a f i n a l d i l u t i o n of 1:100. S t e r i l e PBS conta in ing aminopterin (0.2 mg/ml Sigma A2255) termed A s tock , was added to a f i n a l d i l u t i o n of 1:1000. HAT media allowed the se l ec t ion of fus ion products according to the method of L i t t l e f i e l d (260). 0.2 ml of the supplemented c e l l suspension was added to each well of a 96 well t i s sue cu l ture p late (Flow o 76-023-05). General ly 4.5 plates were used per spleen (10 c e l l s ) fused , with about twice that many thymocytes in 0.2 ml of 20% FCS and HAT cDME medium. Plates were incubated at 37 °C , 10% COg and 95% r e l a t i v e humidity with pe r iod i c i nspec t ion . Unless FCS was suspected of being d e f i c i e n t , the 51 the media was not changed un t i l ELISA t e s t i n g . By th i s p r o t o c o l , 2 - 4 hybrids per well could be observed by day 5. b) Se lec t ion and Cloning of Hybridomas Plates conta in ing hybridomas were screened f o r antigen binding when co lon ies were very l a r g e , and at the point of outgrowing the media (about 10 days a f t e r fus ion and 8 days a f t e r c l on ing ) . Cultures were tested by removing 0.1 ml of medium and adding i t to s t e r i l e Fd coated ELISA p l a t e s . The media was replaced with 0.1 ml of cDME conta in ing 1:100 d i l u t i o n of HT stock and 20% FCS. Wells shown to possess Fd binding a c t i v i t y were 5 t rans fe r red to 2.3 ml of medium conta in ing 2 x 10 thymocytes/ml in cDME supplemented with 1:100 HT stock and 20% FCS, in 24 well t i s sue cu l ture p lates (Linbro 76-033-05). The c e l l s were cu l tured un t i l growth was con f luen t , and supernatants were tested f o r Fd and KLH binding a c t i v i t y . Ce l l s from wel ls showing s p e c i f i c antibody to Fd were cloned from 10 to 1.25 c e l l s per well by doubling d i l u t i o n in cDME conta in ing thymocytes and 20% FCS. Ce l l s were usua l l y cloned 3-5 times before they were assumed to be pure and s t ab l e . A l l cDME used in fus ion and c lon ing was made wi th in one day of use. c) Asc i tes Production Mice were given 0.5 ml of 2, 6, 10, 14 - tetramethylpentadecane (Pr istane) (A ldr i ch T2280-2) at l eas t 3 days p r i o r to i n j e c t i on of hybridoma c e l l s . 24 hours p r i o r to i n j e c t i on mice were i r r ad i a t ed with 500 rad using a ^ C o gamacell (AEC). 10^ to lO'' v i ab le c e l l s were given i/p in PBS to each mouse. Asc i tes f l u i d conta in ing antibody usua l l y developed wi th in 14 days, and was drained every other day. It should be noted that random bred mice i r r ad i a t ed and given hybridoma c e l l s were f a r super ior to inbred pure or F. s t r a ins in asc i t es product ion. 52 Ce l l s from cu l tures or asc i t es were frozen at -70°C in cDME conta in ing 20% FCS and 10% DMSO (F isher D-128) by standard procedure, and stored in cryopreservat ion v i a l s (Nunc 1.8 ml ) . Ce l l s were t rans fe r red to l i q u i d a f t e r 24 hours at -70°C. 4) Production of A n t i - i d i o t y p i c Reagents a) P u r i f i c a t i o n of Anti-Fd Ant ibodies Anti-Fd antibody was prepared from the serum of AKR, RF, A , and BIO.BR mice hyperimmunized with p u r i f i e d f e r r edox in . Mice were immunized on day 0, 30 and 80 r e spec t i v e l y , and blood pooled from 12-15 animals on days 101 and 104. The pooled sera was frozen un t i l use. Anti-Fd antibody was p u r i f i e d by immunoabsorption on Fd-Sepharose, and e lu t i on with 0.1M HC1 into enough 3M T r i s to neu t ra l i ze the ac id (35 ul/ml HC1). The e luted f r a c t i ons were pooled and concentrated by pressure (Amicon YM 10) then d ia l yzed into PBS. The p u r i f i e d ant ibodies were stored f rozen . b) Immunization and Bleeding of Rabbits Female rabbi ts were hyperimmunized with 100 ug of p u r i f i e d anti-Fd antibody in 50% CFA. Each animal was in jec ted with 0.1 ml subcutaneously in each shoulder and haunch (0.4 mis t o t a l ) . Animals were bled on days 7, 15 and 21 fo l lowing t e r t i a r y or subsequent immunizations by e i t he r p i e r c ing the marginal ear v e i n , or by card iac puncture. Blood from the three bleeds was pooled and stored f rozen . c) P u r i f i c a t i o n of A n t i - i d i o t y p i c Antiserum Anti-mouse Ig a c t i v i t y in the sera of rabbi ts immunized with anti-Fd mouse antibody was removed by repeated absorption of the rabb i t antiserum over mouse immunoglobulin (MIg) immunoabsorbents as fo l l ows : 53 4.0 mis of PBS conta in ing 0.5% sodium azide was added to 16.0 ml of rabb i t serum. The serum was repeatedly absorbed over a 10 ml MIg column (a g i f t of Rakesh S ingha i ) . Columns were washed repeatedly with 0.1M HC1 to remove bound RaMIg. The RaMIg was saved and used to prepare enzyme conjugates f o r ELISA. A f t e r a va r i ab le number of passages over the heterologous MIg column, the rabb i t serum was passed over a 5.0 ml immunoabsorbent conta in ing MIg s p e c i f i c f o r the mouse s t r a i n against which the rabbi t had been immunized. A f t e r numerous absorpt ions , the a n t i - i d i o t y p i c rabbi t sera were assayed f o r RaMIg a c t i v i t y on the ELISA. In th i s way, anti-AKR/J i d i o t ype , f o r example, was absorbed 20 times over the heterologous and 5 times over the s t r a i n s p e c i f i c columns, d) Concentrat ion of A n t i - i d i o t y p i c and Control Normal Rabbit Sera Fol lowing immunoabsorption, the f r a c t i ons conta in ing absorbed sera were pooled (about a 3-fold d i l u t i o n of o r i g i na l concentrat ion) and concentrated by p r e c i p i t a t i o n with a f i n a l concentrat ion of 45% ammonium sulphate as descr ibed above. The d ia l yzed immunoglobulin in PBS was she l l f rozen and l y o p h i l i z e d . The l y o p h i l i z e d powder was resuspended in 10 ml of 50% g lycero l conta in ing 0.2% sodium azide to a f i n a l concentrat ion of 50% g lycero l in PBS/azide. To th i s was added 1.0 ml of NMS of the s t r a i n against which the an t i- id io type was produced. Control NRS was prepared by t es t ing non-immune rabb i t serum fo r anti-MIg a c t i v i t y , then adding an equal volume of g l y c e r o l . To th i s was added normal B10.BR sera and azide to a f i n a l concentrat ion of 10% NMS and 0.2% az ide . These reagents were kept at -20°C before and a f t e r use. II. Biochemical Methods A) Reagent P u r i f i c a t i o n 54 Acrylamide (5521), methylene b is acrylamide (P8383) and TEMED (8178) were obtained from Eastman. T r i s (T1378), s i l v e r n i t r a t e (S-6506), SDS (L-5750), bromthymol blue (B-8630), T r i t on X-100 (X-100), sodium hydroxide (S5881), and g lyc ine (G6761) were obtained from Sigma. Ace t i c ac id (A-38-S), g lycero l (G-33), sodium borohydride (S-678), sodium carbonate (S-263), formaldehyde (F-79), urea (U-15), phosphoric ac id (A-260) and ammonium peroxydisulphate (A-682) were obtained from F i she r . D i t h i o t h r e i t o l (DTT-161-0610), agarose (162-0125), AG-11A8 mixed bed ion exchange res in (142-7834) and c e l l u l o s e gel f i l m (165-0922) were obtained from Bio Rad. Ampholines pH 3.0-10.0 were obtained from LKB. Only products from Bio Rad and LKB were e lec t rophores i s grade. 30% stock so lu t ions of acrylamide were p u r i f i e d by s t i r r i n g f o r one hour with 10 g/1 of AG-118 ion exchange res in at room temperature. The so lu t ions were then f i l t e r e d through a 0.45 micron f i l t e r and stored at 4 ° C . 500 g of urea were d isso lved in 1 1 of 70% ethanol at 50°C and s t i r r e d with 20 g/1 of AG-118 ion exchange res in f o r one hour. The so lu t ion was suct ion f i l t e r e d through Whatman 1 MM f i l t e r paper and l e f t at -20°C overnight . The c r y s t a l s were washed with 1 1 of -20°C 95% ethanol fol lowed by 500 ml of ether under suc t i on . The c r y s t a l s were packed and suct ion dr ied f o r 20 minutes then a i r dr ied overnight at room temperature (261). 1.5 M T r i s pH 8 .8 , 0.5 M T r i s 6 .8 , 10% SDS and gel so lu t ion conta in ing 12% acrylamide f o r PAGE or 4% acrylamide fo r IEF were f i l t e r e d through 0.45 micron f i l t e r s . The gel so lut ions were stored at 4 °C f o r up to a week before use. PAGE running buf fe r conta in ing 9.0 g T r i s , 43.2 g g lyc ine and 1.5 g of SDS per 1.5 1 was f i l t e r e d through a 1.2 micron f i l t e r before use (262) and occas iona l l y reused once without r e f i l t e r i n g . 55 The above p u r i f i c a t i o n procedures and the use of 5mM DTT in the sample b u f f e r , rather than 2-mercaptoethanol, dramat ica l l y reduced sta ined gel background, the presence of the 55K a r t i f a c t , and the intense co lo ra t ion around 20 K. These p u r i f i c a t i o n s , new e lec t rophores i s p l a t e s , longer e q u i l i b r a t i o n times f o r IEF tube gels in the sample buf fe r p r i o r to running in the second dimension, and e l iminat ion of the agarose over lay d id not e l iminate the v e r t i c a l l i n e a r t i f a c t s seen in the 2-D gels sta ined with the Sammon's Method (263). These l i nes were not observed in gels sta ined with e i t he r Coomassie B lue , or another s i l v e r s ta in ing method (264), and may be due to the greater s e n s i t i v i t y of the Sammon's s i l v e r s t a i n . B) Mini column Immunoadsorbents 1) Preparat ion Mini-immunoadsorbents were produced as descr ibed by Pearson and Anderson (265). Columns conta in ing 25 or 50 ul of Fd-Sepharose were packed in to a 0.25 ml p ipet te t i p (Bio Rad) and held with a wisp of co t ton . This p ipet te t i p was inser ted in to a 1000 ul p ipet te t i p (Evergreen S c i e n t i f i c ) which was placed wi th in a 5 ml d isposable p l a s t i c tube (Falcon 2003). 2) Use of Columns Sera , asc i t es f l u i d , or fus ion supernatants conta in ing anti-Fd a c t i v i t y were added to the top of the mini-column and centr i fuged at 200 x g f o r 3 minutes. The sera were cyc led twice. The column was then washed as fo l l ows : Once with 200 ul of PBS, fol lowed by 200 ul of Buf fer 1 (0.15M NaCl , 0.02 M T r i s pH 7 .5 , 1% T r i t on X-100 - a g i f t of Rob Shipman), then 200 ul of Buf fer 2 (0.15 M NaCl , 0.02 M T r i s pH 8 .8 , 0.1% SDS, 1% T r i t on X-100 - a g i f t of Rob Shipman), fol lowed by three washes with Buffer 3 (0.15 M NaCl , 0.02M T r i s pH 6 .8 , 0.2% SDS, 1% T r i t on X-100 - a g i f t of Rob Shipman) and f i n a l l y once with 200 ul of d i s t i l l e d water. The minicolumn was spun dry 56 at 400 x g f o r 1 minute, and then the column was removed from the tube and b lo t ted dry . The column was then placed in a 1.5 ml p l a s t i c tube (Eppendorf) held wi th in a 15 ml p l a s t i c tube (Falcon 2007). Bound antibody was e luted with 3 x 75 ul a l iquots of 0.1 M HC1. The e luted antibody was immediately f rozen at -70°C and l y o p h i l i z e d . The column was washed with PBS and stored wet in PBS conta in ing 1% sodium azide at 4 °C un t i l reuse. C) SDS Polyacrylamide Gel E lec t rophores is Proteins were assayed by PAGE by a method s im i l a r to that descr ibed by Laemmli (266). B r i e f l y : Samples were d i l u t ed in sample buf fe r conta in ing 2.3% SDS, 0.065M T r i s pH 6 .8 , 10% g l y c e r o l , 5 mM DTT and bromthymol b lue . Samples were heated at 100°C f o r 5 minutes in an o i l f i l l e d block heater (P ierce 189000). Stacking was done in 3% acrylamide at 50V f o r 90 minutes, and the e lec t rophores i s was performed f o r approximately 1100 V-h at e i t he r 75 V or 225 V with coo l ing at 4 ° C . A l l gels were 1.5 mm th i ck . Genera l l y , less than 5 ug of prote in was added to each sample w e l l . D) 2-Dimensional E lec t rophores is 2-D e lec t rophores i s was done by the method of O ' F a r r e l l (267) with modi f i ca t ions as descr ibed by Ingman-Baker and Candido (268) or Anderson (269) as fo l l ows : Glass tubes: (Bio Rad 12.5 cm x 0.2 cm diameter) were f i l l e d with gel so lu t ion conta in ing 4% acrylamide (30:1.8 mono:bis ) , 4.5 M urea , 2% T r i t on X-100, 2% pH 3-10 Ampholines, and polymerized with TEMED and ammonium persulphate . Polymerizat ion was allowed to proceed f o r at l eas t 3 hours and the tubes were covered in pa ra f f i n and stored at 4 ° C . Tubes were warmed at room temperature f o r 1 hour before use and prefocussed in 500 ml of 0.03 M NaOH (well degassed) at the anode, and 3 1 of 0.01 M phosphoric 57 ac id at the cathode in a Bio Rad model 155 tube gel e l e c t rophore t i c apparatus. Prefocussing was ca r r i ed out at 200 V f o r 15 minutes, 300 V f o r 15 minutes and 400 V f o r 30 minutes. Anode buf fe r was replaced p r i o r to sample add i t i on . The l y o p h i l i z e d sample was resuspended in 25 ul of SDS sample buf fe r without dye, and heated at 100°C fo r 5 minutes. Samples were well cooled and 25 ul of 4.5 M urea , 5 mM DTT, and 2% Ampholines 3-10 was added. The samples were vortexed b r i e f l y then cen t r i fuged . 50 ul of sample was added to the tops of the IEF tubes through anodic bu f f e r . Gels were focussed at 400 V f o r 17 hours then 800 V f o r 1 hour, with coo l ing at 4 ° C . Tube gels were c a r e f u l l y extruded with water pressure and stored in 5 ml of SDS PAGE sample buf fe r at -20°C. P r i o r to running in the second dimension, gels were thawed by incubat ion f o r 45 minutes at room temperature. Tube gels were added to the top of the PAGE gel and sealed with a hot so lu t ion of 1% agarose, 2.3% SDS, 0.065 M T r i s pH 6 .8 , 10% g lycero l and 5 mM DTT. Gels were stacked and run as descr ibed above, un t i l the marker dye had advanced 14 cm into the running gel (about 1200-V-h). E) S i l v e r S ta in ing PAGE gels were sta ined by the method of Sammon's (263). The only dev iat ions from the publ ished method were the reduct ion of f i x i n g time in 25% and 10% ethanol to 1 hour, and s ta in ing time in s i l v e r n i t r a t e to 1 hour. Stained gels were dehydrated from the sodium carbonate so lu t ion in 50% e thano l , 10% ace t i c a c i d , and 10% g l y c e r o l . Gels were preserved by drying onto c e l l u l o s e membranes as per manufacturers i n s t r u c t i o n s . 58 Chapter 3. Major Idiotypes in The Murine Ant i-Ferredoxin Response I. Results A) S p e c i f i c i t y of Ant i- id io types Young mice were immunized with e i the r Fd or KLH and boosted as explained in Methods (Chapter 2 ) . Sera from the 3 ° day 21 and 24 Fd-immune bleeds were pooled from B10.BR/SnJ, AKR/J, A/J , and RF/J mice s t r a i n s . The pools were immunopurified on Fd-Sepharose and the p u r i f i e d ant ibodies were used to immunize r abb i t s . Serum from hyperimmune rabbi ts was p u r i f i e d to produce a n t i - i d i o t y p i c reagents and used f o r i n h i b i t i o n studies with ant i se ra from var ious s t ra ins of mice (see Table I). The e f fec t i veness of the an t i- id io types was determined by i n h i b i t i n g the anti-Fd binding c a p a b i l i t i e s of the pooled sera . It should be noted that th i s assay depends on the s t e r i c hindrance of bound an t i- id io type competing f o r antigen keeping the antibody from binding to the ELISA p l a t e . Some i d i o t y p i c determinants may be too f a r away from the antigen binding s i t e to in te r rup t antigen b ind ing . Thus, the i n h i b i t i o n assay used here may represent a lower proport ion of id iotype expression than i s a c tua l l y present. As can be seen from Table I, the reagents were prepared against mouse sera possessing d i f f e r e n t heavy chain and l i g h t chain a l lo t ype markers, usua l l y in terpre ted to represent genet ic divergence. A l l of these s t ra ins were high responders to Fd and produced over 80% C - determinant s p e c i f i c antibody (250). Consequently, one would expect most of the an t i - id io type to i n h i b i t C - d i rec ted an t ibod ies . The a b i l i t y of the an t i- id io types to s p e c i f i c a l l y i n h i b i t the immunizing ant ibodies to which they were ra i sed i s shown in Figure 1. As can be seen, the ant i- id io types are qu i te s p e c i f i c to the anti-Fd response and d id not i n h i b i t the mu l t i s t r a i n anti-KLH pool to a s i g n i f i c a n t degree. The r e l a t i v e l y steeper 59 Table 1 Cha rac t e r i s t i c s of Mouse S t ra ins U t i l i z e d in the Experiments Described in  T h i s Thesis Table 1 cons is ts of data that have been known to a f f e c t id io type production in other systems (as reviewed p rev ious l y ) . A l l mice used produced more than 20 ug of ant i-Fd antibody per ml of serum, hence t he i r high responder s ta tus . Igh a l lo types are determined by the IgG 0 a l l o t ype and TgK a l lo types were determined by i s o - e l e c t r i c f ocuss ing . As can be s e e n , a l l mice produce ant ibodies predominantly d i rec ted to the C determinant of Fd. 60 Table I - Cha rac t e r i s t i c s of Appropriate S t ra ins Reference S t ra in H-2 Igh IgK E f l Ef2 Fd response N:C Ratio C3H/HeJ k j b a High 5 :95 CBA/J k j b a High 15 :85 AKR/J k d a a High 10 :90 B10.BR/SnJ k b b a High 15 :85 A/J a e b a High ND RF/J k c a a High 10 :90 CE/J k f b b High 10 :90 ST/bJ k g b a High 20 :80 C57BR/cdJ k a b a High ND C58/J k a a b High 10 :90 Data from references 56, 250, 251, 271 and 277. 61 Figure 1: S p e c i f i c i t y of A n t i - i d i o t y p i c Sera on Pooled Anti-Fd or Anti-KLH Sera Ant i-Fd or anti-KLH serum was pooled from 12-15 ind iv idua l mice and assayed in quadrupl icate f o r i n h i b i t i o n of binding to antigen by an t i - id io t ype . The an t i- id io type was used to i n h i b i t the ant i-Fd ant ibodies from the mouse s t r a i n to which i t was d i r e c t ed . In th i s way the s p e c i f i c t y of id io type binding i s i l l u s t r a t e d . The standard er rors of i n h i b i t i o n of binding of Fd ( • ) or KLH (A) were never above 10 percent. DILUTION X io-» 63 t i t r a t i o n curves seen with the anti-A/J and anti-BIO.BR id iotypes are probably due to the use of a n t i - i d i o t y p i c sera obtained from an e a r l i e r response (3 ° - 4 ° ) than that from the anti-AKR or anti-RF a n t i - i d i o t y p i c a n t i s e r a , which were obtained a f t e r 6 or 7 immunizations. From ELISA data (not shown), i t was found that greater than 99% of the anti-mouse Ig a c t i v i t y was removed from a l l of the a n t i - i d i o t y p i c an t i se ra fo l lowing the mu l t ip le absorpt ions on MIg columns, as descr ibed in Methods. The amount of mouse antiserum used in the i n h i b i t i o n assay was that required to give an approximate in the ELISA of 0.5 in one hour at room temperature. This value was usua l ly in the middle of the t i t r a t i o n curve , but not necessar i l y the t i t r a t i o n po in t . It was thought that in th i s way a constant amount of Ig would be bound to the p l a t e , a f f i n i t y e f f e c t s would be minimized s ince there was s t i l l antigen excess , and the value of 0.5 was near the middle of the l i n e a r range of s e n s i t i v i t y of the spectrophotometer, permit t ing more accurate ca l cu l a t i ons or i n h i b i t i o n . It should be noted that in over 15 independent assays of the control s e r a , the standard e r ro r was in the range of 2% i n h i b i t i o n , demonstrating the r e l i a b i l i t y of th i s method (data not shown). In these i n h i b i t i o n assays , a l l negative values have been regarded as zero percent i n h i b i t i o n . A very important observat ion a r i ses from the data on Figure 1. The maximal i n h i b i t i o n values seem to plateau near 75%. Thus, even a f t e r 7 immunizations, the rabbi t does not respond to up to 25% of the immunizing i d i o t ypes . The most obvious ra t iona le i s that there are many poorly represented id io types in the pooled se ra , poss ib l y as a r e su l t of the N-determinant (10 - 20% of ant i-Fd response) s p e c i f i c response. Since these id io types are present in small concent ra t ions , there may not be enough of any p a r t i c u l a r id iotype to appropr ia te ly immunize the r abb i t . At 64 two of p o s s i b i l i t i e s may account f o r these observat ions . The ind iv idua l may express a few major id iotypes and many minor ones, or a few immunoglobulins from high t i t r e sera may dominate the ant i-Fd binding of the p o o l , and the id io types ava i l ab le to immunize the r a b b i t , thus b ias ing the observat ions . The d i f f e r e n t p o s s i b i l i t i e s may be d is t ingu ished by observing the i n h i b i t i o n of ind i v idua l an t i s e r a . B) I nh ib i t ion of Anti-KLH A c t i v i t y of Indiv idual Sera by Anti-Fd  Idiotypes Mice immunized with KLH were bled on day 15 a f t e r 2° immunization, and the ant i sera were used in ind iv idua l i n h i b i t i o n assays. These resu l t s are shown in F igure 2. As can be seen, the maximum i n h i b i t i o n s are less than 25%, with most i n h i b i t i o n s being less than 10%. The high background of anti-B10.BR id io type observed in Figure 1 i s not seen at the ind iv idua l l e v e l ; in f a c t , none of the an t i- id io types have a d isproport ionate background, as the mean backgrounds are with in 2 - 5 % i n h i b i t i o n (see Appendix 1 ) . The C57BR/cdJ mice appear to be more sens i t i ve to i n h i b i t i o n , but the small sample s i ze l i m i t s conc lus ions . Since the l eve l s of i n h i b i t i o n were so sma l l , i t was assumed that the i n h i b i t i o n of anti-KLH binding was due to non-spec i f i c f a c t o r s , and these data were used as the non-spec i f i c background f o r s t a t i s t i c a l purposes (see Appendix 1) . A second experiment was done in which B10.BR/SnJ mice were i n d i v i d u a l l y marked and fol lowed through t h e i r response to KLH. These data are shown in Figure 3. As can be seen, the i n h i b i t i o n s are again qui te low, and do not change great l y over t ime. There was no s i g n i f i c a n t d i f f e rence between the BIO.BR anti-KLH i n h i b i t i o n s and background, and no s i g n i f i c a n t change occurs over t ime. In conc lus ion , the ant i- id io types have been shown to poorly i n h i b i t anti-KLH se r a , from at l eas t two independent experiments. S p e c i f i c i t y of A n t i - i d i o t y p i c Sera on Indiv idual Anti-KLH Sera Indiv idual secondary anti-KLH sera from d i f f e r e n t s t ra ins of mice were i nh ib i t ed from binding to KLH in t r i p l i c a t e by four anti-Fd i d i o t y p i c reagents. The percent i n h i b i t i o n i s represented on the v e r t i c a l axis and the resu l t s f o r each mouse are represented on the hor izonta l a x i s . Ve r t i ca l alignment of i n h i b i t i o n s with d i f f e r e n t an t i- id io types def ine the i d i o t y p i c p r o f i l e of a s ing le mouse serum. The standard er rors of i n h i b i t i o n s were never above 10 percent. anti- AKR anti- RF anti- A/J anti- BIO.BR 100 75 50 25 75 50 25 75 50 25 75 50 25 . L i ff Ill .i..r M. mm mm M.i III ,\..