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Characterization of a T lymphocyte-derived, antigen-binding molecule with suppressive activity Chu, Nelson Randall 1989

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CHARACTERIZATION  OF A T LYMPHOCYTE-DERIVED  ANTIGEN-BINDING MOLECULE WITH SUPPRESSIVE ACTIVITY By NELSON RANDALL CHU B.Sc, M.Sc,  Queen's U n i v e r s i t y , 1980 Queen's U n i v e r s i t y , 1983  A THESIS SUBMITTED IN PARTIAL FULFILLMENT THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Microbiology  We accept t h i s t h e s i s as conforming t o the r e q u i r e d  standard  THE UNIVERSITY OF BRITISH COLUMBIA NOVEMBER 198 9 ® Nelson  R a n d a l l Chu, 1989  In presenting this thesis in partial fulfilment  of the  requirements for an advanced  degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or  by his  or  her  representatives.  It  is  understood  that  copying  or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department of The University of British Columbia Vancouver, Canada Date  DE-6 (2/88)  ABSTRACT  Regulation action these a  of suppressor T c e l l s cells  soluble  such  to  analog of the c e l l  the small,  has  system  that  Ag.  One i n t r i g u i n g  protein,  t o study TsF.  Previously,  s h a r e s many antigen  A Fd-specific  a soluble  possessed both Ag-binding a c t i v i t y  linked and on  supernatant  mediator,  FdllF  t h e a n t i - F d Ab r e s p o n s e  material  immunosorbent  from  assay.  that  FdllF  preferential spleen  cell  binding culture  ( F d ) , was u s e d  experiments  was  and t h e a b i l i t y The  Fdll.  to  TsF-specific  f o rboth the recovery of  of FdllF  by t h e enzymebiological,  was d o n e w i t h  of FdllF.  and l i q u i d - p h a s e  Fd, and  determinant  was d e v i s e d w h i c h  suggested  network  known as F d l l F ,  that  emphasis  I t was  found  demonstrated  o f t h e Ag.  showed t h a t  s u p p r e s s e d Ab p r o d u c t i o n i n a c o n c e n t r a t i o n - d e p e n d e n t Additional  as a  nonresponsive t o  'F u r t h e r immunochemical,  to the carrier assay  response  suppressor c e l l  i n mice.  the Ag-binding properties  bound t o s o l i d -  The immune  SN a n d i t s d e t e c t i o n  biochemical characterization describing  an Ag-  (SN) o f t h e T s h y b r i d o m a ,  a n t i b o d y , B16G, was u s e d  FdllF-enriched  (TsF):  of i t s properties,  are genetically  i n the culture  monoclonal  aspect of  (Ag) a n d s u p p r e s s  ferredoxin  found  suppress  by t h e  suppressor factor  o f TsF t o Ts a r e unknown.  bacterial  i n part,  The e x a c t m o l e c u l a r n a t u r e o f T s F a n d  been d e s c r i b e d i n mice t h a t  this  i s mediated,  of T cell  to bind free  immune r e s p o n s e .  relationship  model  (Ts).  i s the description  as t h e a b i l i t y  specific the  o f t h e immune r e s p o n s e  A  FdllF  manner.  the suppressive effect  was  Ag-specific.  The i d e n t i f i c a t i o n o f t h e A g - b i n d i n g m o l e c u l e was  a t t e m p t e d by t h e f r a c t i o n a t i o n o f F d l l F - e n r i c h e d m a t e r i a l u s i n g high performance  gel filtration  under non-reducing c o n d i t i o n s ) . p o l y p e p t i d e o f about identified  o r p r e p a r a t i v e SDS-PAGE ( r u n U s i n g SDS-PAGE, a u n i q u e ,  30k r e l a t i v e m o l e c u l a r mass  as t h e A g - b i n d i n g m o i e t y o f F d l l F .  single  ( M ) was r  The p o s s i b l e  r e l a t i o n s h i p of t h i s moiety t o other i d e n t i f i e d m a t e r i a l s i s discussed.  iv T A B L E OF  CONTENTS  ABSTRACT  i  T A B L E OF CONTENTS  i i v  L I S T OF T A B L E S  v i i  L I S T OF F I G U R E S  v i i i  ACKNOWLEDGEMENTS  x  ABBREVIATIONS  x i  INTRODUCTION Suppression Properties Summary  a n d t h e Immune R e s p o n s e of T cell  1  Suppressor Factors  T h e Immune R e s p o n s e t o F e r r e d o x i n Mechanisms o f Immunoregulation  as a Model  2 11 f o rt h e Study o f 13  The c h o i c e o f F d a s a m o d e l a n t i g e n E p i t o p e m a p p i n g a n d H-2 l i n k e d r e s p o n s i v e n e s s Summary Idiotype-anti-idiotype interactions Summary B16G a n d t h e D e v e l o p m e n t Summary Purpose  Animals  Lines  Conventional Antigens  . .  14 15 18 19 22 23 27 28  METHODS  and S o l u t i o n s  Experimental Cell  hybridoma  of the Investigation  M A T E R I A L S AND Buffers  of the Fdll  t o Fd  29 29 29  and Monoclonal A n t i b o d i e s  30 32  V  Enzyme-linked Immunization Enrichment In v i t r o  Immunosorbent  33  of Animals  of FdllF  Measurement  Suppression  Assay  35  using  Immunoaffinity  of FdllF  Suppressive  o f t h e Serum A n t i b o d y  Sodium Dodecyl  Sulphate  High Performance  Activity  Response u s i n g  Polyacrylamide  .  . . . .  FdllF  35 37  . .  Gel Electrophoresis  39 40  Gel Filtration  P r e p a r a t i v e Sodium Dodecyl Electrophoresis Two-dimensional  Chromatography  41  Sulphate  Polyacrylamide  Gel 42  Gel Electrophoresis  ( I E F x SDS-PAGE)  . . .  43  RESULTS Recovery  of FdllF  Immunochemical  from C u l t u r e  Supernatants  45  Properties of FdllF  45  Biological Properties of FdllF Development o f an a n t i b o d y c u l t u r e assay f o r t h e measurement of suppression C o r r e l a t i o n between b i o a c t i v i t y and a n t i g e n - b i n d i n g activity of F d l l F Suppression of anti-Fd antibody levels i n vivo . . . . Biochemical Analysis of FdllF Introduction Analysis of F d l l F using high performance g e l f i l t r a t i o n : c o r r e l a t i o n between a n t i g e n - b i n d i n g and suppressive activity I d e n t i f i c a t i o n o f t h e a n t i g e n - b i n d i n g m o l e c u l e u s i n g SDSPAGE  57 57 61 69 74 74  75 81  DISCUSSION B16G  as a Probe f o r T Suppressor F a c t o r  Immunochemical Biologic  Properties of FdllF  Properties of FdllF  91 .  93 96  vi Biochemical Analysis of F d l l F  101  Summary  106  LITERATURE CITED  108  APPENDIX  THE COMPOSITIONS OF BUFFERS AND SOLUTIONS  . . .  117  vii L I S T OF  I.  The b i n d i n g  of FdllF  TABLES  t o Fd  52  II. Comparison of t h e suppressive c a p a b i l i t y i n two a s s a y s o f c e l l u l a r f u n c t i o n . III. A b i l i t y of various fractions eluting c o l u m n t o i n h i b i t t h e i n v i t r o Ab r e s p o n s e cells  of FdllF  and A10F 68  f r o m F P L C S u p e r o s e 12 o f Fd-immune s p l e e n 82  v i i i L I S T OF F I G U R E S 1.  Binding  o f B16G t o F d l l F  2. Binding of FdllF d e t e c t i o n w i t h B16G  adsorbed t o polystyrene  t o Fd adsorbed t o polystyrene  3. The r e a c t i v i t y o f B16G a n d t h e i r r e l e v a n t FdllF 4.  The b i n d i n g  5. Antigenic s p e c i f i c mAb 6.  Inhibition  of soluble Fd t o FdllF  mAb,  wells  wells  and i t s 49  CAMAL-1,  i n fluid-phase  a n a l y s i s o f Fd and i t s analogs  46  using  with 50  . .  53  determinant54  of FdllF  binding  by F d and i t s analogs  . .  56  7. D e t e c t i o n o f a n t i - F d A b i n t h e SN o f s p l e e n c e l l c u l t u r e s p r e p a r e d f r o m B10.D2 m i c e t h a t w e r e i m m u n i z e d w i t h C - m o l e c u l e a n d Fd t w i c e 58 8. R e l a t i o n s h i p between Absorbance o f a n t i - F d Ab  a t 4 0 5 nm v s . c o n c e n t r a t i o n 60  9A. S p e c i f i c i t y o f a n t i - F d Ab f r o m Fd-immune cultures f o r Fd analogs ' 9B. mice  cell 63  S p e c i f i c i t y o f serum a n t i - F d Ab f r o m Fd-immune f o rFd analogs  10. The r e l a t i o n s h i p binding activity 11.  spleen  of suppressive  S p e c i f i c i t y of the suppressive  activity of FdllF activity of FdllF  12. Suppressive a c t i v i t y of a f f i n i t y - e n r i c h e d F d l l F BW5147 c o n t r o l m a t e r i a l u s i n g t h e " r e v i s e d " p r o t o c o l 13. X-Y p l o t s h o w i n g a n t i - F d A b l e v e l s a f t e r treatment w i t h F d l l F and Ag 14. X-Y p l o t s h o w i n g a n t i - K L H A b l e v e l s a f t e r treatment w i t h F d l l F and Ag 15.  SDS-PAGE  of eluates  o f animals  B10.D2 63 t o i t s Ag64 . .  67  compared t o . . . 70  before  arid 72  of animals  f r o m B16G a f f i n i t y  before  and 73  columns  . . .  76  16. P r o t e i n p r o f i l e s ( 2 8 0 nm) o f B 1 6 G e l u a t e s d e r i v e d f r o m m e d i u m ( A ) , BW5147 ( B ) , a n d F d l l F (C) SN f r a c t i o n a t e d o n a S u p e r o s e 12 c o l u m n u s i n g F P L C 17.  Ag-binding  a c t i v i t y o f FPLC f r a c t i o n s  obtained  from  78  FdllF  IX  (•),  BW5147  (a),  a n d medium  (O) e l u t i o n p r o f i l e s  18. SDS-PAGE a n a l y s i s o f FPLC t o 19 i n c l u s i v e  f r a c t i o n s (shown  o f F i g . 16  79  i n F i g . 17) 16 80  19. A g - b i n d i n g a c t i v i t y o f DC10 a n d F d l l F m a t e r i a l e l u t e d f r o m s l i c e s o f a 12% p r e p a r a t i v e SDS-PAGE g e l r u n u n d e r n o n - r e d u c i n g conditions 84 20. PAGE o f m a t e r i a l f r o m s e l e c t e d f r a c t i o n s o f F i g . 19 u s i n g 10% SDS g e l r u n u n d e r r e d u c i n g c o n d i t i o n s  a 85  21. T w o - d i m e n s i o n a l g e l e l e c t r o p h o r e s i s o f t h e 80k M o f medium a n d F d l l F  89  r  proteins  X  ACKNOWLEDGEMENT S I w o u l d l i k e t o t h a n k A n t h e a Tench Stammers, f o r b o t h p e r f o r m i n g t h e experiments t h a t complemented those p r e s e n t e d here and h e r h e l p f u l comments a n d s u g g e s t i o n s . S p e c i a l t h a n k s s h o u l d a l s o go t o Agnes Chan a n d J o a n S h e l l a r d f o r t h e i r d i s c u s s i o n s a n d s u g g e s t i o n s a b o u t some o f t h e t e c h n i c a l a s p e c t s o f t h e work. I would a l s o l i k e t o acknowledge J a n North, Mark C u r r y , and Mary MacDonald f o r t h e i r u s e f u l d i s c u s s i o n s about the project. I h a v e b e e n f o r t u n a t e t o r e c e i v e e n o r m o u s s u p p o r t f r o m b o t h my families during t h i s time. The knowledge, e n c o u r a g e m e n t a n d g u i d a n c e r e c e i v e d f r o m my s u p e r v i s o r , J u l i a L e v y w i l l a l w a y s b e remembered. L a s t l y , I t h a n k my w i f e , E s t h e r , f o r h e r u n d e r s t a n d i n g a n d b e l i e f i n me.  xi ABBREVIATIONS  Ag  antigen  Ab  antibody  BSA  bovine  CS  contact  CTL  cytotoxic  DNP  2,4-dinitrophenyl  ELISA  enzyme-linked  FACS  fluorescence-activatedc e l l  FCS  fetal  FPLC  high performance  Fd  ferredoxin  GAT  1-glutamic  HPLC  high performance  i.v.  intravenous *  serum  albumin  sensitivity T  calf  cell  immunosorbent  sorter  serum gel filtration  acid^-l-alanine^-l-tyrosine  .  .  liquid  chromatography  .  IEF  isoelectric  KLH  keyhole  MHC  major h i s t o c o m p a t i b i l i t y  mAb  monoclonal  M  relative  r  assay  focusing  limpet  hemocyanin complex  antibody  molecular  mass  NP  4-hydroxy-3-nitrophenyl  OFd  peroxyformic  PBS  phosphate b u f f e r e d  RaMIg-AP  a conjugate  acetyl  acid-oxidized ferredoxin saline  of rabbit  immunoglobulin  anti-mouse  and a l k a l i n e  phosphatase  xii s.c.  subcutaneous  SDS-PAGE  sodium dodecyl gel  SN  sulphate  polyacrylamide  electrophoresis  supernatant  TCR  T cell  receptor  Th  T helper  Ts  T suppressor  TsF  T cell  Monoclonal  Specificity  cell cell  suppressor  factor  antibody B16G  invariant  CAMAL-1  leukemia-associated  16a  carboxyl-terminal  6-90  amino-terminal  Abbreviations forms  marked w i t h  Journal  were t a k e n  o f Immunology  determinant  factor  of ferredoxin  of ferredoxin  were c o n s i d e r e d  i n the text.  as  standard  The f o r m s o f  from t h e standard  ( J . Immunol.  suppressor  antigen  determinant  an a s t e r i s k  and were n o t d e f i n e d  abbreviations  region of T c e l l  list  p u b l i s h e d by t h e  1 9 8 8 . 140 ( 4 ) . )  1 INTRODUCTION  Suppression  a n d t h e Immune  The m a n n e r i n w h i c h following is  stimulation  known t h a t  (Th)  these  t h e a/R  cells  capability.  from  that  understood.  T helper  cells  The s t u d y o f Th h a s b e e n of i t sclonally  It  aided  distributed  and t h e a b i l i t y  t o clone  T cells  the inability  by t h e  with suppressive to identify  Ts h y b r i d o m a s o r c e l l  lines  may p o s s e s s  £> c h a i n  has l e d t o t h e  an a l t e r n a t e  type of  (Hedrick e t a l . 1985). spite  of this,  received  p r o g r e s s h a s b e e n made  o f t h e Ts s u b s e t .  much a t t e n t i o n ,  suppressor factors  a p p e a r t o s h a r e many and  by both  r e c e p t o r (TCR),  suppressor c e l l s  characterization  cell  (Ts).  i n cloning  Furthermore  rearrangements  has  i s modulated  itself  t h e study o f s u p p r e s s i o n has been hampered  difficulty  suggestion  (Ag) i s n o t w e l l  of the structure T cell  regulates  directly.  contrast,  relative  In  by a n t i g e n  and T suppressor c e l l s  receptor,  TCR  t h e immune s y s t e m  t h e response  by t h e e l u c i d a t i o n  In  Response  the capacity  described  at both  a consensus molecular  One  intriguing  i s the description  (TsF):  soluble  analogs  of i t sproperties,  t o suppress  reached  nature and a c t u a l  such  aspect  o f t h e Ts  as Ag  concerning their  T  that  specificity TsF have  and b i o c h e m i c a l l e v e l s ;  relationship  that  of Ag-specific  t h e immune r e s p o n s e .  the functional  has n o t been  i nthe  precise  t o t h e Ts.  been  however,  2 Properties  The both  of  T cell  e x i s t e n c e of Ag-binding  documented. spleen Ag  induction  and  suppressive  Initially,  adjuvant. of  Ts.  TsF  possible  of the  suppressive  m a t e r i a l t h a t was  response  was  carrier-specific  primed mice would not made i m m u n e t o DNP  conjugated  (BGG).  m i c e w o u l d now  effect  response.  suppressive  donor locus,  and  TsF  known as  Immunochemical Ag  and  by  an  Only the  restricted:  the  (KLH)  the to  an  analysis indicated  antiserum  raised  not  the  to  of the  identity  Major  (2,4-  Furthermore,  carrier,  of  mice  BGG-primed  anti-DNP  Ab was  between the a certain  TsF  KLH-  bovine  from  to  the  from  response  for suppression  the  the  this  hapten  o f K L H - s p e c i f i c TsF  that the  was  in  extract prepared  required, at  (see b e l o w ) ,  showed  extract at  extract obtained  was  the  response  carrier,  a different  i e . genetic  favored  the  a n t i - D N P Ab  activity  carrier  contained  immunogen.  suppression  recipient  I-J  or  hapten-carrier  extract  and  s i n c e an  suppress  gamma g l o b u l i n  genetically  thymus  (1975)  (Ab)  spleen  directed to  p o r t i o n of the  Tada  antibody  demonstrated that the  DNP)  crude  and  the  of  substantially  immunization  given  limpet hemocyanin  The  as  Takemori  and  This  dinitrophenyl,  prepared  immunized w i t h the  (using adjuvant)  keyhole  is  anti-hapten  same t i m e .  case,  activity  method of  example,  when m i c e were  conjugate  was  derived m a t e r i a l capable  from mice t h a t were p r i m e d w i t h  This  For  suppression  Factors  soluble T-cell  extracts obtained  without  that  Suppressor  also  TsF  gene  occur.  c o u l d be  bound  Histocompatibility  by  3 Complex  (MHC)  o f t h e TsF  donor.  L a t e r w o r k showed t h a t A g - s p e c i f i c TsF e x p r e s s e d  determinants  t h a t w e r e e n c o d e d by t h e I - J s u b r e g i o n , a g e n e t i c l o c u s t h a t mapped s u p p o s e d l y t o w i t h i n t h e I r e g i o n o f t h e m u r i n e (Murphy e t a l . 1976; o r H-2  MHC  Tada, T a n i g u c h i , and D a v i d 1976).  The  c o m p l e x , i s l o c a t e d on chromosome 17 o f t h e mouse.  gene p r o d u c t s f r o m t h e I s u b r e g i o n s , I-A and fundamental  The  I-E, p l a y a  r o l e i n g o v e r n i n g immune r e s p o n s i v e n e s s b e c a u s e Ag i s  r e c o g n i z e d by t h e Th o n l y i n a s s o c i a t i o n w i t h t h e s e proteins,  MHC  a l s o known as c l a s s  I I antigens.  cell-surface  Paradoxically,  m o l e c u l a r g e n e t i c a n a l y s i s had d e m o n s t r a t e d  that  I - J e n c o d e d gene  p r o d u c t s c o u l d not have a c t u a l l y o r i g i n a t e d from w i t h i n t h e I region  (Kronenberg  e t a l . 1983).  I - J p r o d u c t ( s ) remains  At present, the o r i g i n  unknown b u t  I - J determinants,  of the  as  r e c o g n i z e d by a n t i s e r a o r m o n o c l o n a l  a n t i b o d i e s (mAb), a r e  u n d e r s t o o d as m a r k e r s  (Murphy 1987;  f o r Ts a n d TsF  Dorf  still  and  B e n a c e r r a f 1985) . The  H-2  l i n k e d nonresponder  t o s p e c i f i c Ag has o f b o t h Ts a n d  s t a t u s of c e r t a i n murine  strains  f r e q u e n t l y been a s s o c i a t e d w i t h t h e p r o d u c t i o n  TsF.  F o r e x a m p l e , H-2^  a n i m a l s do n o t mount an  Ab  6 0  response  to the synthetic terpolymer, 1-glutamic a c i d  alanine^^-l-tyrosine''"^ t o be m e d i a t e d  by Ts  covalently  This nonresponsiveness  (Kapp e t a l . 1974) .  became r e s p o n s i v e t o GAT was  (GAT) .  was  However, H-2^  t h a t thymus and  (GAT-MBSA).  spleen extracts  found  mice  when t h e y w e r e i m m u n i z e d w i t h GAT  l i n k e d t o m e t h y l a t e d BSA  (1976) d e m o n s t r a t e d  -1-  that  Kapp e t a l . from  GAT-  4 primed mice if  the  suppressed the  e x t r a c t was of  addition  extract to  stimulated  a n t i - G A T Ab  with  by  the  an  irrelevant  of  Ag.  not  studies  achieved  by  and  have just  be  and  Gershon  d i s t i n g u i s h e d by  glycoproteins properties. induced Lyl~2  +  Lyl,2  +  both Ly  the  regulatory  Ts.  secreted  an  Lyl 2  +  type  markers)  of  and  Lyl 2~,  that  caused  reported  to  least  of  +  one  or  cells,  that  was a  Taniguchi other  cell  cell  could  surface  cells,  that  suppressive that  activity,  could  be  further  expression  that  I-J )  Tokuhisa cell  and  +  the  inducer  Lyl 2~ +  for Ly2  factor  T  cell  subsets  of  of  T  was  functional  targeted  type  was  mediator.  targets  and  Ag.  suppression  being  second  to  I-J  suppression  cells  reported  was  shown  expressed  inducer  exert  that  response  immobilized  T  their  were t h e  i n turn, produced  at  TsF  vitro  the  was  in vitro  mice  In  cells  suppression  I - J determinant  a l . (1981a,b)  suppression.  that  Ag.  shown by  spleen  TsF  I-J  +  that  A g - s p e c i f i c TsF  cells,  was  In general,  I - J , suppressor  cells,  Yamauchi et  as  cell-mediated  expression  to their  being  cells.  -  T  effector cytotoxic/suppressor  cells  of  bound by  r e s t i n g nonimmune T c e l l s  (cytotoxic  TsF  GAT-specific  of  i t s own  They d e s c r i b e d  according  by  (1979) p r o p o s e d t h a t  (called  classified  same t i m e  suppress the  c o u l d be  type  GAT-MBSA-immunized  several phenotypically different  each producing  Cantor  the  Specificity  shown t h a t  one  of  c u l t u r e of  L i k e KLH-TsF,  accomplished through subsets,  a primary  GAT-TsF t o  encoded determinants Other  at  production  GAT-MBSA.  inability  response  administered  suppression of  Ab  +  cells cells.  (effector (1979) was  TsF)  also  required,  in  5 addition  to the  suppression (effector that Ts  the  one  of the  Ts)  was  producing  a n t i - h a p t e n Ab the  c e l l s be  of  p r i m e d w i t h Ag.  suppressing  According  to  through  different Thus,  T cell  primary  level  secreted  TsF^  of  structure  of the  molecule  ability in  TsF.  donor.  TsF-^  initial  priming  the  or  presence TsF^  would  second  inducer of  first  Just  T cell  the  TsF  other (1986)  of the order  receptor  Ts  (TS2)  i n several aspects:  TS2  (Ts-^) .  type  TsF.  cells  and  also  NP-binding  antigenic site  of the  Ab  have  d e m o n s t r a t e d by i t s (CS)  response  to  a l l o g e n e i c with the  TsF^  i n animals.  during  the  also induced In t h i s  d i d not  way,  require  anti-idiotypic  and  NP  TsF  second TsF-^  is  the  s e c r e t e d TSF2 t h a t d i f f e r e d  T S F 2 was  was  of  These  I - J determinants  i f administered  This  4-  c e l l s induced  s t r u c t u r e s can  w i t h Ag.  d e s c r i b e d above.  e x o g e n o u s Ag.  Ts  possess  or  well.  suppression  spleen  sensitivity  suppress animal  as  f o r the  system,  a c t i v i t y was  syngeneic  were  three phenotypically  order  can  required effector  they  Ag  cell  and  once t h e s e  Ag-combining  contact  only  other  associated with the  as Ab  TsF-^  This  s e c r e t e d i t s own  expressed  l o c a t e d w i t h i n the  suppress  Dorf  least  determinants  mice t h a t were e i t h e r  level  at  to  hapten  that each  determinants.  to  of  known as  (idiotype),  idiotypic  (NP)  t h a t b o u n d NP,  idiotypic  and  cause  o f Ag,  of mice w i t h NP-coupled  Ts  expressed  determinants  acetyl  subsets  presence  response  Sherr,  a cascade  immunization  Ab  to  A g - s p e c i f i c TsF However,  i n the  the  Hausman,  hydroxy-3-nitrophenyl achieved  response.  t a r g e t of the  were combined w i t h TsF,  capable  A g - s p e c i f i c TsF,  bound  from to  a  6 anti-NP Ab i e . t h e i d i o t y p i c determinants expressed response  I - J encoded determinants o f p r e v i o u s l y primed  o f t h e Ab.  animals.  Furthermore,  but were only a c t i v a t e d by T s F -  expressed  I - J encoded and i d i o t y p i c determinants  T s F  2  Lastly,  third  3  bound t o NP, o f t h e NP-  The f i n a l t a r g e t o f the TsF^ was t h e T c e l l  r e s p o n s i b l e f o r mediating CS.  Once N P - s p e c i f i c T s F ^ i n t e r a c t e d  with NP, t h e s u p p r e s s i o n o f other CS responses vicinity  i t was  at the same time as Th d u r i n g Ag  exposure,  b i n d i n g moiety.  2  and c o u l d suppress t h e CS  g e n e t i c a l l y r e s t r i c t e d f o r suppressive a c t i v i t y . order Ts (TS3), were primed  T s F  occurred  (bystander s u p p r e s s i o n ) .  i n t h e immediate  I t i s not known i f  a c t u a l p h y s i c a l c o n t a c t between the c e l l types i s r e q u i r e d f o r the e x p r e s s i o n o f s u p p r e s s i v e a c t i v i t y . determinants Ts  2  However t h e i d i o t y p i c  o f Ts^ and Ts-^ and the a n t i - i d i o t y p i c  s p e c i f i c i t y of  (TSF2) c o u l d be viewed as a p o s s i b l e way o f b r i d g i n g a l l  t h r e e suppressor components t o g e t h e r . have been proposed  Other  and are reviewed by Tada  suppressor pathways (1984).  A more d e t a i l e d d e s c r i p t i o n o f t h e f u n c t i o n a l p r o p e r t i e s and m o l e c u l a r composition o f TsF became p o s s i b l e with t h e development of  T c e l l hybridomas and c l o n e s .  Factor-secreting T c e l l  hybridomas and c l o n e s served as homogeneous and continuous sources f o r t h e b i o c h e m i c a l study o f TsF.  I t has been e s t i m a t e d  t h a t TsF i n |ig q u a n t i t i e s can be r e c o v e r e d from t h e hybridoma supernatant al.  (SN) i n s t e a d o f i n ng q u a n t i t i e s from mice  (Krupen et  1982). Ts hybridomas were c r e a t e d v i a the f u s i o n o f the BW5147  7 thymoma w i t h expression  spleen cells  o f Ts m a r k e r s  Kapp, Araneo, spleen Ts  cells  markers,  Fresno  and  specific  expressed  extracted  a relative  the  TsF  extracts  determinant-bearing Taniguchi,  Th  cloned This  Takei,  effector  Ts  line  (M ) SN  effector  MHC  express TsF  was  Ag-specific  hybridoma  (eg.  that plates,  genetically  filtration  that  Saito,  A  form)  an A g - b i n d i n g one  showed t h a t  or  this and  and  TsF  comparison  of  cellular  particular  TsF  an I - J  Tada,  1979;  for i t s a b i l i t y to act directly response,  was  Nakauchi  phenotypically KLH-TsF  determinants.  I t was  also  Unlike  from  the  i t d i d not  the  S h e r r , and clearly  a  et a l . 1984).  different i n that  on  prepared from  f o r i t s expression of a c t i v i t y  ( T s F ^ , Hausman,  i n action.  (IL-2)  1980).  restriction  mentioned  from  suppressive material  demonstrated  monoclonal  I - J coded  the  1979;  bound t o Ag-coated  (secreted  ( K i t a m u r a e t a l . 1984; TsF  a Ts  of 42k-68k.  r  (Taniguchi,  Tada  Tada  interleukin-2,  Gel  +  chains: one  and  (or)  (or) t h e e x p r e s s i o n of  Ag-binding,  I - J .  so c a l l e d  mentioned  demonstrate  was  form)  and  TsF,  KLH-specific  not  and  as t h e  t o s u p p r e s s t h e a n t i - K L H Ab  previously  did  and  prepared from  (cytoplasmic  and  c l o n e s were d e r i v e d  from  ie.,  m o l e c u l a r mass  composed o f two  +  produced  spleen cells,  material  activity  et a l . 1984).  same p r o p e r t i e s  in activity,  had  Lyl  activity  Nakauchi  f o r KLH,  the  from  restricted  An  f o r Ts  Ts  I - J encoded d e t e r m i n a n t s and  demonstrated  was  1980).  then propagated using  e t a l . 1981;  TsF,  f o r Ts  (eg. T a n i g u c h i , S a i t o ,  Clevinger  enriched and  enriched  and i t  other  Dorf  1986)  shown t h a t  i t the  8 simultaneous to  presence  and  TsF  were r e q u i r e d  for suppression  occur. A  GAT-specific  (Kapp,  Araneo,  et  a l . , 1976);  and  TsF  and  same p r o p e r t i e s  from  a Ts  Clevinger  hybridoma  1980).  as t h e m a t e r i a l i e . , b i n d s Ag,  the  GAT-MBSA was GAT.  The  able  T cell  extracted  effector  In t h i s  be  cells  Lyl TsF  Ts  resulted  hybridomas  practically  ability Krupen Araneo, affinity  have  one  SN was  (1980,  response was Lyl  of T c e l l  see  two  were  active  above)  rounds  26  was  of  also  Ag-primed  cells with  b a n d a t 24k  factor,  based  content,  calculated  M  r  was  material  into  of b i o a c t i v e  bioactivity  5.2  x  10  TsF  the of  .  and  A  SN.  Kapp,  t o homogeneity.  observed.  TsF.  f o r the  SDS-PAGE u n d e r  7 as  selected  on  Ag  proliferation.  g of protein).  analyzed using  with  containing  Using  o f r e v e r s e - p h a s e HPLC ug  In  shown d i r e c t l y  the GAT-specific  (containing  purification was  primed  added t o c u l t u r e s  H P L C , t h e y r e c o v e r e d 1.8  material  conditions,  secrete  (Kapp  determinants,  of mice  of Ag-primed  purified  chromatography,  6 1 of crude  purified  was  a l l cases, hybridomas  Clevinger  spleen c e l l s  f a c i l i t a t e d the biochemical study of  e t a l . (1982) and  response  inhibition  to constitutively  ion-exchange from  i n the  the  t o GAT-MBSA-primed c e l l s .  proliferative  incubation  reported  demonstrated  from  c a s e , t h e t a r g e t 'of t h e T s F  since  been  o f K i t a m u r a e t a l . (1984)  to suppress the T c e l l  cells.  and  when TsF  KLH-TsF  also  expresses I - J encoded  proliferative  inhibited  has  T h i s TsF  suppresses the primary response  addition,  In  o f Ag  and  material  When  the  reducing  final total  protein  to  9 Using  t h e same G A T - s p e c i f i c T s h y b r i d o m a ,  isolated bioactive specific expressed using the  mRNA f r o m  i t and achieved  GAT-TsF.  The t r a n s l a t e d  T cell  I - Jdeterminants,  affinity  a lack  i nM  system  glycosylating Molecular also  used  GAT-TsF.  r  reverse-phase  protein.  spot,  contained material were  composition  Effector  lying  Kapp,  structure  GAT-TsF  was a p p l i e d  determinants.  single  result  since the  d i dn o t possess  .  TsF s p e c i f i c  a n d Webb  from  f o r GAT h a s  1986).  This TsF  the previously  was a d i s u l p h i d e - l i n k e d  linkage),  described heterodimer.  non-reducing/reducing  was r e c o v e r e d .  t o a reverse-phase  Suppressive  one c h a i n was shown  effector  (24k M ) as a  SDS-PAGE,  o f f the diagonal of the g e l (indicating  a disulphide  30k M  HPLC a n d SDS-PAGE,  of the factor,  o f an e f f e c t o r  (Turck,  i n molecular  resolved.  about  I t  t o homogeneity  reticulocyte)  activity  mixed t o g e t h e r and added t o c u l t u r e .  J  GAT-  They e x p l a i n e d t h e  modification  (rabbit  both  Ab r e s p o n s e .  a n d when p u r i f i e d  When a n a l y z e d b y t w o - d i m e n s i o n a l one  suppressed  activity.  been determined  differed  product  (1982)  translation of  with the secreted material  r  of carbohydrate  translating  cell-free  and t h e anti-GAT  chromatography,  T s F r e s o l v e d a s a 19k M  discrepancy of  proliferation  Wieder e t a l .  Like  SDS-PAGE, b o t h  the single  chain factor,  bioactivity  two p r o t e i n s  was r e s t o r e d when t h e y  were  F o l l o w i n g HPLC s e p a r a t i o n , chain displayed I -  c h a i n s were  c h a i n GAT-TsF,  TsF were g l y c o s y l a t e d .  When t h e  HPLC c o l u m n ,  t o b i n d Ag w h i l e t h e other  Using  thati t  resolved at  both  components o f  However,  i n contrast tothe  was l o s t  following  neuraminidase  10 treatment. Work w i t h of  KLH-TsF h a s p r o g r e s s e d  GAT-TsF.  Taniguchi  e t a l . (1982),  (described by Taniguchi, specific  Takei,  f o r KLH, o b t a i n e d  translated a chain  expressing  was  recovered  using  and Tada  manner t o t h a t  a Ts h y b r i d o m a  1979, s e e above)  s e v e r a l mRNA s p e c i e s  by t h e f r o g oocyte  and  i n a similar  that,  when  system y i e l d e d an A g - b i n d i n g  I - J determinants.  when t h e t w o c h a i n s  Suppressive  chain  activity  were combined and t e s t e d i n  culture. Saito  and Taniguchi  two-chain  (1984) b i o c h e m i c a l l y  KLH-specific  bound Ag and expressed to The J  TsF.  27k M  chain  r  reagents.  The n o n - c o v a l e n t l y  I - J determinants.  b o t h K L H a n d t o a mAb  characterized the  specific  associated  The 45k M  f o ra constant  Suppression  monoclonal  reagents.  Although  extensive  region  of the anti-hapten from  purified  t o homogeneity.  specific  f o r1-tyr-p-azophenyltri-methyl-ammonium  anti-TMA Ab. subjected and  high  specificities  Webb e t a l . ( 1 9 8 9 )  TsF t h a t  have  been  described  a TsF  (TMA).  bound Ag, expressed  material  f r o m t h e Ts h y b r i d o m a  t o (NH^^SO^ p r e c i p i t a t i o n , performance  gelfiltration  with was  IEF, reverse-phase  to yield  I t was  both I - J  and t h e c r o s s - r e a c t i v e i d i o t y p e a s s o c i a t e d Extracted  was  t h e GAT a n d K L H  other  as an i n d u c e r  different  anti-I-  i m m u n o a d s o r b e n t s made w i t h t h e  work h a s been done w i t h  TsF w i t h  bound  of TsF.  Ab r e s p o n s e  systems,  determinants  chain  bound t o both p o l y c l o n a l and monoclonal  demonstrated by TsF recovered  described  r  chains  HPLC,  a major b i o a c t i v e  11 peak at 62k M M .  .  Using SDS-PAGE, a major peak was  observed  They noted t h a t the b i o c h e m i c a l c h a r a c t e r i s t i c s of T M A - T s F  were very s i m i l a r t o t h a t of s i n g l e - c h a i n G A T - T s F . demonstrated t h a t a mAb authors of  at 2 6k  suggested  suppressor  r a i s e d t o G A T - T s F bound t o T M A - T s F .  t h a t both f a c t o r s belonged  Fresno  chain, Ag-binding express  The  t o the same f a m i l y  (as opposed t o a hybridoma)  has  et a l . (1981) recovered a 70k M , s i n g l e -  f a c t o r from a L y 2 3  I - J determinants.  I t was  the T h and r e q u i r e d the presence  +  T c e l l clone.  p u r i f i e d using gel  ion-exchange chromatography and IEF.  suppressive  they  inducer p r o t e i n s .  A TsF i s o l a t e d from a Ts c l o n e been r e p o r t e d .  I n fact,  I t d i d not filtration,  T h e t a r g e t of t h i s TsF  was  of A g f o r the e x p r e s s i o n of  activity.  Summary  A v a r i e t y of p r o p e r t i e s have been a t t r i b u t e d t o TsF. Initially, response,  such f e a t u r e s as the s p e c i f i c s u p p r e s s i o n of the A b the c a p a b i l i t y t o b i n d A g , the e x p r e s s i o n of I - J  encoded determinants  and the requirement  for genetically  r e s t r i c t e d c e l l u l a r i n t e r a c t i o n s were d i s c o v e r e d u s i n g extracts.  cellular  These f i n d i n g s were v e r i f i e d and extended u s i n g Ts  hybridomas and the TsF produced by them.  Indeed, the  cascade model f o r the s u p p r e s s i o n of the NP Sherr, and Dorf 1986,  see above) was  response  linear (Hausman,  based on data o b t a i n e d u s i n g  hybridomas and t h e i r r e s p e c t i v e f a c t o r s made from the t h r e e  orders  of  Ts:  suppression circuits its of  own  the  was  with type  the  TS2  and  achieved  TsF  Since  possible  cell  of  was  the  TsF  and  of  Ts  cell  can  to  idiotype-anti-idiotype help  to  insure that  interact. dependent  Although and  w o u l d be  explains  how  response. possible  HPLC,  the  express  other  T  study  and  TsF  using  at  TsF  the  different  ion-exchange  in various  material.  TsF  has  been  molecule.  The  one  chain  the  could  i n the  pathway  cell,  control  through  could  factors  responses  suppress  Ag-  immediate  suppression).  This  a polyclonal T  molecular  level.  separation  to  was  yield as  Several  techniques  cell  IEF  and  homogeneous and  a one  chain  or  two  b i o a c t i v e , b o u n d Ag  a non-Ag-binding  displayed  I - J determinants.  and  Both  have been r e p o r t e d  of as  the  two  chain  chains  either  have  reverse-phase  of  chain  TsF  including,  consisted  and  also  would  i n the  The  bioactivity  one  the  I - J encoded determinants. Ag-binding  (eg.  determinants,  and  or  which  expressed  an  target  h y b r i d o m a s h a v e a l s o made i t  isolated TsF  The  e f f e c t o r f u n c t i o n was  chromatography,  combinations,  Ts  or  elaborating  restriction  cells  cell  pathways  subset  suppressive  (bystander  F a c t o r - s e c r e t i n g Ts to  next  Genetic  of  that  e f f e c t o r TsF).  idiotypic  appropriate  suppressed  Ts  r e c o g n i t i o n was  suppression  cell  effector T  under  network.  a monoclonal  been p u r i f i e d affinity  the  was  cell  specific,  vicinity  TsF,  e i t h e r the  path,  suggested  interacting  inducer  function that  way  Other work  through  (eg.  A g - s p e c i f i c TsF  mediated the  TSg.  each p h e n o t y p i c a l l y d i s t i n c t of  terminal  Th).  Ts^,  bioactive chain  and  structure  were  chain  that  required  non-covalently  for  13 associated from  or disulphide-linked.  at least  translation types  M e s s e n g e r RNA h a s b e e n  two d i f f e r e n t Ts h y b r i d o m a s  of bioactive  o f TsF have been  TsF.  and used t o achieve  B o t h t h e one c h a i n  synthesized  using  prepared  i n vitro  and two  chain  translation  techniques. A precise because  description  of the variety  o f t h e TsF molecule i n both  i t s reported  properties.  reflection  o f t h e d i f f e r e n t Ag systems under  mechanisms a t work Despite on  i t s structure  Immune  Mechanisms  protein  will  cloning  i s a  the labile  of the suppressor i s unknown.  o f TsF, a  be r e a c h e d  only  o f a gene(s)  consensus by  encoding  an  TsF.  Response t o F e r r e d o x i n  as a Model  f o r t h e Study of  of Immunoregulation  i n this laboratory.  as a d e f i n e d Ag.  study,  of a l l three,  only  immune r e s p o n s e t o f e r r e d o x i n  extensively served  and f u n c t i o n  and  of results  devoted t o the study  t e s t i n g eg. molecular  Ag-specific  The  the complexity  or a combination  the effort being  stringent  The  Whether t h e v a r i e t y  o f t h e TsF molecule,  difficult  molecular  functional  nature  has been  model  (Fd) h a s b e e n  studied  Collectively, the findings  f o r t h e immune r e s p o n s e t o a  have  natural  14 The  choice  Fd  o f F d as a model  i s a small  (5500 M ) m o l e c u l e p r o d u c e d b y  pasteurianum which the  electron  functions  transport  well-characterized (Rabinowitz  antigen  as an e l e c t r o n - a c c e p t o r  pathway  of that  biochemically  1972).  using  gene c l o n i n g  1985)  and i t s three-dimensional  using  X-ray  protein  i s distinguished  residues, two  the sulfur  iron-sulfur  protein.  techniques  present In  i n both halves  reason  amino-acid  cysteine  coordination  make u p t h e c e n t r a l  pairs  determined  55  of eight  form  that  and Rabinowitz  has been This  confirmed  bonds  core  F d was c r e a t e d  has a two-fold  of amino-acid  with  of the through  axis  residues  a  of  that are  knowledge o f i t s biochemistry,  there  was  f o r c h o o s i n g Fd as a model Ag f o r t h e study o f mechanisms.  Ag o f d e f i n e d  distinct  advantages  Firstly,  natural  Stated  structure.  over  proteins  mechanisms e l u c i d a t e d implications.  simply,  This  type  some o f t h e o t h e r a r e what  by i t s study  Fd i s a  natural  of molecule  has two  e x p e r i m e n t a l Ag.  i s e n c o u n t e r e d b y t h e immune  system under p h y s i o l o g i c a l conditions  based  o f F d was  1974).  by t h e presence  that  t o homogeneity  of the molecule.  t o having  immunoregulatory protein  (Jensen  protein i n  I t has been  Mullenbach,  structure  as t h e p r o t e i n  a n d 13 i d e n t i c a l  addition  another  (Graves,  I t has been h y p o t h e s i z e d  gene d u p l i c a t i o n e v e n t symmetry  sequence  atoms o f w h i c h ,  clusters,  organism.  and p u r i f i e d  The a m i n o - a c i d  crystallography  Clostridium  and t h e r e f o r e  would presumably  Such c o n c l u s i o n s  cannot  any have  necessarily  broad be  drawn  15 from  synthetic  defined or  primary  the  interact with  will  be  protein  the  Ag.  the  below,  or  from  limited  of  only  a  these has  of  of  Ag  of  Having  the  has  the  Ag.  interpreted  GAT,  precise  number  a  defined  as  selected  epitopes.  a prototype  involved  of  As for  natural  the  use  However the  i n terms  of  structure  cell-dependent  Fd  of  immunodominance:  immunodominant  T  are  like  l i m i t e d number a r e  been u s e f u l  H-2  linked responsiveness  were aimed at  Fd  using  guinea pigs.  number o f  alter  protein  and  studies  modification not  Knowledge  potentially large  forms  or  proteins  synthetic  study  clones.  of  natural  of  data  the  have  response  to  Ag.  determinants cells  the  work w i t h  synthetic  E p i t o p e mapping  Initial  f o r the  Ag,  Fd  the  always been r e l a t e d to native  DNP-KLH.  immune r e s p o n s e t o  Some o f  derivatized  the  of  identification  of  opposed to  lymphocyte  described  study  as  is vital  e x p r e s s e d by  the  Secondly,  like  phenomenon i n w h i c h ,  allows  Ag.  structure,  structure  epitopes  the  or  derivatized antigens  antigenic  to  haptens  of  the  (1971),  using  treated  Fd  the  I t was  cysteine of  and  since  residues  antiserum  the  raised to  Further  work,  antiserum  raised to  synthetic  peptides  by  antigenic  and  peroxyformic of  Fd  major  demonstrated that  antiserum  a l . 