\ ml ML m. mm. mm m% m\ m tri .It.l .11 A/J AKR RF CBA C58 C3H C57BR 67 Figure 3 Inh ib i t ion of Anti-KLH Sera from Ind iv idua l l y Marked B10.BR Mice Sera from the anti-KLH response of B10.BR mice were tested f o r i n h i b i t i o n of binding to KLH in t r i p l i c a t e at var ious times in the immune response. Percent i n h i b i t i o n s f o r each an t i- id io type are represented on the v e r t i c a l axis while the resu l t s f o r the ind iv idua l mice at var ious times in the response are represented h o r i z o n t a l l y . The v e r t i c a l alignment of the bars represent the i n h i b i t i o n of a s ing le animal with the four an t i - id io t ypes . Standard er rors of i n h i b i t i o n are less than 10 percent. CO l w day 28 2° day 15 3° day 15 25 75 anti-RF 50 25 75 anti-A/J 5o 25 25 _fLi.it J r.t .LiLf.jl.1 nlJtil \1 ..IJ—I -L.lJJJ INDIVIDUAL BIO.BR anti- KLH SERA 69 C) Inh ib i t i on of Anti-Fd Indiv idual Sera Indiv idual a n t i s e r a , taken at var ious times in the immune response from the group of mice used to immunize the r a b b i t s , were tested with the an t i - i d i o t ypes . These data are shown in Figure 4. The sca t te r p lo ts show that a major i ty of the mice produced s i g n i f i c a n t amount of i d io t ype . No apparent change over time was observed. As w e l l , the amount of i n h i b i t i o n does not appear to co r re l a te with the anti-Fd t i t r e of AKR, RF, or B10.BR mice (data not shown). A/J mice, on the other hand, that expressed high proport ions of i n h i b i t a b l e i d i o t y p e , produced high t i t r e sera . In th i s case only the large amounts of ind iv idua l id iotypes assoc iated with high t i t r e sera were capable of e l i c i t i n g an immune response in the r abb i t . Thus, only AKR, RF, and B10.BR mice produce a major c ross-react i ve id io type that i s expressed in most s e r a , and no co r r e l a t i on between id iotype expression and t i t r e were seen, whereas high t i t r e sera of A/J mice reacted bet ter with A/J an t i- id io type than low t i t r e sera . It should be noted that some an t i se ra d id not react with an t i - i d i o t ype . Presumably, these sera were composed of a va r i e t y of e i t he r minor i n t r a s t r a i n , or ind iv idua l i d i o t ypes . It i s i n t e r e s t i ng to compare the resu l t s of ind iv idua l sera with the resu l t s using pooled sera (see Table I I ) . At a 1:100 an t i- id io type d i l u t i o n , the pooled sera showed about 75% i n h i b i t i o n . I f the means of the ind iv idua l i n h i b i t i o n s are taken, the r e su l t i s sometimes less than ha l f of the pooled r e s u l t s . The t i t r e of the sera (determined both by amount and a f f i n i t y ) probably has a major a r t i f a c t u a l e f f e c t on the pooled sera r e s u l t s . Thus, i n h i b i t i o n s using pooled sera should not be used to quant i fy the behavior of a populat ion of ind iv idua l an t i s e r a . Figure 4 I nh ib i t ion of Indiv idual Anti-Fd Sera with Appropriate Ant i- id io type Indiv idual ant i-Fd ant i-sera from the four s t ra ins of mice to which ant i- id io types were prepared were i nh ib i t ed from binding to Fd in t r i p l i c a t e by t h e i r appropriate an t i - i d i o t ype . Percent i n h i b i t i o n i s represented v e r t i c a l l y and the resu l t s fo r ind iv idua l mice at var ious times in t h e i r ant i-Fd response are represented ho r i zon -t a l l y . Ve r t i c a l alignment does not represent the same ant ibody. Thres -hold values of i n h i b i t i o n fo r each an t i- id io type (as def ined in Appendix 1) are ind icated on the hor izonta l a x i s . As be fore , the standard er rors of i n h i b i t i o n were less than 10 percent. 71 AKR 19.3 RF 19.7 A/J 17. 100 80 60 40 20 0 100 80 60 40J 20 0 100 80 60, 40. 20. BIO.BR 100. 80 60, 40 20 18.8 • » » • m • X t : l°doyl5 I °day2 l 2 °day7 2 °day l5 3 ° day7 3° day 15 72 Table II A Summary of Pooled Versus Individual Ant ibodies Inh ib i ted  by Various Ant i- id io types This tab le ind ica tes the d i f f e rence between the pooled and mean ind iv idua l serum i n h i b i t i o n s of the anti-Fd ant ibodies tested with t h e i r respect ive an t i- id io types at the same d i l u t i o n of an t i - id io t ype . These data were obtained from Figures 1 and 4 r e spec t i ve l y . The data are expressed as percent i n h i b i t i o n . 73 Table II - Summary of Pooled Versus Indiv idual Anti-Fd Inh ib i t ions S t ra in A n t i - i d . D i l . Pooled Serum Mean Indiv. No. Ind. AKR 1/100 71.6 39.4 14 RF 1/100 74.9 36.1 14 A 1/100 81.0 44.0 11 B10.BR 1/100 79.7 71.9 15 74 The resu l t s of Figure 4 show that most mice of the groups used to immunize rabbi ts possess ant i-Fd sera bearing i d i o t y p i c determinants, recognized by rabb i t an t i - id io t ype . These determinants are observed in primary s e r a , and the frequency of t h e i r expression appears to be constant throughout the response. Since the mice were not i n d i v i d u a l l y marked, nothing can be sa id about the k ine t i c s of each ind iv idua l mouse. As w e l l , one cannot recogn ize , sa fe l y a major c ross-react ive id io type in these mice s ince each serum may have produced ant ibodies to i t s own ind iv idua l p r o f i l e . This i s un l i ke l y s ince many sera are un inh ib i t ab le with an t i - i d i o t ype . A new group of mice well marked and fol lowed i n d i v i d u a l l y throughout t he i r response would add some very useful in format ion , but the best experiments would involve an t i- id io type d i rec ted against the anti-Fd response of a s ing le mouse, which may not be poss ib le due to the low amounts of ant i-Fd produced even in high responder animals (251). D) K ine t i cs of Idiotype Expression New mice of each i d i o t y p i c representat ion were marked and immunized with a d i f f e r e n t preparat ion from that used p rev ious l y . These mice were bled at i n t e r va l s during t h e i r response and tested as before f o r i n h i b i t i o n of binding to Fd. Thus, even though the mice used were immunized with a d i f f e r e n t Fd p repara t ion , ELISA assays were done using the same Fd preparat ion as used p rev ious l y . The resu l t s of these experiments are shown in Figure 5. One can observe that the frequency of sera conta in ing id io type in AKR, RF, and BIO.BR mice i s qui te h igh , whi le very few s t r a i n A mice are i nh ib i t ed with anti-A/J an t i - i d i o t ype . Thus, AKR, RF and BIO.BR mice d e f i n i t e l y produce major c ross-react i ve i d i o t y p i c f am i l i e s but s t r a i n A mice appear to make many anti-Fd i d i o t y p e s , very few of which are shared between the two independent groups of mice. Figure 5 K ine t i cs of Inh ib i t ion of Anti-Fd Sera from Ind iv idua l l y Marked Mice with Appropriate Ant i- id io type  Indiv idual ant i-Fd ant i-sera from four s t ra ins of mice were tested in t r i p l i c a t e with t h e i r appropriate an t i- id io type at var ious times of the anti-Fd response. Percent i n h i b i t i o n f o r each stage of the response i s represented on the v e r t i c a l axis whereas the ind iv idua l resu l t s of each ind i v idua l id io type-ant i - id iotype pa i r of the four s t ra ins i s represented on the hor izonta l a x i s . The i n h i b i t i o n resu l t s of the same mouse at var ious times in i t s anti-Fd response can be fol lowed v e r t i c a l l y . As be fore , the standard er rors of i n h i b i t i o n were less than 10 percent. 100, 80. 60 40, 20. 0. I00J 80. 60. 40, 20. 0 100. 80, 60. 40, 20. 0, 100. 80. 60. 40. 20J 0 l l— t t— i -1 LU Ll.fLfi l-..jjffc j J i L u J . [II •1 i l l njuu RF AKR A/J BIO.BR 77 There appears to be a r i s e in the frequency of AKR animals producing id io type which i s very h ighly s i g n i f i c a n t (see Chapter 4 ) . The other responses do not vary s i g n i f i c a n t l y over t ime. This phenomenon not seen in the e a r l i e r experiments (Figure 4) may be a t t r i bu tab l e to the d i f f e r e n t batches of Fd immunogen. The f i r s t batch contained many aggregates, while the commercial preparat ion was qui te homogeneous. Thus, the form of the antigen may have in f luenced development of the reper to i re in AKR mice. As w e l l , the form of the ant igen ic Fd may have caused the production of d i f f e r e n t id io types in the second experiment, lowering the amount of id iotypes recognized by the a n t i - i d i o t y p i c reagents. Such a phenomenon would account f o r the A/J r e s u l t s . Equal ly poss ib le i s the theory that many id iotypes ex i s t in the anti-Fd response in mice, and i t i s un l i k e l y that a l l of the id iotypes present in the f i r s t group of mice w i l l be expressed by the second group of mice, regardless of the form of the ant igen. The most l i k e l y explanat ion f o r the d i f fe rences between f igures 4 and 5 i s probably a combination of these p o s s i b i l i t i e s . II. D iscuss ion In order to inves t iga te i d i o t y p i c populat ions in mouse s e r a , i t i s important to have s p e c i f i c a n t i - i d i o t y p i c reagents as probes. Several ways to produce these have been documented in the l i t e r a t u r e . One may immunize another species with e i t he r pooled pure antibody (219, 272, t h i s work), pure antibody from a s ing le animal (110, 203, 273) or monoclonal antibody (194, 274). As w e l l , one may immunize another s t r a i n of mice with the antibody (51) or even t ry to ra i se an t i- id io type in normal, syngeneic mice (119). Once obta ined, these reagents must be rendered f ree of any non- id io typ ic anti-immunoglobulin a c t i v i t y by absorpt ion (273) or 78 appropr iate se l ec t i on of a monoclonal an t i- id io type (275). The an t i - id io type may be assayed by d i r e c t binding to id iotype (16) , by i n h i b i t i o n of binding of l abe l l ed an t i- id io type from id iotype (276) or by i n h i b i t i o n of binding of id iotype to antigen (194, 203, 219, t h i s work). It was comforting to see that almost exact ly the same immunization protocol (272) and assay f o r id io type used in th i s work (194) have been prev ious ly used by other i n ves t i ga to r s . The ra t iona le f o r the immunization protocol was that the xenogeneic determinants of mouse Ig would provide help to the i d i o t y p i c determinants, y i e l d i n g a high t i t r e , f a s t response to even poorly represented i d i o t ypes . The disadvantage was that exhaustive (and tedious) absorpt ions were required to remove the i n t e r f e r i n g , non-spec i f i c RaMIg a c t i v i t y . Some s l i g h t ( 1%) a c t i v i t y could not be absorbed and was probably due to the response to denatured MIg, which could not be removed by absorp t ion , and was ampl i f i ed as a consequence of the nature of the ELISA assay. This a c t i v i t y was presumably mopped up by the added NMS, and did not se r ious l y a f f e c t s p e c i f i c i t y . The assay f o r i n h i b i t i o n of binding of anti-Fd to Fd was chosen f o r i t s s i m p l i c i t y , r e l a t i v e l y low background and theore t i ca l s p e c i f i c i t y f o r determinants in the v i c i n i t y of the antigen binding region of anti-Fd antibody. The data in Chapter 3 show that four s p e c i f i c a n t i - i d i o t y p i c reagents were success fu l l y prepared. Mice of AKR, RF and B10.BR s t ra ins produce an i d i o t y p i c fami ly that i s r e s t r i c t e d in va r i e t y enough to be present in two independent samples of Fd immune animals. The an t i- id io type produced from A/J ant i-Fd sera seems to react well with the id iotypes represented by high t i t r e s e r a , and does not f u l l y represent the i d i o t y p i c reper to i re of the anti-Fd response of t h i s s t r a i n . 79 Inspection of the k i ne t i c data (Figure 5) shows that the frequency of mice expressing AKR id iotypes change over t ime, compared to RF, B10.BR, or A/J. Probably some degree of i d i o t y p i c maturation e x i s t s , such as w i l l be discussed in Chapter 4. One can see d i f f e rences in the maximal i n h i b i t i o n of pooled s e r a , and the mean i n h i b i t i o n s of the ind iv idua l sera (Table I I ) . This i s not s u r p r i s i n g , as no account had been made of the a f f i n i t y or concentrat ion of the ind iv idua l ant ibodies in the poo l . Consequently, one would expect a large d i f f e rence between the pool and ind iv idua l serum mean i n h i b i t i o n s , s ince low a f f i n i t y or concentrat ion anti-Fd an t ibod ies , standardized i n d i v i d u a l l y , would be swamped out in the poo l . Thus, one should use ind i v idua l sera rather than pooled sera to look at complex i d i o t y p e s , or f o r q u a n t i f i c a t i o n , therefore proper ly represent ing the serum c h a r a c t e r i s t i c s of the s t r a i n . I dea l l y , one could immunize 15 rabbi ts with antibody from ind i v idua l mice, then, using s tandard ized, equiva lent amounts of an t i - i d i o t ype , make a p o o l , which would be used to tes t the i d i o t y p i c reper to i re of the s t r a i n . Needless to say, th i s would be both time consuming and probably not even poss ib le in the Fd system due to the well documented poor production of antibody even in high responder s t r a i n s . A quick way to determine i f antibody concentrat ion of responding mice i s a f a c to r in determining i n h i b i t i o n s i s to look f o r a co r r e l a t i on between t i t r e and i n h i b i t i o n . Such a co r r e l a t i on was observed only in A/J r e s u l t s . In conc lus ion , the data support the p o s s i b i l i t y that mul t ip le id io types ex i s t in the anti-Fd response. Mu l t ip l e id iotypes in the immune response to macromolecules have been already discussed in Chapter 1, and w i l l be fu r the r reviewed in Chapter 4. 80 Chapter 4. I n te rs t ra in Cross-react ive Idiotypes in the Ant i-Ferredoxin Response  1) Results A) I n te r s t r a in Cross-react ive Idiotypes in the Pooled Anti-Fd Response T e r t i a r y an t i se ra of mice immunized with Fd were pooled f o r each s t r a i n and tested f o r i n h i b i t i o n of Fd binding (see Table I f o r s t ra ins ) with each of the four a n t i - i d i o t y p i c reagents discussed in Chapter 3. A rough est imat ion of id iotype sharing between s t ra ins may be made by comparing i n h i b i t i o n curves of pooled se ra . The data from Figures 6, 7, and 8 show that there i s indeed some c ros s- reac t i v i t y of i d i o t y p i c reper to i re between s t r a i n s . AKR an t i se ra express BIO.BR i d i o t y p i c determinants, C58 ant i se ra express AKR and A/J i d i o t ypes , BIO.BR ant i se ra express AKR i d i o t ypes , and C57BR ant i se ra express A/J i d i o t ypes . RF/J ant i sera probably express some AKR i d i o t y p e s , though not very many. Thus, a strong i nd i ca t i on of c ros s- reac t i v i t y of i d i o t y p i c reper to i res between s t r a ins e x i s t s , although at l eas t three p o s s i b i l i t i e s may account f o r th i s observat ion . Many id iotypes may be present in each response, of which some cross-react between s t r a i n s . A second p o s s i b i l i t y may be that a small number of id iotypes ex i s t of which only one or two c ross-reac t , but these are dominant. F i n a l l y , a small amount of high t i t r e sera may be c ross- reac t ing , and a r t i f i c i a l l y in f luences the leve l of pool i n h i b i t i o n , as d iscussed in Chapter 3. As be fore , one must tes t ind i v idua l sera to determine the true i d i o t y p i c p i c ture - despi te the massive increase in complexity compared to that of working with pooled se ra . Figure 6 Inh ib i t ion of Pooled Te r t i a r y Anti-Fd Sera from AKR/J, RF/J, A/J and B10.BR/SnJ Mice  Te r t i a r y ant i-Fd sera from the ind ica ted mouse s t r a ins were pooled from 12-15 animals and tested in t r i p l i c a t e f o r i n h i b i t i o n of binding to Fd by the ind ica ted d i l u t i o n s of an t i - id io t ype . The standard er rors are represented by the v e r t i c a l bars . 82 Figure 7 Inh ib i t ion of Pooled Te r t i a r y Anti-Fd Sera from C3H/HeJ, ST/bJ, C58/J and CE/J Mice  Te r t i a r y ant i-Fd ant i sera were pooled from 12-15 ind i v idua l s of the ind ica ted mouse s t r a i n and tested f o r i n h i b i t i o n of binding to Fd in t r i p l i c a t e with various d i l u t i o n s of the ind icated an t i - i d i o t ype . The standard er rors are represented by the v e r t i c a l bars . 84 O ffi Z o ui a. 100. 90. 80 70 60 50 40 30 20 10 0 100 90 80 70 60 50 40 30 20 10 0 AKR onti-id BIO.BR anti-id A/J anti-id RF/J anti-id C3H N 4 - M I T ST/bJ C58 CE 2 4 8 16 32 64 I 2 4 8 16 3264 DILUTION X I0"2 Figure 8 Inh ib i t ion of Pooled Te r t i a r y Anti-Fd Sera from C57BR/cdJ Mice Te r t i a r y ant i-Fd a n t i s e r a , pooled from 9 C57BR/cdJ mice, were tested in t r i p l i c a t e f o r i n h i b i t i o n of binding to Fd by various d i l u t i o n s of the ind ica ted an t i - id io t ype . Percent i n h i b i t i o n i s represented on the v e r t i c a l a x i s . The standard er rors are represented by v e r t i c a l bars . C57BR AKR anti-id BIO.BR anti-id A/J anti - id RF anti-^id I 2 4 8 16 32 64 DILUTION X 10-2 87 B) I n te r s t r a in Cross-react ive Idiotypes in Indiv idual Anti-Fd Sera The sera of mice immunized with the o r i g i na l Fd preparat ion were tested f o r i n h i b i t i o n at var ious times during the response with the a n t i - i d i o t y p i c reagents discussed in Chapter 3. The resu l t s are shown in Figures 9 - 16. The responses of AKR, RF, and BIO.BR to t h e i r own an t i - i d i o t ypes , are a lso i l l u s t r a t e d f o r comparative purposes on l y , and have already been discussed in Chapter 3. The standard er rors were very r a re l y above 10% and thus ind i v idua l er rors are not marked. The data a re , as expected, qui te complex and w i l l be discussed sequen t i a l l y . Figure 9 shows the i d i o t y p i c response of AKR mice. As prev ious ly d i s cussed , and expected, most of the mice are i nh ib i t ed with the anti-AKR id io t ype . Mu l t ip l e examples e x i s t , however, of strong c ross- reac t i v i t y with the ant i- id io types of other s t r a i n s . In f a c t , a l l an t i- id io types used were capable of i n h i b i t i n g some ind iv idua l se ra . With AKR mice the frequency of c ros s- reac t i v i t y appears the same throughout the response, although th i s cannot be i n d i v i d u a l l y proven with the unmarked mice used in th i s experiment. In t h i s experiment (and in a l l other experiments) one observes some sera that cross-react with more than one a n t i - i d i o t y p e , as well as some sera that are not i nh ib i t ab l e with any an t i - i d i o t ype . Some sera i n h i b i t i o n s add up to greater than 100% cumulative i n h i b i t i o n with d i f f e r e n t an t i - i d i o t ypes , implying the presence of the same i d i o t y p i c determinants recognized by d i f f e r e n t an t i - i d i o t ypes . The un inh ib i t ab le sera show that the ant i- id io types are s p e c i f i c f o r i d i o t y p i c determinants, but that the four reagents do not def ine the to ta l ant i-Fd response in mice. S t a t i s t i c a l analyses are seen in Table III. The resu l t s with anti-AKR and anti-RF i n h i b i t i o n frequencies are s t a t i s t i c a l l y s i g n i f i c a n t , whi le i t i s qui te obvious that some mice are making A/J and BIO.BR Figure 9 Inh ib i t ion of Indiv idual Anti-Fd Sera from AKR/J Mice  Indiv idual ant i-Fd ant i sera from AKR mice obtained at var ious times in t h e i r ant i-Fd responses were tested in t r i p l i c a t e fo r i n h i b i t i o n of binding of Fd by the var ious an t i - i d i o t ypes . Percent i n h i b i t i o n i s represented by the v e r t i c a l a x i s . Data f o r a s ing le serum can be fol lowed v e r t i c a l l y . Standard er rors were less than 10 percent i n h i b i t i o n . 1 ° , DAY 15 1°, DAY 21 2 ° , DAY 7 2 ° , DAY 15 3°, DAY 7 3 ° , DAY 15 75 anti-AKR 50 25 75 anti — RF/J 50 25 75 anti-A/J 50 25 75 anti-BIO.BR 50 25 Ik t L . I L . . i l l l l l JlL 11 i l u 4U. J r ^ f — f i INDIVIDUAL AKR an t i - Fd SERA Figure 10 Inhibition of Individual Anti-Fd Sera From RF/J Mice  Individual anti-Fd antisera from RF/J mice obtained at various times in their anti-Fd responses were tested in t r i p l i c a t e for inhibition of binding of Fd by the various anti-idiotypes. Percent inhibition is represented by the vertical axis. Data for a single serum can be followed vertically. Standard errors were less than 10 percent. I • , DAY 15 1°, DAY 21 2% DAY 7 2% DAY 15 3 ° , DAY 7 3 ° , DAY 15 75 anti-AKR 50 25 75 anti- RF/J 50 25 . 75 anti-A/J 50 25 . 75 anti-BI0.BR 50 25 ill 11 Jl l lillll f . . . l f JL i.l.lfr U L J J INDIVIDUAL RF/J a n t i - F d SERA Figure 11 Inh ib i t ion of Indiv idual Anti-Fd Sera from A/J Mice  Indiv idual ant i-Fd ant i se ra from A/J mice obtained at var ious times in t h e i r anti-Fd responses were tested in t r i p l i c a t e f o r i n h i b i t i o n of binding of Fd by the var ious an t i - id io t ypes . Percent i n h i b i t i o n i s represented by the v e r t i c a l ax i s . Data f o r a s ing le serum can be fol lowed v e r t i c a l l y . Standard er rors were less than 10 percent. 75 anti-AKR 50 25 75 anti-RF/J 50 25 75 anti -A/J 50 75 antl-Bl0.BR 50 25 , DAY 15 1°, DAY 21 2 ° , DAY 7 2 ° , DAY 15 3° , DAY 7 3 ° , DAY 15 25 , A i 1 1 -l.it A ,f..n i. Jhl l.Hi.li..f, i.fil.1 LUi LIU INDIVIDUAL A/J an t i -Fd SERA Figure 12 Inh ib i t ion of Indiv idual Anti-Fd Sera from B10.BR/SnJ Mice  Indiv idual ant i-Fd ant i sera from B10.BR mice obtained at var ious times in t h e i r ant i-Fd responses were tested in t r i p l i c a t e f o r i n h i b i t i o n of binding of Fd by the various an t i - i d i o t ypes . Percent i n h i b i t i o n i s represented by the v e r t i c a l a x i s . Data f o r a s ing le serum can be fol lowed v e r t i c a l l y . Standard er rors were less than 10 percent. 1 ° , DAY 15 1°, DAY 21 2 ° , DAY 7 2° , DAY 15 3°, DAY 7 3 ° , DAY 15 75 anti-AKR 50 4 25 75 anti-RF/J 50 25 75 anti-A/J 50 25 . 75 anti-BI0.BR 50 25 . i . r l . j . l . IN •t.l.if.l, ..i..n,.f . i . , n . 1 uL JL llllL d l l l INDIVIDUAL BIO.BR anti- Fd S ERA Figure 13 Inh ib i t ion of Indiv idual Anti-Fd Sera from C58/J Mice  Indiv idual ant i-Fd ant i se ra from C58/J mice obtained at var ious times in t h e i r anti-Fd responses were tested in t r i p l i c a t e f o r i n h i b i t i o n of binding of Fd by the various an t i - i d i o t ypes . Percent i n h i b i t i o n i s represented by the v e r t i c a l a x i s . Data f o r a s ing le serum can be fol lowed v e r t i c a l l y . Standard er rors were less than 10 percent. 1°, DAY 15 1°, DAY 21 2 ° , DAY 7 2 ° , DAY 15 3°, DAY 7 3 ° , DAY 15 75 anti-AKR 50 25 75 anti-RF/J 50 25 75 anti-A/J 50 25 75 anti-BI0.BR 50 25 M1_ lull W i l l iLuk ll-.I.. Ill f , l , . l l . .n. l , l u L I, i.-i-iii—.i i l l J U L . i l l I L L INDIVIDUAL C58 an t i -Fd SERA Figure 14 Inh ib i t ion of Indiv idual Anti-Fd Sera from C57BR/cdJ Mice  Indiv idual ant i-Fd ant i sera from C57BR/cdJ mice obtained at var ious times in t he i r ant i-Fd responses were tested in t r i p l i c a t e f o r i n h i b i t i o n of binding of Fd by the var ious an t i - i d i o t ypes . Percent i n h i b i t i o n i s represented by the v e r t i c a l a x i s . Data f o r a s ing le serum can be fol lowed v e r t i c a l l y . Standard er rors were less than 10 percent. CTl , DAY 15 1°, DAY 21 2 ° , DAY 7 2°, DAY 15 3°, DAY 7 3 ° , DAY 15 75 anti-AKR 50 25 75 anti— RF/J 50 25 75 anti-A/J 50 25 75 anti-BlO.BR 50 25 A AL 1 JUL U l LL t.fil.rii i f a l l l i L 1. ,i-•f i f .1 ll tflli.LI JLiL INDIVIDUAL C57BR a n t i - F d SERA Figure 15 Inh ib i t i on of Indiv idual Anti-Fd Sera from C3H Mice Indiv idual ant i-Fd ant i se ra from C3H mice obtained at var ious times in t h e i r ant i-Fd responses were tested in t r i p l i c a t e f o r i n h i b i t i o n of binding of Fd by the var ious an t i - i d i o t ypes . Percent i n h i b i t i o n i s represented by the v e r t i c a l ax i s . Data f o r a s ing le serum can be fol lowed v e r t i c a l l y . Standard er rors were less than 10 percent. , DAY 15 1°, DAY 21 2 ° , DAY 7 2 ° , DAY 15 3°, DAY 7 3 ° , DAY 15 75 anti—AKR 50 25 . 75 anti-RF/J 50 25 . 75 anti-A>(J 50 25 . 75 anti-BlOBR 50 25 I., l i i i . i II...lt .1 ll nil jjffil.r lirflLL ilrlJiiL. . 1 . . . . . rf.rr.tL  i It l l.j r f1.1 1 .  iLliAl INDIVIDUAL C3H an t i -Fd SERA Figure 16 Inh ib i t ion of Indiv idual Anti-Fd Sera from CBA/J Mice  Indiv idual ant i-Fd ant i se ra from CBA/J mice obtained at var ious times in t h e i r ant i-Fd responses were tested in t r i p l i c a t e f o r i n h i b i t i o n of binding of Fd by the various an t i - i d i o t ypes . Percent i n h i b i t i o n i s represented by the v e r t i c a l ax i s . Data f o r a s ing le serum can be fol lowed v e r t i c a l l y . Standard er rors were less than 10 percent. 2° day 8 2° day 15 3° d a y 8 anti-AKR anti— RF anti- A/J anti—BIO.BR 50 25 . 75 . 50 25 75 50 25 25 J 9 k 1.1,1 LlLiLjtJ rJ..I .Tt?M..f ..Jr.. . I f ..f... -»---t -.I.tfJil.T ..IT t ,nit , . t . . -h INDIVIDUAL CBA anti-Fd SERA 104 i d i o t y p e s , although the frequency of i n h i b i t i o n of these mice with those two ant i- id io types i s not s t a t i s t i c a l l y d i f f e r e n t from the KLH con t ro l s . Thus, some AKR mice produce i d i o t y p i c determinants that are seen in other s t r a i ns by r a b b i t s , though, as well some AKR sera are not i n h i b i t a b l e with any of the four reagents. There seems to be no h ighly s p e c i f i c cross-r e a c t i v i t y with any s ing le s t r a i n , and no obvious change in the frequency of i n h i b i t i o n over t ime. The resu l t s f o r RF/J mice are shown in Figure 10. There i s a s i g n i f i c a n t frequency of c ross- reac t i v i t y with AKR and A/J id iotypes but not with those of B10.BR (see Table I I I ) . At l eas t one RF/J serum reacts f a r above background with the anti-B10.BR id io t ype . Aga in , un inh ib i t ab le sera and cumulative i n h i b i t i o n s greater than 100% are seen with RF/J s e ra , as was the case with AKR se ra , with up to four d i f f e r e n t an t i - i d i o t ypes . This s t rongly impl ies the presence of mu l t i -s t r a in determinants on the same molecule. No obvious changes over time are seen. A/J ind iv idua l sera were tested with the resu l t s shown in Figure 11. A s i g n i f i c a n t frequency of A/J sera are i n h i b i t a b l e by anti-RF/J i d i o t y p e , but not by anti-AKR or anti-B10.BR. Genera l l y , A/J mice showed poor cross-r e a c t i v i t y with other i d io t ypes . This i s in agreement with the resu l t s discussed in Chapter 3, in which i t i s l i k e l y that A/J mice express many id iotypes in the response to Fd. Figure 12 shows the resu l t s with B10.BR se ra . A very s t r i k i n g cross-r e a c t i v i t y i s seen between B10.BR and AKR i d i o t y p i c r epe r to i r e s . Almost 105 Table III A Summary of the Reac t i v i t i e s of Various Mouse Stra ins  to Four Anti-Fd Ant i- id io types This tab le shows the s t a t i s t i c a l comparisons of the var ious s t ra ins of mice with d i f f e r e n t : an t i - id io t ypes . The percent frequency of response, time averaged to secondary Day 15 i s given in a column fo r each an t i - i d i o t ype , fol lowed by the "P" value of a s t a t i s t i c a l comparison with the KLH control (as c a r e f u l l y explained in Appendix 1) . These data merely summarize the graphica l r esu l t s of Figures 9 to 16 and 17 to 20. 106 Table III - Summary of S t a t i s t i c a l Comparisons f o r Chapters 3 and 4 Test S t ra in % AKR P % RF P % A/J P % BIO.BR P 1 AKR 73.5 < .005 34.0 £.025 32.1 NS 30.2 NS 1 RF 30.0 < .05 66.0 <.005 45.3 5.025 17.0 NS 1 A 18.5 NS 35.2 <.025 64.8 1.005 3.8 NS 1 BIO.BR 58.9 1.005 11.1 NS 4.5 NS 94.5 1.005 1 C58 58.2 i .005 20.9 NS 52.3 1.005 7.6 NS 1 C57BR 10.0 NS 16.7 NS 80.0 £.005 26.7 NS 1 C3H 33.3 1.025 13.3 NS 20.0 NS 15.6 NS 1 CBA 46.5 i.005 0.0 NS 0 NS 4.5 NS 1 Mix (KLH) 6.4 — 6.4 — 9.7 — 9.7 2 AKR 55.8 <.005 0.0 NS 0 NS 26.9 NS 2 RF 50.0 1.005 83.3 1.005 39.6 ^.025 27.1 NS 2 A/J 26.7 NS 18.3 NS 16.7 NS 21.7 NS 2 BIO.BR 58.3 1.005 35.0 1.025 23.3 NS 58.3 i .005 BIO.BR(KLH) 4.8 NS 16.6 NS 9.5 NS 4.1 NS NS = P>.05 (Not S i gn i f i c an t ) 107 60% of B10.BR mice are s i g n i f i c a n t l y i nh ib i t ed by anti-AKR id io type (see Table I I I ) . Anti-BIO.BR i s a f a i r r e f l e c t i o n of the reper to i re in the B10.BR animals , as almost 95% make a s i g n i f i c a n t amount of recognized i d i o t ype . Many ant i se ra express both AKR and B10.BR id io types with cumulative i n h i b i t i o n s approaching 200%. One serum i s 90% i n h i b i t a b l e with anti-AKR, 90% i nh ib i t ab l e with anti-RF/J and 95% i n h i b i t a b l e with anti-B10.BR. This i s very strong c i rcumstant ia l evidence that the three s t ra ins express the same i d i o t y p i c determinants, on the same molecule. Why i s i t that B10.BR i s i n h i b i t a b l e with anti-AKR, whereas the reverse i s not the case? The eas ies t explanat ion i s that the reper to i re of B10.BR animals i s qu i te r e s t r i c t e d , and overlaps with AKR. AKR reper to i re may be l a r g e r , thus l ess l i k e l y to be i nh ib i t ed with anti-B10.BR reagents. Another i n t e r es t i ng observat ion i s the change in frequency of AKR id io type expression in the B10.BR response, over t ime. Th is change i s h igh ly s i g n i f i c a n t (p < .005 , Df. =5) and occurs during the 50 day gap between the 2° and 3 ° responses. Strangely enough, there i s no change in the frequency of anti-B10.BR i n h i b i t i o n , and no change in the mean anti-B10.BR i n h i b i t i o n . Thus the anti-AKR and anti-B10.BR i d i o t y p i c reagents do not always recognize the same determinants. As the anti-B10.BR an t i- id io type was made to the l a t e 30 se ra , i t understandably would not recognize the l o s t AKR id iotypes and would thus not great ly i n h i b i t AKR ant i-Fd sera . The concept of i d i o t y p i c maturation w i l l be discussed l a t e r . In conc lus i on , Figure 12 shows that a high proport ion of B10.BR mice express AKR i d i o t y p e , but th i s frequency changes over t ime. These resu l t s may be due to an overlap between unequal s i ze r e p e r t o i r e s , or to a loss of AKR determinants of B10. BR ant ibodies before the l a t e t e r t i a r y response. 108 The resu l t s of experiments with C58 mice are shown in Figure 13. A high frequency of these mice express i d i o t y p i c determinants recognized by both anti-A/J and anti-AKR/J i d io t ypes . Not a l l C58 id iotypes can be def ined with the four reagents, though sera i nh ib i t ed with anti-A/J are almost completely i n h i b i t e d , poss ib l y implying a r e s t r i c t e d i d i o t y p i c response in these mice. No obvious changes in i n h i b i t i o n frequency over time are observed. C57BR/cdJ mice have an i n t e r es t i ng pattern of i n h i b i t i o n , as shown in Figure 14. Almost a l l sera were h igh ly i nh ib i t ed by anti-A/J i d i o t ype . Almost no r e a c t i v i t y i s seen with any other an t i - id io t ype . Thus, the data from Figures 13 and 14 show that though the anti-A/J id io type w i l l not i n h i b i t d i f f e r e n t groups of A/J mice, C58 and C57BR ant i se ra are h igh ly i n h i b i t a b l e at high frequency with th i s reagent. C3H and CBA mice may be discussed together as they d i sp lay almost i den t i ca l patterns of express ion , shown in Figures 15 and 16. Both groups are s i g n i f i c a n t l y i nh ib i t ed by anti-AKR i d i o t y p e , and are un inh ib i t ab le with any other reagent. The amount of i n h i b i t i o n with anti-AKR i s qui te low. P l a i n l y , the major i ty of the i d i o t y p i c response of these two s t r a ins cannot be def ined by the four a n t i - i d i o t y p i c reagents. Although the ind iv idua l i n h i b i t i o n data i s qui te complex, several gene ra l i t i e s can be made concerning the presence of c ross-react i ve i d i o t y p i c determinants in the var ious high responder s t r a i n s . A l l of the s t ra ins tested cross-react with at l e as t one of the four an t i - i d i o t ypes , though not a l l with the same reagent. This pattern i s almost always constant over the durat ion of the response. Some sera cannot be def ined by any of the four an t i - i d i o t ypes , and some sera probably contain ant ibodies 109 with determinants shared by many s t r a i n s , though th i s cannot be d e f i n i t i v e l y proven by these experiments. C) K ine t i cs of Indiv idual I n te rs t ra in Cross-Reactive Idiotype Expression Mice were i n d i v i d u a l l y marked and immunized by the protocol d iscussed in Chapter 3. The resu l t s from AKR mice are shown in Figure 17. As can be seen, not much c ross- reac t i v i t y i s observed with other i d io t ypes . Thus, the s l i g h t degree of c ross- reac t i v i t y seen with the anti-RF/J id iotype in Figure 9 cannot be reproduced. There does appear to be some c ros s- reac t i v i t y with BIO.BR, of the same order as observed e a r l i e r (see Table I I I ) . Th is i s important, s ince e a r l i e r experiments showed strong c ros s- reac t i v i t y between BIO.BR and AKR i d i o t ypes . The k ine t i c s show changes in only the frequency of AKR i d i o t y p e , which w i l l be discussed in the next chapter. The data from RF/J animals f u l l y corroborate e a r l i e r experiments. F igure 18 shows an even higher frequency of i n h i b i t i o n with anti-RF/J id iotype and with anti-AKR id iotype than that found in r esu l t s presented in Figure 10. The frequency of i n h i b i t i o n with anti-A/J id iotype i s almost unchanged (see Table I I I ) . A l s o , no s i g n i f i c a n t frequency of i n h i b i t i o n with anti-B10.BR id iotype i s observed. A s t a t i s t i c a l ana lys is of the change in i n h i b i t i o n frequency over time (Table IV) shows no s i g n i f i c a n t d i f f e rences between sample per iods . A/J sera tested f o r i n h i b i t i o n exh ib i t the pattern shown in Figure 19. Although the time averaged data showed no s i g n i f i c a n t i n h i b i t i o n frequencies (Table III) above the KLH c o n t r o l s , inspect ion of the data shows that most primary sera are somewhat i n h i b i t e d , e spec i a l l y with anti-RF/J i d io t ype . When these data are tested f o r changes over time Figure 17 Inh ib i t ion of Anti-Fd Sera from  Ind iv idua l l y Marked AKR/J Mice: K ine t i cs of the Response Anti-Fd ant i se ra from marked AKR/J mice obtained at var ious times in t h e i r anti-Fd responses were tested in t r i p l i c a t e f o r i n h i b i t i o n of binding to Fd by the var ious an t i - i d i o t ypes . Percent i n h i b i t i o n at var ious times in the response i s represented by the v e r t i c a l a x i s . Data from a s ing le animal can be fol lowed v e r t i c a l l y . Standard er rors were less than 10 percent i n h i b i t i o n . day 28 day 15 day 15 day 15 75. 50. 25, 75 50J 25 75. 50. 25. 75. 50. 25. ff„„ttf.L.— .fiffl.jl. i l •T. ii •.hi. ..1 LllMiJ. Lit .f. II „ f infill _f . . .lil..t . .l Ink I [[ill 1, . . . . . i J l L . anti-AKR anti- RF anti-A/J anti— BI O.BR INDIVIDUAL AKR anti-Fd S ERA Figure 18 Inh ib i t ion of Anti-Fd Sera from  Ind iv idua l l y Marked RF/J Mice: K ine t i c s of the Response Anti-Fd ant i sera from marked RF/J mice obtained at var ious times in t he i r ant i-Fd responses were tested in t r i p l i c a t e f o r i n h i b i t i o n of binding to Fd by the var ious an t i - i d i o t ypes . Percent i n h i b i t i o n at var ious times in the response i s represented by the v e r t i c a l ax i s . Data from a s ing le animal can be fol lowed v e r t i c a l l y . Standard er rors were less than 10 percent i n h i b i t i o n . !• day 28 2° day 15 3° day 15 4 ° day 15 75 50 fl..1 Ii ml iriLt.... ..Ltlhir JU All Lit ...i h Jil Frl. LA. Ut.,.,1 #11 ttfl | l t Wtflfrt.-f anti-AKR anti— RF anti-A/J INDIVIDUAL RF anti-Fd SERA anti - BIO.BR 114 Table IV A Summary of the Change in Idiotype Expression over Time of 4 S t ra ins of Mice This table i s a composition of the data of Figures 3, 17, 18, 19 and 20. For each s t r a i n and an t i- id io type i nd i c a t ed , the f r a c t i o n of sera i nh ib i t ed above threshold are l i s t e d . The value of the Chi-square t es t ing f o r no change in i n h i b i t i o n frequency over time-, and the corresponding "P" value are g iven. The tes ts were performed as descr ibed in Appendix 1. 115 Table IV - Summary of K ine t i cs of Idiotype Expression S t ra in Ant i- id io type 1° 2 ° 3 ° 2 X P AKR/J AKR 2/13 11/14 9/14 11.8 5.005 II RF 0 0 0 ND NS II A 0 0 0 ND NS II BIO.BR 3/13 4/14 5/14 0.52 NS RF/J AKR 4/12 6/12 6/12 0.90 NS II RF 9/12 10/12 9/12 0.32 NS II A 4/12 3/12 5/12 0.75 NS II BIO.BR 2/12 4/12 4/12 1.11 NS A/J AKR 7/15 2/15 3/15 4.77 NS II RF 9/15 1/15 1/15 15.40 5.005 II A 6/15 1/15 2/15 5.84 NS II BIO.BR 6/15 4/15 2/15 2.73 NS BIO.BR AKR 8/15 9/15 9/15 0.19 NS II RF 2/15 5/15 10/15 9.26 5 .01 II A 1/15 2/15 6/15 5.84 NS II BIO.BR 8/15 11/15 9/15 1.32 NS BIO.BR(KLH) AKR 1/14 0/14 1/14 1.05 NS n RF 3/14 0/14 3/14 3.50 NS II A 2/14 1/14 1/14 0.55 NS II BIO.BR 1/14 1/14 0/14 1.05 NS NS = P< .05 ND = Not Done Figure 19 Inh ib i t ion of Anti-Fd Sera from  Ind i v idua l l y Marked A/J Mice: K ine t i cs of the Response Anti-Fd ant i se ra from marked A/J mice obtained at var ious times in t he i r ant i-Fd responses were tested in t r i p l i c a t e f o r i n h i b i t i o n of binding to Fd by the var ious an t i - i d i o t ypes . Percent i n h i b i t i o n at var ious times in the response i s represented by the v e r t i c a l a x i s . Data from a s ing le animal can be fol lowed v e r t i c a l l y . Standard e r rors were less than 10 percent i n h i b i t i o n . day 28 50 day 15 day 15 day 15 75 50 25 75 , 25 25 —Lit II IL 1.IM L J.l..l.t. I lIllLi. fjLLlffflf i-fff_i.rLi.lt f f-itTl-L .tftir[..,jii tJIfr. Jfffi. Jlfl....r.!ti. . 1 - 1 f Ilr..L.j. .JiiiJii.. t..Jf ..l.ll anti - AKR anti - RF anti— A/J anti-BIO.BR INDIVIDUAL A/J anti-Fd SERA 118 (Table IV) , there i s s i g n i f i c a n t dropoff of the frequency of i n h i b i t i o n with anti-RF/J i d io t ype . Although the frequency data i s not s t a t i s t i c a l l y s i g n i f i c a n t f o r other an t i - i d i o t ypes , one can see that the cumulative amount of i n h i b i t i o n decreases a f t e r the primary bleed f o r a l l an t i - i d i o t ypes . These observations seem to ind ica te that a change in id io type expression occurs during the maturation of the immune response of A/J mice to Fd. This change i s i d i o t y p i c p r o f i l e over time w i l l be re fe r red to as i d i o t y p i c maturation f o r s i m p l i c i t y , and w i l l be discussed in the next chapter. The data from BIO.BR an t i se ra are shown in Figure 20. These data completely reproduce the f ind ings of e a r l i e r experiments (Figure 12) although the frequency of expression of the BIO.BR id iotype i s somewhat lower. Exact ly the same frequency of i n h i b i t i o n with anti-AKR id io type i s seen as be fore , and, in a d d i t i o n , a s i g n i f i c a n t degree of c ros s- reac t i v i t y with RF/J id io types i s present (Table I I I ) . This c ro s s- reac t i v i t y changes s i g n i f i c a n t l y over t ime, though, in contrast to the e a r l i e r experiments, the frequency of AKR id iotype expression appears constant. Though not s t a t i s t i c a l l y s i g n i f i c a n t (P> .1) some evidence may a lso ex i s t f o r an increase in A/J expression over t ime. Thus, several i n t e res t i ng points emerge from the data of the second set of experiments. Genera l l y , with a few except ions , once an animal i s making a c ross-reac t i ve i d i o t ype , i t continues to make i t throughout the response. The exceptions are with some BIO.BR and A/J animals , which exh ib i t examples of i d i o t y p i c maturat ion, causing not iceable changes in the patterns of c ros s- reac t i v i t y over t ime. The cross r e a c t i v i t i e s observed in the f i r s t sets of experiments are a lso observed in the second s e t , with the exception of the A/J data . Indiv idual sera may express more than one cross-react i ve i d i o t y p i c f am i l y , poss ib l y with the determinants Figure 20 Inh ib i t ion of Anti-Fd Sera from  Ind iv idua l l y Marked B10.BR/SnJ Mice: K ine t i cs of the Response Anti-Fd ant i sera from marked B10.BR/SnJ mice obtained at var ious times in t h e i r ant i-Fd responses were tested in t r i p l i c a t e f o r i n h i b i t i o n of binding to Fd by the various an t i - i d i o t ypes . Percent i n h i b i t i o n at var ious times in the response i s represented by the v e r t i c a l ax i s . Data from a s ing le animal can be fol lowed v e r t i c a l l y . Standard er rors were less than 10 percent i n h i b i t i o n . 1° day 28 2°day 15 3°day 15 4° day 15 75 . 50 . 25 . 75 . 50 25 . 75 . 50, 25 i 75 . 5Q, 25. ialkil LULLII if..i.-..lJ.i LLLLIL ML LLO JMMMtl 1 •t.t..ttT.I.I ii.-t.i. M anti-AKR anti-RF anti— A/J anti—BIO.BR INDIVIDUAL BIO.BR anti-Fd SERA 121 on the same molecule conserved across a l l of the s t r a i n s . As w e l l , some sera cannot be i nh ib i t ed by any of the an t i - id io t ypes . D) E f f e c t of H-2 Genes on Idiotype Expression The sera of mice used in e a r l i e r experiments mapping to ta l and determinant s p e c i f i c responses to Fd in recombinant s t ra ins of mice were assayed f o r i d i o t y p i c s p e c i f i c i t y . These mice had been immunized by Dr. L. S ikora with her preparat ion of Fd (251). Figure 21 shows the e f f e c t of H-2 l inked genes on id io type express ion. Sera from B10.BR mice show the c l a s s i c pattern of i n h i b i t i o n with both anti-AKR and B10.BR an t i - i d i o t ypes . B10.S and SJL share the same H-2 reg ion , but only B10.S i s i nh ib i t ed with anti-A/J an t i - i d i o t ype . Obviously more genes than H-2 are involved in that p a r t i c u l a r i d i o t y p i c express ion. BIO, B10.A(3R) and B10.S(9R) do not have a high frequency of response with the expression of any of the four i d i o t y p e s , whereas BIO.A and B10.A(4R) have a reasonably high expression of B10.BR i d i o t y p i c determinants. S u r p r i s i n g l y , BIO.A express B10.BR id iotypes but not those of AKR. Some B10.A(2R) and B10.A(5R) are a lso able to express B10.BR id io t ypes . Though these data are from small sample s i z e s , they are very i n t e r e s t i n g . H-2 l inked genes do a f f e c t id io type express ion , as a l l of these BIO responding congenics would be expected to produce at l e as t B10.BR i d i o t y p i c determinants. This i s not seen, instead a va r i e t y of patterns of id io type expression are seen. Genera l l y , mice with H-2K/IA l o c i (251) seemed to produce the best expression of the B10.BR i d i o t y p e , though the data of B10.A(5R) and B10.A(3R) are not conc lus ive due to the small sample s i z e . S t ra ins with H-2K/IA'3 have members that are s t i l l able to express B10.BR i d i o t ypes . No explanat ion can be found f o r the B10.S expression of Figure 21 Inh ib i t ion of Secondary Anti-Fd Sera from  Various S t ra ins of  Mice D i f f e r i n g at H-2 Loci Indiv idual ant i-Fd sera from the secondary responses of mice d i f fe r ing- at H-2 l o c i were tested f o r i n h i b i t i o n of binding to Fd by var ious an t i - id io t ypes . The v e r t i c a l axis represents percent i n h i b i t i o n using the ind ica ted an t i - i d i o t ypes . Data from the same serum can be fol lowed v e r t i c a l l y . Standard e r rors were less than 10 percent i n h i b i t i o n . 75 . anti-AKR 50. 25, 75 . anti-RF 50 , 25 . 75 , anti-A/J 50. 25 75 J anti-BIOBR 50 25 J 1 r t . t f f IlJL -tl_-J l 1 ft... t t l In IT.. II I L 1 I L J L Ik Mi BIO.BR BIO.S SJL C57BL/I0 BIO.A BiO.A(2R) BI0.A(3R) BI0.A(4R) BI0.A(5R) BIO.SOR) INDIVIDUAL ANTI - Fd SERA 124 A/J i d i o t y p e , as th i s r e su l t i s not seen in B10.S(9R) or SJL mice. Probably other non H-2 l inked genes and poss ib l y other H-2 l inked genes are capable of i n f luenc ing the i d i o t y p i c response. In summary, the phenomenon of i d i o t y p i c c ro s s- reac t i v i t y i s both widespread and reproducib le in the anti-Fd antiserum from high responder mice. The to ta l i d i o t y p i c reper to i re of the anti-Fd response in mice i s not def ined by the four a n t i - i d i o t y p i c reagents used, implying a large amount of d i v e r s i t y . B10.BR mice appear to be the most r e s t r i c t e d of the s t r a ins i nves t iga ted . The expression of th i s id iotype appears to be inf luenced by both H-2 and non-H-2 l inked genes. II. D iscuss ion Sera from mice immunized with Fd were tested f o r the presence of four d i f f e r e n t i d i o t ypes . Two separate experiments were done with AKR/J, RF/J, A/J , and B10.BR/SnJ an t i s e r a . One experiment was done with C58/J, CBA/J, C57BR/cdJ and C3H/HeJ mice. These experiments y i e lded several i n t e r es t i ng r e s u l t s . A l l s t r a ins tested contained members whose ind iv idua l an t i se ra reacted with at l eas t one " f o r e i g n " an t i - i d i o t ype , and usua l l y representat ive i n h i b i t i o n was observed with a l l an t i - i d i o t ypes . Some s t r a ins had a very s i g n i f i c a n t frequency of c ross- reac t i v i t y such as B10.BR with AKR, C58 with AKR and A/J , RF with AKR and A/J , C57BR with A/J , and C3H and CBA with AKR. An i n t e r es t i ng trend can be observed from Table III. Ant i se ra from most s t ra ins could be i nh ib i t ed with ant i AKR i d i o t y p e , few s t ra ins cross-reacted extens ive ly with A/J or RF/J and only B10.BR were i n h i b i t a b l e with anti-B10.BR an t i - i d i o t ype . It would be des i rab le to f i nd some common ground to expla in these patterns of c r o s s - r e a c t i v i t y . Table V 125 has been assembled to show some add i t iona l information between the s t r a ins concerning some genes of immunological i n t e res t and Figure 22 es tab l i shes the r e l a t i onsh ip between the heavy chain l o c i by v i r tue of t h e i r Ig2a c ros s- reac t i v i t y (56,277). The c ross-react i ve ant i-Fd id iotype data has been summarized in the r e l a t i onsh ip diagram shown in Figure 23. No co r r e l a t i on between a l lo type i den t i t y and expression of i n t e r s t r a i n c ross-react i ve id iotypes can be seen. As w e l l , no co r r e l a t i on between id iotype sharing and breeding h i s to ry (278) can be d i sce rned , with one except ion. CBA/J and C3H/HeJ are i den t i ca l at the l o c i shown in Table V and are re la ted gene t i c a l l y by v i r tue of t he i r common breeding h i s t o r i e s (278). A computer search of a l l known mouse gene a l l o t y p e s , cross-matched with the id io type c ros s- reac t i v i t y patterns may show an eventual pa t t e rn , but such a monumental undertaking i s beyond the scope of th i s t h e s i s . Instead, a b r i e f d i scuss ion comparing th i s work to f ind ings reported in other prote in systems i s in order . As prev ious ly d i s cussed , the i d i o t y p i c response to prote ins i s much more complex than the response to haptens. Idiotypes to HEL were shown to be present in the anti-HEL response of a l l mouse s t ra ins t e s t ed , with no co r r e l a t i on to H-2 or Igh a l l e l e s (206) and such id iotypes were a lso present on antibody d i rec ted to d i f f e r e n t determinants of HEL (208). Diverse s t r a i n or species i d i o t y p i c c ross- reac t i v i t y i s seen in the anti-GAT response (153, 154) in which ant ibodies to d i f f e r e n t determinants can a lso share the same id iotypes (165). Idiotypes produced in the a n t i - i n s u l i n response are shared by d iverse s t ra ins of mice, although one pub l i c id io type may occur in greater amounts in mice of Igh a a l l o t ype 126 Table V A Summary of A l lo types of Mice at Loci of  Immunological Interest This tab le represents the a l lo types of mice at heavy chain and l i g h t chain l o c i compiled from the ind ica ted references . 127 Table V - A l lo types of Mice at Loci of Immunological Interest Reference S t ra in lg2a lg2b i g i lgM E f l Ef2 lgA AKR/J d d a NT a a d RF/J c a a c b a d A/J e e a c b a d B10.BR/SnJ b b b b b a b C58/J a a a a a b a C3H/HeJ j a a NT b a a CBA/J j a a NT b a a CE/J f f a NT b b f St/bJ a a a a b a a C57BR/cdJ a a a a b a a Data from references 56, 271 and 277 NT = Not Tested Figure 22 Simple Relatedness Tree of Immunoglobulin  Heavy Chain A l lo types in Mice This diagram i s a representat ion of the relatedness of the heavy chain l o c i of mice compiled from a se ro log i ca l i nves t iga t ion of t h e i r heavy chain a l lo t ype immunological s p e c i f i c i t i e s as de ta i l ed in references 56 and 277. CTl C\J THEORETfCA L ANCESTRAL Ig 2a ALLOTYPE C57BR C3H C58 Figure 23 Re la t ionship of Various Anti-Fd Id io typ ic  Famil ies This i s a simple r e l a t i onsh ip diagram i l l u s t r a t i n g the d i s t r i b u t i o n of id iotypes among the s t ra ins of mice used in t h i s t h e s i s . The data are a compi lat ion of the resu l t s of Chapter 4. 