1969).  rabbit  (OFd)  rabbit  determinants  binding  (Nitz et  both  mapping the  to  Ag-primed Fd  a  acid  native the  contained  T  molecule  unmodified  K e l l y and  peroxyformic representing  Levy acid-  various  did  16 sequences Ab  i n the Fd molecule  activity  was  directed  showed t h a t  to either  essentially  a l l of the  the amino-terminal  heptapeptide  (NH^-ala-tyr-lys-ile-val-asp-ser-COOH,  determinant)  or the c a r b o x y l - t e r m i n a lpentapeptide  val-gln-glu-COOH, the  amino-terminal  cell of  known  determinants  in  bridge.  was  The  shown u s i n g s y n t h e t i c sequences synthetic  guinea pigs since would  as  as t o t h e i n t a c t  Hull  1973).  primed  cells  proliferate  Evidence  p r o v i d e d when i t was  two  separately primed  inbred was  and Levy  strains  H-2  Ag  both  consisting  o b t a i n e d from  t o be  immunogenic  these  primed  to the respective  (Levy e t a l . 1972; K e l l y ,  demonstrated  and  and Levy  carried  Levy,  congenic  and  response  only a mixture  of the  recognize the (DNP)  Ab  1975).  out a genetic a n a l y s i s  and determined  peptides  C-determinant-  DNP-OFd i n a n a n t i - h a p t e n  (1980)  Using  that  p o p u l a t i o n s would  Kilburn,  of mice  linked.  of  together v i a a repeating  the N-determinant  cell  complex,  (Feldmann,  Sikora  ability  s y n e r g i z e d f o r t h e g e n e r a t i o n o f a Th c e l l  hapten-carrier  N-  (NH^-ala-pro-  peptides  peptides proved  i n response  that  was  response  linked  the T cells  animals well  The  as  and c a r b o x y l - t e r m i n a l p e p t i d e s t o a c t as T  t h e immunodominant  glycine  as C - d e t e r m i n a n t ) .  known  that  strains  t h e Ab  o f mice,  using  response  t o Fd  the response  was  v mapped high  t o t h e I-A s u b r e g i o n .  responders,  animals An  H-2  '  mice  were d e s i g n a t e d as  important  confirmed  H-2  were  identified  as i n t e r m e d i a t e r e s p o n d e r s  and  nonresponders.  s t u d y by S i k o r a ,  and extended  animals  Weaver,  and Levy  t h e p r e v i o u s work t h a t  (1982)  described the  as H-2  determinant mapping o f Fd. antigenic which Fd  They  were g e n e r a t e d by e n z y m a t i c  tripeptide,  leaving  C-determinant, C-determinant  intact.  prepared  from  observation  determinants.  Monoclonal  the N-determinant bind  as b a s i s  directed  antibodies  of comparison  with  I t was  also  anti-Fd  the  digests  the  leaving  intact.  anti-Fd  the  A  antiserum  earlier  exclusively  t o t h e two  specificity  for either  were produced  and C-determinant  e q u a l t o i t st o t a l  including  confirming the  enzyme-digested  determinant.  the N-determinant  was  or C-determinant  to the respective  appropriate  was  activity  with  of  terminal  the N-determinant,  mice,  cleavage  3 amino a c i d s ,  d i d not react  high responder Ab  the  of the molecule,  the terminal  including  molecule  that  by removing  o f two  analogs  Trypsin  Carboxypeptidase A p a r t i a l l y  by removing  digested  digestion.  the remainder  of the molecule,  doubly  Fd consisted  determinants, through t h e use o f s t r u c t u r a l  destroys the N-determinant  rest  showed t h a t  and shown t o  molecule bearing the observed that  reactivities  reactivity.  between s t r a i n s  t h e sum  o f an  antiserum  This finding  of mice that  of  was  used  were k  responsive  t o Fd.  The h i g h r e s p o n d i n g s t r a i n s  Ab,  of which,  the  intermediate responders  the  I - A s u b r e g i o n ) , o n l y 25 t o 5 0 % o f t h e t o t a l  specific It  was  60 t o 8 0 % was  f o r the found  responsive  produced  t o the C-determinant.  (expressing  Of  t h e b and s a l l e l e s a n t i - F d Ab  at was  C-determinant.  that  trypsin-digested  directed  (I-A )  Fd  injection (also  t o subsequent  of nonresponder  referred  mice  t o as C-molecule)  challenges of Fd  (Sikora  (H-2  )  made  and Levy  with them 1984).  18 Furthermore, t r e a t e d Fd  i f the mice were primed with carboxypeptidase A(also r e f e r r e d t o as N-molecule) and then c h a l l e n g e d  with C-molecule,  t h i s r e s u l t e d i n an a c c e l e r a t e d a n t i - F d  response as compared t o c o n t r o l animals t h a t r e c e i v e d immunization determinant t h a t was  only.  I t was  C-molecule  p o s t u l a t e d t h a t a second o v e r l a p p i n g  r e s i d e d w i t h i n the N-determinant  heptapeptide region  "masked" by the a l a - t y r - l y s t r i p e p t i d e , but was  i n the case of C-molecule. primed mice,  Interestingly,  f o r the  l e s s than 10% of the a n t i - F d Ab was  C-determinant  Ab  exposed  C-molecule-  s p e c i f i c f o r the  (see F i g . 9B) .  Summary  Fd was  chosen as a model Ag because  p r o t e i n with defined structure.  i t i s a small,  Initial  natural  s t u d i e s i d e n t i f i e d the  amino-terminal heptapeptide and the c a r b o x y l - t e r m i n a l p e n t a p e p t i d e as the only a n t i g e n i c determinants or e p i t o p e s of the molecule. T cells.  These determinants were r e a c t i v e with both Ab  and  T h i s suggested t h a t e i t h e r e p i t o p e had the p o t e n t i a l  f o r b e i n g a s s i g n e d a hapten or c a r r i e r f u n c t i o n . a n t i b o d i e s were r a i s e d t o e i t h e r determinant  Monoclonal  and through the use  of analogs h e l p e d t o v e r i f y the o r i g i n a l e p i t o p e mapping of Fd. The  immune response t o Fd was  determinant  linked  s e l e c t i v i t y of the a n t i - F d Ab response was  r e l a t e d t o the haplotype 2  observed t o be H-2  (genotype)  and also  of the responding mouse.  animals were i d e n t i f i e d as h i g h responders and 60 t o 80% of  H-  t h e Ab r e s p o n s e animals  was d i r e c t e d  t o the C-determinant.  were d e s i g n a t e d as i n t e r m e d i a t e responders  o f t h e Ab was s p e c i f i c  f o r the C-determinant.  to  Fd, expressed by t h e nonresponder  by  priming the animals with  analog  of Fd.  was d i r e c t e d  Less  than  by  associated system  a n d 25 t o 5 0 %  Lastly,  mice,  tolerance  c o u l d be  broken  the trypsin-digested  10% o f t h e a n t i - F d Ab o f t h e s e  animals  interactions  and examining with  t o Fd has also  each  been  idiotypic-anti-idiotypic  the anti-Fd  response.  other v i a sets  components  of antigenic  t h e V r e g i o n s o f A b o r TCR  (idiotype),  o f t h e immune that  determinants i s now  pursued  interactions  The c o n c e p t  as an a u t o r e g u l a t o r y n e t w o r k w i t h  recognize by  C-molecule,  s t u d y o f t h e immune r e s p o n s e  defining  H-2  '  t o t h e C-determinant.  Idiotype-anti-idiotype  The  H-2  expressed  generally  accepted. Network were  first  interactions  influenced  t h e immune r e s p o n s e  r e v e a l e d t h r o u g h t h e u s e o f an a n t i - i d i o t y p i c  (rabbit  antiserum)  anti-Fd  mAb,  Fd.  that  specific  Fd-1.  The i d i o t y p e  f o rthe idiotype  T h i s mAb  identified  i s specific  t o Fd  reagent  e x p r e s s e d by t h e  f o r t h e N-determinant  by t h e a n t i - F d - 1 reagent  of  represented k  a major high and  idiotype  responders Levy  expressed and H-2  S  i n a n t i - F d Ab o b t a i n e d f r o m b o t h  intermediate responders  (Weaver,  H-2  Sikora,  1982).  Weaver e t a l . (1983)  described T cells,  which  expressed the  20 Fd-1  idiotype, that  B10.BR with  (H-2 )  helped  animals.  k  anti-Fd-1  plus  t o regulate  This  the anti-Fd  was done b y t r e a t i n g B10.BR T  complement  and then  adoptively  them t o s u b l e t h a l l y i r r a d i a t e d B10.BR r e c i p i e n t s . were  immunized w i t h  was  a significant  the  animals  reagent  received  treated  o f Fd-1 i d i o t y p e - b e a r i n g  the anti-idiotypic  reagent.  i n the antiserum  treatment.  of  there  i n those  also  anti-Fd-1  suggested that  of expression  was a s i g n i f i c a n t animals  existed  idiotype-bearing important  the  was b e i n g  d i d n o t change w i t h  that  suggested t o the authors  T cells  This  T cells  t o nonimmune B10.BR m i c e  immunization,  This  with  Ab i n  a  influenced of anti-  o f t h eFd-1  the anti-idiotype  When F d - 1 , t h e mAb d i s p l a y i n g t h e F d - 1 i d i o t y p e , w a s  administered  production  There  Interestingly, the level  N - d e t e r m i n a n t Ab o r t h e p e r c e n t a g e idiotype  The r e c i p i e n t s  i n anti-C-determinant-specific T cells  cells  transferring  f o r Ab p r o d u c t i o n .  as compared t o c o n t r o l animals.  population by  that  Fd and monitored  increase  Ab r e s p o n s e o f  days p r i o r t o F d  increase  had received  that  and that  seven  t h e mAb  an a n t i - i d i o t y p i c  Ab  injection. population  t h e i n t e r a c t i o n between t h e  and t h e a n t i - i d i o t y p e bearing  i n the regulation  i n anti-Fd  of the anti-Fd  T cells  Ab r e s p o n s e  was i n B10.BR  animals. Singhai 2, t h a t  et a l .  was s p e c i f i c  anti-idiotypic reagent  (1984)  another  f o r t h e C-determinant  reagent  was a d m i n i s t e r e d  immunized w i t h  characterized  to i t .  When t h i s  t o B10.BR m i c e ,  anti-Fd  mAb, F d -  o f F d and p r e p a r e d an  anti-Fd-2  idiotype  that  subsequently  were  F d , t h e a n t i - F d Ab r e s p o n s e was e n h a n c e d i n  treated  animals  a s compared t o c o n t r o l  groups.  The l e v e l o f  a n t i - C - d e t e r m i n a n t Ab h a d i n c r e a s e d , a s was o b s e r v e d al.  (1983).  detected  U n l i k e t h e Fd-1 i d i o t y p e ,  i n B I O . B R immune s e r a .  administration were t h e n Ab  animals.  investigated  Fd-2  further  cells  into  t o nonresponder  by Singhai,  with either  irradiated  recipients  were  production  o f a n t i - F d Ab.  Both  by adoptively  was  o f the Fd-2-depleted  with as  Fd.  transferred  suspension  into  transferring  the Fd-2-depleted  idiotype  that  maintenance  remaining The  responded t o  that  o f these  ofT cells  and t h e  were t h e n animals  both populations o f T c e l l s idiotype  o f the nonresponder  challenged negligible  who r e c e i v e d e i t h e r This  e x p r e s s i n g t h e Fd-2  were r e q u i r e d  status  that  anti-Fd-2-  were  o rthe anti-Fd-2-depleted T c e l l s .  and t h e anti-Fd-2  or anti-  f o r the  a mixture  suspension  recipients,  The a n t i - F d Ab l e v e l s  They  o f n o n r e s p o n s i v e n e s s was  compared t o t h e other immunized r e c i p i e n t s  suggested  the  groups o f animals  Reconstitution  (1985).  Fd-2 a n d c o m p l e m e n t  B10.D2 r e c i p i e n t s .  demonstrated  depleted  that  i n the production of anti-Fd  immunized w i t h F d and monitored  the F d challenge.  composed  B10.D2 m i c e ,  Hoffmann, and Levy  anda d o p t i v e l y  syngeneic  was n o t  T h i s a b r o g a t i o n o f n o n r e s p o n s i v e n e s s was  B10.D2 T c e l l s  a n dcomplement  t h e Fd-2 i d i o t y p e  More i n t e r e s t i n g l y , t h e  immunized t o Fd, r e s u l t e d  i n these  treated  o f anti-Fd-2  b y Weaver e t  i n H-2  f o r the  animals.  22 Summary  Idiotype  was d e f i n e d  as t h e s e t o f a n t i g e n i c  expressed by t h e V regions idiotypic  i n t e r a c t i o n s were  idiotype-expressing antisera  against  these  intermediate  idiotype-bearing  cells  idiotype-bearing  worked  i n concert  animals. network  Results  bearing  t o help  i d i o t y p e was  governed  itself as w e l l .  regulate  t o t h e animal  an  anti-Fd-1-  I t was s p e c u l a t e d  Fd-2 i d i o t y p e )  responsiveness  t o Fd.  responsiveness  ensued  resulted.  high  also  that  suggested that  t h e Ab r e s p o n s e  complete  from  was c a u s e d b y t h e  of idiotype-bearing  almost  The F d -  appeared t o  The o b s e r v a t i o n  When t h e t w o p o p u l a t i o n s  nonresponsiveness  that  that  populations i n B10.BR  from t h e i n v e s t i g a t i o n o f a second  was d e p l e t e d ,  immunization.  tested  and a n t i - i d i o t y p e - b e a r i n g c e l l  two p o p u l a t i o n s  T cells  population  returned  existed  i n t e r a c t i o n (designated  showed t h a t  This  o f B10.BR T c e l l s  o f Fd-1 i d i o t y p e  anti-idiotypic  of sera  i n a n t i - F d Ab i n B10.BR a n i m a l s  administration  the  animals.  t h e a n t i - F d Ab r e s p o n s e .  enhancement  mAb a s t h e  were r a i s e d i n r a b b i t s .  i n the majority  responder  anti-Fd  Affinity-purified  idiotypes  expressed by a population regulate  Idiotypic-anti-  investigated using  reagents.  1 i d i o t y p e was e x p r e s s e d and  o f A b o r TCR.  determinants  immune  i n B10.D2  mice  and a n t i - i d i o t y p e When  either  following  were combined, and restoration of  23 B16G  and t h e Development  Another  area of interest  development for  screening  tumor  tumor  workers  have a l s o  immune r e s p o n s e Fisher  Further factor, that  to this  and K r i p k e  In order t o extend our  i s lethal  hybridoma,  days)  investigation  of studies  (Takei,  described  well  a P815-specific was q u i c k l y  CTL overwhelmed  and K i l b u r n  factor  suppressive  appeared activity  with  an  1976).  t h e i n v o l v e m e n t o f Ts i n t h e  and o t h e r tumors and North  (Granstein  e t a l . 1984;  1985).  l e dto the identification  t o P815  after  o f Ts c o r r e l a t e d  Levy,  from t h e thymocytes  (CTL) r e s p o n s e  when  soon  of a  of P815-bearing  was c a p a b l e o f s u p p r e s s i n g t h e s y n g e n e i c i n v i t r o  T cell  on t h e  t o DBA/2 m i c e  phase  Fdll,  B16G.  I t was shown t h a t  The a p p e a r a n c e  1982; M i l l s  extractable  of suppressive  as a r e s u l t  However t h i s  i n tumor growth  f o rboth the  mastocytoma P815 i s a  (three t o f i v e  was e v i d e n t .  specificity  B16G h a s b e e n u s e d  low doses.  t h e emergence o f Ts.  Other  The  The m u r i n e  tumor model which  inoculation  acceleration  having apparent  t o Fd, a T c e l l  was d e v e l o p e d  system.  has been t h e  Ag and s c r e e n e d u s i n g  at relatively  response by  that  itself  characterized injected  laboratory  i m m u n o a f f i n i t y columns.  against  B16G  hybridoma  and t h e i s o l a t i o n  o f t h e immune r e s p o n s e  was r a i s e d  P815  e p i t o p e on TsF.  o f Ts h y b r i d o m a s using  studies  i n this  a n d u s e o f B 1 6 G , a mAb  an i n v a r i a n t  material  of the Fdll  (Takei,  t o have a degree  Levy,  and K i l b u r n  o f tumor  c o u l d be absorbed  soluble mice, cytolytic 1978).  specificity.  out of solution  by  The passage  24 over  a P815-Sepharose  Attempts In  at purification  order  Stammers, specific  t o study and Levy  mAb,  isolated  (1983)  B16G.  B16G by  f r o m w h i c h B16G was d e r i v e d . B16G was t e s t e d b o t h  that  T cell  DBA/2 s p l e e n  B16G n o t o n l y  than  inactivation  cells  to irradiated  as w e l l .  of a regulatory were  of a regulatory  incubated  With t h e a v a i l a b i l i t y purposes,  a T cell  thymoma l i n e ,  was r e c o v e r e d  Sepharose  columns.  from  t o DBA/2 m i c e ,  growth,  but not that  Panning over"  population.  f o rs e l e c t i o n  from t h e murine  i n vivo  and Levy  1985).  w i t h P815 A10 f a c t o r  e i t h e r B16G- o r P 8 1 5 -  When A 1 0 F w a s a d m i n i s t e r e d  (i.v.)  effect  B16G was r e s p o n s i b l e f o r  stimulated  SN u s i n g  enhancing  from t h e spleen  Stammers,  by  on o r "panned  h y b r i d o m a , A 1 0 , was c r e a t e d  (Steele,  cells  T cell.  o f a " T s F " - s p e c i f i c mAb  BW5147 a n d T c e l l s  membrane e x t r a c t s (A10F),  T cell  animals  B16G was r e s p o n s i b l e f o r  (suppressor?)  Bl6G-coated p l a t e s , demonstrated that removal  This  That  substantiated  from B16G-treated  suggested that  where T c e l l s  Once i t was and i n v i t r o .  t u m o r , M-1  controls.  T s F was  inhibited the  histoincompatible  d i duntreated  proliferation  experiments,  the  i n vivo  was n o n s p e c i f i c a l l y i m m u n o e n h a n c i n g was a l s o noting  Maier,  and c h a r a c t e r i z e d t h e TsF-  o f P815 tumor b u t t h e syngeneic  proliferation  the  were n o t s u c c e s s f u l .  f a c t o r s more d i r e c t l y ,  developed  o f DBA/2 m i c e w i t h  responded b e t t e r  on  material  column.  A f f i n i t y - e n r i c h e d thymocyte-derived  and cloned,  Pretreatment  of this  tumor-specific  used t o immunize mice  growth  column b u t n o t by a c o n t r o l  intravenously  i tcaused t h e enhancement o f L 1 2 1 0 o r M-1 t u m o r .  o f P815 tumor  I n a d d i t i o n , A10F  25 d i d not enhance or suppress the T c e l l p r o l i f e r a t i v e a l l o g e n e i c response o f DBA/2 spleen c e l l s .  The i n a b i l i t y o f AlOF t o a f f e c t  the growth o f two u n r e l a t e d tumors or the T c e l l  response o f  DBA/2 c e l l s t o h i s t o i n c o m p a t i b l e c e l l s i n d i c a t e d t h a t i t might be tumor-specific. Molecular A10F  c h a r a c t e r i z a t i o n revealed that  r e s o l v e d as 140k, 80k and 45k M  SDS-PAGE under r e d u c i n g c o n d i t i o n s 1985;  r  degradation  f o r "aggregation"  m o i e t i e s when analyzed by  (Steele, Stammers, and Levy  Chan 1988; Chan et a l . 1988).  propensity  affinity-enriched  The 80k M  t o the l a r g e r M  r  s p e c i e s had the  r  s p e c i e s or  t o a s m a l l e r s i z e o f approximately  30k M .  under non-reducing c o n d i t i o n s of A10F showed t h a t a c t i v i t y was a s s o c i a t e d with the 140k and 80k M 45k  and 32k M  forms showed marginal  r  SDS-PAGE  suppressive forms while the  r  bioactivity.  Biosynthetic  35 l a b e l i n g o f A10 c e l l s with t h a t the 80k M  r  S-methionine and c y s t e i n e r e v e a l e d  form was a c t u a l l y s e c r e t e d i n t o the SN and  f o r m a l l y r u l e d out the p o s s i b i l i t y t h a t the 80k M was a medium or FCS a r t i f a c t .  r  forms i n d i c a t e d t h a t  shared t h e same sequence while the 30k M d i f f e r e n t amino-terminal sequence.  r  polypeptide  they  had a  Because o f the p o s s i b i l i t y  t h a t sequence data c o u l d have been d e r i v e d from assignment o f these  A10F moiety  Amino-acid sequencing o f the  amino-terminal o f the 140k and 80k M  impurities,  r  co-migrating  sequences t o the TsF molecules  c o u l d not be done. Much i n f o r m a t i o n has been gathered t o Fd.  Of p a r t i c u l a r i n t e r e s t  about the immune response  i s the e x i s t e n c e of an i d i o t y p e -  26 anti-idiotype as p a r t to  Fd  of the suppressor network  t h e knowledge  hybridoma primed  was  made u s i n g  Because  unlike  that  screened  Sepharose  The  affinity  columns.  material  a n a l y z e d by material 80k  FdllF  M  r  and  This  from e i t h e r  Ag  was  Fd, flux the  p r e s u m a b l y by studies.  events associated  reaction. Quin-2.  Fdll Seconds  cells after  were  known as F d l l F ,  binding could  from B16G-Sepharose  hybridoma  M  r  one  as w e l l  with  a cell  were  loaded with  was  When  as  at  a number  the  of  was  and  Fd  were  Ab  groups  actually  provided  of calcium  surface  while  migrating  concomitantly,  TCR,  Fd-  the material  can  be  FdllF-enriched  When F d l l F  Fdll  or  plates  d i d not.  conditions,  flux  the exposure  known,  molecules capable of  Evidence that  intracellular  was  cells  compared t o t h e c o n t r o l  a cell-associated  The  hybridoma  columns.  s u p p r e s s e d as o r A10F.  et a l .  the case whether  response  PBS  Levy  30k  t o F d - i m m u n e DBA/2 m i c e  receiving  Chan, N o r t h , and  unique bands,  o r B16G  cell Fd-  reducing  administered was  a T  from  the hybridoma  irrelevant  another at about  response  obtained  material,  major  functions  immune  bound t o Fd-coated ELISA  SDS-PAGE, u n d e r  a p p e a r e d as two  the  existed,  T cells  chromatography  f r o m an  other minor bands. recovered  secreted  Ts  of the F d l l  f o r the a b i l i t y to secrete  a n d B16G.  control  Chu,  hybridoma,  which  mice.  BW5147 a n d  t h e n o m i n a l Ag  level,  to control  Fd-specific  (Steele,  o f t h e A10  r e c o v e r e d by  about  that  B10.D2 a n i m a l s  1987).  Fd  at the T c e l l  i n n o n r e s p o n d e r B 1 0 . D 2 H-2  With  to  interaction,  ions  recognize  by  calcium  i s one  of  receptor-ligand  the calcium-sensitive  of F d l l  cells  t o Fd,  dye,  Quin-2  27 fluorescence  was d e t e c t e d ,  intracellular expressed  calcium  indicating  an i n c r e a s e  and t h e presumed b i n d i n g  on t h e s u r f a c e  i n the level of  o f F d by  FdllF  of the cell.  Summary  The the  s t u d y o f t h e immune r e s p o n s e t o a s y n g e n e i c t u m o r  impetus  response allow  f o r t h e development  t o t h e P815 tumor,  the growth  activity  specific  thymocytes  allowed  Ts p l a y  o f t h e tumor. f o r P815 were  o f P815-bearing mice  hybridoma.  Interestingly,  the selection  was a n a l y z e d u s i n g  proteins  of similar  hybridomas.  Soluble  initially  was e l u t e d  advantage  that  hybridoma  When  this  of the Ag-binding properties thesis.  i t s e l f but  binding  of FdllF  offered  of  Bl6G-reactive  reducing  conditions,  t h e A10 o r F d l l  of FdllF  was i d e n t i c a l column.  o v e r t h e A10  was t h e a v a i l a b i l i t y o f t h e n o m i n a l A g o f F d l l . study  suppressive  and t h e recovery  SDS-PAGE, u n d e r  of the dual  and  f r o m t h e A10  B16G was n o t A g - s p e c i f i c  from t h e Fd- o r Bl6G-Sepharose the Fdll  role  with  were r e c o v e r e d from e i t h e r  As a c o r o l l a r y  In the  prepared from  and then l a t e r  a n d B16G, t h e SDS-PAGE a p p e a r a n c e it  extracts  TsF from t h e s e c e l l s .  material  r  B16G.  t h e predominant  o f Ts h y b r i d o m a s  apparently Ag-specific  M  o f t h e mAb,  provided  of FdllF  The  t o Fd whether major  hybridoma  Indeed, t h e  i s a major  subject  of  28 Purpose  of  the  Investigation  T h a t F d l l F was  suppressive  suggested  i t might  be  mentioned  group  proteins  study into  the  likely  to  response.  with  classified  FdllF further with nature  Furthermore,  FdllF  of  at  of  an  T  the  emphasis  the  and  along  known as hope of  to  the  immunochemical, describing  of  Ag  p r o m p t e d us  molecules.  the  of  FdllF  was  anti-Fd  a detailed examination and  to  additional insight  properties  biological, the  bind  previously  This  providing  understanding  work c o m p r i s e s  to  the  cell-derived, Ag-binding  present  on  able  with  TsF.  i n v e s t i g a t i o n i n t o the  contribute The  in vivo  biochemical  Ag-binding properties  of  of  levels FdllF.  29 M A T E R I A L S AND  Buffers  The in  and  METHODS  Solutions  compositions of the buffers  t h e Appendix,  Experimental  and s o l u t i o n s  THE C O M P O S I T I O N S  OF B U F F E R S AND  Cell  They were u s e d between  development  description product,  of the Fdll  were  of the University  of British  6 a n d 12 w e e k s o f a g e .  FdllF  originally  made  production  have  been  described 1987).  of FdllF  The F d l l  cell  sorter  was f o u n d t o b e Thy 1  e x p r e s s e s b o t h H-2  +  four  (FACS)  preliminary  of i t s secreted  previously  f o r at least  d  fusion  and a  i n 1985 a n d h a s r e m a i n e d  fluorescence-activated hybridoma  hybridoma  of the immunological properties  Stammers, N o r t h and Levy  by  mice  1  Lines  The  its  x DBA/2)F  from t h e Animal Care Unit  Columbia.  SOLUTIONS.  Animals  B10.BR, DBA/2, B10.D2 a n d ( B a l b / c purchased  used are l i s t e d  (Steele,  hybridoma stable  with  years.  Chu,  was respect  As a s s e s s e d by  analysis,-the  and F23.1 " -  The  to  Fdll  hybridoma  k a n d H-2  o f t h e BW5147 c e l l  Ag.  T h e DC10 h y b r i d o m a  line  with  T cells  was  obtained  derived from  30 DBA/2 m i c e p r i m e d t o control  i n these  phosphoribosyl from the  a  l e u k e m i a - a s s o c i a t e d Ag  studies.  The  transferase  American  hypoxanthine  negative  Type C u l t u r e  lines producing  the  were  created  laboratory  described Levy was  (Weaver,  1982). also  1985).  The  Sikora,  cell  produced The  tissue and  in this  in this  shown t o  service  be  MO),  at  37°C,  Conventional  The  mAb,  in a  and  16a,  of  10%  C0  6-90  DBA/2)F^  an  Immediately before of  5 X  animals.  MD).  Two  respectively,  previously Weaver,  and  mAb,  Stammers,  Type C u l t u r e  B16G  and  Levy  by  the  Collection  were m a i n t a i n e d Grand  CPSR-3,  were r a i s e d  10^  cells  Three to cells,  the  0.5  (Pristane,  inoculation  gamma r a d i a t i o n  6-90,  obtained  l i n e s were t e s t e d  A l l cells  (FCS,  was  TsF-specific  (Gibco,  a  Island,  Sigma,  St.  ascites  by  in  NY) Louis,  Antibodies  i n j e c t i o n of  Tetramethylpentadecane  rads  cell  line  Sikora,  (Maier,  American  B16G  i n j e c t i o n of  intraperitoneal  the  as  incubator.  2  and  (Balb/c  the  the  Monoclonal  approximately  receiving  Fdll  c a l f serum  injecting X  and  and  1982;  laboratory  E a g l e ' s medium  fetal  16a  producing  used  (Rockville,  have been  Levy  mycoplasma-free.  Dulbecco's Modified 10%  mAb, and  and  line  B W 5 1 4 7 , DC10  culture  containing  anti-Fd  was  guanine  BW5147 c e l l  Collection  cell  and  ml  as  intraperitoneally f i v e days p r i o r  to  mice were p r e t r e a t e d of  with  2,6,10,14-  Aldrich,  with  into  cells,  (Gamma-cell, Atomic  Milwaukee, the  mice  Energy  WI).  received of  Canada,  250  Ottawa,  Ont.).  10  after  days  collection at  used  of fluid,  (SN)  The c e l l s  cells  additional  mice,  and stored  was c o l l e c t e d ,  precipitate  a t 4°C.  with constant  solution at  the pellet  The s u p e r n a t a n t  Los Angeles,  was g e n t l y  solution to  The s o l u t i o n  the precipitation  resuspended  supernatant  CA).  into  Monoclonal  To  t o t h e mAb  a t 4°C u s i n g 1 4 , 0 0 0 M  was d i v i d e d  -70'C.  nitrogen.  w a s s p u n a t 9 0 0 0 g f o r 30 m i n a t 4 ° C .  changes),  (Spectrum,  o r added t o d i m e t h y l  a t room t e m p e r a t u r e .  equal t o the o r i g i n a l  ( 2 x 2 1  i n PBS  stirring,  a t 4°C o v e r n i g h t t o c o m p l e t e  volume  resuspended  s a t u r a t e d ( N H ^ ^ S O ^ was  stirred  centrifugation,  pelleted  fraction,  50% s a t u r a t i o n  precipitate  and d e b r i s were  When a p p r o x i m a t e l y 2 5 m l o f  achieve  The  about  after the  a n e q u a l v o l u m e o f PBS was a d d e d t o i t .  t h e gamma g l o b u l i n  added drop-wise,  was i n i t i a t e d  Immediately  were e i t h e r  a n d FCS a n d f r o z e n i n l i q u i d  was p o o l e d  fluid  of cells.  the ascitic  to inoculate  sulfoxide  of the ascitic  the introduction  4 0 0 g f o r 10 m i n .  and  SN  Tapping  o f mAb. Following  i n PBS t o a  and d i a l y z e d r  was  cut-off  a g a i n s t PBS  dialysis  Following dialysis,  tubing  t h e mAb  50 | i l , 1 a n d 5 m l a l i q u o t s  Ab p r e p a r a t i o n s were t h a w e d  and frozen  immediately  before use. Affinity-purified  C A M A L - 1 , a mAb s p e c i f i c  a s s o c i a t e d A g , was u s e d supplied  by Joan  over  leukemia-  p r e p a r a t i o n , a n d was  Shellard.  Affinity-purified for  as a c o n t r o l  f o r a  B10.BR a n t i - F d Ab was u s e d  t h e measurement  o f Ab.  a Fd-Sepharose  column.  Antiserum After  as a standard  was p r e p a r e d  washing  and passed  t h e c o l u m n , A b was  32 eluted  using  0.1  t h e n made i n t o concentration  M HC1,  neutralized, dialyzed against  a l i q u o t s which o f t h e Ab  method m o d i f i e d  were f r o z e n  s o l u t i o n was  a t -20'C.  PBS The  measured a c c o r d i n g  f r o m L o w r y e t a l . (1951)  and  (see below  protein to  for  a  details).  Antigens  Fd  isolated  f r o m C.  (374805,  Calbiochem,  Fraction  V,  NJ)  and  Sigma),  cytochrome  reconstituted aliquoted, OFd,  and  N-,  La  (Type  at  CA),  water  solution hr  on  by  a t 1 mg/ml.  ice.  a d d i n g an  After  c e n t r i f u g a t i o n , the p e l l e t  pi  of  46  mM  Trypsin  protein  ratio  2  the  (w/w;  p r e p a r e d by  stirring  18.75  KLH  (BSA,  Freehold,  were  acid  was  allowed  of  1 mg/ml,  p e l l e t e d at  was  ice-cold  8.1,  100%  ug  u.g i n 30  continued  i n a sealed  overnight vial.  1 mM  TCA  to a  o f TCA-Fd with  11.5  enyzme  to  HC1)  was  for 1 10  ethanol.  was  in  prepared  720  mM  added  a t room t e m p e r a t u r e  N-molecule  Fd  vacuum.  supplemented  p i of  had  (TCA-Fd).  11,600 g f o r  d r i e d under  d i s s o l v i n g 750  that  to continue  ( T R T P C K , W o r t h i n g t o n ) , a t a 1:40  d i g e s t i o n was  gentle  Sigma),  e q u a l volume o f 20%  washed once w i t h  T r i s - H C l b u f f e r , pH  CaCl .  Worthington,  to a concentration  Precipitation  The  was  serum albumin  trichloroacetic  min.  C-molecule  was  Sigma),  were p r e p a r e d from n a t i v e Fd  I n s o l u b l e p r o t e i n was  pellet  F7629,  -20°C.  C-molecule  produced  bovine  ( L y s f 8D8,  been p r e v i o u s l y denatured w i t h T C A - F d was  ( t y p e V,  I I I , C-2506,  distilled  stored  and  Jolla,  lysozyme C  with  pasteurianum  and  with by  33 resuspending 750 pH 7.5,  ug of TCA-Fd i n 720  supplemented with 0.5 M NaCl.  (COAPMS, Worthington) was (w/w;  u l of 25 mM  Tris-HCl buffer,  Carboxypeptidase  added at a 1:25  enzyme t o p r o t e i n  30 ug i n 40 u l of 10% L i C l ) and d i g e s t i o n was  o v e r n i g h t at room temperature vial.  A ratio  continued  with g e n t l e s t i r r i n g i n a s e a l e d  F o l l o w i n g o v e r n i g h t i n c u b a t i o n , both d i g e s t s were  a l i q u o t e d and s t o r e d at -20"C.  OFd was  prepared as d e s c r i b e d by  Tanaka et a l . (1964) by resuspending 750 formic a c i d and 67 u l of p e r f o r m i c a c i d .  ug of TCA-Fd i n 33 u l of Performic a c i d  was  prepared by mixing nine p a r t s of formic a c i d with one p a r t of ^2^2 for  f  o  r  2.5  NaCl-dry  ^  h  r  a t  r  o  o  m  temperature.  The Fd s o l u t i o n was  hr at -10"C, maintained at t h i s temperature i c e bath.  The r e a c t i o n was  30%  reacted  with a 5 M  stopped by l y o p h i l i z i n g  the  protein.  Enzyme-linked  Immunosorbent  Assay  The amount of a n t i - F d Ab present i n the s p l e e n c e l l was  measured u s i n g ELISA.  Dynatech, A l e x a n d r i a , VA)  P o l y s t y r e n e ELISA w e l l s were coated with 100  culture  (Immulon I,  u l of Fd  prepared at 1 ug/ml i n b i c a r b o n a t e b u f f e r , o v e r n i g h t , at under humid c o n d i t i o n s .  37'C,  A f t e r washing, 100 u l  SN were added t o the w e l l s and allowed t o r e a c t f o r 2 hr at  37*C under humid c o n d i t i o n s . 100  solution  Between each i n c u b a t i o n step, the p l a t e s  were washed t h r e e times u s i n g PBS-Tween. of  SN  The p l a t e s were washed again and  u l of r a b b i t anti-mouse i m m u n o g l o b u l i n - a l k a l i n e  phosphatase  34 (RaMIg-AP,  J a c k s o n , West G r o v e ,  PBS-Tween, were added same c o n d i t i o n s  and  PA) , u s e d a t 2 u g / m l ,  allowed  as above.  to react  A f t e r washing,  nitrophenyl  phosphate  105,  i n diethanolamine buffer  Sigma) FdllF  plates  was  measured  concentrations  containing  2 h r a t 37'C  t h e T s F - s p e c i f i c mAb,  at  18  ug/ml,  above.  F d was  incubated solution For  o f 16  a t 4°C was  After  Various  f o r 2 hr.  The  amount o f f r e e  immunoassays,  an automated  Ont.)  s e t a t 405  nm.  gently  the substrate The  (Multiskan,  and t h e average v a l u e  was  added  by  fluidof Fd, were  shaken  and  F d l l F remaining i n  was  plates Flow,  Each ELISA determination  ul  a  the ELISA described  conditions.  ELISA reader  was  100  previously.  C d i l u t e d i n PBS,  mixture  wells  described  concentrations  of F d l l F s o l u t i o n , used at  under humid  material  washing,  to Fd-coated wells  ELISA.  at  to Fd-coated  The  using  triplicate  or fractionated  ug/ml.  a l l three  104-  Fd-coated  were u s e d as m e n t i o n e d  then determined using  3 h r a t 37'C  ELISA.  d i l u t e d i n P B S - T w e e n , was  or the c o n t r o l , cytochrome volume  buffer,  added.  u l volumes  of F d l l F binding using  p-  f o r 1 hr at the conditions  substrate  measured  t o an e q u a l  concentration  to  and  inhibition  analogs,  added  incubated  RaMIg-AP  The  its  and  the  F d l l F or control material  conditions.  B16G,  u l of  solid-phase  i n 100  under humid  100  were  d i l u t e d i n PBS,  of  phase  using  F d l l F were added  f o r 1 hr under  (one t a b l e t / 5 m l  were p r e p a r e d as above.  various  for  also  substrate  diluted in  was  above.  developed f o r 1 were  read  Mississauga, performed i n  used f o r data c a l c u l a t i o n s .  35 Immunization  of  Animals  B10.D2 a n i m a l s by  a boost  w i t h Fd.  molecule  i n PBS  Freund's  adjuvant  of  0.1  ml.  (s.c.)  were immunized t o Fd  prepared  The  i n the  The  (H37  mice were p r i m e d as  Ra,  a  on  secondary  immunization  exception  that  50  Freund's  adjuvant  opposite  flank  ug  was  (Difco)  and  both  site  and  with  C-  complete volume  subcutaneously 21  identical using  days,  the  manner w i t h  the  incomplete  injection  injection.  in a similar  of  in a total  After  of  ug  followed  was  i n the  Hyperimmune  manner w i t h  secondary  the  immunizations  were  Fd.  Animals 21  were days  i m m u n i z e d w i t h 50  later.  administration  accomplished KLH  Enrichment  by  was  for  giving  t h e Ag  lysozyme and  w i t h Fd  route  a  of  was  chromatography.  and  KLH  was of  the  ug/mouse.  using Immunoaffinity  material  f o l l o w e d by  separately to opposite sides  a d m i n i s t e r e d a t 50  FdllF-enriched  of  Fd.  o f DBA/2 a n i m a l s  of F d l l F  immunoaffinity  ug  Antigen emulsion  were as  Co-immunization  mice.  side.  first  the primary  MI)  administered  the  of the  exception  boost  emulsion  were.prepared  were p r e p a r e d  with  was  w i t h 50  Detroit,  g i v e n i n an  B10.BR a n i m a l s that  (v/v)  the ventral  of Fd  to that  1:1  Difco,  immunization  flank  u s i n g C-molecule  Chromatography  recovered from  the  Cultures of F d l l  SN  using  hybridoma  cells  were grown t o a d e n s i t y of about 5 X 10 Typically,  t o 1 X 10 /ml.  2 1 of c e l l s were grown f o r each l o t of F d l l F .  Supernatant was r e c o v e r e d by c e n t r i f u g a t i o n u s i n g a S o r v a l l rotor  (Dupont, Newtown CT) spun at 4,300 g f o r 20 min.  Supernatant was pumped onto a column Rad,  GS-3  (2.5 x 23 cm, Econo, B i o -  Richmond, CA) at a flow r a t e o f 60 ml/hr,  B16G-Sepharose CL-4B beads. (Pharmacia,  Montreal, P.Q.)  c o n t a i n i n g 5 ml of  B16G was coupled t o Sepharose u s i n g CNBr  (F964-5,  P h i l l i p s b u r g , NJ) as d e s c r i b e d by Cuatrecasas  CL-4B  Baker, (1970).  However,  j u s t b e f o r e SN was pumped onto the column, the beads were p r e c o n d i t i o n e d with 5 ml of 0.1 M HC1 f o l l o w e d by washing with 200 ml of PBS.  Once the SN had been loaded, the beads were  washed w i t h PBS at a r a t e of 100 ml/hr u n t i l the absorbance (280 nm) o f the e f f l u e n t was l e s s the 0.005.  P r o t e i n was e l u t e d  u s i n g 10 ml o f 0.01 M HC1. The e n t i r e 10 ml o f e l u a t e was c o l l e c t e d and then n e u t r a l i z e d by adding 1.5 M T r i s - H C l , pH 7.5, 100 u l at a time.  T h i s was c o n t i n u e d u n t i l the pH o f the  s o l u t i o n reached 7.5. immediately  d i a l y z e d a g a i n s t PBS and then c o n c e n t r a t e d u s i n g  microconcentrators Danvers,  F o l l o w i n g n e u t r a l i z a t i o n , the s o l u t i o n was  MA).  (30k M  r  c u t - o f f , Centricon-30, Amicon,  A f t e r c o n c e n t r a t i o n , a l i q u o t s of F d l l F - e n r i c h e d  m a t e r i a l were prepared u s i n g p o l y p r o p y l e n e tubes and then at  -70"C.  frozen  I f the sample was t o be used the next day f o r  p r e p a r a t i v e , non-reducing SDS-PAGE or h i g h performance g e l filtration ul,  a n a l y s i s , then i t was c o n c e n t r a t e d t o 500 u l or 200  r e s p e c t i v e l y , and s t o r e d at 4°C u n t i l use.  Samples used f o r  37 biological instead FdllF  o f PBS.  The  protein  (1951). water.  of  The p r o t e i n  was t h e n m i x e d  containing  standard  In v i t r o  were p r e p a r e d  o f 49 p a r t s  shaken  t h e tube  from  and  screen.  distilled  was s h a k e n  and  and p o r t i o n s  of this  reagent  f o r 30 m i n .  water  solution  and  to 1 were  after  (Fisher, Fairlawn, immediately.  The  At t h e end of t h i s  w a s r e a d a t 7 5 0 nm.  A series  2.5 t o 50 u g o f p r o t e i n ,  time,  o f BSA  was u s e d f o r  curve.  Measurement  immunization  made i n t o  was  (Na2C03 s o l u t i o n )  immediately  of FdllF  Suppressive  Fd-immune B10-D2 m i c e w e r e k i l l e d booster  i n 200 u l o f  Two m i l l i t r e s  mixture,  colour of the solution  material  w i t h 2 0 0 u l o f 1 N NaOH.  citrate  v/v.  developed  a t 4°C.  o f 2% Na2CO-3 i n d i s t i l l e d  i n a ratio  Again,  was t h e n  solutions, the  were c o n d u c t e d  5 m i n , 200 u l o f 1 N P h e n o l  were added.  the  concurrently with  o f t h e method o f Lowry e t a l .  i n 1% s o d i u m  were mixed  1640  A l l procedures,  s a m p l e was d i s s o l v e d  (CuSO^ s o l u t i o n ) ,  reaction  was p r e p a r e d  s e t o f equipment.  