131 RELATIONSHIP OF IDIOTYPIC FAMILIES 132 (244). This pub l i c id io type can be shared by antibody d i rec ted to d i f f e r e n t s p e c i f i c i t i e s (244) as can pub l i c id iotypes in the anti-H-2K system (279), the anti-SWM system (194), the rabb i t anti-hemoglobin system (280), and even the ARS hapten system (45). Another example of an i n t e r s t r a i n c ross-react i ve id iotype i s seen in the response to TGAL. C57BL/10 i d i o t y p i c determinants are seen on a wide var ie ty of anti-TGAL ant ibodies (238). I n t e res t i ng l y , a s im i l a r type of one way c ross- reac t i v i t y seen in th i s work with the B10.BR and AKR responses i s a lso seen in the TGAL system. C3H.SW mice produce anti-TGAL ant ibodies i n h i b i t a b l e with anti-BlO i d i o t y p e , yet the C3H.SW id iotype i s only found in C3H.SW sera (240). This i s explained i f the BIO i d i o t y p i c reper to i re i s much la rger than and includes the C3H.SW r epe r to i r e . A l t e r n a t i v e l y , the BIO mice may not be making the C3H.SW id iotype at the stage of the response at which they were t e s t e d , but are capable of expressing those determinants at an e a r l i e r or l a t e r stage. Some examples of i d i o t y p i c maturation are evident in the data presented in Chapters 3 and 4. Other systems where th i s phenomenon may occur are NP (88), ARS (281), and HEL (208). The HEL system (208) shows an increase in the frequency of IdX over t ime, whereas the ARS system (281) and the NP system (88) show that the predominant id iotype i s expressed more poorly as the response progresses. An i n t e res t i ng co r re l a t i on between IgM and CRI expression at the leve l of the precursor B c e l l has been shown. IgM PFC remain associated with the CRI but IgG PFC undergo a substant ia l decrease in the frequency of CRI express ion. This disappearance of CRI co r re l a tes with an up to 10 f o l d increase in ARS a f f i n i t y , showing that the id io type maturation of IgG PFC may be a f f i n i t y dr iven (281). I f id io type 133 maturation e x i s t s , one would expect to see an increase in detectable id iotype frequency over time since the an t i- id io type i s d i rec ted to t e r t i a r y determinants. Such examples are seen in the AKR i d i o t y p i c and in the BIO.BR cross-react i ve RF i d i o t y p i c responses (Table IV). The A/J id io type appears to change with respect to a l l reagents. D i f ferences in th i s maturation between the two experiments may be due to d i f fe rences in presentat ion of the two d i f f e r en t Fd prepara t ions , promoting d i f f e r en t regulatory c i r c u i t s , or to a large A/J ant i-Fd reper to i re and many regulatory methods so that no two groups of A/J mice respond a l i k e . The l a s t p o s s i b i l i t y appears un l i ke l y based on observations with other ant igen ic systems. The best explanat ion f o r i d i o t y p i c maturation i s that i t i s a f f i n i t y dr iven (62, 281). This explanat ion assumes that i d i o t y p i c determinants are very c lose l y re la ted to paratopic determinants. Many pub l i ca t ions show that th i s assumption i s not always cor rec t (165, 208, 244, 279, 280). No sa t i s f a c to r y explanation accounts f o r a l l cases of i d i o t y p i c maturat ion, inc lud ing those observed in th i s work. Probably many mechanisms inc lud ing a f f i n i t y , ease of genet ic rearrangement and mutat ion, and T c e l l r egu l a t i on , and au to-ant i- id io typ i c regulat ion may account f o r these phenomena. In summary, the data of th i s chapter show that extensive i n t e r s t r a i n i d i o t y p i c c ross- reac t i v i t y ex i s t s in the murine anti-Fd response. No co r r e l a t i on between known genet ic markers and i d i o t y p i c pattern i s obvious, but H-2 and non-H-2 genes seem to play a ro le in in f luenc ing the expression of at l eas t the BIO.BR id io t ype . Several cases of change in id iotypes expressed over time have been observed. Not a l l of the poss ib le anti-Fd id io types can be def ined by the four ant i- id io types used. A thorough 134 discuss ion of the concepts of id io typy and c ross- reac t i v i t y i s needed. Such top ics w i l l be discussed in a l a t e r chapter. 135 Chapter 5. Id io typ ic Charac te r iza t ion of Monoclonal Ant i-Ferredoxin Ant ibodies 1.) Results A) S p e c i f i c i t y of Monoclonal Anti-Fd Ant ibodies Monoclonal ant ibodies were produced from hybridomas r esu l t i ng from a fus ion of anti-Fd hyperimmune splenocytes from C57BR mice, and NS-1 as descr ibed in Methods. The splenocytes of the mice were pooled fo r the f u s i ons . One of the mice was a high producer of A/J i d i o t ype , whereas the other was a low producer (data not shown). It was hoped that the monoclonal ant ibodies produced would r e f l e c t the immune response of the donor mice. Six c e l l l i nes were i so l a t ed from 42 anti-Fd pos i t i v e fus ion products by v i r tue of t h e i r s p e c i f i c r e a c t i v i t y f o r Fd over KLH. Since 11 Fd-binding c e l l l i nes prev ious ly i so l a t ed showed poor s p e c i f i t y of antigen b i nd ing , i t was necessary to e s t ab l i sh the s p e c i f i c i t y of these monoclonals. A comparison between Fd and KLH binding in the ELISA assay i s shown in Figure 24. One can see the very high s p e c i f i c i t y of the monoclonal ant ibodies f o r Fd , although the a c t i v i t y of H21 i s qui te low. Since th i s hybridoma proved d i f f i c u l t to c lone , i t was not used f o r fu r ther experiments. B) Determinant Charac te r iza t ion of Monoclonal Anti-Fd Antibodies i The % N-determinant and % C-determinant charac te r iza t ions were performed as prev ious ly descr ibed (250). The i n h i b i t i o n percentages were ca l cu la ted as f o l l ows , where 0% control i s the un inh ib i ted serum b ind ing : 136 Figure 24 : S p e c i f i c i t y of C57BR Anti-Fd  Monoclonal Ant ibodies Monoclonal antibody supernatants were tested f o r binding to Fd (A) or KLH (•). Standard er rors were less than 10 percent. Hybridomas are designated by numbers 1 to 34, and u t i l i z e d at the ind ica ted d i l u t i o n s . 137 A B S O R B A N C E a t 4 0 5 n m 138 % I = 1 - (N or C) i n h i b " - Fd i nh i b " X 100% 0% control - Fd i nh i b " The r esu l t s of these experiments are shown in Figure 25. One can see that a l l of the C57BR monoclonals are i nh ib i t ed much more with the C-determinant fragment, except f o r Fd-1, which i s known to be N-speci f ic (250), and H4. This antibody probably recognizes a conformational determinant that i s destroyed in the enzymatic d iges t ion of Fd required to produce the N or C- determinants. The C57BR monoclonals i so l a ted fo l low k the cha r a c t e r i s t i c s of H-2 mouse sera in that most of them are d i rec ted to the C-determinant. Consequently, one may expect t h e i r i d i o t y p i c p r o f i l e to represent some of the serum pat terns . C) Id io typ ic Charac te r iza t ion of Monoclonal Ant i-Ferredoxin Ant ibodies F ive C57BR monoclonal an t ibod ies , Fd-1 (from c e l l s donated by Dr. M. Weaver) and RIO and R32 (donated by R. Singhai) were tested in the i n h i b i t i o n assay. The resu l t s are shown in Figure 26. Note that Fd-1, HI, and H5 do not react with any of the an t i - id io t ypes . Though Fd-1 has been proposed as a major id iotype in the anti-Fd response of BIO.BR mice (252) and i s present in over 30% of the anti-N response, i t s net amount would be at best only 5% of the to ta l anti-Fd response. Thus, i t i s probably present in such low amounts in BIO.BR anti-Fd sera that i t would f a i l to immunize a r abb i t . This observat ion i s in agreement with the r esu l t s of Chapter 4 in which congenic mice which produced a high anti-C d i rec ted response appeared to be more i n h i b i t a b l e with anti-B10.BR id io t ype . The HI and H5 monoclonals, though C-determinant s p e c i f i c , bear i d i o t y p i c determinants that do not cross-react with any of the four tes t s t r a i n s . The i n t e res t i ng resu l t s can be observed from the data of H16 and H34. These two monoclonals are very s t rongly i nh ib i t ed by A/J an t i - id io t ype . Figure 25: Determinant S p e c i f i c i t y of C57BR Anti-Fd Monoclonal Ant ibodies A 1:10 d i l u t i o n of hybridoma supernatant was incubated with a fragment of Fd bearing e i the r the C or N determinant. The percent i n h i b i t i o n of Fd binding i s represented by the v e r t i c a l ax i s . The standard er rors are ind icated by the v e r t i c a l bars . 140 PERCENT INHIBITION • mm • • X i 5? 8 s I O z 4k X 0) X 1 CO o --4 X OI 4k Figure 26: Inh ib i t ion of Monoclonal Anti-Fd Ant ibodies with Various Ant i- id io types A d i l u t i o n of monoclonal antibody corresponding to 0.5 A ^ g / h o u r was incubated with an t i- id io type and tested f o r i n h i b i t i o n of binding to Fd. Each panel ind icates a separate monoclonal antibody and four an t i - i d i o t ypes , with the v e r t i c a l axis represent ing percent i n h i b i t i o n . The standard er rors are as ind icated by the v e r t i c a l bars . H4 H34 AKR RF A/J BR AKR RF A/J BR H5 x±ff_L 6 - 9 0 rH RIO R32 m_ t ± _ RF A/J BR AKR RF A/J BR 143 Such a s i t ua t i on i s observed with ind iv idua l C57BR anti-Fd sera (Chapter 4 ) . In a d d i t i o n , RIO and R32 are s t rongly i nh ib i t ed with both anti-AKR and anti-B10.BR an t i - i d i o t ypes , again r e f l e c t i n g cha r a c t e r i s t i c s seen commonly in ind iv idua l BIO.BR anti-Fd sera . H4 i s i nh ib i t ed by anti-AKR a n t i - i d i o t y p e , aga in , an id iotype occas iona l l y expressed in the C57BR serum response. Thus, three of f i v e monoclonals bear i d i o t y p i c determinants observed in the C57BR serum anti-Fd response. Two BIO.BR monoclonals a lso show cha r a c t e r i s t i c s of ind iv idua l parental se ra . Another very important conclus ion can be gained from H16, H34, RIO and R32. These monoclonals are i nh ib i t ed by more than one an t i - id io t ype . This observat ion confirms the data from Chapter 4, in which i t was postulated that major c ross-react ive i d i o t y p i c determinants of d i f f e r e n t s t ra ins were present on the same molecule. There fore , the use of monoclonal ant ibodies has strongly coroborrated e a r l i e r observat ions. The same phenomena seen with the serum response are observed at the c e l l u l a r , indeed molecular , l e v e l . Major c ross-react ive i d i o t y p i c determinants are shared between d i f f e r e n t s t ra ins and several of these determinants can be present on the same molecule. II. Discuss ion The preceding two chapters have dea l t with the expression of id io types on serum immunoglobulins. These observations may be quite d i f f e r en t from these at the leve l of the B c e l l (41). I f the B c e l l s producing a ce r ta in id io type are r e l a t i v e l y few in number, compared to a large suppressed popu la t ion , one would not expect to see serum patterns produced from a small number of antigen binding B c e l l s . In f a c t , 3 of 5 C57BR, and 2 of 3 144 B10.BR hybridomas exh ib i t the same cross-react i ve patterns as serum ant ibod ies . These data ind ica te that a few dominant clones may determine the serum id io type pattern in these s t r a i n s . The data from the monoclonals a lso ind ica tes the u n i v e r s a l i t y of some id io t ypes . Since both A/J and RF/J determinants are found on one C57BR hybr id , and B10.BR and AKR determinants are found on B10.BR hybr ids , there must be shared major id iotypes in these s t r a i n s . As w e l l , the monoclonals serve as good contro ls fo r the s p e c i f i c i t y of the a n t i - i d i o t y p i c reagents. Figure 26 shows almost no i n h i b i t i o n of HI and H5 by any an t i - id io t ype . Since monoclonal ant ibodies cons i s t of only one s p e c i f i c immunoglobulin (by de f i n i t i on ) one would expect any non-spec i f i c RaMIg e f f e c t to se r ious l y impair the monoclonal b ind ing . Several other systems have f i r s t been def ined by serum id iotypes and then have been fu r ther character ized with monoclonals. This approach has been taken in at l eas t the ARS (15) , HEL (207), NP (282), and TGAL (240) systems. These monoclonal ant ibodies have been very useful probes in determining the s t ructure of i d i o t y p i c determinants, and the regu la t ion of id io type express ion. 145 Chapter 6. Two-Dimensional E lec t rophore t i c Ana lys is of Ant i-Ferredoxin Ant ibodies 1.) Results Although the se ro log i ca l data s t rongly ind ica ted the presence of mul t ip le id iotypes in the mouse anti-Fd response, some biochemical evidence was des i reab le . Several ways to examine t h i s problem were a va i l ab l e . Since immunoglobulin heavy and l i g h t chains have r e l a t i v e l y constant molecular weights, the only way to resolve the d i f f e r e n t chains would be to use i s o e l e c t r i c focuss ing ( IEF) . Standard f l a tbed IEF has been used in many s i tua t ions to examine immuloglobulin heterogeneity (52, 115, 128). Such a s i t ua t i on usua l l y provides qu i te complex data because the IEF pattern i s inf luenced by va r i a t i on in the heavy, l i g h t and carbohydrate chain res idues , producing many bands, even with monoclonal ant ibodies (252). Ana lys is of immunoglobulins by two dimensional gel e lec t rophores i s (2-D) represents the best r eso lu t ion and s e n s i t i v i t y . The advantage of th i s method would be the a b i l i t y to compare the l i g h t or heavy chains of d i f f e r e n t samples to see i f any i den t i t y e x i s t e d , poss ib ly i nd i ca t i ng a s t ruc tura l basis f o r i d i o t y p i c c r o s s- r ea c t i v i t y . The o r i g i na l technique was to pur i f y small amounts of ind iv idua l sera by immunoabsorption, d igest the charged carbohydrate with neuraminidase and reduce, denature, e lectrophorese and detect the prote ins with a s e n s i t i v e , s i l v e r based prote in s t a i n . Though neuraminidase d iges t ion improved the charge smear of the heavy chains (data not shown), s a t i s f a c to r y reso lu t ion was s t i l l not achieved. There fore , l i g h t cha ins , though probably not represent ing major i d i o t y p i c 146 s p e c i f i c i t i e s , were the only r e a l i s t i c biochemical r e f l e c t i o n of d i v e r s i t y . Poss ib le improvements to th i s technique w i l l be discussed l a t e r . Figure 27 shows the 2-D pattern of 1.0 ul of normal mouse serum. Obv ious ly , some kind of p u r i f i c a t i o n i s v i t a l to analyze the immunoglobulins. In Figure 27, the v e r t i c a l dark streaks represent s t a in ing a r t i f a c t s . Figure 28 shows the 2-D s ta in ing pattern of mini column p u r i f i e d Fd-1 monoclonal anti-Fd ant ibody. The charge heterogeneity of the heavy chain i s observed at the top l a b e l . As w e l l , mu l t ip le spots can be seen represent ing l i g h t chain heterogeneity . Mu l t ip l e spots can a lso be observed in the 2-D ana lys is of other ant i-Fd monoclonals (data not shown). Although deamidation or carbamylation could account fo r the d i f f e r e n t charges, no completely cons is tent explanat ion has yet been of fered (265). The extra heterogeneity of such monoclonal l i g h t chains may make in te rp re ta t i on of the actual number of d i f f e r e n t l i g h t chains d i f f i c u l t . Figures 29, 30 and 31 show the patterns of immunopurified immunoglobulin chains from 50 ul of pooled ant i-Fd an t i s e r a . The label ind ica tes the heavy and l i g h t cha ins . Other spots ind ica te contaminants or standard prote in carbamylation t r a i n s . The resu l t s f o r the RF/J serum pool (data not shown ) i s very s i m i l a r to that of AKR/J. These resu l t s ind ica te that many l i g h t chains are expressed in the anti-Fd responses of a l l four s t r a i n s , even accounting f o r charge heterogeneity of s ing le molecules. No obvious spots were shared between s t r a i n s , though quant i ta t i ve ana lys is would be bet ter performed on an ISO-DALT system (265). Ana lys is of ind iv idua l sera genera l ly y i e lded poor detect ion of l i g h t cha ins , poss ib l y due to a d iverse va r i e t y of l i g h t chains with poor prote in representat ion of each. Figure 32 shows the best example of l i g h t chain 147 Figure 27 Two Dimensional Gel E lec t rophores is of Normal Mouse Serum 1.0 ul of normal mouse serum was analyzed by two-dimensional e lec t rophores is and s i l v e r s ta in ing f o r p ro te ins . The ampholines were from pH 3-10 and the s lab gel was 12% acrylamide. 148 normal mouse serum 149 Figure 28. Two-dimensional Gel E l e c t r o - phoresis of Monoclonal Ant i-Fd Antibody  (Fd-1) 10 ul of Fd-1 was p u r i f i e d on a Fd-Sepharose minicolumn, denatured and reduced, resolved by IEF with pH 3-10 ampholines, separated into components on a 12% acrylamide s lab gel and the prote ins were detected by s i l v e r s t a i n i n g . 150 3 10 pH H chain L chain m o n o c l o n a I anti - F d ( F d - 1 ) 151 Figure 29. Two-Dimensional Gel E lec t rophores is of Pooled Anti-Fd Ant ibodies  from AKR/J Mice 50 ul of anti-Fd an t i se ra pooled from 15 AKR mice was p u r i f i e d on a Fd-Sepharose mini column, denatured and reduced, resolved by IEF with pH 3-10 ampholines, separated into components on a 12% acrylamide s lab gel and the prote ins were detected by s i l v e r s t a i n i n g . A K R o n t i - F d p o o l 153 Figure 30. Two Dimensional Gel E lec t rophores is of Pooled Anti-Fd Ant ibodies  from A/J Mice 50 ul of ant i-Fd ant i sera pooled from 15 A/J mice was p u r i f i e d on a Fd-Sepharose mini column, denatured and reduced, resolved by IEF with p H 3 r l 0 ampholines, separated into components on a 12% acrylamide s lab gel and the prote ins were detected by s i l v e r s t a i n i n g . A / J anti - Fd pool 155 Figure 31. Two Dimensional Gel E lec t rophores is of Pooled Anti-Fd Ant ibodies  from B10.BR/SnJ Mice 50 ul of ant i-Fd ant i se ra pooled from 15 BIO.BR mice was p u r i f i e d on a Fd-Sepharose mini column, reduced and denatured, resolved by IEF with pH 3-10 ampholines, separated into components on a 12% acrylamide s lab gel and the prote ins were detected by s i l v e r s t a i n i n g . Figure 32. Representative Two-Dimensional Gel  E lec t rophores is of an Indiv idual BIO.BR  Anti-Fd Serum 20 ul of ant i-Fd antiserum from a s ing le BIO.BR animal producing a high leve l of both BIO.BR and AKR id iotypes was p u r i f i e d on Fd-Sepharose. The e lu ted antibody was denatured and reduced, resolved by IEF with pH 3-10 ampholines, separated in to components on a 12% acrylamide s lab gel and the prote ins were detected by s i l v e r s t a i n i n g . BIO.BR a n t i - F d indiv idual s e r u m 159 r e s t r i c t i o n in an ind iv idua l BIO.BR serum, s t rongly c ross-react ive with AKR. Aga in , several l i g h t chain spots can be observed. Thus, the resu l t s show that several dominant l i g h t chains ex i s t in the ant i-Fd response of a l l inbred s t r a ins t e s t ed . No obvious i den t i t y between l i g h t chains of d i f f e r e n t s t r a ins could be observed. II. D iscuss ion Since these mice produce such small amounts of s p e c i f i c ant ibodies in t h e i r serum, detect ion of a l l l i g h t chains being manufactured i s impossible by these procedures. The resu l t s show that several immunodominant l i g h t chains are produced, averaging to at l eas t about 2 to 3 l i g h t chains per mouse, as observed. This number i s only a f r a c t i on of the number of l i g h t chains involved in Fd b ind ing , and ce r t a i n l y does not r e f l e c t a l l of the v a r i a b i l i t y observed in the se ro log i ca l da ta , and as such must only be considered an i nd i ca t i on that more than one l i g h t chains are involved in the anti-Fd response in these s t ra ins of mice. The 2-D method of ana lys is i s very useful but fu r ther work must go into i t to proper ly inves t iga te the c ross- reac t i v i t y of ind iv idua l se ra . It would be very hard to improve the s e n s i t i v i t y of th i s system by d i r e c t s t a i n i n g . If 20 ug/ml of ant i-Fd antibody i s produced by high responder mice (251) and 50 ul of serum is absorbed, then about 1 ug of immunoglobulin i s appl ied to the g e l , of which s ta inab le l i g h t chain represents about one-third (by molecular weight ) . I f 15 spots are v i s u a l i z e d , then the s ta in i s s t a in ing about 20 ng! This number represents a conservat ive leve l of s e n s i t i v i t y . Aside from using large samples (which defeats the purpose of the microassay) , very few options are a v a i l a b l e . D i rec t iod ina t ion of the antibody on the Fd immunoadsorbent was done 160 and worked s a t i s f a c t o r i l y (data not shown) but the 2-D pattern d i f f e r e d from the uniodinated pa t te rn . This may be due to Chloramine T ox idat ion or the pKa change of the ty ros ine phenol ic group upon iod ina t ion (283). Thus, t h i s method w i l l not be u s e f u l . As w e l l , the l i g h t chains label poorly s ince they appear to be protected by the immunoadsorbent (data not shown). Unfor tunate ly , the best method of increas ing s e n s i t i v i t y i s by b l o t t i n g onto n i t r o c e l l u l o s e and detect ing with immunoperoxidase (284) or iodinated antibody (285). This would be extremely time consuming. Poss ib ly i t would be less complex to d i r e c t l y analyze the l i g h t chains by IEF (286), fol lowed by b l o t t i ng and detect ion (284, 285). Though detect ion of small amounts of prote in on f l a tbed IEF by Sammon's s i l v e r s t a in ing i s almost non-existent (data not shown), b l o t t i n g of the l i g h t chains onto n i t r o c e l l u l o s e would al low very sens i t i v e detect ion (284). Neuraminidase was not found to be cons i s ten t l y e f f e c t i v e at removing charged carbohydrate from immunoglobulin (data not shown). As w e l l , most neuraminidase preparat ions contain proteases. A bet te r method may be incubat ion with Endoglycosidase H (per. comm. Dr. 0. Wi t te , UCLA). This enzyme cleaves the carbohydrate s ide chain d i s t a l to the f i r s t N-acetyl-glucosamine residue (287). T h e o r e t i c a l l y , t h i s enzyme should t o t a l l y e l iminate the s i a l i c ac id charge heterogeneity (288) however, problems have been encountered at c leav ing charged s ide chains (287). Perhaps a change in react ion condi t ions may circumvent th i s problem. In conc lus ion , the resu l t s of Chapter 6 show that at l eas t 3 d i f f e r e n t l i g h t chains are expressed in ind iv idua l mice responding to Fd (assuming the usual 2-3 spots per l i g h t cha in , and not the worse case scenario observed from Figure 28). 161 These l i g h t chains vary from mouse to mouse and do not appear to determine i d i o t y p i c express ion , though the super ior r e p r o d u c i b i l i t y of an ISO-DALT system would be essent ia l f o r t r u l y quant i t a t i ve comparisons. Bet ter detect ion and the reso lu t ion afforded by pure IEF separat ions are necessary f o r accurate quant i ta t ion of to ta l anti-Fd l i g h t chain express ion . Nonetheless, the r esu l t s of chapter 6 are va luable in demonstrating that many l i g h t chains are produced in the mouse anti-Fd response, and t h i s biochemical evidence cor re la tes qu i te n i ce l y with the se ro log i ca l data . 