stock solutions  added t o t h e p r o t e i n  NJ)  a g a i n s t RPMI  concentration of the B16G-eluted  CuSO^.S^O  exactly  material  chromatography,  This solution  each  were d i a l y z e d  by a m o d i f i c a t i o n  separate  part  Control  affinity  determined  0.5%  i n vitro  using a duplicate  including  Two  testing  single  The c e l l s  with Fd. cell  13 t o 15 d a y s  Spleens  suspensions  Activity  following the  were removed by passage  were washed t h r e e t i m e s  aseptically  through  u s i n g RPMI  a wire  1640 ( 1 0 -  38 601-24,  Flow,  penicillin  "Original"  with  VA)  supplemented with  and streptomycin  respectively, a)  Mclean,  Gibco)  (100 u n i t s / m l  (4192,  a n d 100  ug/ml,  the cells  were  a n d s p u n a t 400 g.  protocol:  A f t e r washing,  F d which had been p r e v i o u s l y  filter  10% FCS a n d  Gelman, Ann A r b o r ,  sterilized MI).  Prior  using  pulsed  a 0.22  um  t o p u l s i n g , about  1  7 X 10  cells  Fd.  were p l a c e d  into  a separate  tube  and d i d not r e c e i v e  P u l s i n g c o n d i t i o n s w e r e 250 ug F d / 1 . 5 m l m e d i u m / s p l e e n .  pulsing of  medium was  20%.  10%  CO2  supplemented with  The p u l s e d atmosphere  and unpulsed  washing,  cells  f o r 2 hr using  were resuspended g e n t l y the cells  twice  to a concentration  final  concentration  FdllF  o r c o n t r o l m a t e r i a l was a d d e d  cultured  f o r seven  SN w e r e c a r e f u l l y measured b)  a t 37°C  at this  i n a 10% C 0  at a concentration  continued treated  Cells  were p u l s e d  as d e s c r i b e d  2 hr.  suppression  formula:  X  Sigma)  to a  time. 2  The c e l l s  atmosphere. samples  were  Culture were  initially  with  FdllF  A t t h e e n d o f 2 h r , F d was above.  A f t e r washing,  as above b u t n o t c o - c u l t u r e d w i t h  Percent the  f o r another  After  ELISA.  c o n t r o l m a t e r i a l , instead of Fd.  added  cells  and p l a t e d out as 2 ml c u l t u r e s .  using  protocol:  The  o f 2.5  (M 6 2 5 0 ,  removed on d a y 7 a n d u n d i l u t e d  f o r Ab c o n t e n t  "Revised"  or  days  tubes.  the 2 hr period.  2-mercaptoethanol  o f 50 uM,  concentration  were c u l t u r e d a t 37'C i n a  polypropylene  during  were a d j u s t e d  10 / m l , s u p p l e m e n t e d w i t h  FCS t o a f i n a l  The  f o r both protocols  Pulsing the cells  was were  FdllF. was c a l c u l a t e d  using  39  ( (Ab  level  of Fd-pulsed Ab  The  Ab  level  background FdllF  unpulsed  s u b t r a c t e d from  cultures  prior  of the  to the  the  Fd  and  tail  21  responders divided group Mice  KLH  21  days  later  (>0.5) o r  into  FdllF,  c o n t a i n i n g an were t h e n  intravenously  anti-Fd  and  each  and  plates,  serum  Tween.  The  calculation  The  ranked,  PBS  number o f  Prior  samples were d i l u t e d  data  at  Similar  1/100  as  10  were  groups w i t h  each  "low"  ug A l O F  were a s s e s s e d  hours, later  as  paired  at  and an  PBS  days  addition  as  responders. or  to their  were r e c o r d e d  from  "high"  for several  1/100  bled  They  They were b l e d seven  animal.  co-immunized  were t h e n  " h i g h " and  recover  responses  suppression.  (<0.5) t o F d .  FdllF,  and  FdllF  then  "treatment"  ug  the  Fd-pulsed  of percent  u s i n g ELISA,  equal  allowed to  as  of the  and  H-  cultures)  100%  used  animals  responders  10  x  Response u s i n g  and  KLH.  was  levels  A10F,  a n t i - K L H Ab  individual  RESULTS).  and  "low"  (i.v.),  w i t h Fd  the  later.  of F d l l F  cultures)  w i t h C-molecule  given either  boosted  for  days  level  culture  Serum A n t i b o d y  DBA/2 m i c e w e r e p r i m e d with  - Ab  of Fd-pulsed  of the  and  Suppression  level  cultures  1/200  v a l u e s were o b t a i n e d at b o t h  and  using (see  dilutions.  the  samples  to the  "X-Y"plot  and  ELISA PBS-  40 Sodium Dodecyl  Sulphate Polyacrylamide Gel  B16G-eluted FPLC and  samples  mini-Protean  gels based  II slab  the manufacturer's  acetone  precipitation  consisting Toronto, mM  of  10%  Ont.),  were heated  5 to  used  about  lane.  10%  for the  gel buffers  Protein  instructions. and  then  mM  Tris-HCl  2%  the  Bio-Rad).  f o r 4 min.  Prior Between  When a p r o t e i n  analysis.  glacial  acetic  heating  the  continued  another the  T)  (1970) .  The  according  (w/v;  sample  B44244,  to  loading,  1 to  2  ug  by  buffer BDH,  bromophenol b l u e the  of  and  10  samples  protein  d e t e r m i n a t i o n was  not  unconcentrated  APPENDIX f o r f u r t h e r  using silver  fixative,  removal  made, sample  details  the  oven  staining  g e l was  fixed  f o r a minimum o f  g e l was The  water.  a t room t e m p e r a t u r e .  process  i n 100  ml  g e l was  After  30  rehydrated i n  f o r 1 min.  The  based i n 100  was  ml  of  10%  Rehydration  by was  mixture  and  rehydrated for  the water  of d i s t i l l e d  on  min.  assisted  of the methanol-acetic acid  with distilled  equilibrated  The  acid  acid:10% methanol.  the  5 min  g e l was  acetic  g e l i n a microwave  by  replacement  of  (%  used  i n SDS  volume of the  See  was  from  solutions.  glacial  removal  SDS  buffer,  bands were v i s u a l i z e d  methanol:10%  After  prepared  Samples were c o n c e n t r a t e d  solubilized  (w/v),  of the t o t a l  and  (Bio-Rad)  t h e method o f Wray e t a l . (1981). 50%  fractions  t h e method of Laemmli  g e l apparatus  (DTT,  100'C  loaded per  between was  to  on  glycerol  62.5  dithiothreitol  were  individual  p r e p a r a t i v e SDS-PAGE w e r e a n a l y z e d u s i n g 10%  polyacrylamide  to  and  Electrophoresis  was  water  removed, containing  33  u l o f 100  mM  DTT.  oven f o r 1 min.  The  time, ul  continued  developing  o f 37%  water, gel  s o l u t i o n was  f o r 15 min  reagent,  formaldehyde  was  was  t h e g e l was  i n a microwave  r e p l a c e d w i t h 100  ml  prepared.  a t room t e m p e r a t u r e .  c o n s i s t i n g o f 40 ml  The  AgNO^ s o l u t i o n was  r i n s e d w i t h a b o u t 20 ml  of d i s t i l l e d  of developing reagent.  ml  During  of  t h e bands began t o appear.  r e a c t i o n was  20 ml  o f 2.3  distilled  Lastly,  water f o r storage u n t i l  200  the  Immediately  replaced with  until  acid.  and  rinsed with  The  M citric  this  distilled  water.  remainder of the developing reagent. The  Sigma).  p o u r e d o f f and  t h e g e l was  T h i s was  of  15% ^ 2 0 0 ^  (F-79B, F i s h e r ) i n 200  f o l l o w i n g the removal of the water, 20 ml  heated  w a t e r c o n t a i n i n g 1 ml o f 10% AgNOj (S 6506,  distilled T h i s was  Again,  r e a c t i o n was  about  the continued  stopped  with  g e l s were t r a n s f e r r e d t o  dried using a slab gel  dryer  (Bio-Rad).  High Performance Gel  Filtration  B 1 6 G - e l u t e d F d l l F was filtration  f r a c t i o n a t e d by h i g h p e r f o r m a n c e g e l  u s i n g the Pharmacia chromatography system  S a m p l e s w e r e c o n c e n t r a t e d t o a v o l u m e o f 200 0.22  um  filter  (SLGVL04S, M i l l i p o r e ,  a S u p e r o s e 12 c o l u m n ml/min. run. unit.  B e t w e e n 150  (HR  M a t e r i a l was  ul, filtered  onto  10/30, P h a r m a c i a ) a t a f l o w r a t e o f  0.25  nm  Bedford,  ug o f t o t a l was  MA)  and  using a  loaded  t o 200  A b s o r b a n c e a t 280  (FPLC).  p r o t e i n were l o a d e d  per  r e c o r d e d u s i n g t h e FPLC i n t e g r a t o r  e l u t e d w i t h PBS  and  48  0.5  ml  fractions,  42 equivalent  t o one column volume,  calibrated  using  M  the following M  ; IgG, 150k M ;  BSA,  r  were h e l d  a t 4°C u n t i l  Fractions  assayed  filtration  using  Preparative  were c o l l e c t e d . markers:  r  thyroglobulin,  66k M ;  c y t o c h r o m e C,  assayed  f o r Ag-binding  r  forbiologic activity a 0.22  um  filter  Sodium Dodecyl  The c o l u m n  were  12.4k M  was  669k  Fractions  r  activity. sterilized  by  (Millipore).  Sulphate Polyacrylamide  Gel  Electrophoresis  B 1 6 G - e l u t e d F d l l F was PAGE.  The P r o t e a n  according of  I I slab  consisting  material  were  and bromophenol blue.  2% SDS  lanes  APPENDIX  f o r f u r t h e r d e t a i l s about  circulating until  t h e dye f r o n t  After  electrophoresis,  and  each piece  containing 4°C, MD,  (% T) p o l y a c r y l a m i d e  were r u n a t c o n s t a n t polyethylene  glycol.  gel buffers  current  sample  Tris-HCl  gel.  the g e l lanes and p l a c e d  See  and s o l u t i o n s .  ( 3 5 itiA) a n d c o o l e d was  MD)  with  continued  w e r e c u t i n t o 5 mm  slices  into polypropylene  tubes  After  e l u t i o n f o r 18 h a t  was t r a n s f e r r e d t o a m i c r o d i a l y s i s u n i t  BRL, G a i t h e r s b u r g ,  buffer  1 cm o f t h e e n d o f t h e g e l .  0.75 m l o f 0.05 M NH^HCC^.  the material  micrograms  6 2 . 5 mM  Electrophoresis  reached t o within  was m i n c e d  used  hundred  i n SDS  (w/v),  was  SDS-  The s a m p l e s w e r e r e s o l v e d i n  adjacent  The g e l s  a 12%  Two  solubilized  (w/v),  preparative  (Bio-Rad)  instructions.  o f 10% g l y c e r o l  using  fractionated using  g e l apparatus  t o manufacturer's  F d l l F a n d DC10  buffer  also  and d i a l y z e d  against  RPMI  (1200  1 6 4 0 f o r 18  43 h.  A second  the  e l u a t e s were  Ag-binding  round  apparatus  water 0.IM  (RBS-35,  were  water  first  (B10290,  r e d u c i n g SDS-PAGE  (IEF x  followed.  f o rthe first Pierce,  analysis  by d i s t i l l e d  Modifications  water.  IL) and r i n s e d The t u b e s  particulate  dimension  gel solution  with tap  matter  suction.  Immediately  were  Prior  bath  by adding  following and  (TEMED; B i o - R a d )  urea  3, [ ( 3 -  (CHAPS), a n d  t o g e t h e r and then  to dissolve  was d e g a s s e d  (APS; Bio-Rad)  the urea.  heating Any  ( 0 . 4 5 urn, 4 1 8 4 ,  f o r 10 t o 15 m i n u s i n g  the addition  of  water  ammonium  N,N,N',N'-Tetramethylto the g e l solution,  t h e tube  c a s t e d and a l l o w e d t o p o l y m e r i z e f o r 2 t o 3 h r .  t o sample  catholyte  by  acrylamide solution,  was r e m o v e d b y f i l t e r i n g  The s o l u t i o n  ethylenediamine  boiled i n  followed  was p r e p a r e d  LKB, Pharmacia)  i n a 37'C w a t e r  Gelman).  persulphate  listed  and d r i e d .  (pH 3 . 5 - 1 0 ,  mixture  slab  are  were t h e n  w i t h t a p water,  BDH), H P L C - q u a l i t y w a t e r ,  ampholines  gels  and  were washed i n  cholamidopropyl)-dimethylammonio]-1-propanesulfonate  the  out and  SDS-PAGE)  dimension  Rockford,  f o r 30 m i n a n d r i n s e d  distilled The  (Bio-Rad)  followed HC1  was c a r r i e d  p r o v i d e d w i t h t h e m i n i - P r o t e a n I I IEF and  Glass tubes  detergent  f o rboth  Gel Electrophoresis  Instructions  below.  combined  and d i a l y s i s  ELISA determinations.  Two-dimensional  gel  of elution  loading,  were p r e p a r e d .  degassed Excess  solutions  unpolymerized  o f a n o l y t e and g e l was  removed  from the t o p of the s o l i d i f i e d bubbles  were d i s c a r d e d .  of  the tube  in  20  gels  and f i l l e d  underlaying  the catholyte.  application  o f 30  marker  along  a tube  M 5768,  ( lmg/ml,  with was The  Sigma),  o f t h e pH  out  a t 500  V  m i n a n d 1500  During  the IEF stage,  length) washed for  out with  loading.  extruded  distilled  water  and M  r  weight  performed  as d e s c r i b e d .  gel  buffers The t h r e e cm  into  water.  The  estimates  into  each  were  were  Focusing  gels  was  (used  carried  min.  were c a s t e d . gels  Thirty (5  mm  i n  the wells  standards  were  were  prepared  g e l was  of the second dimension  loaded  sample b u f f e r  SDS  with  and e l e c t r o p h o r e s i s  See APPENDIX f o r f u r t h e r d e t a i l s  DTT.  was about  and s o l u t i o n s . tube  sections a tube  standards  by  by t h e  blank  of IEF, each tube  into the well  ug  6 p i o f M e t h y l -Red  polymerization,  At the completion  length-wise  gels  each  10  (about  sample g e l s  of IEF, s t a c k i n g  Following  Molecular  pH  SDS  a n d e q u i l i b r a t e d f o r 3 m i n i n 2 x SDS  gel  1  12%  f o r 90  connected t o  protected  and two o t h e r  V  trapped a i r  a microsyringe  The  gradient).  the conclusion  were poured.  s a m p l e was  gel containing  t h e measurement  minutes before  with  Sample  using  buffer.  for  f o r 10  catholyte.  loaded  u l of overlay  with  Any t u b e s  Sample r e s e r v o i r s were  u l o f sample b u f f e r )  focussed  gels.  gels  without  and each group  containing  250  samples were e x t r u d e d o f 3 x 1 cm g e l p i e c e s  u l of HPLC-quality,  ampholines were e l u t e d w e r e made b y  of the  tubes.  and c u t i n t o was  degassed  f o r 2 h r a t room  i m m e r s i n g pH t e s t s t r i p s  placed distilled  temperature. (pH 5 - 1 0 ,  BDH)  45 RESULTS  Recovery  The  of FdllF  from Culture  SN o f o v e r g r o w n  FdllF,  were p a s s e d  Supernatants  cultures of Fdll  over  using was  aliquots for  phosphate buffered  dialyzed against and frozen  immunoreactivity. FCS,  (PBS),  FdllF  neutralized with  PBS a n d c o n c e n t r a t e d ,  a t -70"C.  protein determination,  After extensive  saline  0.1 N H C 1 a n d i m m e d i a t e l y  then  containing  a column o f S e p h a r o s e CL-4B beads t o  w h i c h B16G was c o v a l e n t l y l i n k e d . column w i t h  hybridoma  washing was  ofthe  eluted  Tris-HCl. divided  Samples were t a k e n  from  I t  into each  batch  SDS-PAGE a n a l y s i s a n d m e a s u r e m e n t o f  C o m p l e t e t i s s u e c u l t u r e medium i n c l u d i n g 10%  o r SN f r o m t h e f u s i o n p a r e n t  BW5147 o r D C 1 0  hybridoma,  a n o t h e r BW5147 f u s i o n p r o d u c t  o f unknown s p e c i f i c i t y  prepare  a d u p l i c a t e B16G-Sepharose  control material with  were used t o column.  Immunochemical P r o p e r t i e s o f F d l l F  The  recovery  of FdllF  be  monitored using  of  FdllF  a solid-phase  A f t e r washing, (IgG2a)  Various  B16G a n d an i r r e l e v a n t  when s o l i d - p h a s e  phosphatase  columns  with  m a t e r i a l was a d s o r b e d  mAb  rabbit  (RaMIg-AP). at a  could  concentrations  adsorbed t o ELISA  were added and d e v e l o p e d  immunoglobulin-alkaline that  ELISA.  a n d DC10 c o n t r o l m a t e r i a l w e r e  overnight. subclass  from t h e immunoadsorbent  Fig.  wells  o f t h e same anti-mouse 1 shows  46  Fig.  1.  Binding  o f B16G t o F d l l F  adsorbed t o polystyrene  wells.  F d l l F a n d DC10 m a t e r i a l w e r e a d s o r b e d t o E L I S A w e l l s a n d d e t e c t e d w i t h B 1 6 G , u s e d a t 18 u g / m l . The E L I S A was d e v e l o p e d w i t h R a M I g A P , u s e d a t 0.5 u g / m l . T h e i r r e l e v a n t mAb, C A M A L - 1 , u s e d a t t h e s a m e c o n c e n t r a t i o n a s B 1 6 G , b o u n d t o n e i t h e r F d l l F o r DC10 material. (•) F d l l F + B 1 6 G ; + CAMAL-1.  (-) F d l l F  + CAMAL-1;  (O) D C 1 0  + B16G;  (•)  DC10  47 concentration phase F d l l F  of  but  6.6 not  CAMAL-1, d i d n o t Representative The  with  bind  data  E L I S A was  phase Fd.  ug/ml,  B16G  DC10  to  e i t h e r the  of three  assay  test  system,  Fd-coated plates.  detected  B16G  where v a r i o u s plates. its  and  binding  concentrations  to  Fd.  The  m e d i u m - d e r i v e d B16G Essentially observed. cellular  Thus  (DC10  B16G-binding Fig.  3  is a  compared t o  results  the  FdllF  isolated  of  18  and  of  from three  solid-  of  FdllF  then  both  curve  Fd-coated  FdllF  F i g . 2A  in spite  is  with  of  the FdllF. was from  reactivity  preparation,  in  the  i t s reactivity  i t s Ag-binding  in  site  and  intact. where an  irrelevant  FdllF.  concentrations,  different  with  show any  detect  B16G  to  shows a t y p i c a l  react  CAMAL-1 f o r F d l l F  r e a c t i o n of  was  were added t o  of m a t e r i a l were employed these  FdllF  bound F d l l F  CAMAL-1 w e r e u s e d  ug/ml t o  of  concurrently  suggested that  B16G  shown.  eluted control material  c o n t r o l experiment  even at  e x c e e d e d by  given  layer.  control preparation  a l s o d i d not  were  binding  concentrations However,  ELISA  B16G.  concentrations nonspecific  SN)  site  from the  mAb,  concentrations  c o n t r o l of  prepared  shows t h a t  f o r any  "sandwich"  negative  reactivity  F i g . 2B SN  assay. the  no  are  F i g . 2A  s t i l l  DC10  binding  FdllF  solid-  irrelevant  or  various The  of  could  eluate  FdllF  the  RaMIg-AP.  E v i d e n t l y , B16G  The  experiments  were added t o with  strongly with  material.  also used to  In t h i s  reacted  and  I t can  (ie. >  be  4  are  seen  when  that  high  ug/ml).  nonspecific  FdllF.  was  equivalent  began o n l y  the  experiments  at  mAb  signal  Representative shown.  was  48  F i g . 2. Binding of F d l l F its detection with B16G.  t o Fd  adsorbed  to polystyrene  wells  and  F d was a d s o r b e d t o E L I S A w e l l s a t 1 u g / m l . M a t e r i a l s e l u t e d from B16G a f f i n i t y c o l u m n s w e r e a d d e d t o s o l i d - p h a s e F d a t v a r i o u s c o n c e n t r a t i o n s a n d t h e n d e t e c t e d w i t h B16G a n d RaMIg-AP, u s e d a t t h e s a m e c o n c e n t r a t i o n s a s f o r F i g . 1. A. F d l l F (•) c o m p a r e d t o m e d i u m c o n t r o l ( • ) . r e s u l t s from seven d i f f e r e n t p r e p a r a t i o n s are B.  Representative shown.  F d l l F (•) c o m p a r e d t o t h e c e l l u l a r SN c o n t r o l DC10 (•). R e s u l t s (mean + SEM) f r o m 3 s e p a r a t e p r e p a r a t i o n s a r e s h o w n .  49  0.00  0.25  0.50  1.00  2.00  4.00  8.00  16.0  ug/ml Factor I  :  o-i  0.00  1  1  0.05  1.00  ug/ml Factor  —  , 2.00  1 4.00  50  OH  0.00  Fig. with  ¥  1.00  3. The r e a c t i v i t y FdllF.  1 2.00  ug/ml Factor  ¥  1  4.00  8.00  o f B16G a n d t h e i r r e l e v a n t  F d l l F a n d DC10 m a t e r i a l w e r e a d d e d t o F d - c o a t e d concentrations. They were d e t e c t e d w i t h e i t h e r u s e d a t 18 u g / m l , a n d R a M I g - A P . (-) B 1 6 G + D C 1 0 ; (•) B 1 6 G + F d l l F ; (•) C A M A L - 1 + F d l l F .  (O) C A M A L - 1  mAb,  CAMAL-1,  wells at various B16G o r CAMAL-1, + DC10;  51 FdllF  showed  o t h e r Ag. Fd,  The  selectivity  data presented  serum albumin,  cytochrome only.  C were adsorbed  The b i n d i n g o f F d l l F  FdllF.  of free  o f Fd were  FdllF  ELISA.  F i g . 4 shows  inhibit  t h e amount  C y t o c h r o m e C,  used  consequently  data  fine  inhibition  were mixed FdllF.  assay  used. was  measured as b e f o r e  that  increasing  of FdllF  also  shown.  are  wells  amount  concentrations of Fd could  protein, FdllF  plates.  d i d not bind t o  binding.  b i n d i n g was  t o Fd.  o f unbound F d l l F  Representative  expressing  and a t h i r d  was  then  this  either  analog, amount  OFd, of  be measured u s i n g  ELISA.  analogs  was  analogs  were adsorbed  assay,  the antigenicity  confirmed u s i n g Fd d e t e r m i n a n t - s p e c i f i c  determinants.  FdllF,  examined u s i n g  Analogs,  or the C-determinant,  to performing this  specific  and t h e  "sandwich"  at various concentrations with a constant  The  of  shown.  of FdllF  and analogs  Various  amount  i n the  binding t o Fd-coated  as t h e c o n t r o l  specificity  "sandwich"  Prior  mAb  was  bound t o Fd  any b i n d i n g  added t o Fd-coated  d i d not i n h i b i t  the N-determinant  the  then  when  protein, FdllF  e l u a t e s showed  were  that  incubated with a constant  of three experiments  The  iron-containing  t o Fd i n s o l u t i o n  T h e m i x t u r e was  amount  I demonstrate  to separate wells,  f o r t h e Ag t h a t  concentrations  i n Table  and another  None o f t h e o t h e r B16G  preference  and  f o r F d i n b i n d i n g when c o m p a r e d t o  t o ELISA wells  f o r the amino-terminal F i g . 5 shows  that  and then  of the mAb.  The  incubated with the  and c a r b o x y l - t e r m i n a l  the amino-terminal  determinant  52 Table I.  The b i n d i n g of F d l l F to Fd.  Fd or other antigens were coated onto ELISA w e l l s at 1 ug/ml. F d l l F or c o n t r o l m a t e r i a l s were added at 4 ug/ml and d e t e c t e d w i t h B16G and RaMIg-AP. R e p r e s e n t a t i v e data of t h r e e d i f f e r e n t experiments are shown. HSA:  human serum albumin  Source of B16G e l u a t e  Adsorbed a n t i g e n  Fd  HSA  cytochrome C  Fdll  .567  .090  . 103  medium  .060  . 015  . 087  DC10  . 050  . 000  BW5147  . 013  . 035  53  701  •  60-  •  60-  c o  a  40-  •/  rcent  •C C  30-  • Q.  20-  • 10-  ^  • • '  • •  0 -  Fig.  4.  .312  .625  • 1.25 2.5 ug/ml Inhibitor  •  1  5  The b i n d i n g o f s o l u b l e Fd t o F d l l F  10  i n fluid-phase.  F d l l F was mixed with v a r i o u s c o n c e n t r a t i o n s o f Fd (•) o r cytochrome C (•) p r i o r i t s a d d i t i o n t o Fd-coated w e l l s . FdllF was d e t e c t e d as f o r F i g . 2.  54  F i g . 5. Antigenic analysis determinant-specific mAb.  of Fd  and  i t s analogs  using  N-molecule (N), C-molecule ( C ) , OFd, F d , KLH a n d BSA w e r e c o a t e d o n t o E L I S A w e l l s a n d t h e n i n c u b a t e d w i t h t h e F d - s p e c i f i c mAb, 16a a n d 6-90, b o t h u s e d a t 2 u g / m l . 1 6 a i s s p e c i f i c f o r Cd e t e r m i n a n t , t h e c a r b o x y l - t e r m i n a l e p i t o p e o f F d a n d 6-90 i s s p e c i f i c f o r N-determinant, t h e a m i n o - t e r m i n a l e p i t o p e of Fd. M o n o c l o n a l a n t i b o d i e s were d e v e l o p e d w i t h RaMIg-AP.  55  s p e c i f i c mAb, analog  6-90,  bound t o both the carboxypeptidase A - t r e a t e d  (N-molecule)  and Fd.  I t d i d not b i n d t o the  t r e a t e d analog, known as C-molecule bovine serum albumin  (BSA).  d e t e r m i n a n t - s p e c i f i c mAb, was  a s l i g h t degree  of the C-molecule  of b i n d i n g t o N-molecule  not complete  Incomplete  OFd was  u s i n g the analogs.  and a s m a l l contaminated  with  and 16a.  Typical  are shown.  f i n e s p e c i f i c i t y of F d l l F b i n d i n g was  determined  10%  16a d i d not b i n d t o e i t h e r  r e a c t i v e with both 6-90  r e s u l t s of seven d i f f e r e n t experiments The  (approximately  d i g e s t i o n of N-molecule has been  noted p r e v i o u s l y i n t h i s l a b o r a t o r y . K L H or BSA.  and Fd; t h e r e  T h i s probably i n d i c a t e d t h a t  of the N-molecule p r e p a r a t i o n was  the u n d i g e s t e d Ag.  K L H or  In c o n t r a s t , the c a r b o x y l - t e r m i n a l  carboxypeptidase A d i g e s t i o n was percentage  or the i r r e l e v a n t Ag,  16a bound t o C-molecule  signal).  trypsin-  subsequently  F i g . 6 shows t h a t C-molecule  was  the most e f f e c t i v e i n h i b i t o r of F d l l F b i n d i n g c a u s i n g 50% i n h i b i t i o n at .312 f o l l o w e d by OFd.  ug/ml.  Fd was  the next b e s t  inhibitor  More than 15 times the amount of N-molecule  r e q u i r e d t o achieve the l e v e l of i n h i b i t i o n observed with molecule  and Fd.  In f a c t , the amount of N-molecule t h a t  r e q u i r e d suggested t h a t i n h i b i t i o n was  Cwas  most l i k e l y due t o the  contamination of the p r e p a r a t i o n with u n d i g e s t e d Fd.  The  i n d i c a t e t h a t F d l l F i s s p e c i f i c f o r the C-determinant  of Fd.  R e p r e s e n t a t i v e data from t h r e e d i f f e r e n t experiments In summary, F d l l F was B16G  data  are shown.  shown t o b i n d t o both Fd and the  which allowed i t s q u a n t i f i c a t i o n ,  was  at l e a s t at the  mAb,  56  0  J  O  .312  Fig.  6.  Inhibition  Q  .625  1  —Q  1.25  1 2.50  ug/ml Inhibitor  of FdllF  A  5.00  1— 10.0  b i n d i n g by Fd and i t s analogs.  F d l l F w a s m i x e d w i t h v a r i o u s c o n c e n t r a t i o n s o f N - m o l e c u l e (o), Cm o l e c u l e ( • ) , O F d ( • ) , F d ( • ) , o r c y t o c h r o m e C (A) p r i o r t o i t s addition t o Fd-coated wells. F d l l F w a s d e t e c t e d a s i n F i g . 2.  57 immunochemical  level.  Fd  t h e use o f analogs  and through  specific studies  F d l l F was shown t o b i n d  i t was d e t e r m i n e d t o b e  f o rthe carboxyl-terminal examined  Biological  Development  region  some o f t h e b i o l o g i c a l  Properties  preferentiallyto  of Fd.  The n e x t  properties  set of  of FdllF.  of FdllF  o f an a n t i b o d y  culture  assay  f o r t h e measurement  of  suppression  Having  established  t h e immunochemical p r o p e r t i e s  was o f i n t e r e s t t o e x a m i n e study  Several  in  the cells  to  drive  culture. of can  this  t h e Ab r e s p o n s e results  immunization  be seen t h a t  Ab  were  with  one was  immunized  t o insure  In addition,  chosen. twice  that  the cells  i t was  f o r Ab p r o d u c t i o n  i n vitro.  amount o f F d i s  by s p l e n i c antigen-presenting  despite  found  Ag j u s t p r i o r t o c u l t u r i n g  a sufficient  and "processed"  Typical  animals  enough s t i m u l u s period,  before  of spleens  had t o be p u l s e d  During the pulsing pinocytosed  were t r i e d  enough Ab i n v i t r o .  t o provide  an i n v i t r o  To  developed.  the harvesting  synthesize  order  was  the C-molecule-primed  Fd before  would that  assay  different protocols  Initially, with  some o f i t s b i o l o g i c p r o p e r t i e s .  t h e e f f e c t o f F d l l F on t h e Ab r e s p o n s e ,  production  of FdllF, i t  the lack  of soluble  (of s i xd i f f e r e n t c u l t u r e  and p u l s i n g p r o t o c o l  Ab was e a s i l y d e t e c t e d  a r e seen  cells  Ag i n experiments)  i n Fig.  i n t h e SN f r o m  7.  day 5  It  58  0.9-,  CD impulsed ^ fd pulsed 5  6  7  Day  F i g . 7. D e t e c t i o n o f a n t i - F d A b i n t h e SN o f s p l e e n cell c u l t u r e s p r e p a r e d f r o m B10.D2 m i c e t h a t w e r e i m m u n i z e d w i t h molecule and Fd t w i c e .  C-  S p l e e n c e l l s were i n c u b a t e d w i t h e i t h e r F d o r medium f o r 2 h before culturing. SN w e r e c o l l e c t e d f i v e , s i x , a n d s e v e n d a y s a f t e r i n i t i a t i o n o f c u l t u r e . A b c o n t e n t o f SN w a s m e a s u r e d u s i n ELISA.  59 cultures to  day  served was  using  6 and as  ELISA.  were done t o  their  Ab  spleens  make t h e  effects  of  FdllF. cells  Eventually,  were p u l s e d  Ag,  and  containing  cells  a n t i - F d Ab  comparison  of  the  or  reached  plate  concentration  shown i n F i g . as  a  source  approximately it  could  8.  be  with  with  were  Fd,  linear  in  vitro  tried  and  a measure until  the  were  previously and  boosted  washed f r e e  SN  were c o l l e c t e d  ELISA. of  that eluted  A  suppression.  maximal  absorbance  assumed t h a t  0.1  to  on  one.  bound t o from  affinity-purified from  of  FdllF-  r e l a t i o n s h i p between absorbance o f Ab  where  FdllF.  cells  seven days.  as  vivo  was  either control material,  allowed  M o u s e Ab of  Fd  Spleen  with  FdllF in  were a l s o  (to e s t a b l i s h immunity)  approximately  curve p l o t t i n g the  used  selected.  l e v e l s served  the  the  experiments  one  This the  Once i t  experiments  In with  to  l e v e l s were measured u s i n g  d e v e l o p m e n t was  and  "susceptible"  medium f o r  Substrate  A  were t r e a t e d  were p u l s e d  cultured  material  in vitro,  suppression.  f r o m Fd-immune B10.D2 m i c e t h a t  once.  cells  ELISA measurement.  concurrently  m e t h o d was  The  the  more  through  medium p u l s e d  produced  amount o f  cells  with  and  be  continued  were removed f o r c u l t u r i n g .  C-molecule  soluble  containing  Preliminary  one  production  f o r the  could  immunized w i t h Fd  Ab  hyperimmune a n i m a l s  to  prepared  culture  control  that  thought  spleen  fact,  maximize the  modification,  the  A  background  established  before  7.  day  In  1.0  Fd-coated  wells  a Fd-Sepharose material.  The  directly  is  column curve  absorbance u n i t s .  a b s o r b a n c e w o u l d be  produced  was was  Therefore  60  F i g . 8. R e l a t i o n s h i p between c o n c e n t r a t i o n o f a n t i - F d Ab.  Absorbance  a t 4 05  nm  vs.  A f f i n i t y - p u r i f i e d a n t i - F d A b was i n c u b a t e d o n F d - a n d K L H - c o a t e d plates. A b was d e t e c t e d u s i n g R a M I g - A P . R e a c t i o n was d e v e l o p e d f o r 2 h. Fd:  (•)  KLH:  (O)  61 r e l a t e d t o the c o n c e n t r a t i o n of Ab t h a t was a c t u a l l y p r e s e n t i n the SN.  The s p e c i f i c i t y of the a f f i n i t y - p u r i f i e d Ab f o r Fd was  shown by i t s l a c k of r e a c t i v i t y with KLH. t h r e e experiments  R e p r e s e n t a t i v e data of  are shown.  The s p e c i f i c i t y o f the a n t i - F d Ab s e c r e t e d i n t o t h e SN f o r the Fd analogs and u n r e l a t e d Ag was then determined.  In F i g . 9A i t  can be seen t h a t e s s e n t i a l l y a l l o f the Ab was s p e c i f i c f o r the amino-terminal determinant. the C-molecule immunization determinant  The r e l a t i v e l a c k o f Ab d i r e c t e d t o  was c o n s i s t e n t with the f i n d i n g t h a t  of  B 1 0 . D 2  C-molecule  animals induces l i t t l e Ab d i r e c t e d t o t h a t  (data not shown).  The SN d i d not r e a c t with the  i r r e l e v a n t Ag, KLH and BSA.  For comparison,  a c t i v i t y o f Fd-immune  animals i s shown i n F i g . 9B.  serum a c t i v i t y  B 1 0 . D 2  the serum Ab  i s q u i t e s i m i l a r t o t h a t o f the SN.  i n c r e a s e o f Ab r e a c t i v e with N-molecule  The apparent  over Fd was p r o b a b l y due  t o d i f f e r e n c e s i n b i n d i n g of Fd or N-molecule some n o n s p e c i f i c b i n d i n g of serum Ab.  The  Indeed,  t o ELISA p l a t e s or the n o n s p e c i f i c  b i n d i n g a c t i v i t y o f the serum, as shown by i t s r e a c t i v i t y w i t h KLH, i s g r e a t e r than t h a t o f the SN  ( c f . Fig. 9A) .  C o r r e l a t i o n between b i o a c t i v i t y and a n t i g e n - b i n d i n g a c t i v i t y of FdllF  Fig.  1 0 i s the "dose-response"  suppressive a c t i v i t y i n v i t r o  curve of F d l l F  ( i e . the response)  correlating with Ag-binding  62  F i g . 9A. cultures  S p e c i f i c i t y o f a n t i - F d Ab f o r Fd analogs.  f r o m Fd-immune s p l e e n  cell  SN f r o m s e v e n d a y c u l t u r e s w e r e h a r v e s t e d a n d A b l e v e l d e t e r m i n e d u s i n g ELISA. Typical results of s i x d i f f e r e n t cultures are shown. N:  N-molecule  F i g . 9B. B10.D2 m i c e  C:  C-molecule  S p e c i f i c i t y o f serum f o r Fd analogs.  a n t i - F d Ab  from  Fd-immune  A b l e v e l a t a 1/400 d i l u t i o n o f s e r u m was d e t e r m i n e d u s i n g R e s u l t s (mean + SEM) f r o m t h r e e s e p a r a t e s e r u m s a m p l e s a r e N:  N-molecule  C:  C-molecule  ELISA. shown.  Antigen  64  F i g . 10. The r e l a t i o n s h i p i t s Ag-binding activity.  of  suppressive  activity  of  FdllF  to  S u p p r e s s i v e a c t i v i t y i s e x p r e s s e d as p e r c e n t suppression. P e r c e n t s u p p r e s s i o n was c a l c u l a t e d b y c o m p a r i n g A b l e v e l s o f c u l t u r e s c o n t a i n i n g F d l l F to the l e v e l s measured i n the c o n t r o l culture. A g - b i n d i n g a c t i v i t y i s e x p r e s s e d as t h e s a n d w i c h E L I S A value of the F d l l F l o t t e s t e d . The p o o l e d d a t a f r o m s e v e n l o t s a r e shown. L o t s 1 t o 5 were a s s a y e d a t two o r t h r e e c o n c e n t r a t i o n s w h i l e l o t s 6 and 7 w e r e a s s a y e d a t o n l y one concentration. F o l l o w i n g i t s e l u t i o n f r o m B16G a f f i n i t y columns, e a c h l o t was m e a s u r e d f o r p r o t e i n c o n t e n t , A g - b i n d i n g activity, and b i o a c t i v i t y . A n e g a t i v e c o n t r o l s a m p l e was prepared c o n c u r r e n t l y w i t h e a c h F d l l F l o t . T h i s s a m p l e was u s e d a s t h e r e s p e c t i v e negative c o n t r o l f o r the d e t e r m i n a t i o n of net Agb i n d i n g a c t i v i t y and b i o a c t i v i t y .  65 activity  as m e a s u r e d by  "sandwich" ELISA  Response v a l u e s were e x p r e s s e d u s i n g t h e Ab  level  as p e r c e n t  suppression  curve  i s a c o m p i l a t i o n of r e s u l t s  l o t s of F d l l F . and A g - b i n d i n g r e l a t e d and 4 and  The  c a n be  7 d i d not  suggest  from seven  t h a t t h e two  ascribed to FdllF.  The  60%.  different bioactivity are  data representing  conform w i t h the m a j o r i t y of o b s e r v a t i o n s . i s unknown.  One  were c o m p a r a t i v e l y  possibility  low.  d i s p r o p o r t i o n a l l y high percentage observed  allow  activities  l e v e l s p r o d u c e d by t h e u n t r e a t e d , F d - p u l s e d  experiments  Ag-binding  FdllF.  c u r v i l i n e a r r e l a t i o n s h i p between  activity  r e a s o n (s) f o r t h i s Ab  as  r a t h e r than t o t a l p r o t e i n concentration to of  (calculated  as c o m p a r e d t o  Dose v a l u e s w e r e e x p r e s s e d  comparison of the data from d i f f e r e n t batches The  dose).  of the c u l t u r e c o n t a i n i n g F d l l F  the untreated c u l t u r e ) . activity,  ( i e . the  that the  lots The  i s that the  total  c u l t u r e s i n these  This r e s u l t e d i n a  of suppression.  s u p p r e s s i o n p r o d u c e d by F d l l F  A l m o s t c o m p l e t e s u p p r e s s i o n was  I t was  d i d not  also exceed  eventually achieved  f o l l o w i n g a r e v i s i o n t o t h e c u l t u r i n g p r o t o c o l (see b e l o w , F i g . 12) . None o f t h e c u l t u r e s c o n t a i n i n g F d l l F  showed an  p r o d u c t i o n as c o m p a r e d t o u n t r e a t e d c o n t r o l s . w i t h n e g a t i v e c o n t r o l SN were p r e p a r e d of F d l l F . (lot these  4)  B16G  in parallel  i n c r e a s e i n Ab e l u a t e s made w i t h each l o t  Of t h e n e g a t i v e c o n t r o l p r e p a r a t i o n s , o n l y one  o f t h e s e v e n d e m o n s t r a t e d any  cultures,  the decrease  negative control material.  was  lot  suppressive a c t i v i t y .  c o r r e c t e d f o r the e f f e c t of  For the  66 FdllF-mediated the  a n t i - F d Ab r e s p o n s e .  three  different  cells,  this  binding  response  BW5147 c o n t r o l  work,  the T cell  was d o n e .  hybridoma,  t o t h e murine  1985).  decrease  The m a t e r i a l ,  i n specific  contained  FdllF.  That assay  at  I t also  d i d not  cell  from  a r e shown.  "specificity" of  another product  P815 ( S t e e l e ,  u s i n g B16G-Sepharose.  derived  Stammers, and  from  P815-primed  the culture When A l O F  mice,  when F d l l F  that  was o n l y s u p p r e s s i v e when u s e d  "criss-cross"  was  that  was a d d e d t o c u l t u r e s  t h e r e was a d e c r e a s e  when u s e d  SN  t h e r e was a  l y s i s as compared t o c u l t u r e s  By c o n t r a s t ,  two o f these  ofthe  ( i e . medium a n d  A10F, i s r e c o v e r e d from  a n d A 1 0 F was o n l y a c t i v e  Table I I .  d i d not possess Ag-  materials  as compared t o t h e c u l t u r e s  i s , FdllF  results  the anti-Fd  A10, i s s u p p r e s s i v e f o r t h e CTL  prepared  Fd-immune s p l e e n c e l l s , cultures  In brief,  mastocytoma,  a f f i n i t y chromatography  the  which  addressing the i n vitro  added t o CTL c u l t u r e s  of  material,  d i dsuppress  Representative data of three experiments  bioactivity  response  by  of FdllF  s u p p r e s s i v e a c t i v i t y a n d was r e p r e s e n t a t i v e  Additional  Levy  when F d l l F ,  was a d d e d t o l y s o z y m e - i m m u n e  generated by t h e other c o n t r o l  SN).  from  concentrations,  a c t i v i t y , i s shown f o r c o m p a r i s o n .  demonstrate  FdllF  F i g . 11 s h o w s t h a t  same p r e p a r a t i o n  response.  DC10  shown t o b e s p e c i f i c f o r  c o n c e n t r a t i o n - d e p e n d e n t s u p p r e s s i o n was n o t o b s e r v e d .  However, Ab  s u p p r e s s i o n was a l s o  with  i n Ab l e v e l s  of  c o n t a i n e d A10F. i n t h e Ab  culture  t h e CTL a s s a y .  experiments  The  a r e shown i n  67  so-i  to I 0.00  Fig.  11.  I  1  1  .147(1) .486(2) Net antigen-binding activity & (ug/ml)  Specificity  of the  suppressive  activity  1.10(4)  of  FdllF.  A s i n g l e p r e p a r a t i o n of F d l l F , used at t h r e e d i f f e r e n t concentrations, was.added t o c u l t u r e s o f lysozyme-immune and Fd-immune c e l l s .  cells  A n t i - l y s o z y m e Ab l e v e l s o f c u l t u r e s c o n t a i n i n g F d l l F (•) were c o m p a r e d w i t h t h e " u n t r e a t e d c o n t r o l c u l t u r e . The p e r c e n t s u p p r e s s i o n i s shown. A l s o i n c l u d e d i s t h e r e s p o n s e o b t a i n e d w i t h BW5147- c o n t r o l m a t e r i a l (O) when a d d e d t o t h e l y s o z y m e immune c e l l s . I t d i d n o t p o s s e s s any A g - b i n d i n g a c t i v i t y and was added t o the c u l t u r e s at t h e p r o t e i n c o n c e n t r a t i o n s t h a t are indicated. To d e m o n s t r a t e t h a t t h e same p r e p a r a t i o n o f F d l l F was a c t i v e , i t was a d d e d t o Fd-immune c e l l s . The p e r c e n t s u p p r e s s i o n o f t h e s e c u l t u r e s , a t t h r e e c o n c e n t r a t i o n s , a r e shown (•).  68 Table I I . Comparison A10F i n two a s s a y s o f  of the suppressive c a p a b i l i t y cellular function.  of F d l l F  and  F d l l F and A10F were e a c h added t o c u l t u r e s c o n t a i n i n g e i t h e r P 8 1 5 - s p e c i f i c CTL o r Fd-immune s p l e e n c e l l s . F o r t h e P815 CTL a s s a y , d a t a a r e e x p r e s s e d as p e r c e n t l y s i s and p e r c e n t suppression f o r the culture. P e r c e n t s u p p r e s s i o n was calculated by c o m p a r i n g p e r c e n t l y s i s o f t h e c u l t u r e s c o n t a i n i n g a f f i n i t y enriched material to the untreated control culture. For the a n t i - F d Ab a s s a y , d a t a a r e e x p r e s s e d a s Ab l e v e l o f t h e c u l t u r e ( A b s o r b a n c e 4 0 5 nm) a n d p e r c e n t s u p p r e s s i o n . Percent suppression was c a l c u l a t e d b y c o m p a r i n g t h e A b l e v e l o f c u l t u r e s c o n t a i n i n g a f f i n i t y - e n r i c h e d material to the untreated control culture. The r e s u l t s f r o m two s e p a r a t e e x p e r i m e n t s a r e shown. Ab c u l t u r e measurements were o b t a i n e d u s i n g t h e " r e v i s e d " p r o t o c o l (see M A T E R I A L S AND METHODS). A:  material  added at  1 ug/ml  B:  Material  %  no  A l OF FdllF  no  FdllF  1.5  ug/ml  lysis  CTL  Anti-Fd  % suppression  Ab  level  %  Ab  suppression  24.5  -  .197  0  100  22  10.7  15  -  .183  7.1  .143  27.4  B  factor  A10F  at  A  factor  Expt.  added  Assay P815  Expt.  material  .301  5.6  62.4  .280  7  14.5  3.3  .100  67  69 The  "dose-response"  curve  amount o f s u p p r e s s i o n activity  60%.  