162 Chapter 7. Impl icat ions of Results and Further Experiments 1. Thesis Discuss ion The work discussed in the preceding chapters was ca r r i ed out to def ine a new prote in i d i o t y p i c system. Several important questions must be addressed before th i s work i s complete. In th i s chapter four important top ics w i l l be d i s cussed , fol lowed by a b r i e f summary of the work of th i s t h e s i s , and several suggested experiments. A) The T Ce l l Receptor Even though i t i s not a major theme of t h i s t h e s i s , i t would be appropriate at t h i s time to b r i e f l y mention the T c e l l receptor . The r e l a t i onsh ip between T and B c e l l id iotypes has been extens ive ly studied (28, 75, 134, 222) and much evidence ex i s t s f o r se ro log i ca l c r o s s - r e a c t i v i t y . However, much data ex i s t s to demonstrate that T c e l l s do not express immunoglobulin genes (80, 180, 181). How then do anti-B c e l l id iotypes recognize T c e l l s ? At l eas t two p o s s i b i l i t i e s e x i s t . T and B c e l l receptors may be d i f f e r e n t , but i f they recognize the same ep i tope , they may bind the an t i- id io type i f i t has residues that are s t r u c t u r a l l y s im i l a r to the recognized ep i tope. This hypothesis w i l l be discussed in more de ta i l l a t e r . Another p o s s i b i l i t y i s that the T c e l l receptor and immunoglobulin (the B c e l l receptor) are separate but s im i l a r p ro te ins . Thus they may share id iotypes by v i r tue of s i m i l a r prote in s t ruc tu re . Some experimental evidence may support t h i s hypothesis . Monoclonal ant ibodies s p e c i f i c f o r ce r ta in T c e l l l i nes or hybridomas can be used to pur i f y molecules cons i s t ing of two disulphide-1 inked g lycoprote ins of 37,000 -50,000 molecular weight (289, 290) which may cons i s t of va r i ab le and constant regions (319, 320). Molecular gene t i c i s t s have i so l a t ed cDNA 163 from mouse mRNA that hybr id izes to genomic regions that are rearranged in T c e l l s and not B c e l l s , and vary f o r d i f f e r e n t antigen s p e c i f i c i t i e s , suggesting that the nuc le i c ac id sequence coding f o r one chain of the T c e l l receptor has been i so l a t ed (321). Examination of the nuc le i c ac id sequences of t h i s putat ive receptor chain gene ind ica tes that i t shows s i g n i f i c a n t homology to immunoglobulin heavy chains with a d i s t i n c t arrangement in to constant and va r i ab le regions (322). Furthermore, a Canadian group has cloned and sequenced a human mRNA s p e c i f i c f o r mammalian T c e l l s . The prote in from th i s cDNA is estimated to have a molecular weight of about 35,000 and shows extensive homology to immunoglobulin l i g h t cha ins , inc lud ing the pos i t ions of the re levant cyste ine residues (323). These resu l t s are very support ive of a c lose s t ruc tu ra l s i m i l a r i t y of B and T c e l l r ecep to rs , which may expla in data i nd i ca t i ng such i d i o t y p i c c r o s s - r e a c t i v i t y . B) What Const i tutes Idiotype? As mentioned before (1 , 2 ) , id io type has been def ined as the ant igen ic s p e c i f i c i t i e s of ant ibodies produced by an ind iv idua l or group of i nd i v idua l s in response to a given ant igen. This desc r ip t i on was modif ied to def ine id io type as the set of epitopes (or ant igen ic determinants) d isp layed by the va r i ab le regions of a set of antibody molecules , in a paper proposing the formal Network Theory of the regu la t ion of the immune response (3) . In t h i s theory , i nd i v idua l ant igen ic determinants on the va r i ab le region of an antibody were c l a s s i f i e d as paratopes, when they were assoc iated with the antigen binding s i t e and id iotopes when they were outs ide i t . Regulation occurred by events associated with the recogni t ion of id iotopes by paratopes of other antibody molecules. These in te rac t ions could r e su l t in changes in the recogniz ing and recognized populat ions . 164 Importantly, d i s t i n c t i o n was made between the paratopes and the id io topes . Since a paratope from the antibody s p e c i f i c f o r an antigen could recognize id iotopes of other an t ibod ies , an " i n te rna l image" of the antigens was ava i l ab le wi th in the immune system i t s e l f . The presence of exogenous antigen thus changed the dynamic equ i l i b r ium of the network, r e su l t i ng in the phenomena of the immune response to that ant igen. Unfor tunate ly , no real desc r ip t i on of the phys ica l proper t ies of e i the r i d i o t ypes , or paratopes was o f f e r ed . Only one paratope was proposed per Fab arm. The theory was very e legant , and some very support ive evidence ex i s t ed . Idiotypes were s e r o l o g i c a l l y def ineable (1 , 2) but were not always assoc iated with an antigen binding s p e c i f i c i t y (291). Unfor tunate ly , the d i s t i n c t i o n between paratopic and i d i o t o p i c determinants became somewhat b lur red by the f i nd ing of m u l t i - s p e c i f i c i t y of an t ibod ies . Hybrids of ant ibodies to two d i f f e r e n t haptens could bind both at the same time but in d i f f e r e n t loca t ions (292). True m u l t i - s p e c i f i c ant ibodies were found that could bind two separate haptens (293, 294). When f luorescence quenching techniques were used by some i n ve s t i g a to r s , as much as 1.4 nm ex is ted between the binding s i t e s (294). When rabbi ts were immunized with an antigen X, a small percentage bound an unrelated hapten Y. When the animals were boosted with Y, the frequency of d i s t i n c t ant ibodies binding both X and Y rose by 80 times (295). As w e l l , c r y s t a l l i z a t i o n data from hapten binding myeloma prote ins showed a r e l a t i v e l y huge c l e f t binding a t i ny hapten (296). It i s not conceptual ly d i f f i c u l t to imagine a large prote in being able to bind two d i f f e r e n t l igands -- th i s i s observed f requent ly with enzymes. A ra t iona l modi f i ca t ion f o r i n te rp re t ing antibody in te rac t ions was proposed. The en t i r e va r i ab le region of the antibody could serve as a " s t i c k y end" with no d i s t i n c t i o n between recogniz ing and 165 and being recognized (297). Thus, no real d i s t i n c t i o n would be made concerning paratopes and i d i o topes , which would ex i s t only as def ined by the p a r t i c u l a r system of i n t e r e s t . Such a d e f i n i t i o n would expla in why paratopes and id iotopes need not be assoc iated (165, 194, 208, 244, 279, 280, 298) as well as why they could be associated (15, 92, 299, th i s work). Accord ing l y , the formal Network Theory was modif ied to def ine the pa ra tac t i c epitope as a binding reg ion , and the para tac t i c id iotope as a bound region of ant ibody, which would be only def ined in a p a r t i c u l a r system as a convenient reference (300). As w e l l , a poss ib le id iotope could cons is t of a c l e f t or protuberance conformat iona l l y , or an amino ac id conf igura t ion chemical ly (300). Much work has been done attempting to def ine the s t ructure of the id iotopes f o r id iotypes of d i f f e r e n t an t ibod ies . Such attempts have been reviewed here e a r l i e r , and elsewhere (64, 98, 301). Only the amino ac id cor re la tes of very simple i d i o topes , from very simple systems have been def ined (112, 116, 302). As w e l l , d i r e c t observat ion has l o c a l i z e d id iotopes to the amino terminus of the Fab fragment (303). By d e f i n i t i o n , id iotopes are as complex as prote in ep i topes , the s t ructures of which are cur rent l y being revea led. As new, high power, computer a ss i s t ed X-ray d i f f r a c t i o n techniques are used, and data accumulated, i d i o t y p i c determinants w i l l be def ined chemica l ly . C) What Const i tutes C ross- reac t i v i t y and S p e c i f i c i t y ? In order f o r bonding to take place between an epitope and an antibody an appropriate complementarity must e x i s t . As with enzyme b ind ing , i o n i c , hydrophobic, and polar bonding forces can a l l be u t i l i z e d to produce a high 166 binding constant f o r the epitope (304). The spa t i a l arrangement of the hapten, as well as the appropriate complementary funct iona l groups of the hapten and antibody determine b ind ing . These groups may be continuous or may be brought together by the conformation of the molecules (305). Thus, molecules of qui te d i f f e r e n t primary s t ructure may cross react by v i r tue of conformational determinants, as observed f o r morphine and enkephalins (306). As c ros s- reac t i v i t y of ant ibodies to d i f f e r e n t haptens by v i r tue of recogni t ion at the same s i t e or multi-hapten binding at d i f f e r e n t s i t e s i s well recognized (292, 293, 294), one would postulate that mu l t i - i d i o t yp i c c ro s s- reac t i v i t y would be poss ib l e . An example of t h i s i s seen in the T15 system (86). The concept of c ross- reac t i v i t y has been e legant ly explored (307). "True" c ross- reac t i v i t y may be def ined as competit ion of two determinants at the same s i t e , with d i f f e r e n t binding constants . Thus, one hapten may completely i n h i b i t the other from b ind ing . The second type , or "shared r e a c t i v i t y " i s a r e a c t i v i t y to d i f f e r e n t parts of a p ro te in . Thus, with heterologous s e r a , the d i f f e r e n t populat ions could not a l l be i nh ib i t ed with a fragment of the prote in or another prote in with some shared determinants (307). True c ross- reac t i v i t y would be observed with monoclonals; shared with heterologous sera . I f one postulated that a l l ant ibodies are m u l t i - s p e c i f i c , but that only a very few w i l l c ross-react with any other antigen (307) at one t ime, then even though a l l of the ant ibodies are i n d i v i d u a l l y m u l t i - s p e c i f i c , the serum appears s p e c i f i c . Such a s i t ua t i on i s probably common with an an t i-pro te in response. 167 C ross- reac t i v i t y w i l l be discussed with respect to several mechanisms. A small amount of c ro s s- reac t i v i t y between some id iotypes may ex i s t by v i r t ue of the m u l t i - s p e c i f i c i t y of ant ibodies (307), s ince some antibody in the system w i l l bind any ant igen. This w i l l be compounded by assuming a " s t i c k y end" model of antibody binding (297) but should s t i l l only account f o r a small percentage of the c ross- reac t i v i t y with an an t i- id io type represent ing id io type of another s t r a i n . An a l t e rna t i ve explanat ion of i d i o t y p i c c ross- reac t i v i t y invokes the " i n t e rna l image" model (300). In t h i s model, c ross- reac t i v i t y ex i s t s because the an t i- id io type binds to the paratope of the id io type bearing immunoglobulin, thus s imulat ing the antigen ep i tope . One may be i n c l i n ed to re jec t t h i s hypothesis at f i r s t g lance , expecting a l l s p e c i f i c antigen binding s p e c i f i c i t i e s to be i nh ib i t ed by an an t i - i d i o t ype , and, indeed, th i s would be expected in simple hapten systems. The response to p ro t e ins , however, i s usua l l y complex, with many determinants involved (150, 195). I f the response i s d i rec ted to many ep i topes , and the an t i- id io type i s the " i n te rna l image" of only a few, dominant ep i topes , one would expect that the ind i v idua l sera would be i nh ib i t ed in a manner r e f l e c t i n g t h e i r an t i -ep i top i c content. Thus, varying degrees of c ross- reac t i v i t y would be expected un i ve r sa l l y among a l l sera d i rec ted against the ant igen. This r e su l t would be cons is tent with observations from the GAT (153, 154), i n s u l i n (244), lysozyme (206) and Fd ( th is work) systems. I f such a r e su l t i s not seen with a l l s t r a ins (or species) one may postulate that though the an t i- id io types r e f l e c t the " i n t e rna l images" of the immunizing s t r a i n , the other s t r a ins respond to d i f f e r e n t ep i topes . Indeed, t h i s r e su l t has often been observed (reviewed in 147) but i s usua l l y cor re la ted to the H-2 168 haplotype. Nonetheless, much more ep i top i c heterogeneity v i a top i ca l determinants may e x i s t , but would be very d i f f i c u l t to detec t . Thus, the " i n t e rna l image" theory has qui te p l aus ib l e grounds f o r support in a prote in system, backed with some evidence (300). Such a theory explains very n i ce l y the presence of B c e l l c ross-react i ve i d i o t y p i c determinants on T c e l l s , in l i g h t of the previous lack of s t ruc tura l data . This theory does not expla in c ross-react ive id io types that do not co r re l a te with antigen s p e c i f i c i t y (194, 207, 244, 280) or gene t i c a l l y mapped id io types in the hapten systems (24, 66) . In f a c t , i t has been observed that the " i n te rna l image" model of i d i o t y p i c c ross- reac t i v i t y i s ra re l y v a l i d , s i n c e , qui te often immunization with an t i - id io type does not produce much antibody capable of binding antigen (308). This i s thought to be as a r e su l t of the frequency of B c e l l s bearing the o r i g i na l i d i o t y p i c determinants. I f the o r i g i na l id io type i s determined by one or very few germline genes, immunization with an t i- id io type i s l i k e l y to produce antigen b ind ing , id io type pos i t i v e antibody (309), whereas i f the id io type i s determined by many germl ine.genes, or p a i r i n g , as in many prote in systems, then an t i - id io type w i l l cause the production of many ant ibodies that bear i d i o t y p i c determinants, but do not bind antigen (309). Another mechanism to expla in c ross-react i ve id io types may be by v i r tue of shared germline V genes. This mechanism has been shown to account f o r the major id io types in PC (66) , ARS (24) , dextran (113), NP (101), GAC (130) and Nase (220) systems. The Nase and GAT systems have shown the presence of mu l t ip l e germline genes. The wide i n t e r s t r a i n i d i o t y p i c shar ing seen in other systems (153, 154, 206), and in th i s prote in system may be due to the conservat ion of ce r ta in V genes across a l l s t ra ins of 169 mice, c e r t a i n l y not an unreasonable p o s s i b i l i t y cons ider ing that a l l inbred s t r a ins may have been der ived from a more l im i t ed gene pool than o r i g i n a l l y thought (310, 311). It i s i n t e r e s t i ng to t r y to in te rp re t the work of t h i s thes i s in the context of the various explanat ions f o r i d i o t y p i c c ross- reac t i v i t y prev ious ly d i scussed . Predominant id iotypes ex i s t in the AKR, RF, and BIO.BR s t r a i n s , but not a l l ant i-Fd producing mice bear c ross-react ive i d i o t y p i c ant ibodies in t h e i r an t i s e r a . Thus, i f there i s a genet ic reason f o r the i n t r a and i n t e r s t r a i n c r o s s - r e a c t i v i t i e s observed, i t s e f f e c t i s not tremendously important in the response. It must be noted that the an t i- id io types were prepared using t e r t i a r y ant i se ra from 10-15 mice. One would expect most poss ib le epitope s p e c i f i c i t i e s to be present . Genetic reasons f o r the i d i o t y p i c c r o s s - r e a c t i v i t i e s observed would very n i ce l y expla in the resu l t s i f many genes, or gene combinat ions, were capable of y i e l d i n g anti-Fd an t ibod ies . Thus, the r e su l t i ng an t i- id io types would recognize mainly a few of the dominant i d io t ypes . I f each ind iv idua l mouse expressed only a few members of the to ta l set of poss ib le i d i o t ypes , and overlaps ex is ted between s t r a i n s , then the v a r i a b i l i t y of id io type expression and the i n t r a and i n t e r s t r a i n c r o s s - r e a c t i v i t i e s , represented in Figure 27 would be exp la ined. Some biochemical data ex i s t s to support the hypothesis of many poss ib le id iotypes (see Chapter 6 ) . In terpreta t ion of these resu l t s with the " i n te rna l image" model i s more d i f f i c u l t . These c r o s s - r e a c t i v i t i e s could be explained i f the a n t i - i d i o t y p i c ant ibodies were f unc t i ona l l y s i m i l a r to an t igen , and thus represented some of the dominant epitopes of Fd. The va r i a t i on in c ro s s- r eac t i v i t y could be explained by microheterogeneity in terms of 170 epitope recogni t ion by the var ious anti-Fd se ra . T rad i t i ona l evidence argues against th i s view. Careful molecular genet ic evidence supports the genet ic inher i tance of id io typy (24, 66, 130). Idiotypes can cross-react when assoc iated with d i f f e r e n t antigen s p e c i f i c i t e s (194, 207). As w e l l , in t h i s system, a pool of at l eas t 10 mice should represent almost a l l of the poss ib le epitopes recognized by the antibody response to Fd. In a d d i t i o n , the Fd molecule i s very sma l l , with most antibody d i rec ted to one determinant (or region loca t ion at the carboxy terminus) (251). With these fac ts one would expect to see greater c r o s s - r e a c t i v i t y , i f the an t i - id io type i s simply mimicking antigen ep i topes . In s i tua t ions where such mimickry i s i n ves t i ga ted , very l i t t l e seems to be found (308). In sho r t , though i t would be d i f f i c u l t to disprove e n t i r e l y , epitope " d i sgu i s e " by the a n t i - i d i o t y p i c ant ibodies probably does not play a major ro le in the observed c r o s s - r e a c t i v i t i e s presented in these r e s u l t s . The " i n t e rna l image" theory , however, appears to be a good explanat ion f o r the presence of B c e l l i d i o t y p i c determinants on T c e l l s . Thus, the genet ic inher i tance of V genes coding f o r i d i o t y p i c determinants seems to be the best explanat ion f o r c ross-react ive id io types in the Fd system. Somatic mutation during the course of antibody maturation may give r i s e to high t i t r e antibody recognized by the a n t i - t e r t i a r y (anti-Fd) i d io t ypes . Presumably, d i f f e r e n t regulatory mechanisms and many V genes could expla in the v a r i a b i l i t y in id iotype express ion , though the " i n te rna l image" model cannot be ru led ou t , and may expla in a proport ion of the c ross- reac t i v i t y observed. 171 D) Id io typ ic Regulation The mechanisms by which antibody l eve l s in v ivo are regulated are l a rge l y unknown. The most accepted explanat ion at present was proposed by Jerne (3) . Much data ex i s t s to support the formal Network Theory and has been reviewed extens ive ly elsewhere (98, 297, 309, 312, 313). Obv ious ly , some regulatory phenomena are occuring in mice in jec ted with Fd. It i s assumed that regu la t ion occurs in a s im i l a r manner in other animals — probably by a network of i d i o t y p i c i n t e r a c t i ons . These in te rac t ions are usua l l y thought to involve T c e l l s (133, 179), although B c e l l s are capable of regu la t ing antibody express ion , perhaps by v i r t u r e of the recogni t ion of d i f f e r e n t segments (314). It i s not the aim of t h i s thes i s to explore the regulatory aspects of the Fd system, which i s the subject of ongoing work by o thers . Instead, an exploratory inves t iga t ion of expressed id iotypes in the Fd system was undertaken with the hope that a r e l a t i v e l y r e s t r i c t e d response ex i s t ed . Such an idea was considered poss ib le in l i g h t of the small s i z e , and the determinant uni formity of the response in H-2 s t ra ins of mice. However, t h i s was not the case. As has been shown, the i d i o t y p i c response to Fd appears complex, with some relatedness not iced between d i f f e r e n t s t ra ins of mice. This relatedness i s probably due to conservat ion of germ l i n e i d i o t y p i c markers, although the " i n t e rna l image" model of c ro s s- reac t i v i t y (300) cannot be ru led out . The reagents exhaust ive ly p u r i f i e d , the many anti-Fd ant i se ra c o l l e c t e d , and the hybridomas produced f o r these experiments can be put to use, however, in the continued inves t iga t ion of ant i-Fd id iotypes in inbred mice. 172 II. Further Experiments Many experiments need to be done to f u l l y def ine the expression of id io types in the Fd system. These w i l l be l i s t e d in order of p r i o r i t y or s i m p l i c i t y . 1) Although the data of t h i s thes i s ind ica te that many id iotypes are expressed in the Fd response, not much i s known about t h e i r dominance. To t r y to determine t h i s , columns of monoclonal ant ibodies bearing react ive i d i o t y p i c determinants to one or more ant i- id io types should be used to absorb the a n t i - i d i o t y p i c r e a c t i v i t y . Very good evidence ex i s t s f o r the dominance of that id io type i f most of the an t i- id io type i s absorbed. 2) I f a monoclonal antibody possessing dominant i d i o t y p i c determinants i s found, a n t i - i d i o t y p i c reagents to that monoclonal antibody should be prepared. This an t i- id io type could be used to re-screen the ind iv idua l sera to estimate the importance of that i d io t ype . 3) With the anti-monoclonal i d i o t ypes , c e l l mixing studies a f t e r id iotype or an t i- id io type treatment and recons t i tu t i on may def ine the importance of these id iotypes in r egu la t i on . 4) As w e l l , in v ivo studies with id iotype or a n t i - i d i o t y p i c reagents should be done to charac ter ize important regulatory c h a r a c t e r i s t i c s . 5) As not a l l ant i-Fd id iotypes can be def ined with the four an t i - i d i o t ypes , perhaps another an t i- id io type to CBA or C3H anti-Fd ant ibodies should be made. 6) The four a n t i - i d i o t y p i c reagents could be in jec ted in to mice. I f the mice began to produce Ab capable of binding Fd, good evidence f o r the ro le of the " i n t e rna l image" model in i d i o t y p i c c ro s s- reac t i v i t y may be obta ined, as well as evidence fo r V gene r e s t r i c t i o n (309). 173 7) Western b lo ts of IEF or 2-D separated ant ibodies and detect ion with l abe l l ed subclass s p e c i f i c ant ibodies would be useful to assoc iate i d i o t y p i c c ro s s- reac t i v i t y with antibody i sotype . 8) As w e l l , the use of more sens i t i ve Western b l o t t i n g , the ISO-DALT system (269) and modi f i ca t ions discussed in Chapter 6 may permit the i d e n t i f i c a t i o n of common H or L chains in ind iv idua l sera d i sp lay ing i n t e r s t r a i n c ross-react ive i d io t ypes . 9) Since the carboxyl terminus of Fd may exh ib i t some sequence homology Is to cytochrome c (315), and H-2 mice are high responders to cytochrome c i t would be i n t e r es t i ng to determine i f anti-cytochrome c id io types cross-react with anti-Fd id io type i n , f o r example, the BIO.BR s t r a i n . Such a r e su l t may imply the expression of s im i l a r germ l i n e V genes. 10) I f a very useful monoclonal id io type probe can be found, then cV^ and cV^ probes can be r ead i l y prepared from the mRNA. These could be used to screen non-idiotype producing responders or non-responder s t r a ins to determine i f the c a p a b i l i t y to react to Fd i s there . For example, although evidence ex i s t s that non-responders in the Fd system can be induced to respond (255), the resu l t s of the ARS system show that the absence of the CRI germline gene determines the lack of CRI expression in mice (24). 11) More work i s needed to def ine the i d i o t y p i c response of B10.D2 or DBA non-responders t h a t , through regulatory manipulat ion, are capable of responding to f e r r edox in . 12) A mixture of the four ant i- id io types should be made, and used to i n h i b i t sera that are not i nh ib i t ab l e over background with any s ing le i d i o t ype . The cumulative e f f e c t may i n h i b i t well over background, showing that at l e as t some of the id io types are c ross- reac t i ve . 