that  itself cells  then At  The c e l l s  co-cultured  attained  This  were p u l s e d  processing.  with  original  a n t i - F d Ab u s i n g  Further culture  approached  FdllF  the  later.  After  incubated factor.  in  another  suppressive  with  spleen  f o r Ag  the culture  soluble  Fd and  f o r seven SN w e r e  modifications  days.  assessed  data  of anti-Fd  which pulsing  introducing  Fd 2 h  p l a t e d and  r e c e i v i n g any a d d i t i o n s  of  as t h e i n t h e o r i g i n a l  (of three  d i f f e r e n t experiments)  e f f e c t was BW5147  concentrationcontrol  material  concentration.  antibody  f o l l o w i n g experiment  included  were washed,  a maximum o f 8 5 % .  a t any  t o t h e Ab  e f f e c t s by F d l l F ,  F d l l F and then  the suppressive  and reached  Suppression  u s e d Fd-immune  Fd t o allow  showed t h a t  2 h, t h e c e l l s  Representative  not suppressive  The  first  12 s h o w t h a t  dependent was  period,  T h e d a y 7 SN w e r e a s s e s s e d  Fig.  assay  (or control material)  f o r seven days without  protocol.  d i dnot exceed  of F d l l F o r o f t h e assay  The c h a n g e s t o t h e p r o t o c o l  Fd-immune c e l l s  of  ELISA.  yielded  100%.  t h e assay  were t h e n washed t o remove  development work  protocol  with  f o r2 h with  t h e end o f t h e incubation  for  t h e maximal l e v e l  was a l i m i t a t i o n  was unknown. that  However,  was e v e r  Whether t h i s  10 i n d i c a t e d t h a t t h e  a c h i e v e d was d e p e n d e n t o n t h e A g - b i n d i n g  o f t h e sample.  suppression  of Fig.  levels i n vivo  was d e s i g n e d t o t e s t t h e e f f e c t o f  70  F i g . 12. S u p p r e s s i v e a c t i v i t y o f a f f i n i t y - e n r i c h e d F d l l F c o m p a r e d t o BW5147 c o n t r o l m a t e r i a l u s i n g t h e " r e v i s e d " p r o t o c o l . V a r i o u s c o n c e n t r a t i o n s o f e i t h e r BW5147 o r F d l l F m a t e r i a l w e r e f i r s t a d d e d t o Fd-immune s p l e e n c e l l s f o l l o w e d b y t h e i n v i t r o p u l s i n g w i t h F d . P e r c e n t s u p p r e s s i o n was d e t e r m i n e d a s f o r F i g . 10. (•), Fd-immune c e l l s p u l s e d w i t h F d l l F ; p u l s e d w i t h BW5147 c o n t r o l m a t e r i a l .  ( • ) , Fd-immune  cells  71 FdllF H-2  on t h e a n t i - F d Ab r e s p o n s e animals,  d  DBA/2  control  with Fd.  animals  of their  groups  either  recover  s.c. and  The  are presented  i s indicated  line.  o f Ag boost  days post majority PBS  adjuvant.  and then  days  within  later  each  had a decrease  group  of animals  from  both  allowed  and t h e a n t i - F d  treatment  were  animal.  and t h e post A decrease  treatment i n Ab  a s d a t a b e l o w t h e "no  that received FdllF  i n circulating  In contrast,  number  where t h e p r e t r e a t m e n t  on t h e y - a x i s .  out of s i x animals  into  w i t h F d and KLH,  individual  appears  They  equal  o r PBS i . v . , were  and a f t e r  on t h e x a x i s  treatment  treatment.  The  divided  E a c h d a t a p o i n t r e p r e s e n t s one a n i m a l .  shows t h a t f i v e time  made  according to the  boosted  a s a n X-Y p l o t  serum Ab l e v e l  change"  and then  f o r each  i splotted  following  10 u g A l O F  before  serum Ab l e v e l  response  responders  were b l e d seven  as p a i r e d samples  data  and ranked  The a n i m a l s  hours  a n t i - K L H Ab r e s p o n s e s  Freund's  were  one h a d an a p p r o x i m a t e l y  10 u g F d l l F ,  f o rseveral  immunized w i t h t h e  t o F d as measured u s i n g ELISA.  t h a t each  The a n i m a l s  assessed  .  response  low and high responders.  received to  later  as low o r h i g h  such  strain of  f o l l o w e d by an  A l l immunizations  (s.c.) u s i n g complete  were d e s i g n a t e d  of  time.  were b l e d 21 d a y s  magnitude  three  w i t h C-molecule  The m i c e were a l s o  Ag, KLH a t t h i s  subcutaneously  using another  mice.  DBA/2 m i c e w e r e p r i m e d immunization  i n vivo,  anti-Fd  F i g . 13 at the Ab  seven  a s shown i n F i g . 14, t h e  groups  showed an i n c r e a s e d Ab r e s p o n s e .  of animals  receiving  T h e X-Y p l o t  A10F o r  shows t h e  72  0.82-1  0.48-  0.44  « O  a © C cd  0.40-  01  0.36  A  o  n  0.32  <  0.28  0.24  0.20 0. 20  0.24  0.28  0.32  0.36  Absorbance  F i g . 13. and a f t e r  0.40  0.44  0.48  0.62  (pre)  X-Y p l o t s h o w i n g a n t i - F d A b l e v e l s t r e a t m e n t w i t h F d l l F and Ag.  of animals  before  A n i m a l s were immunized w i t h C-molecule and t h e n co-immunized w i t h F d a n d KLH. O n c e i m m u n i t y was e s t a b l i s h e d , t h e y w e r e b o o s t e d w i t h F d a n d K L H a n d g i v e n F d l l F , PBS o r c o n t r o l m a t e r a l , i . v . . Matched p a i r s of s e r a were o b t a i n e d from t h e i n d i v i d u a l a n i m a l s t h a t were b l e d b e f o r e and a f t e r t r e a t m e n t . Symbols r e p r e s e n t the x-y c o o r d i n a t e s o f p r e t r e a t m e n t a n d p o s t t r e a t m e n t s e r u m Ab l e v e l s f o r i n d i v i d u a l a n i m a l s . A d e c r e a s e i n Ab l e v e l i s p l o t t e d a s a s y m b o l b e l o w t h e "no c h a n g e l i n e " . A n i m a l s were t r e a t e d PBS (• ).  with either  FdllF  (•), A10F  (•)  or  73  Absorbance (pre)  F i g . 14. and a f t e r See  X-Y p l o t s h o w i n g a n t i - K L H Ab l e v e l s t r e a t m e n t w i t h F d l l F and Ag.  of animals  F i g . 13 f o r e x p l a n a t i o n .  A n i m a l s were t r e a t e d w i t h PBS ( • ) .  either  FdllF  (•), A10F  (•)  or  before  results  from  t h e same a n i m a l s , o n l y t h i s  response  i s plotted.  increase  i n a n t i - K L H Ab whether  together, for  The l a r g e  the data reveal  that  t h e a n t i - F d Ab r e s p o n s e  majority treated FdllF  a n d a n o t h e r Ab r e s p o n s e  reactive  material  To  was t e s t e d  i n vitro  used  t o show t h a t  and i n v i v o . FdllF  concentration-dependent suggested  effect KLH  that  treatment  mice  t o have  four  Taken  specificity  Ag.  times.  control (B16G-  Representative  A spleen c e l l  was  assay  manner.  Data  from  Ag c h a l l e n g e .  other  the availability FdllF,  of both  experiments In vivo,  o f Fd-immune  AlOF  The  anti-  by any o f t h e t r e a t m e n t s . an immunochemical  and  biological  of the FdllF  Ag-  possible.  of FdllF  Introduction  When t h e e l u a t e s  from  H-  a n d PBS h a d no  response.  biochemical analysis  m o l e c u l e ( s ) w a s now  Analysis  As c o n t r o l s ,  and  i n a  was A g - s p e c i f i c .  of t h e secondary  was n o t a f f e c t e d  demonstrated  was d e v e l o p e d  s u p p r e s s e d t h e Ab r e s p o n s e  on t h e development  Biochemical  of FdllF  s u p p r e s s e d t h e serum Ab r e s p o n s e  means o f m e a s u r i n g binding  activity  the suppressive effect  following  Ab r e s p o n s e With  appears  or FdllF.  t o an i r r e l e v a n t  i n vivo,  the biologic  both  2  w i t h AlOF  showed an  a r e shown).  summarize,  FdllF  of animals  when c o m p a r e d t o B 1 6 G - e l u t e d  material  results  t i m e t h e a n t i - K L H Ab  B16G c o l u m n s were a n a l y z e d u s i n g  SDS-  PAGE, run under r e d u c i n g c o n d i t i o n s , a c h a r a c t e r i s t i c  and  r e p r o d u c i b l e banding  staining.  Fig.  p a t t e r n was  15 shows the r e s u l t  experiment.  observed  with s i l v e r  from a r e p r e s e n t a t i v e F d l l F  recovery  Equal amounts of p r o t e i n (1 t o 2 ug) were loaded  i n t o each w e l l .  A f f i n i t y chromatography y i e l d e d only p a r t i a l l y  p u r i f i e d p r e p a r a t i o n s of F d l l F s i n c e bands resembling probably  o t h e r serum components c o - e l u t e d with F d l l F .  predominant bands always appeared. unique band at approximately approximately 80k M  r  80k M  was  r  30k M  Of primary .  and  Two  other  i n t e r e s t was  a  In a d d i t i o n , a band at  r o u t i n e l y recovered.  L e s s e r amounts of  m a t e r i a l a l s o appeared i n the lane c o n t a i n i n g the sample  d e r i v e d from n o n - F d l l SN, BW5147 or DC10 the "80k"  SN.  or "30k"  approximately Fig.  BSA  80k M  (Note: M r  r  whether i t was  prepared  from medium,  Subsequent r e f e r e n c e s i n the t e x t to  p r o t e i n s r e f e r t o the p r o t e i n at and the p r o t e i n at approximately  30k M  r  of  15, r e s p e c t i v e l y . )  From t h i s s t a r t i n g p o i n t , e f f o r t s were d i r e c t e d at the b i o c h e m i c a l i d e n t i f i c a t i o n of the Ag-binding The  aim was  moiety of F d l l F .  to f u r t h e r r e s o l v e the F d l l F - e n r i c h e d m a t e r i a l from  the B16G-Sepharose column and i d e n t i f y the molecule(s) t h e i r Ag-binding  and b i o l o g i c  by  both  activities.  A n a l y s i s of F d l l F u s i n g h i g h performance g e l  filtration:  c o r r e l a t i o n between a n t i g e n - b i n d i n g and s u p p r e s s i v e  High performance g e l f i l t r a t i o n  activity  (FPLC, Pharmacia,  Fig.  15.  SDS-PAGE of e l u a t e s from B16G a f f i n i t y  columns.  E l u a t e s d e r i v e d from F d l l SN (A) or medium c o n t a i n i n g 10% FCS (B) were analyzed u s i n g a 10% SDS g e l run under r e d u c i n g c o n d i t i o n s . P o s i t i o n of M markers are i n d i c a t e d . A c t u a l M equals number shown m u l t i p l i e d by 1000. The predominant bands at 80k and 30k M were c o n s i s t e n t l y obtained with F d l l SN. Bands were developed by s i l v e r s t a i n i n g . T y p i c a l r e s u l t s of s i x d i f f e r e n t p r e p a r a t i o n s of F d l l F (and c o n t r o l m a t e r i a l ) are shown. r  r  77 Superose it  12  allowed  column) fast  of denaturing dissociation The  avoid  of  non-covalently  fraction  each  was  FdllF  sample.  v o l u m e was  performance a m o u n t was  the  (Representative peak  of  presence  r e v e a l e d by In  over  addition,  a  results  and  SDS-PAGE two  45k  present  in fraction  binding  activity.  appeared  i n both  sample  M  by  activity  r  r  Lesser control  17)  medium,  void  v o i d volume data  and This 16  to  are  of  the large 22)  shown i n F i g .  as 17.  shown.)  i n the  F r a c t i o n s 16  quantities  loaded  coincided exactly with  identified  They d i d not  lanes,  of  at the  (fractions  profile,  bands unique t o  16.  reducing  Pharmacia).  determinations  band  ( F i g . 18C).  (The  experimental  provided  (fraction  30k  M  on  of three  the  amounts o f p r o t e i n were  FdllF  based  Ag-binding  80k  under  the  the  80k  consistent with  fraction  19  FdllF  appear t o of  to  are  sample  r  The the as  shown. were  contribute to M  to  was  using  FPLC p r o t e i n p r o f i l e s  Equal  first  column  activity  SDS-PAGE r u n  several fractions  immunoreactivity of  by  the  l a r g e p r o t e i n peak b e g i n n i n g  specifications  by  for Ag-binding  shows t h e  calculated  use  B16G-  chromatographed  loaded.  from the  the  cause  from the  m a t e r i a l was  material. A  spread  eluates  After  analyzed  without  since  associated materials.  of p r o t e i n s .  assayed  recovered  c o l u m n was  suggested  FdllF  F i g . 16  BW5147, a n d  native molecules  C o n t r o l m a t e r i a l was  E L I S A and  conditions.  s e p a r a t i o n method chosen  m a t e r i a l s were t h e  columns.  "sandwich"  the  of the  cross-contamination  Each  first  c o n d i t i o n s or b u f f e r s which would  washed w i t h e l u e n t ,  for  the  recovery  starting  Sepharose  was  material  Ag-  78  TG  IgG BSA  cytochrome C  F i g . 16. P r o t e i n p r o f i l e s ( 2 8 0 nm) o f B 1 6 G e l u a t e s derived f r o m m e d i u m ( A ) , BW5147 ( B ) , a n d F d l l F (C) SN f r a c t i o n a t e d o n a S u p e r o s e 12 c o l u m n u s i n g F P L C . Enhanced r e c o v e r y o f m a t e r i a l a t t h e v o i d volume d e r i v e d from F d l l F SN i s s h o w n . E q u i v a l e n t a m o u n t s o f p r o t e i n ( 1 8 8 ug) w e r e loaded and chromatographed according t o t h e conditions described i n M a t e r i a l s and Methods. R e l a t i v e m o l e c u l a r mass m a r k e r s a r e indicated. M a r k e r s i n c l u d e d t h y r o g l o b u l i n (TG) , 6 6 9 k M ; I g G , 1 5 0 k M ; B S A , 6 6 k M ; a n d c y t o c h r o m e C, 1 2 . 4 k M . I n t o t a l , 48 f r a c t i o n s (0.5 ml) were c o l l e c t e d . Protein traces are f o r f r a c t i o n s 15 t o 4 7 . T y p i c a l d a t a o f f i v e d i f f e r e n t f r a c t i o n a t i o n s a r e shown. r  r  r  r  79  13  15  17  19  21  23  28  Fraotlon no.  27  29  31  33  F i g . 17. A g - b i n d i n g a c t i v i t y o f FPLC f r a c t i o n s o b t a i n e d f r o m F d l l F (•), BW5147 (•), and medium (O) e l u t i o n p r o f i l e s o f F i g . 16. A g - b i n d i n g a c t i v i t y was m e a s u r e d as f o r F i g . 2. One h u n d r e d m i c r o l i t r e s o f e a c h f r a c t i o n were a d d e d t o F d - c o a t e d p l a t e s and d e v e l o p e d w i t h B16G. E L I S A v a l u e s a r e t h e mean o f d u p l i c a t e determinations. A p r e l i m i n a r y FPLC e x p e r i m e n t h a d shown t h a t Agb i n d i n g a c t i v i t y was n o t d e t e c t a b l e i n t h e r e g i o n o f f r a c t i o n s 35 t o 48. Representative r e s u l t s of three d i f f e r e n t determinations a r e shown.  80  16  17  18  19  16  A  B  F i g . 18. SDS-PAGE a n a l y s i s 17) 16 t o 19 i n c l u s i v e .  17  18  19  16  17  18  19  C  o f FPLC f r a c t i o n s  (shown i n F i g .  T e n p e r c e n t SDS g e l s c o n t a i n i n g r e d u c i n g a g e n t w e r e d e v e l o p e d b y silver staining. T w e n t y - f i v e m i c r o l i t r e s from e a c h f r a c t i o n were c o n c e n t r a t e d and e l e c t r o p h o r e s e d . Position of M markers are indicated. Actual M e q u a l s number shown m u l t i p l i e d b y 1000. R e p r e s e n t a t i v e d a t a o f t h r e e a n a l y s e s a r e shown. r  A:  medium  control  B: BW5147 m a t e r i a l  C:  FdllF  81 previous FdllF  results.  (or t o the  fractions A  T h e r e w e r e no control  t h a t were  FdllF  for  bioassay.  for  testing.  production  with  material  from t h i s  M  was  r  bands  at  seen.  between  , not  Together,  suppressive moiety  recovered M  30k  and  Identification  Although  of  be  from the  run  along  the  a fresh set  collecting  FdllF  of  fractions  sample were s e l e c t e d  suppression  seen t h a t maximal sample.  fractionation  with with  data  suppressive  of  Ab  the  suppression  Therefore  profile  fractions  reducing an  80k  related  suggest  a number o f  (Table  III)  v o i d volume of the  when t h e  directly  these  percent  and  material eluting  made u p  to  activity.  However, gels  of  from each  from the  ( F i g . 17)  column.  45k  of  t h a t were c o - c u l t u r e d w i t h  18  recovered  using  intent  20  I t can  suppressive  polyacrylamide-SDS  to  region of the  Thus, A g - b i n d i n g  filtration  cells  fraction  were  bands unique  remainder  conducted  I I I shows t h e  spleen  occurred  material  i n the  eluates with the  material.  demonstrated  was  F r a c t i o n s 15  by  visible  analyzed.  Table  fractionated  30k  samples)  second FPLC experiment  BW5147 a n d  other  M  r  gel  were a n a l y z e d  agent,  a unique band  protein.  f r o m B16G  were  also  Ag-binding,  i s composed of  identifiable  peptides  a high  with  M  r  80k.  of  FPLC  the  antigen-binding molecule  fractionation  provided  of  Additional  to Ag-binding,  that the  by  using  more e v i d e n c e  SDS-PAGE  relating  M  r  82  Table I I I . A b i l i t y o f v a r i o u s f r a c t i o n s e l u t i n g from FPLC S u p e r o s e 12 c o l u m n t o i n h i b i t t h e i n v i t r o A b r e s p o n s e o f F d immune s p l e e n c e l l s . P e r c e n t s u p p r e s s i o n was c a l c u l a t e d b y c o m p a r i n g l e v e l s o f Ab f o r m a t i o n i n c u l t u r e s i n c u b a t e d w i t h eluted materials to levels detected i n untreated controls. A t o t a l o f 475 u l f r o m e a c h f r a c t i o n were a d d e d t o t h e c u l t u r e s . C u l t u r e s c o n t a i n i n g t h e f r a c t i o n a t e d m a t e r i a l were i n c u b a t e d f o r s e v e n d a y s . Ab c u l t u r e m e a s u r e m e n t s were o b t a i n e d u s i n g t h e " o r i g i n a l " p r o t o c o l ( s e e M A T E R I A L S AND M E T H O D S ) . Representative r e s u l t s o f t h r e e d i f f e r e n t c u l t u r i n g e x p e r i m e n t s a r e shown.  Fraction  No.  Percent BW5147  suppression FdllF  15  -8  0  16  6  9  17  2  -5  18  6  53  19  13  -7  20  -10  13  83 Ag-binding indicate  activity  and b i o a c t i v i t y  which molecule(s)  with  was a c t u a l l y  FdllF,  i td i d not  responsible  f o rAg-  binding. Another using  preparative  eluates slab  attempt  of FdllF  gel.  material  eluted  activity.  end  SDS-PAGE  reducing  of activity  is  exactly  were unique The g e l s  were l o c a t e d  the  80k M  Fig.  r  20).  detected.  Two  or  a chemical lane  they  resolved  faint  resolved  were s i l v e r  samples,  with  distinct  bands e l u t e d i factually  d i d not  12 t o 14 region  r  from  was d e t e c t e d  4 of FdllF  were v i s i b l e  r  20.  with  i n lanes were  appearance  sample  This  m a t e r i a l (see  Whether they Their  SDS-  region.  bands unique t o F d l l F  a protein  staining  i n this  activity  at slice  was u n k n o w n .  not loaded  Major  material  of slices  a doublet  b a n d s a t 68k M  artifact  19.  o f bands i n t h e 30k M  No A g - b i n d i n g  no o t h e r  f o r Ag-binding  sample as assessed by reducing  " F " a n d "D" o f F i g .  The p r o t e i n FdllF  20.  b y SDS-  12 t o 14 a n d a t t h e  DC10 e l u t e d  the presence  B16G  and t h e  was a n a l y z e d  The a c t i v i t y  to the FdllF  In fact,  f o rboth  that  with  bands t h a t  14,  control  activity.  made  on a 12%  i s shown i n F i g . at slices  was  reagent).  strips  and assayed  20 t o 2 2 ) .  actually  shown i n F i g .  slices  conditions  The A g - b i n d i n g p r o f i l e  coincided  reducing  equal-size  from t h e i n d i v i d u a l  have any A g - b i n d i n g  PAGE.  (runwithout  a n d DC10 SN w e r e e l e c t r o p h o r e s e d  of the g e l (slices  that  the Ag-binding moiety  The g e l was c u t i n t o  PAGE r u n u n d e r  regions  to identify  ("C")  were 13 a n d  protein  i n a  suggested  artifacts. slices  present,  2 0 t o 2 2 o f t h e DC10 o r  were n o t v i s i b l e ; most  likely  84  •M-1  Fraction no.  F i g . 