174 The important question that was the motivat ion f o r a l l of the work of t h i s thes i s i s s t i l l unanswered. Are id iotypes that are conserved in the ant i-Fd response of d i f f e r e n t s t ra ins of mice conserved because they are important regulatory id iotypes? The background work has been done, now is the time to f i n a l l y address th i s quest ion . III. Summary The work of t h i s thes is demonstrates the production of at l eas t 3 major c ross-react i ve i d i o t y p i c f ami l i e s among the response of 3 s t r a ins of mice to f e r r edox in . Whether these id iotypes are r e s t r i c t e d or are repeated " i n d i v i d u a l " determinants cannot be completely def ined at present , however, the evidence ind ica tes that many id iotypes are expressed. Mice of 8 s t ra ins can express i d i o t y p i c determinants that cross-react outs ide t h e i r own s t r a i n . Both H-2 and non-H-2 genes appear to in f luence i d i o t y p i c express ion , but no pattern i s cor re la ted with Igh or Igl a l l o t y p e . Monoclonal ant ibodies s p e c i f i c f o r fe r redox in express i d i o t y p i c determinants capable of react ing with " fo re ign a n t i - i d i o t y p e s . " Poss ib le mechanisms f o r the i n t r a and i n t e r s t r a i n c r o s s - r e a c t i v i t i e s have been d i scussed , and fu r the r experiments have been proposed. This work represents a study of id iotype expression in the Fd system, and was intended to provide a platform from which inves t iga t ions into immune regulat ion at the c e l l u l a r leve l could be launched. 175 BIBLIOGRAPHY 1. Oudin, J . M . , 1963. Une nouvel le for rn^d 'a l lo typ ic des g lobu l ins du se'rum de l a p i n , apparemment l i e e a al fonct ion et l a s p e c i f i t e ' an t i co rps . Compt. Rend. Acad. S c i . 257:805. 2. Kunkel , H .G. , Mannik M., W i l l i ams , R . C , 1963. Indiv idual ant igen ic s p e c i f i c i t i e s of i so l a t ed an t ibod ies . Science 140: 1218. 3. Je rne , N.K., 1974. Towards a network theory of the immune system. Ann. Immunol, 125C:373. 4. Eichmann, K., 1974. Influence of the dose and of the e f f e c to r funct ions of a n t i - i d i o t y p i c antibody on the production of an i d i o t ype . Eur. J . Immunol. 4_:296. 5. Sy, M-S., Back B.A., Dohi , Y . , N i sonof f , A . , Benacerraf , B., and Greene, M.I . , 1979. Induction of suppressor T c e l l s with a n t i - i d i o t y p i c an t ibod ies . J . Exp. Med. 150:1216. 6. Kuettner , M.G. , Wang, A-I . , N i sonof f , A . , 1972. Quant i ta t i ve inves t iga t ions of i d i o t y p i c an t ibod ies . VI. Idiotype s p e c i f i c i t y as a potent ia l genet ic marker f o r the va r i ab le regions of mouse immunoglobin polypept ide cha ins . J . Exp. Med. 135:579. 7. L e fue r t , A-K. , James, R.W., A l l o i d , C , Fu lp ius , B.W., 1982. A monoclonal a n t i - i d i o t y p i c antibody against ant i- receptor ant ibodies from myasthenic se ra . Eur. J . Immunol. 12:790. 8. Matsuyama, T . , Fukumori, J . , Tanaka, H. 1983. Evidence of unique i d i o t y p i c determinants and s im i l a r i d i o t y p i c determinants on human ant i- thyrog lobu l in an t ibod ies . C l i n . Exp. Immunol. 51:381. 9. Bonagura, V .R . , Kunkel , H .G. , Pe rn ia , B., 1982. C e l l u l a r l o c a l i z a t i o n of rheumatoid f a c to r i d io t ypes . J . C l i n . Invest. 69:1356. 10. Szewczuk, M.R., and Campbell , R.J., Loss of immune competence with age may be due to au to-ant i- id io typ i c antibody r egu la t i on . Nature 286:164. 11. Tung, A .S . and N isonof f , A. 1975. I so la t ion from ind iv idua l A/J mice of p-azophenylarsenate ant ibodies bearing a c ross-react ive i d i o t ype . J . Exp. Med. 141:112. 12. Capra, J . D . and N i sono f f , A. 1979. The complete amino ac id sequence of the heavy chain va r i ab le region of anti-p-azophenylarsenate ant ibodies from A/J mice bearing a c ross-react i ve i d i o t ype . J . Immunol. 123:279. 176 13. Capra, J . D . , Tung, A . S . , N i sonof f , A. 1975. S t ructura l studies on induced ant ibodies with def ined i d i o t y p i c s p e c i f i c i t i e s . I. The heavy chains of A/J mouse anti-p-azophenylarsenate ant ibodies bearing the c ross-react ive i d io t ype . J . Immunol. 119:993. 14. Capra, J . D . , Tung, A .S . and N i sonof f , A. 1977. The complete amino ac id sequence of l i g h t chain va r i ab le regions of anti-p-azophenylarsonate ant ibodies from A/J mice bearing a c ross-react i ve i d i o t ype . J . Immonol. 119:993. 15. Lamoyi, E . , E s tess , P., Capra, J . D . and N i sonof f , A. 1980. Heterogeneity of an i n t r a s t r a i n c ross-reac t i ve id iotype assoc iated with anti-p-azophenylarsonate ant ibodies of A/J mice. J . Immunol. 124:2834. 16. Marshak-Rothstein, A . , S e i k i v i t z , M., Margo l ies , M.N., Mudgett-Hunter, M., Ge f te r , M. 1980. Hybridoma prote ins expressing the predominant id iotype of the anti-p-azophenylarsonate response of A/J mice. Proc. Na t l . Acad. S c i . 77:1120. 17. Marshak-Rothstein, A . , Benedetto, J . , K i r s c h , R.L . , Ge f t e r , M.L. 1980. Unique determinants associated with hybridoma prote ins expressing a c ross-react i ve id iotype—frequency among ind iv idua l immune se ra . J . Immunol. 125:1987. 18. Es tess , P., N i sonof f , A . , Capra, J . D . 1979. Ami no-terminal amino ac id sequence ana lys is of the heavy and l i g h t chain va r i ab le regions of monoclonal anti-p-azophenylarsonate ant ibodies from A/J mice d i f f e r i n g with respect to a c ross-react ive i d i o t ype . Mol . Immunol. 16:1111. 19. E s tess , P., Lamoyi, E. , N i sonof f , A. and Capra, J . D . 1980. Framework d i f fe rences in the heavy and l i g h t chain va r i ab le regions of monoclonal anti-p-azophenylarsonate ant ibodies from A/J mice. J . Exp. Med. 151:863. 20. M i l ne r , E.C.B. and Capra, J . D . 1982. V„ f ami l i e s in the antibody response to p-azophenylarsonate: Cor re l a t ion between serology and amino ac id sequence. J . Immunol. 129:193. 21. L a sk in , J . A . , Gray, A . , N i sonof f , A . , Klinman, N.R. and G o t t l i e b , P.D. 1977. Segregation at a locus determining an immunologlobulin genet ic marker f o r the l i g h t chain va r i ab le region a f f ec t s inher i tance of the expression of an i d i o t ype . Proc. N a t l . Acad. S c i . 74:4600. 22. M i l ne r , E.C.B. and Capra, J . D . 1983. S t ructura l ana lys is of monoclonal ant i-arsonate an t ibod ies : i d i o t y p i c s p e c i f i c i f i e s are determined by the heavy cha in . Mol. Immunol. 20:39. 23. Capra, J . D . , S laughter , C , M i l na r , E .C .B . , Es tess , P., Tucker, P.W. 1982. The c ross-react i ve id io type of A-st ra in mice. Sero log ica l and s t ruc tu ra l ana lyses . Immunol. Today 3:372. 177 24. S i e k i v i t z , M. , Huang, S .Y . , Ge f t e r , M.L. 1983. The genet ic bas is of antibody product ion: a s ing le heavy chain va r i ab le region gene encodes a l l molecules bearing the dominant ant i-arsonate id iotype in the s t r a i n A mouse. Eur. J . Immunol. 13:123. 25. Cannon, L.E. and Woodland, R.T. 1983. Rapid and sens i t i v e procedure f o r ass igning i d i o t y p i c determinants to heavy or l i g h t cha ins : app l i c a t i on to id iotopes assoc iated with the major c ross-react ive id io type of A/J anti-phenylarsonate an t ibod ies . Mol . Immunol. 20:1283. 26. Doh i , Y. and N isonof f , A. 1979. Suppression of an id io type and generation of suppressor T c e l l s with id io type conjugated thymocytes. J . Exp. Med. 150:909. 27. Sy M-S., Bach, B.A., Brown, A . , N i sonof f , A . , Benacerraf , B. and Greene, M.I. 1979. Induction of suppressor T c e l l s with id iotype-coupled syngeneic spleen c e l l s . J . Exp. Med. 150:1229. 28. H i r a i , Y. and N i sonof f , A. 1980. Se lec t i ve suppression of the major i d i o t y p i c component of an anti-hapten response by so lub le T ce l l -de r i ved fac tors with i d i o t y p i c or a n t i - i d i o t y p i c receptors . J . Exp. Med. 151:1213. 29. Sy M-S., D i e t z , M.H., Germain, R.N. , Benacerraf , B., Greene, M.I. 1980. Idiotype bearing I-J pos i t i v e suppressor T c e l l s which express a n t i - i d i o t y p i c receptors . J . Exp. Med. 151:1183. 30. Owen, F . L . , Ju S-T., N i sonof f , A. 1977. Presence on i d i o t ype-spec i f i c suppressor T c e l l s of receptors that in te rac t with molecules bearing the i d i o t ype . J . Exp. Med. 145:1559. 31. Bach, B.A. , Greene, M.I . , Benacerraf , B., N i sonof f , A. 1979. Azobenzenarsonate-specif ic suppressor f ac to r ( s ) bear cross--react ive i d i o t y p i c determinants, the expression of which i s l i nked to the heavy chain l inkage group of genes. J . Exp. Med. 149:1084. 32. Sy, M-S., D i e t z , M.H. , N i sonof f , A . , Germain, R.N. , Benacerraf , B., Greene, M.I. 1980. The f a i l u r e of id io type coupled spleen c e l l s to induce unresponsiveness in animals lack ing the appropriate V„ genes i s caused by the lack of idiotype-matched t a rge t s . J . Exp. Med. 152:1226. 33. Haas, W. 1982. Microbio logy 402 lec ture notes. U.B.C. Dept. Mic rob io logy . 34. Goodman, J .W. , Lewis, G .K . , P r im i , D. , Hornbeck, P., Ruddle, N.H. 1980. Antigen s p e c i f i c molecules from murine T lymphocytes and T c e l l hybridomas. Mol . Immunol. 17:933. 35. M i l l e r , J . F . A . P . , Mottrom, P .L . , Thomas, W.R., McKimm-Breschkin, J . , Walker, I.L., Gamble, J . 1982. Antigen s p e c i f i c i t y of continuous T c e l l l i n e s . Curr . Top. M i c r o b i o l . Immunol. 100:249. 178 36. P a c i f i c o , A. and Capra, J . D . 1980. I n i t i a l charac te r ixa t ion of antigen s p e c i f i c T c e l l products that bear a c ross-react i ve id io type and determinants encoded by the murine major h is tocompatab i l i t y complex. J . Exp. Med. 152:1289. 37. C l a r k , A . F . and Capra, J . D . 1982. Ubiquitous non-immunoglobulin p-azobenzenearsonate binding molecules from lymphoid c e l l s . J . Exp. Med. 155:611. 38. Lucas, A. and Henry, C. 1982. Expression of the major c ross-react ive id io type in a primary anti-azobenzenearsonate response. J . Immunol. 128:802. 39. Henry, C. and Lucas, A. 1982. The r e l a t i on of id iotype expression to isotype and a l lo t ype in the anti-p-azobenzenearsonate response. Eur. J . Immunol. 12:175. 40. Moser, M., Leo, 0 . , Hiernaux, J . and Urba in , J . 1983. Id io typ ic manipulat ion in mice: BALB/c mice can express the c ross-react i ve id io type of A/J mice. Proc. Na t l . Acad. S c i . 80:4474. 41. Si g a l , N. 1982. Frequency of c ross-react i ve id io type pos i t i v e B c e l l s in A/J and BALB/c mice. J . Exp. Med. 156:1352. 42. Kaps l a i s , A . A . , Tung, S . , N i sonof f , A. 1976. Re la t ive combining a f f i n i t i e s of anti-p-azophenylarsonate ant ibodies bearing a c ross-react i ve i d io t ype . Immunochemistry 13:783. 43. Roths te in , T . L . and Ge f te r , M.L. 1983. A f f i n i t y ana lys i s of id io type pos i t i v e and id iotype-negat ive ARS-binding hybridoma prote ins and ARS-immune se ra . Mol. Immunol. 20:161. 44. Conger, J . D . , Lamoyi, E., Lewis, G .K . , N i sonof f , A. and Goodman, J.W. 1983. Reversal of the r e l a t i v e dominance of major and minor c ross-reac t i ve id io types in arsonate s p e c i f i c T-independant responses. J . Exp. Med. 158:438. 45. Wysocki, L. J . and Sato, V .L . 1981. The s t r a i n A anti-p-azophenylarsonate major c ross-react i ve id io type fami ly inc ludes members with no r e a c t i v i t y toward p-azophenylarsonate. Eur. J . Immunol. 11:832. 46. E s tess , P., O t an i , F. , M i l ne r , E .C .B . , Capra, J . D . , Tucker , P.W. 1982. Gene rearrangements in monoclonal A/J ant i-arsonate an t ibod ies . J . Immunol. 129:2319. 47. Anderson, P.N. and Potter M. 1968. Induction of plasma c e l l tumors in BALB/c mice with 2, 6, 10,14 - tetramethylpentadecane ( P r i s tane ) . Nature 222:994. 179 48. Leon, M.A. and Young, N.M. 1971. S p e c i f i t y f o r phosphorylchol ine of s ix murine myeloma prote ins react ive with Penumococcus C polysacchar ide and B l i p o p r o t e i n . Biochemistry 10:1424. 49. Po t te r , M. and Lieberman, R. 1970. Common ant igen ic determinants in f i v e of 8 BALB/c IgA myeloma prote ins that bind phosphoryl-cho l i ne . J . Exp. Med. 132:737. 50. Cozenza H. and Kohler , W. 1972. S p e c i f i c i n h i b i t i o n of plaque formation to phosphorylchol ine by antibody against ant ibody. Science 176:1027. 51. Lieberman, R., Po t te r , M., Mushinsk i , E.B., Humphrey, W., Rudikof f , S. 1974. Genetics of a new IgV„ (T15 id io type) marker in the mouse regu la t ing natural ant ibodies to phosphory l-chol ine. J . Exp. Med. 139:983. 52. C l a f l i n , J . L . and Rudikof f , S. 1976. Uniformity in a c lonal r epe r t o i r e : a case f o r a germ l i n e bas is of antibody d i v e r s i t y . Co ld . Spr ing . Harbor. Symp. Quant. B i o l . 41:725. 53. C l a f l i n , J . L . and Davie, J .M . 1974. Species s p e c i f i c b inding c h a r a c t e r i s t i c s of rodent ant i-phosphory lchol ine an t ibod ies . J . Immunol. 113:1678. 54. Rudikof f , S. and Po t te r , M. 1980. A l l e l i c forms of the immunoglobulin heavy chain va r i ab le reg ion . J . Immunol. 124:2089. 55. Lieberman, R., Rud ikof f , S . , Humphrey, W., Po t te r , M. 1981. A l l e l i c forms of ant i-phosphory lchol ine an t ibod ies . J . Immunol. 126:172. 56. Lieberman, R. 1978. Genetics of IgCH (a l lo type) locus in the mouse. Spr inger Sem. Immunopathol. 1 :^7. 57. Gearhart , P.J. , Si g a l , N .H. , Klinman, N.R. 1977. The monoclonal ant i-phosphory lchol ine antibody response in several murine s t r a i n s : genet ic impl i ca t ions of a d iverse r epe r to i r e . J . Exp. Med. 145:876. 58. Kearney, J . F . , B a r l e t t a , R., Quan, Z . S . , Quintans, J . 1981. Monoclonal vs . heterogeneous anti-H8 ant ibodies in the ana lys is of the ant i-phosphory lchol ine response in BALB/c mice. Eur. J . Immunol. 11:877. 59. Pease, L. and C l a f l i n , J . L . 1981. Heterogeneity among T15 id io t ype-pos i t i ve ant ibodies in inbred and wi ld mice. Eur. J . Immunol. 11:662. 60 Gearhart , P.J., Johnson, N.D. , Douglas, R., Hood, L. 1981. Ig G ant ibodies to phosphorylchol ine exh ib i t more d i v e r s i t y than t he i r IgM counterpar ts . Nature 291:29. 180 61. C l a f l i n , J . L , Hudak, S . , Maddelena, A. 1981. D i rec t evidence f o r three d i s t i n c t f ami l i e s of ant ibodies in the murine response. J . Exp. Med. 153:352. 62. Rodwel l , J . D . , Gearhart , P.J. , Karush, F. 1983. A f f i n i t y ana lys i s of monoclonal ant iphosphory lchol ine an t ibod ies . J . Immunol. 130:313. 63. Rud ikof f , S . , G i u s t i , A . M . , Cook, W.D., S cha r f f , M.D. 1982. S ingle amino ac id subs t i tu t i8on a l t e r i n g antigen binding s p e c i f i t y . Proc. N a t l . Acad. S c i . 79_:1979. 64. Rud ikof f , S. 1983. Immunoglobulin s t ructure - funct ion co r r e l a t e s : antigen binding and id io t ypes . Contemp. Top. Mol . Immunol. 9:169. 65. Cory, S. and Adams, J .M . 1980. Delet ions are assoc iated with somatic rearrangement of immunoglobulin heavy chain genes. Ce l l 19:37. 66. Crews, S . , G r i f f e n , J . , Huang, H . , Calame, K., Hood, L. 1981. A s ing le V., gene segment encodes the immune response to phosphory lchol ine : somatic mutation i s cor re la ted with the c lass of ant ibody. Ce l l 25:59. 67. C l a rk , S .H . , C l a f l i n , J . L . , Po t te r , M., Rudikof f , S. 1983. Polymorphism's in anti-phosphocholine ant ibodies r e f l e c t i n g evo lut ion of immunoglobulin f a m i l i e s . J . Exp. Med. 157:98. 68. Cozenza, H . , J u l i u s , M.H. , August in , A . A . , 1977. Idiotypes are va r i ab le region markers: analogies between receptors on phosphorylchol ine s p e c i f i c T and B lymphocytes. Immunol. Rev. 34 :3 . 69. August in , A .A. and Cosenza, H. 1976. Expression of new id io types fo l low ing neonatal i d i o t y p i c suppression of a dominant c lone . Eur. J . Immunol. 6^:497. 70. Cosenza, H . , August in , A . A . , J u l i u s , M.H. 1976. Idiotypes and an t i- id io types as probes in ana lys i s of immunoregulation. Cold Spring Harbor Symp. Quant. B i o l . 41:709. 71. Bottomly, K., Mathieson, B.J., Mos ier , D.E. 1978. Ant i- id io type induced regulat ion of helper c e l l funct ion f o r the response to phosphorylchol ine in adult BALB/c mice. J . Exp. Med. 148:1216. 72. Berek, C. 1983. Ant ibodies s p e c i f i c f o r d i f f e r e n t T15 id iotypes induce neonatal suppression of the T15 id io t ype . Eur. J . Immunol. 13:766. 73. Cosenza, H. 1976. Detect ion of an t i- id io type reac t i ve c e l l s in the response to phosphory lchol ine . Eur. J . Immunol. 6^ : 114. 74. Koh ler , H. 1975. The response to phosphory lchol ine: d i s sec t i ng an immune response. Transplant Rev. 27:24. 181 75. Bottomly, K. and Maurer, P.H. 1980. Antigen s p e c i f i c helper T c e l l s required f o r dominant product in of an id iotype (Th Id) are not under immune response ( Ir ) gene c o n t r o l . J . Exp. Med. 152:1571. 76. Bottomly, K. and Janeway, C A . ( J r ) . 1981. Selected populat ions of a l l o r e a c t i v e T helper c e l l s that lack Th Id , an antigen s p e c i f i c helper T c e l l required f o r dominant production of the T15 id io t ype . Eur. J . Immunol. 11:270. 77. Bottomly, K., Janeway, C A . ( J r . ) , Matheison, B.J . , Mos ier , D.E. 1980. Abscence of an an t igen-spec i f i c helper T c e l l required f o r the expression of the T15 id iotype in mice t reated with an t i -ant ibody. Eur. J . Immunol. 10:159. 78. Kohmo, T . , Yamamoto, H . , M izuoch i , T . , Hamaoka, T. 1981. Detect ion of the dominant expression of the TEPC-15 i d i o t y p i c determinant(s) on phosphorylchol ine s p e c i f i c suppressor T lymphocytes in v i t r o . J . Immunol. 126:1378. 79. Sugimura, K., Nakanish i , K., Maeda, K., Kashiwamura, S-I., Suemura, M., Shiho, 0 . , Yamamura, Y . , Kishimoto, T. 1982. The involvement of two d i s t i n c t subsets of T c e l l s f o r the expression of the IgE c lass s p e c i f i c suppress ion: establ ishment and cha rac te r i za t ion of PC s p e c i f i c , T15 id io t ype-pos i t i ve T hybridomas and IgE c lass s p e c i f i c ant igen-non-speci f ic T hybridomas. J . Immunol. 128:1637. 80. Nakanish i , K., Sugimura, K., Yao i t a , Y . , Maeda, K., Kashiwamura, S-I., Honjo, T . , Kishimoto, T. 1982. A T15 id io t ype-pos i t i ve T suppressor hybridoma does not use the T15 V H gene segment. Proc. Na t l . Acad. S c i . 79:6984. M 81. McNamara, M. and Koh ler , H. 1983. Idiotype recogniz ing T helper c e l l s that are not id io type s p e c i f i c . J . Exp. Med. 158:811. 82. W i l l i ams , K. and C l a f l i n , J . L . 1980. S t ructura l and i d i o t y p i c s i m i l a r i t i e s in a d iverse r epe r to i r e . J . Immunol. 125:2429. 83. Mond, J . M . , Lieberman, R.L . , Inman, J . K . , Mos ier , D .E . , Pau l , W.E. 1977. I n a b i l i t y of mice with a defect in B-lymphocyte maturation to respond to phosphorylchol ine on immunogenic c a r r i e r s . J . Exp. Med. 146:1138. 84. Kenny, J . J . , Wickers, L .S . , Guelde, G . , Scher, I. 1982. Mice expressing the Xid immune defect provide normal help to T15 pos i t i v e B c e l l p recursors . J . Immunol. 129:1534. 85. E t l i n g e r , H.M. and Heusser, C H . 1983. A l t e r a t i on of immunologic funct ion through ear l y ontogenic exper iences. Se lec t i ve a c t i v a t i on of T15-posi t ive B c e l l s in neonatal mice i s re la ted to receptor a v i d i t y and i s independant of thymus func t i on . Eur. J . Immunol. 13:180. 182 86. P i l l emer , E. and Weissman, I. 1981. A monoclonal antibody that detects a V K-TEPC 15 i d i o t y p i c determinant c ross-react i ve with a Thy-1 determinant. J . Exp. Med. 153:1068. 87. Makela, 0. and Kar ja la inen , K. 1977. Inher i ted immunolgobulin id iotypes of the mouse. Immunol. Rev. 34:119. 88. Jack, R.S., Imanishi-Kar i , T . , Rajewsky, K. 1977. Id io typ ic ana lys is of the response of C57BL/6 mice to the (4-hydroxy-3-nitrophenyl) acety l group. Eur. J . Immunol. 7^:559. 89. Imanishi , T . and Makela, 0. 1974. Anti-(4-hydroxy-3-nitrophenyl) acety l of the mouse primary response. J . Exp. Med. 140:1498. 90. Ka r j a l a inen , K. 1980. The major id iotypes in the mouse ant i (4-hydroxy-3-nitrophenyl) acety l (NP) ant ibodies are con t ro l l ed by a l l e l i c genes. Eur. J . Immunol. 10:132. 91. Kar ja la inen , K., Bang, B., Makela, 0. 1980. Fine s p e c i f i c i t y and id iotypes of ea r l y ant ibodies against (4-hydroxy-3-nitrophenyl) acety l (NP). J . Immunol. 125:313. 92. White-Scharf. M.E. and Imanishi-Kar i , T . , 1981. Charac te r iza t ion of the NP id iotype through the ana lys is of monoclonal BALB/c anti-(4-hydroxy-3-nitrophenyl) acety l (NP) an t ibod ies . Eur. J . Immunol. 11:897. 93. White-Scharf, M.E. and Imanishi-Kar i , T. 1982. C ross- reac t i v i t y of the NP and NP i d i o t y p i c responses of BALB/c and C57BL/6 mice to (4-hydroxy-3-nitrophenyl) acety l (NP). Eur. J . Immunol. 12:935. 94. Bothwel l , A . L . M . , Paskind, M., Reth, M. Imanishi-Kar i , T . , Rajewsky, J . , Ba l t imore , D. . 1 9 8 1 . Heavy chain va r i ab le region cont r ibut ion to the NP fami ly of an t ibod ies : somatic mutation evident in a y 2 a va r i ab le reg ion . Ce l l 24:625. 95. Bothwel l , A . L . M . , Paskind, M., Reth, M. Imanishi-Kar i , T . , Rajewsky, K., Ba l t imore , D. 1982. Somatic var iants of murine immunoglobulin l i g h t cha ins . Nature 298:380. 96. Rajewsky, K., Takemori, T . , Reth, M. 1981. Ana lys is and regu la t ion of V gene expression by monoclonal ant ibodies in Monoclonal  Ant ibodies and T Ce l l Hybridomas. (G. J . Hammerling, OT Hammer!ing, and J . F . Kearney, E d i t o r s ) . E lsev ier/North Holland Biomedical Press. Amsterdam, p. 399. 97. Reth, M., Imanishi-Kar i , T . , Rajewsky, K. 1979. Charac te r iza t ion of id iotopes by monoclonal an t i- id io tope an t ibod ies . Eur. J . Immunol. 9:1004. 183 98. Rajewsky, K. and Takemori, T . 1983. Genet ics , expression and funct ion of i d io t ypes . Ann. Rev. Immunol. 1^:569. 99. Takemori, T . , Tesch , H . , Reth, M. , Rajewsky, K. 1982. Induction of id iotope bearing ant ibodies and ana lys is of the id iotope r epe r to i r e . Eur. J . Immunol. 12:1040. 100. D i l d r o p , R., Bruggeman, M., Radbruch, A . , Rajewsky, K., Bayreuther, K. 1982. Recombination between V genes. EMBO J . 1^:635. 101. Loh, D.Y. , Bothwel l , A . L . N . White-Scharf, M.E. , Imanishi-Kar i , T . , Ba l t imore , D. 1983. Molecular bas is of a mouse s t r a i n s p e c i f i c anti-hapten response. Ce l l 33:85. 102. Weinberger, J . Z . , Germain, R.N., Ju S-T, Greene, M.I . , Benacerraf , B., Dor f , M.E. 1979. Demonstration of i d i o t y p i c determinants on suppressor T c e l l s . J . Exp. Med. 150:761. 103. Weinberger, J . Z . , Benacerraf , B., Dor f , M.E. 1980. In teract ion of e f f e c t o r suppressor T c e l l s i s r e s t r i c t e d by H-2 and Igh-V genes. J . Exp. Med. 151:1413. 104. Weinberger, J . Z . , Germain, R.N. , Benacerraf , B., Dor f , M.E. 1980. Role of id iotypes in the suppressor pathway. J . Exp. Med. 152:161. 105. Krawinkel , U . , Cramer, M., Imanishi-Kar i , T . , Jack, R.S., Rajewsky, K., Makela, 0. 1977. Receptors from nylon wool-enriched mouse T lymphocytes lack se ro log i ca l markers of immunoglobulin constant domains but express heavy chain va r i ab le por t ions . Eur. J . Immunol. 7_:566. 1977. 106. Suzuk i , G . , Cramer, M., Hayakawa, K., Okumura, K., Tada, T. 1983. Id io typ ic and f i ne s p e c i f i c i t y ana lys is of a (4-hydroxy-3-ni t rophenyl ) acty l (NP) - s p e c i f i c suppressor T c e l l hybridoma at the l eve l of c e l l surface s t r u c tu r e s , i so l a t ed receptor m a t e r i a l , and funct iona l suppressor f a c t o r . Eur. J . Immunol. 13:711. 107. Bruggeman, M. and Rajewsky, K. 1982. Regulation of the antibody response against hapten-coupled erythrocytes by monoclonal anti-hapten ant i-bodies of var ious i so types . C e l l . Immunol. 71:365. 108. Wa l l , K .A. , F racke l ton , A.R. ( J r . ) , R e i l l y , E.B., Azuma, T . , Chang, T.W., E i s en , H.N. 1983. Quant i ta t ion of anti-NP-(4-hydroxy-3-ni t rophenyl ) acety l id iotype expression on spleen and thymus c e l l s . Eur. J . Immunol. 13:441. 109. Leon, M.A., Young, N.M., M c l n t i r e , K.R. 1970. Immunochemical studies of the react ion between a mouse myeloma macroglobulin and dextrans. Biochem. 9:1023. 184 110. Lundblad, A . , S t ea l e r , R., Kabat, E .A . , H i r s t , J .W. , Weigert , M.G. , Cohn, M. 1972. Immunochemical studies on mouse myeloma prote ins with s p e c i f i c i t y f o r dextran and or levan. Immunochem. 9_:535. 111. Weigert, M.G. , C e s a r i , I .M., Yonkovich, S . J . , Cohn, M. 1970. V a r i a b i l i t y in the lambda l i g h t chain sequences of mouse ant ibody. Nature 228:1045. 112. S c h i l l i n g , J . , C l ev inger , B., Davie, J . M . , Hood, L. 1980. Amino ac id sequence of homogeneous ant ibodies to dextran and DNA rearrangements in heavy chain V-region gene segments. Nature 283:35. 113. Blomberg, B., Gecke ler , W.R., Weigert , M. 1972. Genetics of the antibody response to dextran in mice. Science 177:178. 114. Hansburg, D. , B r i l e s , D .E . , Dav ie , J .M . 1977. Demonstration of mul t ip le id iotypes with va r i ab le expression in several s t r a i n s . J . Immunol. 119:1406. 115. S c h i l l i n g , J . , Hansburg, D. , Dav ie , J . M . , Hood, L. 1979. Ana lys is of the d i v e r s i t y of murine ant ibodies to dextran B1355: N terminal amino ac id sequences of heavy chains from serum ant ibod ies . J . Immunol. 123:384. 116. Newman, B., S u g i i , S . , Kabat, E .A . , T o r i i , M. , C l ev inge r , B .L . , S c h i l l i n g , J . , Bond, M., Davie, J . M . , Hood, L. 1983. Combining s i t e s p e c i f i t i e s of mouse hybridoma ant ibodies to dextran B13555S. J . Exp. Med. 157:130. 117. Ward, R.E . , Kearney, J . F . , Kohler , H. 1981. L ight chain isotypes s e l e c t i v e l y assoc iated with heavy chain id iotypes in T dependant and T independant precursors . J . Immunol 126:146. 118. Ward, R.E . , Kearney, J . F . , Kohler , H. 1981. L ight chain isotypes s e l e c t i v e l y associated with heavy chain id iotypes in T dependant and T independant dextran s p e c i f i c precursors . Nature 292:629. 119. Eichmann, K. 1972. Id io typ ic i den t i t y of ant ibodies to s t reptococca l carbohydrate in inbred mice. Eur. J . Immunol. 2^:301. 120. Eichmann, K. and Berek, C , 1973. Mendelian segregation of a mouse antibody i d io t ype . Eur. J . Immunol. 73:599. 121. Eichmann, K. 1973. Idiotype expression and the inher i tance of mouse antibody c lones . J . Exp. Med. 137:603. 122. Eichmann, K. 1974. Influence of the dose and of the e f f e c t o r funct ions of a n t i - i d i o t y p i c antibody on the production of an id io t ype . Eur. J . Immunol. £:296. 123. Per lmutter , R.M., Hansburg, D. , B r i l e s , D .E . , N i c o l o t t i , R.A. , and Davie , J .M . 1978. Subclass r e s t r i c t i o n of murine ant i-carbodydrate an t ibod ies . J . Immunol. 121:566. 185 124. S lack. J . , Der-Ba l ian , G .P . , Nahm, M., Davie, J .M . 1980. The Ig G plaque-forming c e l l response to thymus independant type 1 and type 2 antigens in normal mice and mice expressing an X-l inked immunodeficiency. J . Exp. Med. 151:853. 125. Krawinkel , U . , Cramer, M., Berek, C , Hammerling, G . , B lack, S . J . , Rajewsky, K., Eichmann, K. 1976. On the s t ructure of the T c e l l receptor f o r ant igen. Co ld . Spring Harbor Symp. Quant. B i o l . 41:285. 126. Berek, C , Tay l o r , B.A., Eichmann, K. 1976. Genetics of the id iotype of BALB/c myeloma S117: mul t ip le chromosomal l o c i f o r genes encoding s p e c i f i c i t y f o r Group A st reptococca l carbohydrate. J . Exp. Med. 144:1164. 127. Nahm, M.H. , C l ev inge r , B .L . , Dav ie , J .M . 1982. A dominant i d i o t y p i c determinant i s located on V^. J . Immunol. 129:1513. 128. B r i l e s , D.E. and C a r r o l l , R.J. 1981. A simple method f o r est imat ing the probable numbers of d i f f e r e n t ant ibodies by examining the repeat f requencies of sequences on i s o l e c t r i c focus ing pat terns . Mol . Immunol. 18:29. 129. Per lmutter , R.M., B r i l e s , D .E . , Greve, J . M . , Dav ie , J .M . 1978. L ight chain d i v e r s i t y of murine ant i s t reptococca l an t ibod ies : IgCH-linked e f f e c t s on L chain express ion. J . Immunol. 121:149. 130. Per lmutter , R.M., K l o t z , J . L . , Bond, M.W., Nahm, M. , Dav ie , J . , Hood, L. 1984. Mu l t ip l e V„ gene segments encode murine an t i -st reptococca l an t ibod ies . J . Exp. Med. 159:179. 131. Braun, D .G . , K indred, B., Jacobson, E.B. 1972. Streptococcal group A carbohydrate ant ibodies in mice: evidence f o r s t r a i n d i f fe rences in magnitude and r e s t r i c t i o n of the response, and f o r thymus dependance. Eur. J . Immunol. 2^:138. 132. B r i l e s , D . E . , Nahm, M. , Mar ion, T . N . , Per lmutter , R.M., Dav ie , J .M . 1982. Streptococcal Group A carbohydrate has proper t ies of both a thymus dependant (TI-2) and a thymus dependent ant igen. J . Immunol. 128:2032. 133. Eichmann, K. and Rajewsky, K. 1975. Induction of T and B c e l l immunity by a n t i - i d i o t y p i c ant ibody. Eur. J . Immunol. 5_:661. 134. Eichmann, K. 1975. Amp l i f i c a t i on of a suppessor T c e l l with a n t i - i d i o t y p i c a c t i v i t y . Eur. J . Immunol. 5_:511. 135. Rajewsky, K. and Eichmann, K. 1977. Antigen receptors of helper T c e l l s . Contemp. Top. Immunobiol. 7_:69. 186 136. Hammer!ing, G . J . , B lack, S . J . , Berek, C , Eichmann, K., Rajewsky, K. 1976. Genetic control of T-helper e e l ! responsiveness to a n t i - i d i o t y p i c ant ibody. J . Exp. Med. 143:861. 137. B lack, S.J. Hammerling, G . J . , Berek, C , Rajewsky, K., Eichmann, K. 1976. S p e c i f i c i t y and heterogeneity of B and T lymphocytes reac t i ve with a n t i - i d i o t y p i c ant ibody. J . Exp. Med. 143:847. 138. R i ch te r , J . 1978. The network idea and the immune response ( in Theoret i ca l Immunology.) G.I. B e l l , A .S . Pere lson, G.H. Pimbley ( Jr . ) Ed i t o r s . Marcel Dekker, Inc. New York, p. 539. 139. Hoffman, G.W. 1980. On network theory and H-2 r e s t r i c t i o n . Contemp. Topics in Immunobiol. 11:185. 140. Morr i son , D.C. and Ryan J . L . 1979. Bac te r ia l endotoxins and host immune responses. Adv. Immunol. 28:293 141. Andersson J . , Mo l l e r , G . , S joberg, 0. 1972. Se lec t i ve induct ion of DNA synthesis in T and B lymphocytes. C e l l . Immunol. 4_:381. 142. Rudback, J . 1974. Immunogenicity of l i popo l ysaccha r ides . In: The Role of Immunological Factors in I n f ec t i ons , A l l e r g i c And  Autoimmune Processes. (R.F. Beers and E. Basset , Ed i tors ) Raven Press , New York. p. 29. 143. S k e l l y , R.R., Ahmed, A . , Munkenbeck, P., Morr i son , D.C. 1973. Immune responses to hapten-1ipopolysaccharide ant igens. III. Genetics of the antibody response to polysacchar ide ant igens. J . Immunogen. 10:237. 144. C o l l e , J-H, Motta, I., Sh indan i , B., T ru f fa-Bach i , P. 1983. Igh-V or c l o se l y l inked gene(s) contro l immunological memory to a thymus-independant ant igen. Nature 301:428. 145. Hiernaux, J . and Bona, C. 1982. Shared id iotypes among monoclonal ant ibodies s p e c i f i c f o r d i f f e r e n t immunodominant sugars of 1 ipopolysacchar ide of d i f f e r e n t Gram-negative bac t e r i a . Proc. N a t l . Acad. S c i . 79:1616. 146. Hiernaux, J . , Schroer , K.R., Baker, P.J. , Rudbach, J . A . , Bona, C. 1982. Study of the id io typy of 1 ipopo lycacchar ide-spec i f i c po lyc lona l and monoclonal an t ibod ies . Eur. J . Immunol. 12:797. 147. S i ko ra , L .K. J . 1983. Genetics of the immune response to f e r redox in : assessment of control at the determinant l e v e l . Ph.D. T h e s i s , U.B.C. Dept. Microbio logy . 148. Merryman, C . F . , Maurer, P .H. , S t imp f l i ng , J . J . 1975. Unigenic and mul t igen ic J region contro l of the immune response of mice to the GAT and GL0-GLT terpolymers. Immunogen. 2;441. 187 149. Dor f , M.E. , Dunham, E.K., Johnson, J . B . , Benacerraf , B. 1974. Genetic control of the immune response: the e f f e c t of non-H-2 l inked genes on antibody product ion. J . Immunol. 112:1329. 150. A t a s s i , M.Z. 1975. Ant igen ic s t ructure of myoglobin: the complete immunochemical antomy of a p r o t e i n , and conclus ions r e l a t i ng to ant igen ic s t ructures of p ro te ins . Immunochemistry 12:423. 151. K e l l y , B. and Levy, J . G . 1971. Immunological studies on the major haptenic peptides from performic ac id ox id ized fer redoxin from C los t r id ium pasteurianum. Biochemistry 10:176. 152. S m i t h - G i l l , S . J . , Wi lson, A . C , Po t te r , M., Prager, E .M., Feldman, R.J., Mainhart , C R . 1982. Mapping the ant igen ic epitope f o r a monoclonal antibody against lysozyme. J . Immunol. 128:314. 153. Ju , S . T . , Benacerraf , B., Dor-fg, M.E-,- 1978. .- Id io typ ic ana lys is of ant ibodies to poly (Glu , A la , Tyr ): i n t e r s t r a i n and i n t e r -species i d i o t y p i c c ross-reac t ions . Proc. N a t l . Acad. S c i . 75:6192. 154. Theze J . and Somrne G. 1979. Charac te r iza t ion of a c ross-reac t i ve id iotype assoc iated with anti-GAT ant ibodies from responder and non-responder mice. Eur. J . Immunol. £ :294. 155. Ruf, J . , Tonne l l e , C , Rocca-Serra, J . , Mo in ie r , D. , P i e r r e s , M., J u , S-T., Dor f , M.E. , Theze, J . and Fougereau, M. 1983. S t ructura l bases f o r pub l i c id io type SDecifictJ.es of-monoclonal ant ibodies d i rec ted against poly (Glu , Ala"™ T y r i U J and poly (Glu , A la ) random copolymers. Proc. Na t l . Acad. S c i . 80:3040. 156. P e t i t , C , G i l b e r t , M., Somrne, G . , L e c l e r q , L . , Mazie, J - C . , Dor f , M.E. , Theze, J . 1982. Ana lys is of a major rat id io type assoc iated with anti-GAT an t ibod ies . Mol . Immunol 19:1139. 157. Ju. S-T. and Dor f , M.E. 1979. Induction of CGAT id io type fo l lowing immunization of var ious synthet ic polymers conta in ing glutamic ac id and t y ros ine . Eur. J . Immunol. 9^:553. 158. J u , S-T., Dor f , M.E. and Benacerraf , B. 1979. Determinant s p e c i f i c i t y and immunoglobulin c lass d i s t r i b u t i o n of CGAT i d i o t ype . J . Immunol. 122:1054. 159. P e t i t , C , Joskov i cz , M. , S tan i s l awsk i , M. , Theze, J . 1979. Res t r i c ted heterogeneity of the anti-GAT response from BALB/c responder mice. Eur. J . Immunol. 9^:922. 160. J u , S-T., P i e r r e s , M., Waltenbaugh, C , Germain, R.N. , Benacerraf , B., Dor f , M .E R n 1979,- Id io typ ic analys i of monoclonal ant ibodies to poly (Glu , A la , T y r i U ) . Proc. N a t l . Acad. S c i . 76_:2942. 161. J u , S-T, P i e r r e s , M. , Germain, R.N. , Benacerraf , B., Dor f , M.E. 1979. I den t i f i c a t i on and s t r a i n d i s t r i b u t i o n of the GA-1 id io t ype . J . Immunol. 123:2505. 188 162. Somme, G . , L e c l e r q , L . , P e t i t , C , Theze, J . 1981. Three types of i d i o t y p i c s p e c i f i c i t e s on BALB/c anti-GAT ant ibod ies . Eur. J . Immunol. _11:493. 163. Ju , S-T., P i e r r e s , M., Germain, R., Benacerraf , B., Dor f , M.E. 1981. Comparison of i n t r a s t r a i n and a l lo t ype associated i d i o t y p i c s p e c i f i c i t e s . J . Immunol. 126:177. 164. Ju , S-T. and D o r f . , M.E. 1981. Genetic mapping of the Gte id io type marker wi th in the Igh-V locus . J . Immunol. 126:183. 165. Ju , S-T., Benacerraf , B., Dor f , M.E. 1980. Genetic control of a shared id iotype among ant ibodies d i rec ted to d i s t i n c t s p e c i f i c i t i e s . J . Exp. Med. 152:170. 166. Somme, G . , Rocca-Serra, J . , L e c l e r q , L . , Moreau, J-L , Mazie, J - C , Mo in ie r , D. , Fougereau, M., Theze, J . 1982. Contr ibut ions of the H and L chains and of the binding s i t e to the i d i o t y p i c s p e c i f i c i t i e s of mouse anti-GAT ant ibod ies . Mol . Immunol. 19:1011. 167. Rocca-Serra, J . , Mazie, J - C , Mo in ie r , D. , L e c l e r cq , L . , Somme, G . , Theze, J . and Fougereau, M., 1982. The l im i t ed d i v e r s i t y of the mouse chains anti-GAT reper to i re does not seem to be not iceably ampl i f i ed among c lass switch. J . Immunol. 129:2554. 168. Rocca-Serra. , J . , Matthes, H.W., Kaar t inen, M., M i l s t e i n , C , Theze, J . , Fougereau, M. 1983. Ana lys is of antibody d i v e r s i t y : VDJ mRNA nucleot ide sequence of four anti-GAT monoclonal an t ibod ies . A paucigene system using a l te rnate D-J recombinations to generate f unc t i ona l l y s i m i l a r hypervar iable reg ions . EMBO J . 2^:867. 169. S c h i f f , C , M i l i l i , M., Fougereau, M. 1983. Immunoglobulin d i v e r s i t y : ana lys i s of the germ-line V„ gene reper to i re of the murine anti-GAT response. Nuc. Ac ids . Res. 11:4007. 170. Rocca-Serra, J . , Tonne l l e , C . , Fougereau, M. 1983. Two monoclonal ant ibodies against d i f f e r e n t antigens using the V„ germ-line gene. Nature 304:353. 171. S c h i f f , C , Corbet , S. , Mi 1 i n i , M., Fougereau, M. 1983. In te rs t ra in conversion of the murine GAT-speci f ic antibody V. reper to i re as analyzed at the germline gene l e v e l . EMBO J . 2^:1771. 172. Tonne l l e , C , Rocca-Serra, J . , MouVifi, A ™ M o m i e r , D. , Fougereau, M. 1983. V. gene fami ly in ( G l u D U A la 1 ™ T y r i U ) in (GAT) - s p e c i f i c ant ibodies that express CGAT (or pGAT) pub l i c i d i o t y p i c s p e c i f i c i t i e s . Protein and mRNA sequencing of e ight monoclonal V k cha ins . J . Exp. Med. 158:1415. 173. Kapp, J . A . , P i e r ce , C.W., de l a C r o i x , F. , Benacerraf , B. 1976. Immunosuppressive f ac to r ( s ) extracted from lympho-id c e l l s ~of non-responder mice primed with L-glutamic ac id -L-alanine -L-tyrosine (GAT) I. A c t i v i t y and antigen s p e c i f i t y . J . Immunol. 116:305. 189 174. Theze, J . , Kapp, J . A . , Benacerraf , B. 1977. Immunochemical proper t ies of the GAT s p e c i f i c suppressor f a c t o r . J . Exp. Med. 145:839. 175. Germain, R.M. and Benacerraf , B. 1978. Induction o f g an t igen s p e c i f i c suppressor T c e l l s (Ts2) in L-Glutamic Ac id -L-Alanine -L-Tyrosine (GAT) responder mice by non-responder-derived GAT-suppressor f a c to r (GAT-TsF). J . Immunol. 121:608. 176. Germain, R.N. Ju , S-T., K ipps , T . J . , Benacerraf , B., Dor f , M.E. 1979. Shared i d i o t y p i c determinants on ant ibodies and T-ce l l-der i ved suppressor f a c to r s p e c i f i c f o r the random terpolymer L-glutamic ac id -Lalanine -L-tyrosine . J . Exp. Med. 149:613. 177. Krupen, K., Araneo, B.A., B r ink , L . , Kapp, J . A . , S t e i n s , S . , Weider, K. J . , Webb, D.R. 1982. P u r i f i c a t i o n and cha rac te r i za t ion o f-a monoclonal T-ce-lJ suppressor f a c to r s p e c i f i c f o r poly (L-Glu -L-Ala - L-Ty r i U ) Proc. Na t l . Acad. S c i . 79;1254. 178. Sorenson, C M . , P i e r ce , C.W., Webb, D.R. 1983. P u r i f i c a t i o n and - Q cha rac te r i za t i on of an L-glutamic ac id -L-alanine -L-tyrosine . (GAT) - s p e c i f i c suppressor f a c to r from genet ic responder mice. J . Exp. Med. 158:1034. 179. Gougeon, M-L., L e c l e r cq , L . , Lowy, I., Bismuth, G . , Somrne. G . , Theze, J . 1982. In v i t r o i n h i b i t i o n of the helper a c t i v i t y of GAT-speci f ic T-ce l l l i nes by a syngeneic a n t i - i d i o t y p i c serum: p re f e ren t i a l e f f e c t on the IgGl response. C e l l . Immunol. 71:254. 180 K r a i g , E. , Kronenberg, M., Kapp, J . A . , P i e r ce , C.W., A b r u z z i n i , A . F . , Sorenson, C M . , Samelson, L . E . , Schwartz, R.H. , Hood, L. 1983. T and B c e l l s that recognize the same antigen do not t ransc r ibe s i m i l a r heavy chain va r i ab le region gene segments. J . Exp. Med. 158:192. 181. Kronenberg, M., K r a i g , E . , S i u , G . , Kapp, J . A . , Kappler , J . , Marrack, D. , P i e r ce , C.W., Hood, L. 1983. Three T c e l l hybridomas do not contain detectable heavy chain va r i ab le gene t r a n s c r i p t s . J . Exp. Med. 158:210. 182. A t a s s i , M.Z. , 1977. The complete ant igen ic s t ructure of myoglobin: approaches and conclusions f o r ant igen ic s t ructures of p ro te ins . In. Immunochemistry of P ro te ins . M.Z. A t a s s i , E d i t o r , Plenum Press , New York. p. 77. 183. Twining, S .S . , Dav id , C . S . , A t a s s i , M.Z. 1981. Mouse ant ibodies in outbred and congenic s t ra ins against spermwhale myoglobin recognize the same ant igen ic s i t e s that are recognized by ant ibodies ra ised in other spec ies . Mol. Immunol. 18:447. 184. Twining, S .S . , Lehmann, H . , A t a s s i , M.Z. 1981. Time dependance study of the antibody response to sperm whale myoglobin: recogni t ion of the ant igen ic s i t e i s unaltered over an extended per iod of immunization. Mol . Immunol. 18:473. 190 185. Berzofsky , J . A . , H i cks , G . , Federko, J . , Minna, J . 1980. Propert ies of monoclonal ant ibodies s p e c i f i c f o r determinants of a prote in an t igen , myoglobin. J . B i o l . Chem. 255:11188. 186. Berzofsky , O .A . , Buckenmeyer, G .K . , H i cks , G . , Gurd, F .R .N . , Feldmann, R.J., Minna, J . 1982. Topographic ant igen ic determinants recognized by moncolonal ant ibodies to sperm whale myoglobin. J . B i o l . Chem. 257:3189. 187. Eas t , J . J . , H u r r e l l , J . G . R . , Todd, P .E .E . , Leach, S.J . 1982. Ant igen ic s p e c i f i c i t y of monoclonal ant ibodies to human myoglobin. J . B i o l . Chem. 257:3199. 188. Infante , A . J . , A t a s s i , M.Z. , Fathman, C.G. 1981. T c e l l clones reac t i ve with sperm whale myoglobin. I so la t ion of clones with s p e c i f i c i t y f o r ind iv idua l determinants on myoglobin. J . Exp. Med. 154:1342. 189. Young, C.R. and A t a s s i , M.Z. 1983. T-lymphocyte recogni t ion of sperm whale myoglobin. Recognit ion of synthet ic peptides car ry ing ant igen ic s i t e 5 by myoglobin primed T c e l l s . J . Immunogen. 10:139. 190. Young, C.R. and A t a s s i , M.Z. 1983. T-lymphocyte recogni t ion by sperm whale myoglobin. S p e c i f i c i t y of T-ce l l recogn i t ion fo l lowing neonatal to lerance with e i t he r myoglobin or synthet ic peptides of an ant igen ic s i t e . J . Immunogen. 10:161. 191. Berzofsky, J . A . 1978. Genetic control of the antibody response to sperm whale myoglobin in mice. Adv. Exp. Med. B i o l . 98:225. 192. Young, C .R . , and A t a s s i , M.Z. 1982. Overcoming genet ic control of antibody response to ant igen ic s i t e s by increas ing the dose of antigen used in immunization. J . Immunogen. £ :343. 193. Young, C.R. , 0 'Conner, G .P . , A t a s s i , M.Z. 1981. Antibody response to myoglobin var iants reveal that gene r e s t r i c t i o n of the antibody responses to myoglobin ant igen ic s i t e s i s dependant on the chemical proper t ies of the s i t e . Immunologic. Comm. 10:483. 194. Kohno, Y . , Berkower, I., Minna, J . , Berzofsky, J . A . 1982. Idiotypes of anti-myoglobin an t ibod ies : shared id iotypes among monoclonal ant ibodies to d i s t i n c t determinants of sperm whale myoglobin. J . Immunol. 128:1742. 195. A t a s s i , M.Z. and Lee, C-I. 1978. The prec ise and en t i r e ant igen ic s t ructure of nat ive lysozyme. Biochem. J . 171:429. 196. Ibrah imi , I.M., Prager, E .M., White, T . J . , Wi lson, A .C . 1979. Amino ac id sequence of C a l i f o r n i a Quail lysozyme. E f fec ts of evo lut ionary subs t i tu t ions on the ant igen ic s t ructure of lysozyme. Biochem. 181:2736. 191 197. Takagaki , Y . , Hirayama, A . , F u j i o , H . , Amano, T . 1980. Ant ibodies to a continuous region of residues 38-54 of hen egg-white lysozyme found in a small f r a c t i o n of anti-hen egg-white lysozyme ant ibod ies . Biochem. 19:2498. 198. Sm i th-G i l l , S . J . , W i l son, A . C , Po t te r , M., Prager, E .M. , Feldmann, R.J., Mainhart , C R . 1982. Mapping the ant igen ic epitope f o r a monoclonal antibody against lysozyme. J . Immunol. 128:314. 199. H i l l , S.W., K ipp, D .E . , Melchers , I., F r e l i n g e r , J . A . , Se rca rz , E.E. 1980. Mu l t ip l e and non-H-2 genes c o n t r o l l i n g the anti-lysozyme response: a l t e rna t i v e gene cons te l l a t i ons can lead to responsiveness. Eur. J . Immunol. 10:384. 200. Se rca rz , E.E. and Metzger, D.W. 1980. Epitope and i d i o tope-spec i f i c c e l l u l a r i n te rac t ions in a model prote in antigen system. Spr inger . Sem. Immunopath. 3:W5. 201. A t a s s i , M.Z. and Sakata, S. 1982. Binding with lysozyme of ant ibodies against surface-s imulat ion peptides represent ing the lysozyme ant igen ic s i t e s . Biochem. J . 201:669. 202. A d o r i n i , L . , Harvey, M.A., M i l l e r , A . , Se rca rz , E.E. 1979. Suppressor and helper T c e l l s are induced by d i f f e r e n t regions of hen egg-white lysozyme in a gene t i c a l l y non-responder mouse s t r a i n . J . Exp. Med. 150:293. 203. Harvey, M.A., A d o r i n i , L . , M i l l e r , A . , Se rca rz , E.E. 1979. Lysozyme-induced T-suppressor c e l l s and ant ibodies have a predominant i d i o t ype . Nature 281:594. 204. Ma i ze l s , R.M., C l a rke , J . A . , Harvey, M.A., M i l l e r , A . , Se rca rz , E.E. 1980. Epitope s p e c i f i c i t y of the T c e l l p r o l i f e r a t i v e c e l l s react predominantly to d i f f e r e n t determinants from those recognized by B c e l l s . Eur. J . Immunol. 10:509. 205. Hirayama, A . , Dohi , Y . , Takagaki , Y . , F u j i o , H . , Amano, T. 1982. S t ructura l r e l a t i onsh ips between c a r r i e r epitopes and ant igen ic epitopes on hen egg-white lysozyme. Immunol. 46:145. 206. Benjamin, C D . , M i l l e r , A . , Se rca rz , E .E . , Harvey, M.A. 1980. A predominant id iotype on anti-hen egg-white lysozyme ant ibodies from diverse mouse s t r a i n s . J . Immunol. 125:1017. 207. Metzger, D.W., M i l l e r , A . , Se rca rz , E.E. 1980. Sharing of an i d i o t y p i c marker by monoclonal ant ibodies s p e c i f i c f o r d i s t i n c t regions of hen lysozyme. Nature 287:540. 208. Metzger, D.W., Furman, A . , M i l l e r , A . , Se rca rz , E.E. 1981. Se lec t ion a f t e r immunization of an IdX marker common to ant ibodies of d i s t i n c t epitope s p e c i f i c i t y . J . Exp. Med. 154:701. 192 209. Metzger, D.W., M i l l e r , A . , Se rca rz , E.E. 1980. Id io typ ic cha rac te r i za t ion of anti-lysozyme hybridomas. Fed. Pro. 39:572. 210. A d o r i n i , L . , Do r i a , G . , R i c c i a rd i -Cas t agno l i , P. 1982. Fine ant igen ic s p e c i f i c i t y and genet ic r e s t r i c t i o n of lysozyme-speci f ic suppressor T c e l l f a c to r produced by rad ia t ion leukemia virus-transformed suppressor T c e l l s . Eur. J . Immunol. 12:719. 211. A d o r i n i , L . , P i n i , C , De San t i s , R., R o b i a t i , F., Do r i a , G . , and R i c c i a rd i -Cas t agno l i , P. 1983. Monoclonal suppressor T c e l l f a c to r d i sp lay ing V„ r e s t r i c t i o n and f i ne ant igen ic s p e c i f i c i t y . Nature 303:704. M 212. M i l l e r , A . , Ch 'ng , L-K., Benjamin, C , Sercarz , E .E . , Brodeur, P., R ib l ey , R. 1983. Deta i led ana lys is of the pub l i c id io type of anti-hen egg-white lysozyme ant ibod ies . In Id io typ ic Networks. Ann. New York. Acad. S c i . in press . 213. Anf insen , C . B . , Cuatrecasas, P., Tan iuch i , H. 1971. Staphylococcal nuclease: chemical propert ies and c a t a l y s i s . In The Enzymes, P.D. Boyer, E d i t o r , Academic Press , New yo rk , p. 177. 214. Sachs, D .H . , Berzofsky, J . A . , P iese tsky , D.S . , Schwartz, R.H. 1978. Genetic control of the immune response to staphylococcal nuclease. Spr inger Sem.Immunopath 1:51. 215. Sachs, D .H . , Schechter, A . N . , Eas t lake , A . , Anf insen , C.B. 1972. An immunological approach to the conformational e q u i l i b r i a of po lypept ides . Proc. Na t l . Acad. S c i . 69:3790. 216. Sachs, D .H . , Berzofsky, J . A . , Fathman, C . G . , P i se tsky , D.S . , Schechter , A . N . , Schwartz, R.H. 1976. The immune response to staphylococcal nuclease: a probe of c e l l u l a r and humoral antigen s p e c i f i c receptors . Cold Spring Harbor Symp. Quant. B i o l . 41:295. 217. P i se tsky , D .H . , Berzofsky, J . A . , Sachs, D.H. 1978. Role of non-H-2 l inked genes in the control of the ant i-nuclease antibody response. J . Exp. Med. 147:396. 218. Berzofsky, J . A . , Schechter, A . N . , Shearer, G.M. , Sachs, D.H. 1977. H-2 l inked control of the r e l a t i v e proport ions of ant ibodies produced to d i f f e r e n t determinants of nat ive nuclease. J . Exp. Med. 145:123. 219. Fathman, C . G . , P i se tsky , D.S . , Sachs, D.H. 1977. Genetic l inkage and s t r a i n d i s t r i b u t i o n of ant i-nuclease i d i o t ypes . J . Exp. Med. 145:569. 220. P i se t sky , D.S. and Sachs, D.H. 1978. Mapping of genes f o r ant ibodies to d i f f e r e n t ant igen ic regions of nuclease. J . Exp. Med. 147:1517. 193 221. M i l l e r , G .G . , Nadler , P.I., Asano, Y . , Hodes, R.J. , Sachs, D.Y., 1981. Induction of id iotype bearing nuc lease-spec i f i c helper T c e l l s by an in v ivo treatment with an t i - i d i o t ype . J . Exp. Med. 154:24. 222. Nadler , P.I., M i l l e r , G .G . , Sachs, D.Y. , Hodes, R.J. 1982. The expression and funct iona l involvement of nuc lease-spec i f i c id io type on nuclease-primed helper T c e l l s . Eur. J . Immunol. 12:113. 223. Sachs, D.Y. , E l-Gami l , M. , M i l l e r , G. 1981. E f f ec t s of in v ivo admin is t ra t ion of a n t i - i d i o t y p i c an t ibod ies . Eur. J . Immunol. 11:509. 224. M i l l e r , G .G . , Nadler , P.I., Hodes, R.J., Sachs, D.Y. 1982. Mod i f i ca t ion of T c e l l ant i-nuclease id iotype expression by an in  v ivo admin is t ra t ion of an t i - id io t ype . J . Exp. Med. 155:190. 225. Makela, 0 . , Kaart inen, M., Pelkonen, J . L . T . , Ka r j a l a inen , K. 1978. Inheritance of antibody s p e c i f i c i t y V. Anti-2-phenyloxazolone in the mouse. J . Exp. Med. 148:1644. 226. Kaar t inen, M., G r i f f i t h s , G .M. , Hamlyn, P .H. , Markham, A . F . , Kar ja l a inen , K., Pelkonen, J . L . T . , Makela, 0 . , M i l s t e i n , C. 1983. Anti-oxazolone hybridomas and the s t ruc ture of the oxazolone i d io t ype . J . Immunol. 130: 937. 227. Z e l d i s , J . B . , R i b l e t , R., Konigsberg, W.H., R ichards , F . F . , Rosenste in , R.W. 1979. Expression of the prote in 315 and 460 i d i o t y p i c determinants in the mouse anti-DNP an t ibod ies . Mol . Immunol. 16:657. 228. Dz ie rzak , E .A . , Janeway, C A . ( J r . ) , Rosenste in, R.W., G o t t l i e b , P.D. 1980. Mapping of genes f o r Id460 expression to the va r i ab le region of immunoglobulin heavy-chain locus and to the va r i ab le region at immunoglobulin K-l ight chain l ocus . J . Exp. Med. 152:720. 229. Dz ie rzak , A . , Rosenste in , R.W., Janeway, C A . ( J r . ) . 1981. Expression of an id io type (Id-460) during an in v ivo ant i-d in i t ropheny l antibody responses. II. Trans ient Id io typ i c dominance. J . Exp. Med. 154:1433. 230. Bona, C , Hooghe, R., Cazenave, P .A. , Leguern, C , Pau l , W.E. 1979. Immunity to anti-MOPC 460 id io type ant ibodies increases the leve l of an t i - t r i n i t ropheny l ant ibodies bearing 460 id io t ypes . J . Exp. Med. 149-.815. 231. Sega l , D.M., Padlan, E .A. , Cohen, G .H . , Rudikof f , S . , Po t te r , M., Dav ies , D.R. 1974. The three-dimensional s t ructure of a phosphorylchol ine-binding mouse immunoglobulin Fab and the nature of the antigen binding s i t e . Proc. Na t l . Acad. S c i . 71:4298. 194 232. Feldmann, R.J . , Po t te r , M., Glaudemans, C .P . J . 1981. A hypothet ical s p a c e - f i l l i n g model of the V-regions of the galactan-binding myeloma immunoglobulin J539. Mol . Immunol. 18:683. 233. Rudikof f , S . , Pawl i ta , M., Pumphrey, J . , Mushinski , E . , Po t te r , M. 1983. Galactan-binding an t ibod ies : d i v e r s i t y and st ructure of i d i o t ypes . J . Exp. Med. 158:1385. 234. Mar ion, T . N . , Lawton ( I I I ) , A.R. Kearney, J . F . , B r i l e s , D.E. 1982. Anti-DNA autoantibodies in (NZB X NZW)F1 mice are c l o n a l l y heterogeneous but share a common id io t ype . J . Immunol. 128:668. 235. Rauch, J . , Murphy, E . , Roths, J . B . , S t o l l a r , B.D., Schwartz, R.S. 1982. A high frequency i d i o t y p i c marker of anti-DNA auto-antibodies in MRL-Ipr/Ipr mice. J . Immunol. 129:236. 236. T ron , F. , Le Guern, C , Cazenave, P-A., Bach, J-F. 1982. In t ras t ra in recurrent id iotypes among anti-DNA ant ibodies of (NZB X NZW)F. hybrid mice. Eur. J . Immunol. J ^ : 761 . 237. Benacerraf , B. and Germain, R.N. 1978. Immune response genes of the major h is tocompatab i l i t y comples. Immunol. Rev. 38:70. 238. P incus , S .H . , Sachs, D .H . , D i c k l e r , H.B. 1978. Production of an t i se ra s p e c i f i c f o r id io type (s ) of murine an t i - (T,G)-A-L an t ibod ies . J . Immunol. 121:1422. 239. J u , S-T., P incus , S .H . , Stocks ( J r . ) , C . J . , P i e r r e s , M. , Dor f , M.E. 1982. Id io typ ic ana lys is of hybridoma ant ibodies to branched synthet i c polymer (Try , glu) A l a , Lys : i d i o t y p i c r e l a t i onsh ip with ant ibodies to l i n e a r random polymer Glu , A la , Tyr ). J . Immunol. 128:545. 240. P incus , S .H . , Stocks ( J r . ) , C . J . , Ewing, L.P. 1982. Monoclonal anti(TG)-A-L an t ibod ies : cha rac te r i za t ion of f i ne s p e c i f i c i t y and id io type express ion. Mol . 1mm. 19:1551. 241. B luestone, J . A . , Eps te in , S . L . , Ozato, K., Sharrow, S .O . , Sachs, D.H. 1981. Expression of anti-H-2K id iotypes on ant ibodies induced by an t i- id io type or H-2K ant igen. J . Exp. Med. 154:1305. 242. Auchincloss ( J r . ) , H . , B luestone, J . A . , Sachs, D.H. 1983. In v ivo an t i - id io type treatment induces id iotype s p e c i f i c helper T c e l l s . J . Exp. Med. 157:1273. 243. B luestone, J . A . , Metzger, J- J . , Knode, M . C , Ozato, K., Sachs, D.H. 1982. Contr ibut ion of i so l a t ed heavy and l i g h t chains to id io type express ion . Mol. Imm. 19:515. 244. Bender, T . P . , Schroer, J . , C l a f l i n , J . L . 1983. Idiotypes on monoclonal ant ibodies to bovine i n s u l i n . I. Two pub l i c id iotypes on ant ibovine i n s u l i n hybridomas def ine i d i o t y p i c a l l y d i s t i n c t f am i l i e s of hybridomas. J . Immunol. 131:2882. 195 245. Mortenson, L . E . , Va l en t ine , R .C . , Carnahan, J . E . 1962. An e lec t ron t ransport f a c to r from C los t r id ium pasteurianum. Biochem. Biophys. Res. Comm. _7:448. 246. Mortenson, L .E . and Nakos, G. 1973. in Iron Su l fu r P ro te ins . 1: 37, Academic P ress , New York. 247. Yasunobu, K.T. and Tanaka, M. 1973. in Iron Su l fu r P ro te ins . II: 29 , Academic P ress , New York. 248. Admam, E .T . , S i eker , L . C , Jensen, L.H. 1973. The Structure of a bac te r i a l f e r r edox in . J . B i o l . Chem. 248:3987. 249. K e l l y , B. and Levy, J . G . 1978. Immunobiologic proper t ies of the major ant igen ic determinants of the fer redoxin molecule. Adv. Exp. Med. B i o l . 98:181. 250. S i ko ra , L . , Weaver, M., Levy, J . G . 1982. The use of unideterminant fragments of fe r redoxin in the genet ic mapping of determinant s p e c i f i c i t y of the immune response. Mol . Immunol. 19:693. 251. S i ko r a , L .K. J , and Levy, J . G . 1980. Genetic control of the immune response to f e r r edox in : l inkage and mapping of T c e l l p r o l i f e r a t i o n and antibody production genes to the MHC of mice. J . Immunol. 124:2615. 252. Weaver, M., S i ko ra , L . , Levy, J . G . 1982. The immune response to f e r r edox in : cha rac te r i za t ion of a major id iotype in serum using monoclonal antibody der ived by c e l l f u s i o n . Mol . Immunol. 19:105. 253. Weaver, M., S ingha i , R., S i ko ra , L . , Levy, J . G . 1983. I d en t i f i c a t i on of an i d i o t y p i c marker of a major regulatory T c e l l of the immune response in BIO.BR mice to f e r redox in . The r e l a t i onsh ip of i d i o t y p i c regu la t ion to conventional hapten c a r r i e r e f f e c t s . J . Exp. Med. 157:285. 254. S i ngha i , R., Weaver, M., S i ko ra , L . , Levy, J . G . 1984. Evidence f o r the presence of id io type bearing regulatory T c e l l s in which id io type expression i s not l inked to e i t he r Igh a l l e l e s or the MHC. (Manuscript submitted f o r pub l i c a t i on ) . 255. S i ngha i , R., Weaver, M. , Levy, J . G . 1984. Regulation of the immune response to fe r redox in by a T c e l l i d i o t y p i c network J . C e l l . Biochem. Supp. 6A. 256. K e l l y , B.S., Levy, J . G . , S i ko ra , L. 1979. The use of the enzyme l inked immunosorbent assay (ELISA) f o r the detect ion and quant i ta t ion of s p e c i f i c antibody from c e l l cu l t u r e s . Immunol. 37:45. 257. Bar ton, R. 1979. Biochemical Research Techniques, p. 284. U.B.C. Department of B iochemistry . 196 258. Cuatrecases, P. and Anf inson , C. 1971. A f f i n i t y Chromatography in Methods in Enzymology. W.B. Jakoby, Ed i t o r . 22:345, Academic P ress , New York. 259. Kohler , G . , Howe, S . C . , M i l s t e i n , C. 1976. Fusion between immunoglobulin secre t ing and non-secreting myeloma c e l l l i n e s . Eur. J . Immunnol. 6^:292. 260. L i t t l e f i e l d , J.W. 1964. Se lec t ion of hybrids from matings of f i b r o b l a s t s vn_ v i t r o and t he i r presumed recombinants. Science 141:709. 261. Candido, E.P.M. personal communication. 1982. 262. Pearson, T . personal communication. 1983. 263. Sammons, D.W., Adams, L .D . , Nishizawa, E.F. 1981. U l t r a sens i t i v e s i l ver-based co lo r s ta in ing of polypept ides in polyacrylamide ge l s . E lec t rophores is 2^:135. 264. Wray, W., Bou l i kas , T . , Wary, V . P . , and Hancock, R. 1981. S i l v e r s t a in ing of prote ins in polyacrylamide g e l s . Ana l . B ioc . 118:197. 265. Pearson, T.W. and Anderson, N.L. 1982. The use of high reso lu t ion two-dimensional gel e lec t rophores is f o r ana lys i s of monoclonal ant ibodies and t h e i r s p e c i f i c ant igens, in Methods in Enzymology. J . J . Langone and H. Van Vunakis, Editors"! 92:196, Academic P ress , New York. 266. Laemmli, U.K. 1970. Cleavage of s t ruc tura l prote ins during the assembly of the head of bacteriophage T4. Nature 227: 680. 267. 0 ' F a r r e l l , P.H. 1975. High reso lu t ion two-dimensional e lec t rophores i s of p ro te ins . J . B i o l . Chem. 250: 4007. 268. Ingman-Baker, J . and Candido, E.P.M. 1979. Two-dimensional e lec t rophores i s in Biochemical Research Techniques, U.B.C. Dept. of B iochemistry , P. 209. 269. Anderson, N.G. and Anderson, N.L. 1978. Two-dimensional ana lys is of serum and t i s sue p ro te ins : mu l t ip le i s o e l e c t r i c f ocuss ing . Ana l . Biochem. 85:331. 270. K l e i n , J . 1975. Biology of the Mouse H i s tocompat ib i l i t y Complex. Spr inger-Ver lag , New York. 271. G ibson, D.M. and MacLean, S.J . 1979. E f2 : A new Ly-3-linked l i g h t chain marker expressed in normal mouse serum immunoglobulin. J . Exp. Med. 149:1477. 197 272. Sherr , D .H . , Ju S-T., Weinberger, J . Z . , Benacerraf , B., Dor f , M.E. 1981. hapten s p e c i f i c T c e l l responses to 4-hydroxy-3-nitrophenyl a c e t y l . VII. Idiotype s p e c i f i c suppression of plaque forming c e l l responses. J . Exp. Med. 153:640. 273. Kuettner, M.G. , Wang A - L . , N i sono f f , A. 1972. Quant i ta t ive inves t iga t ions of i d i o t y p i c an t ibod ies . III. Id io typ ic s p e c i f i c i t i e s as a potent ia l genet ic marker f o r va r i ab le regions of mouse immunoglobulin polypept ide cha ins . J . Exp. Med. 135:579. 274. C l a f l i n , J . L . and Cubber ly , M. 1978. Clonal nature of the immune response to phosphory lchol ine. VI. Molecular uni formity of a s ing le id io type among BALB/c mice. J . Immunol. 121:1410. 275. Cerny, J . , W a l l i c h , R., Hammerling, G. 1982. Ana lys is of T15 id io types by monoclonal an t ibod ies : v a r i a b i l i t y of i d i o t y p i c expression on phosphory lcho l ine-spec i f i c lymphocytes from ind i v idua l inbred mice. J . Immunol. 128:1885. 276. Lamoyi, E . , E s tess , P., Capra, J . D . , N i sonof f , A. 1980. Presence of h igh ly conserved i d i o t y p i c determinants in a fami ly of ant ibodies that cons t i tu te an i n t r a s t r a i n c ross-react i ve i d io t ype . J . Exp. Med. 152:703. 277. Herzenberg, L.A. and Herzenberg, L.A. 1978. Mouse immunoglobulin a l l o t y p e s : desc r ip t i on and spec ia l methodology. In Handbook of  Experimental Immunology. D.M. Weir, E d i t o r , P. 12 .1 , Blackwell S c i e n t i f i c Pub l i c a t i ons , Oxford. 278. Morse, H.C. 1978. Or ig ins of Inbred Mice. Academic P ress , Inc . , New York. 279. B luestone, J . A . , Auch inc loss , H. ( J r . ) , Sachs, D .H . , F i b i , M., Hammerling, G.J . 1983. Ant i- id io types against anti-H-2 an t ibod ies . VI. Detect ion of shared id iotypes among monoclonal anti-H-2 an t ibod ies . Eur. J . Immunol. 13:489. 280. K a r o l , R., R e i c h l i n , M. , Noble, R.W. 1978. Id io typ ic c ros s- reac t i v i t y between ant ibodies of d i f f e r e n t s p e c i f i c i t i e s . J . Exp. Med. 148:1488. 281. Conger, J . D . , Lewis, G .K . , Goodman, J.W. 1981 Idiotype p r o f i l e of an immune response. Contrasts in i d i o t y p i c dominance between primary and secondary responses and between IgM and IgG plaque-forming c e l l s . J . Exp. Med. 153:1173. 282. Reth, M., Hammerling, G . J . , Rajewsky, K. 1978. Ana lys is of the reper to i re of anti-NP ant ibodies in C57BL/6 mice by c e l l f u s i o n . 1. Charac te r iza t ion of antibody f ami l i e s in the primary and hyperimmune response. Eur. J . Immunol. 8^:393. 283. Mor r i son , M. 1980. Lactoperoxidase - cata lyzed iod ina t ion as a tool f o r i nves t iga t ion of p ro te ins . In Methods in Enzymology. 70:214. Academic press Inc . , New York. 198 284. Glynn, P., G i l b e r t , H . , Newcombe, J . , Cuzner, N.L. 1982. Rapid ana lys is of immunoglobulin i s o e l e c t r i c focuss ing patterns with c e l l u l o s e n i t r a t e sheets and immunoperoxidase s t a i n i n g . J . Immunol. Met. 51:251. 285. Towbin, H . , S t aehe l i n , T . , Gordon, J . 1979. E lec t rophore t i c t r ans fe r of prote ins from polyacrylamide gels to n i t r o c e l l u l o s e sheets: procedures and some app l i c a t i ons . Proc. N a t l . Acad. S c i . 76:4350. 286. G ibson, D. 1976. Genetic polymorphism of mouse immunoglobulin l i g h t chains revealed by i s o e l e c t r i c focuss ing . J . Exp. Med. 144:298. 287. Ta ren t ino , A . L . , Plummer, T .H . ( J r . ) , Maley, F. 1974. The re lease of i n t a c t o l igosacchar ides from s p e c i f i c g lyoprote ins by endo-B-N-acetylglucosaminidase H. J . B i o l . Chem. 249:818. 288. N i sono f f , A . , Hopper, J . E . , Sp r ing , S.B. 1975. The Antibody Molecule , p. 194. Academic Press , New York. 289. A l l i s o n , J . P . , Bradley W.M., B loch , D. 1982. Tumor - s p e c i f i c antigen of murine T - lymphoma def ined with monoclonal ant ibody. I. Immunol. 129: 2293. 290. Mever S . C . , Acuto 0 . , Hussey, R., Hodgdon J . C , F i t zge ra ld K .A. , Schlossman S . F . , Reinherz E.L. 1983. Evidence f o r the T3 assoc iated 90K heterodimer as the T-ce l l antigen receptor . Nature 303:808. 291. Oudin, J . and Cazenave, P.A. 1971. S imi l a r i d i o t y p i c s p e c i f i c i t i e s in immunoglobulin f r a c t i ons with d i f f e r e n t antibody funct ions or even without detectable antibody func t i on . Proc. Na t l . Acad. S c i . 68:2616. 292. Bhattacharjee, A .K , and Glaudemans, C .P . J . 1978. Dual binding s p e c i f i c i t i e s in M0PC 384 and 870 murine myeloma immunoglobulins. J . Immunol. 120:411. 293. S t ree fkerk , D .G . , Manjula , B .N. , Glaudemans, C.P . J . 1979. An in te rp re ta t i on of the apparent dual s p e c i f i c i t y of some murine myeloma immunogloblins with i n s u l i n binding a c t i v i t y . J . Immunol. 122:537. 294. R ichards , F . F . , Konigsberg, W.H., Rosenste in, R.W., Varga, J .M . 1975. On the s p e c i f i c i t y of an t ibod ies . Science 187:130. 295. Varga, J .M . Konigsberg, W.H., R ichards , F . F . , 1973. Ant ibodies with mul t ip le binding func t ions . Induction of s ing le immunoglobulin species by s t r u c t u r a l l y d i s s i m i l a r haptens. Proc. Na t l . Acad. S c i . 70:3269. 296. Dav ies , D.R., Padlan, EA . , Sega l , D.M. 1975. Immunoglobulin s t ructures at high r e s o l u t i o n . Contemp. Top. Mol . Immunol. 4:127. 199 297. Hoffman, G.W. 1980. On network theory and H-2 r e s t r i c t i o n . Contemp. Top. Immunobiol. 11:185. 298. B i n i on , S.B. and Rodkey, L.S. 1983. Destruct ion of antibody id io types with u l t ra low concentrat ions of reducing agents. Mol. Immunol. 20:475. 299. Dickerman, J . , C l ev inger , B., Fr iedenson, B. 1981. Loss of an ind i v idua l id io type on chemical mod i f i c a t i on . A strategy fo r ass igning i d i o t y p i c determinants. J . Exp. Med. 153:1275. 300. Je rne , N.K., Roland, J . , Cazenave, P-A. 1982. Recurrent id iotypes and in terna l images. EMBO J . 1^:243. 301. Capra, J . D . and Fougereau, M. 1983. One from column A, one from column B. Immunol. Today 4^:177. 302. Johnson, N . , S lankard, J . , Pau l , L . , Hood, L. 1982. The complete V domain amino ac id sequences of two myeloma inu l in-b ind ing p ro te ins . J . Immunol. 128:302. 303. Roux, K.H. and Metzger, D.W. 1982. Immunoelectron microscopic l o c a l i z a t i o n of id iotypes and a l lo types on immunoglobulin molecules. J . Immunol. 129:2548. 304. Karush, F. 1962. Immunologic s p e c i f i c i t y and molecular s t ruc tu re . Adv. Immunol . 2_:1. 305. R e i c h l i n , M. 1975. Amino ac id subs t i tu t i on and the an t i gen i c i t y of g lobu la r p ro te ins . Adv. Immunol. 20:71. 306. Simon, E.J. 1982. p . l . In Endorphins. J . B . Mal ich and R.M.S. B e l l , E d i t o r s . Marcel Dekker Inc . , New York. 307. Berzofsky, J . and Schechter, A .N . 1981. The concepts of cross-r e a c t i v i t y and s p e c i f i c i t y in immunology. Mol . Immunol. 18:751. 308. Urba in , J . and Wuilmart, C. 1983. Some thoughts on i d i o t y p i c networks and immunoregulation. Immunol. Today 3_:88. 309. Tesch, H . , Takemori, T . , Rajewsky, K. 1983. The immune response against an t i- id io tope an t ibod ies . II. The induct ion of ant ibodies bearing the target id iotope (Ab 3B) depends on the frequency of the corresponding B c e l l s . Eur. J . Immunol. 13:726. 310. F e r r i s , S .D . , Sage, R.D., Wi lson, A .C . 1982. Evidence from mt DNA sequences that common laboratory s t ra ins of inbred mice are descended from a s ing le female. Nature 295:163. 311. F e s t i ng , M.F.W. 1982. Do a l l laboratory mice trace back to a s ing le female? Nature 295:94. 200 312. Urba in , J . , Wuilmart, C , Cazenave, P-A. 1981. Id io typ ic regulat ion in immune networks. Contemp. Top. Mol . Immunol. 8;113. 313. Koh ler , H. 1980. Id io typ ic network i n t e r a c t i ons . Immunol. Today 1:18. 314. Po l l ok , B.A. and Kearney, J . F . 1984. I den t i f i c a t i on and cha rac te r i za t ion of an apparent germline set of au to-ant i- id io typ i c regulatory B lymphocytes. J . Immunol. 132:114. 315. Dayhoff , M.O. 1976. At las of prote in sequence and s t ruc tu re . V o l . 5, supplement 2. p.34. National Biomedical Research Foundation, Washington, D.C. 316. Schor, S. 1968. Fundamentals of B i o s t a t i s t i e s , p. 176. G.P. Putnam's Sons" New York. 317. Spe ige l , M.R. 1961. Theory and Problems of S t a t i s t i c s , p. 203. McGraw-Hill Book Company, New York. 318. Brown, A.R. and N i sono f f , A. 1981. An i n t r a s t r a i n c ross-reac t i ve id io type assoc iated with anti-p-azophenyl arsonate ant ibodies of BALB/c mice J . Immunol. 126;1263. 319. Mclntyre B.W. and A l l i s o n J . P . 1983. The Mouse T c e l l receptor : s t ruc tu ra l heterogeneity of molecules of normal T c e l l s def ined by xenoantiserum. Ce l l 34:739. 320. Kappler J . , Kubo R., Haskins K., White J . , Marrack P. 1983. The mouse T c e l l receptor : comparison of MHC - r e s t r i c t e d receptors on two T c e l l hybridomas. Ce l l 34:727. 321. Hedrick S .M. , Cohen D. I . , Nielsen E .A. , Davis M. 1984. I so la t ion of cDNA clones encoding T c e l l - s p e c i f i c membrance - assoc iated p ro te ins . Nature 308:149. 322. Hedrick S .M. , Nie lsen E .A. , Kavaler J . , Cohen D. I . , David M.M. 1984. Sequence re l a t i onsh ips between putat ive T-ce l l receptor polypeptides and immunologlobulins. Nature 308:153. 323. Yanagi , Y . , Yoshikai Y . , Leggett K., Clark S .P . , Aleksander I., Mak T.W. 1984. A human T eel 1-spec i f ic cDNA clone encodes a prote in having extensive homology to immunoglobulin cha ins . Nature. 308:145. 201 Appendix 1 I) S t a t i s t i c a l Methods E s s e n t i a l l y , qui te simple s t a t i s t i c a l tes ts were used to compare populat ions of id io type producing mice in th i s t h e s i s . Since the d i s t r i b u t i o n s of id iotype producing mice are not normal with respect to percent i n h i b i t i o n , the students t tes t must not be used (316). Consequently, a threshold leve l of i n h i b i t i o n was a r b i t r a r i l y des ignated, and responder frequency was ca l cu l a t ed . The X tes t was used to compare these frequencies (317). II) Threshold of Response KLH immunized mice, represent ing 7 s t r a i n s , were tested with the d i f f e r e n t a n t i - i d i o t y p i c reagents at the 2 ° day 7 per iod in t h e i r response. Since the i n h i b i t i o n s were qui te low, and the mice were responding to a fer redoxin unrelated p r o t e i n , these resu l t s were designated as background. The values f o r a l l mice with each ant i- id io type were averaged and the threshold point was set as T = X KLH + 2 ^KLH^ where: T = threshold *KLH = ^ E A N s „ . u = Standard dev iat ion KLH T AKR 19.3% T, RF 19.7% 17.1% T, BIO.BR 18.8% 202 Since these values were so s im i l a r they were assumed to represent the background leve l of i n h i b i t i o n . Thus, values of i n h i b i t i o n higher than threshold were regarded as pos i t i v e f o r a s p e c i f i c i n h i b i t i o n . I l l ) S t a t i s t i c a l Comparisons 2 X contingency tables were prepared (per. comm. Dr. Siu Dept. of Community Health Sc iences , Un ive rs i t y of Calgary) to t es t s t r a in 2 s p e c i f i c i t i e s . These X values were ca l cu la ted by es t ab l i sh ing the to ta l s t r a i n frequency over a l l sample t imes, and standard iz ing them with the 2 ° day 15 populat ion f o r that s t r a i n . For example: Observed frequency of AKR i n h i b i t i o n was 73.5% over a l l t imes. Standardized to the 2° day 15 populat ion (11 mice ) , 8.09 mice were i n h i b i t e d , 2.91 mice were not i n h i b i t e d . This was compared to 2 of 31 2 p o s i t i v e l y i nh ib i t ed anti-KLH mice. Thus, X = 20.0 with p_.005 where: H o : F A K R = F KLH. By th i s method the s i gn i f i c ance of i n t e r s t r a i n c ros s- reac t i v i t y could be e s t ab l i shed . The k ine t i c s of the i d i o t y p i c response could be analyzed s t a t i s t i c a l l y to determine i f the frequency of i n h i b i t i o n var ied over t ime. Contingency tables were prepared, f o r example: AKR Marked Indiv idual Mice 1° 2° 3° Total Pos i t i ve 2 11 9 22 Negative 11 3 5 19 Total 13 14 14 41 df = 2 X 2 = 11.8 p5.005 Ho: f . o = f-o = f o 0 203 Appendix 2. Summary of Id io typ ic Systems Antigen Idiotype igh i g i Referenc ARS CRI c a k 318 ARS CRI e k 6, 21 PC T15 a k 51, 60 PC C3 b ,e , j k 55 NP NP-a a A 90 NP NP-b b X 88 NP Bl-8 b A 97 Dextran J558 a A 113 Dextran M104E a A 114 GAC A5A e k 120 GAC S117 a k 126 GAC VkGAC various k 127 LPS IdX a l l - 146 GAT CGAT various k 153 GAT pGAT var ious k 154 GAT srGAT-1 a ,c ,e - 162 GAT Gte b - 163 HEL IdX var ious k 206 Nase 5 id io types a k 220 phOx Ox-l var ious k 206 DNP M-460 a k 227, 2 BI not named a ,b ,c - 244 204 Appendix 3 The Structure of Ferredoxin TRYPSIN A L A - T Y R - L Y 5 - I L £ - A L A - A S P - S E R - C Y 5 - V A L - S E R - C Y S - G L Y - A L A - C Y S - A L A - S E R - A L A - C r S - P R O - V A L - A S N - A L A - I L E t f THE N - DETERMINANT C L N I 6 L Y I A S P I S E R I I L E I P H E I THE C- DETERMINANT V * L i * I G L U - G L N - V A L - P R O - A L A - G L Y - V A L - P R O - C Y S - V A L - A S N - A L A - C Y S - A S N - G L Y - C Y S - A S P - I L E - C Y S - T H R - A S P - A L A - A S P J y CARBOXYPEPTIDASE A R e p r o d u c e d w i t h p e r m i s s i o n f r o m 147 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0096398/manifest

Comment

Related Items