1 9 . A g - b i n d i n g a c t i v i t y o f DC10 a n d F d l l F m a t e r i a l e l u t e d f r o m s l i c e s o f a 1 2 % p r e p a r a t i v e SDS-PAGE g e l r u n u n d e r n o n reducing conditions. Two h u n d r e d m i c r o g r a m s e a c h o f a f f i n i t y - e n r i c h e d F d l l F (•) o r DC10 (•) m a t e r i a l w e r e f r a c t i o n a t e d u s i n g p r e p a r a t i v e SDS-PAGE under non-reducing c o n d i t i o n s . Recovered m a t e r i a l from i n d i v i d u a l s l i c e s was t e s t e d f o r A g - b i n d i n g a c t i v i t y a s i n F i g .  85  4  12  13  F D  F D  14  F D  C  F D  F i g . 20. PAGE o f m a t e r i a l f r o m s e l e c t e d f r a c t i o n s o f F i g . u s i n g a 10% SDS g e l r u n u n d e r r e d u c i n g conditions.  19  F r a c t i o n 4 s h o w s t h e 80k M p r o t e i n r e c o v e r e d from b o t h F d l l F and DC10 control material. F r a c t i o n s 12 t o 14, w h i c h e x h i b i t e d A g binding activity ( F i g . 19), s h o w b a n d s u n i q u e t o F d l l F f r o m t h e 30k M region. r  r  F:  FdllF  C:  control  D:  DC10  lane  material showing  silver  staining  "artifact"  86 due  to the inability  < 14k M ) .  of the g e l to resolve  r  Whether t h e m a t e r i a l  activity  was a b r e a k d o w n p r o d u c t  represents unknown.  relationship, It  should  fractionation recovering  three  experiments  bioactivity  material  had a detrimental  cultures.  binds  r  t o both  or  F d a n d B16G i s  to clarify  separate  i t s  preparative  SDS-PAGE  the aim of  without  success.  I t was  t h e r e s i d u a l a m o u n t s o f SDS i n t h e e l u t e d e f f e c t on t h e g r o w t h  An a l t e r n a t i v e e x p l a n a t i o n  fractionation  species  protein.  from g e l s l i c e s ,  that  had destroyed  associated  binding.  other  On o c c a s i o n ,  18C) h a v e b e e n  was t h a t  bioactivity  or more n o n - c o v a l e n t l y  Fig.  r  were p e r f o r m e d w i t h  possible  (eg.  o f t h e 30k M  i f any, t o t h e 30k M that  proteins ( i e .  f o r t h e ELISA  work would be r e q u i r e d  be n o t e d  r  responsible  a different protein that Further  low M  fraction  o f t h e Ab SDS-PAGE  by t h e s e p a r a t i o n  molecules,  one o f w h i c h was A g -  bands unique t o t h e F d l l F  recovered  along  o f two  with  t h e 30k  sample M  r  molecule. The  a p p e a r a n c e o f t h e 30k M  fractions  and t h e m a t e r i a l  preparative moiety  SDS-PAGE  responsible  determine  remainder Fig. and  eluted  r  molecule  of the selected  material  suggested that  activity.  i t was t h e  However, t o  was a c t u a l l y u n i q u e t o F d l l F , i t gel electrophoresis.  The  f r a c t i o n s from t h e FPLC e x p e r i m e n t o f  M a t e r i a l from from  i n b o t h t h e FPLC  from t h e non-reducing  f o r the Ag-binding  t o two-dimensional  18 w e r e u s e d . pooled  protein  fractions strongly  i f t h e 80k M  was s u b j e c t e d  r  fraction  17 o f t h e F d l l F  f r a c t i o n s 17 t o 19 o f t h e m e d i u m  sample sample  87 were of  analyzed  the technique,  t h e 80k  samples were n o t o n l y isoelectric  F i g . 21  i n parallel.  point  M  r  proteins  o f t h e same M  as w e l l .  shows  I t i s generally  Therefore  i n s p i t e of the preponderance  identical  assumed t h a t  would n o t have t h e i d e n t i c a l  control material, and were d e r i v e d  FCS o f t h e c o m p l e t e medium  limits  not from t h e F d l l  two  p i and M .  o f t h e 80k  t h e two p r o t e i n s  mixture.  the  , b u t h a d t h e same  proteins  over  within  f r o m medium a n d F d l l F  different  FdllF  that,  were  M  r  protein  of  probably  hybridoma,  but the  88  F i g . 21. proteins  Two-dimensional g e l electrophoresis o f medium and F d l l F .  o f t h e 80k  M  r  E q u a l a m o u n t s o f 8 0k M m a t e r i a l f r o m b o t h m e d i u m ( p a n e l A) a n d F d l l F ( p a n e l B) w e r e s e p a r a t e d b y I E F u s i n g p H 3-10 ampholytes and r e d u c i n g SDS-PAGE. A f t e r f o c u s s i n g , t h e tube g e l s were l a y e r e d o n t o p o f 12% S D S - P A G E g e l s a n d i n c u b a t e d i n s a m p l e b u f f e r c o n t a i n i n g reducing agent f o r 5 min. Following i n c u b a t i o n , e l e c t r o p h o r e s i s i n t h e s e c o n d d i m e n s i o n was b e g u n c o n c u r r e n t l y f o r both samples. G e l s were f i x e d and s i l v e r stained. I s o e l e c t r i c p o i n t and M c a l i b r a t i o n s a r e i n d i c a t e d . r  89  90 DISCUSSION  I t has been r e p o r t e d t h a t F d l l F , Fdll,  was  r e c o v e r e d u s i n g t h e T s F - s p e c i f i c mAb,  possessed both Ag-binding a c t i v i t y the  a n t i - F d Ab  Levy the  p r o d u c e d by t h e Ts  1987).  response i n v i v o  In t h i s t h e s i s ,  F d l l F was  immunochemical, b i o l o g i c a l ,  The  p r o p e r t y was binding.  this  to  Chan, N o r t h ,  and b i o c h e m i c a l l e v e l s .  of F d l l F  measured u s i n g a s p l e e n c e l l  A g - b i n d i n g and b i o l o g i c  below.  activity  FdllF Ab  c u l t u r e system.  With  Furthermore,  additional  aimed at  Agisolating  By u s i n g g e l f i l t r a t i o n ,  both  were r e c o v e r e d f r o m a h i g h  When n o n - r e d u c i n g p r e p a r a t i v e SDS-PAGE  a single Fdll-associated,  a p p e a r e d t o b i n d Ag,  This  on t h e a n t i - F d  B i o c h e m i c a l c h a r a c t e r i z a t i o n was  Ag-binding moiety of F d l l F .  used,  was  for solid-  of  e x p e r i m e n t s s u g g e s t e d t h a t t h e s u p p r e s s i v e e f f e c t was  peak i e . > 500,000.  at  shown t o s u p p r e s s Ab p r o d u c t i o n i n a  c o n c e n t r a t i o n - d e p e n d e n t manner.  the  and  It  selectivity  used t o determine the f i n e s p e c i f i c i t y  a s s a y , F d l l F was  specific.  suppress  f a c t o r r e a c t e d a l s o w i t h f l u i d - p h a s e Ag.  The b i o l o g i c a l e f f e c t  r e s p o n s e was  FdllF  characterized further  shown t h a t F d l l F b o u n d t o and d e m o n s t r a t e d phase Fd.  B16G.  and t h e a b i l i t y  ( S t e e l e , Chu,  hybridoma,  was  30k M  isolated.  r  M  r  was  p o l y p e p t i d e , which  These f i n d i n g s a r e d i s c u s s e d  91  B16G  as  a Probe  Isolation mAb,  of F d l l F  B16G.  As  developed  f o r T Suppressor  to  A10F,  isolated B16G  has  indicated  i t was  respect  B16G  UV  or TsF^  2  that  B16G  different  lead  t o an  a UV-induced  untreated  controls.  induction  of tumor-specific  association  between d e f e c t i v e  suppression  o f t h e CS  t o UV  suppression  response  radiation as w e l l ,  o f t h e CS  abrogated the effect  was  used  impairment  response o f UV  the  was  and  noted  in with  In a completely UV  exposure  of  mice  function.  compared attributed  Kripke and  i n mice  t h e CS  to  1977).  the  An  systemic  B16G  was  i n the UV-induced  on  to  following  (Yee e t a l . 1 9 8 9 ) .  radiation  This  systems  i n immune  presentation  implicated  to  from UV-treated animals  (Fisher  was  used  B16G  to study  (Noonan e t a l . 1 9 8 1 ) .  was  response  I t i s known t h a t  Ag  then  "class-specific"  of rejection Ts  was  system.  outside  s y n g e n e i c tumor as  Failure  initially  itself  showed t h a t  was  lymphoid c e l l s  was  to study suppression i n  t y p e s o f TsF.  B16G  suppression.  can  TsF-specific  I t was  o f t h e NP  developed, but  transferred  TsF  a tool  useful  to reject  show t h a t  as  e t a l . (1987) TsF  Ab  i n vivo.  only  radiation  exposure  and  The  s u p p r e s s e d t h e CTL  e x p e r i m e n t a l system,  Adoptively fail  was  system.  not  radiation-induced to  not  to recognizing  different  which  of the  INTRODUCTION, B16G  in vitro  Steele but  1  because  tumor  been employed  TsF , that  both  a TsF  systems.  recognized  which  i n the  t o s t u d y t h e P815 active,  o t h e r Ag  possible  mentioned  shown t o be  P815.  was  Factor  used  systemic  In vivo,  response.  to  B16G The  92 investigators suppressive  al.  h a v e u s e d B16G  involved i n the  1989).  Thus,  suppression on  the  used  B16G  m a t e r i a l f r o m Ts  colleagues material  also prepared  B16G  immunoadsorbents  c u l t u r e SN.  f o r the  isolation  been e f f e c t i v e  in several laboratories.  immune r e s p o n s e  f o r the  recovery  Lastly,  r e g u l a t i o n of the  has  of IgE  suppressive  Sehon  and  response  i n the  I t has  recovered  suppressive (Sehon  study  effect  i n v i v o and  molecules  et  of  a modulating  when i t i s a d m i n i s t e r e d of  and  can  in different  be  Ag  systems. The TsF  use  o f Ab  molecule  putative raised heavy  i s not  TCR  an  The  they  constant  region  Sorensen  went on  and  Pierce  o f mAb,  which were used mAb and  Iverson  1985;  i n the  or  was  attempt  Taniguchi  (1982)  found  to bind  chain  prepared  effector  murine  allotypic  Ag-binding (1985)  I n an  f o r the  identify  Ag-binding,  as  of  mAb  TsF.  effector  specific  TsF,  Ferguson  recovery  variable  immunogens.  t h a t were  inducer  analysis.  and  region of to  TsF.  of  f o r the  respectively  t h a t were they  regions  of  Iverson  Ag-binding (Ferguson,  and  Iverson  1986) .  TsF  for functional  Both and  the on  the  KLH-TsF.  Although  Similarly,  identify  Using  determinants a  the  immunoglobulin  either produced  none o f them were c l o n o t y p i c i e . none  with the  isolated  to  the  for inducer  reactive  mAb  antiserum  of  for a constant  e x c l u s i v e t o B16G.  alloantiserum specific  reagent,  battery  specific  isotypes, Tokuhisa  chain.  specific  reagents  any and  a  were of  the  TsF,  colleagues  chain Beaman,  of and  groups used  biochemical  the  93 Immunochemical P r o p e r t i e s  Direct  binding  of  Initially,  however,  binding  B16G  of  to  of  FdllF the  to  data  FdllF,  or  from the  plates. Fd,  but  but  not  reactivity  not  data to  was  The  of  Table  explained  that  the  FdllF  react  concentrations  100% was  with  Fd  independent  was of  Fd.  The  inhibition) not  of Fd  Fd  could  whether Fd  of  any  as  in  well.  bound t o  Ag,  the That  f r o m medium c o n t a i n i n g  FCS  Fd-coated  indicated  nonspecific  the  than  6,  of  FdllF  that  to  FdllF  adsorption  FdllF  to  due  that  FdllF was  a  to  a  the  binding  level  of  particular,  determining  I t was  or  of  to  be  Fd.  quantified  and  1.25  binding  charge e f f e c t s .  Since  binding and  was  inhibition as  well.  It  f o r the  C-determinant  Fd  better  ug/ml to  solid-  approached  FdllF  The  various  to  inhibition  specific  ug/ml v s .  FdllF  solid-phase  of  whether  found that  v e r i f i e d that  C-molecule  FdllF  to  t e s t e d by  conformation  6 that  suggested that due  was  in fluid-  of  (0.312  the This  binding  OFd  was  inhibit  was  "fixed"  observation  simply  demonstrated  in solution.  C-molecule.  shown i n F i g .  inhibitors  was  with  o f Ag  and  ELISA allowed  specific  a variation  showed b i n d i n g  binding  shown i n F i g .  identical  and  surface.  conformation  As  by  With  DC10,  unrelated  ELISA.  1 showed d i r e c t  was  I, which  not  solid-phase  phase Fd.  shown u s i n g  Fig.  hybridoma,  other  possibility  could  of  FdllF  two  protein-coated  was  material prepared  irrelevant  The  Fd  FdllF-coated plates.  immunoassay, A g - b i n d i n g by is,  FdllF  Fd Fd  were for  50%  (or i t s analogs) i s an  acidic  protein  ( i s o e l e c t r i c p o i n t of Fd i s 3.7),  b i n d i n g was  i t was  due t o the net n e g a t i v e charge  solution.  of Fd while i n  I f so, then OFd would be expected t o be a b e t t e r  i n h i b i t o r of F d l l F b i n d i n g as peroxyformic imparted  conceivable that  acid oxidation  an even g r e a t e r n e g a t i v e charge t o the p r o t e i n .  Furthermore,  the i n t e r m e d i a t e i n h i b i t o r y a c t i v i t y of  c o r r e l a t e d with i t s expressed l e v e l of C-determinant as measured with the C - d e t e r m i n a n t - s p e c i f i c mAb,  reactivity,  16a.  seen i n F i g . 5 where the l e v e l of 16a b i n d i n g t o OFd i n t e r m e d i a t e between C-molecule and N-molecule.  OFd  This  was  was  T h i s lends  a d d i t i o n a l support t o the s u g g e s t i o n t h a t F d l l F i s s p e c i f i c f o r the C-determinant  of Fd.  That F d l l F should b i n d t o e i t h e r N- or C-determinant, opposed t o other s i t e s on the molecule, p a t t e r n of Fd a n t i g e n i c i t y . cell  was  as  c o n s i s t e n t with the  I t i s known t h a t a l l of the B and T  i n t e r a c t i o n s with Fd r e p o r t e d so f a r , are f o c u s s e d at the N-  and C-determinants.  The  s i g n i f i c a n c e of the  C-determinant  s p e c i f i c i t y of F d l l F , with r e s p e c t t o r e g u l a t o r y mechanisms, cannot be assessed at t h i s time.  However, i t i s of i n t e r e s t t o  compare F d l l F b i n d i n g with the determinant  s p e c i f i c i t y of the  s e c r e t e d by the c e l l s used i n the s u p p r e s s i o n assay. Fig.  9A,  a l l of the a n t i - F d Ab produced  by the c e l l s  from the C-molecule and Fd-immunized mice was molecule.  The  As shown i n obtained  d i r e c t e d t o N-  l a c k of r e a c t i v i t y with C-molecule was  e x p l a i n e d by the "hidden" e x p r e s s i o n of C-determinant b i n d i n g t o s o l i d - p h a s e , as the a n t i - F d mAb,  Ab  16a  not due t o i t s  ( s p e c i f i c f o r the  95  C-molecule), activity  was a b l e  (Fig. 5).  of the cultured cells  binding  distribution  results  implied that,  suppressed  C-determinant  of  the Fd molecule  Carrier  The p a t t e r n  was c o n f i r m e d  i n vitro,  as t h e c a r r i e r  (Fig. 9B).  C-determinant-specific Ab r e s p o n s e .  was d e m o n s t r a t e d  this  and Kapp  1984).  response Most  i n H-2  of the reported  binding  activity  columns.  ability  to FdllF,  ELISA or t h e determination and s t r u c t u r a l  molecule with in  described  FdllF.  Using  to bind  analogs.  such  of fine  raised insulin  does  they  using  of binding  using  share  detected  and had a M  reducing  agent.  was a l s o  seen w i t h  1986).  detection  I t displayed  t o n a t u r a l p r o t e i n Ag: lysozyme and Kapp  immunochemical  a T cell-derived,  a r a b b i t antiserum,  shown b y F d l l F  (Jensen  shown  b y Cone e t a l . (1987)  b y SDS-PAGE, w i t h  t o immunoadsorbent  specificity  However,  immunochemical p r o p e r t i e s t o F d l l F  specificity  also  A b on t h e immune  as i t s d i r e c t  t h e serum o f d e s e n s i t i z e d mice.  analyzed  o f F d was  A g - s p e c i f i c TsF have e x h i b i t e d Ag-  by t h e i r  similar  (Jensen,  (Weaver e t a l 1983) .  R e l a t i v e l y few examples have  properties  designation  (Takemori and  The h a p t e n - c a r r i e r m o d e l  animals  FdllF  observation.  f o r KLH-TsF  used t o e x p l a i n the e f f e c t s of a n t i - i d i o t y p i c  The  as t h e hapten  1 9 7 5 ) a n d T s i n t h e immune r e s p o n s e t o i n s u l i n  Pierce,  Ag  The  and N-determinant  w o u l d be c o n s i s t e n t w i t h  specificity  o f Ab  by t h e s i m i l a r i t y i n  o f serum Ab o f t h e m i c e  the anti-N-determinant  of  Tada  to bind  These  r  Ag-binding  some  features  the molecule  similar o f about  The f i n e  37k as  Ag  two o t h e r  TsF  ( A d o r i n i e t a l . 1984) a n d i n v e s t i g a t o r s used  96  i m m u n o a d s o r b e n t c o l u m n s made w i t h s t r u c t u r a l r e s p e c t i v e Ag t o d e m o n s t r a t e A more d i r e c t F d l l F was (1988). 30k M to  r  A rabbit antiserum p r o t e i n of F d l l F  o f TsF b i n d i n g .  (known as a n t i - p 3 0 ) was  and  system  and  hybridoma,  as w e l l .  differentiated T cell  did  not express e i t h e r marker.  independent  r a i s e d to the  shown, u s i n g FACS a n a l y s i s ,  and t h e A10  murine T c e l l s  to bind  but t o a p r o p o r t i o n of  Anti-p30  r e a c t e d w i t h t h e CD4  subsets i n a d d i t i o n t o thymocytes R e l a t e d t o t h i s work, b u t  and  that  i n an  l i n e o f s t u d y , A s k e n a s e and c o l l e a g u e s d e s c r i b e d T  c e l l - d e r i v e d , Ag-binding molecules response  t h a t were f o u n d t o  (Ptak e t a l . 1986a,b).  I n an e f f o r t  that T c e l l s  they  found  f r o m nude m i c e w e r e c a p a b l e o f p r o d u c i n g t h i s  binding factor a cloned c e l l  initiate to  c h a r a c t e r i z e the o r i g i n of the f a c t o r - p r o d u c i n g c e l l ,  anti-p30  the  shown by u s i n g an a n t i s e r u m d e v e l o p e d by N o r t h e t a l .  CD8  t h e CS  of  r e l a t i o n s h i p b e t w e e n a d i f f e r e n t Ag  not o n l y F d l l  normal  specificity  analogs  (Herzog e t a l . 1989). line  unpublished).  r e a c t i v e w i t h a n o v e l t y p e o f TCR,  B i o l o g i c P r o p e r t i e s of  with  Whether t h e a n t i s e r u m i s  p o s s i b l y e x p r e s s e d on  "primitive" T cell  Biologic activity  they derived  f r o m nude m i c e t h a t r e a c t e d s p e c i f i c a l l y  (P. A s k e n a s e ,  undifferentiated  Subsequently,  Ag-  subset, remains  an  t o be  seen. -  FdllF  o f F d l l F was  shown i n two  ways.  the a d d i t i o n of F d l l F t o Ag-primed spleen c e l l s p r o d u c t i o n o f a n t i - F d Ab.  T h i s was  Firstly,  suppressed  i n comparison  to  the  cultures  97 containing this  F d t o sheep r e d b l o o d  assay  forming  was d e v i s e d ,  cell  assay,  suppression. cultures for  of spleen  reliable  pulsed  the  source  cells  Therefore  cells  system.  an i n s u f f i c i e n t  B10.D2  Therefore,  of  protocols testing with  were  shown  Fd and then  entire  cells  cells  were  d o s e o f Ag) t o s t i m u l a t e  as part  theeffect  of FdllF  o f t h e immunization  o f F i g . 7 showed t h a t t h e amount  a strong  "signal"  above background, t o  was unknown, before  was d e t e r m i n e d .  i nthis  thesis.  t h emaximal  The r e s u l t s  cells  modified  so that t h e spleen  cells  and  exposed t o Fd, without  level  from two  t h a t were  continuously co-cultured with FdllF ( F i g . 10).  several  The m a j o r i t y o f i n v i t r o  was done w i t h A g - p r i m e d  incubation period  then  o f Ab  Ag-primed  t h e spleen  t o test  had t o be t r i e d  with FdllF  with FdllF  amount  primary  with.  i n t h e assay  suppression  plaque-  (H-2 ) m i c e w e r e u s e d a s  As t h e mechanism o f a c t i o n o f F d l l F variations  spleen  w e r e made i m m u n e t o F d b y p r i m i n g  The d a t a  s e c r e t e d Ab r e p r e s e n t e d  Therefore,  a high  i norder  t h e B10.D2 a n i m a l s  measure s u p p r e s s i o n  a different  of FdllF-mediated  Furthermore, with  feasible to  h a d a l s o shown t h a t  u s i n g ELISA.  o f Ab i n v i t r o .  them w i t h C-molecule. of  produced  f o r t h e assay.  of spleen  a syngeneic  protocol,  cells.  f o r t h e measurement  (ie. cultured briefly  production  experience i n  t o replace theconventional  measurement  the  Past  i t was n o t t e c h n i c a l l y  P r e l i m i n a r y experiments  would be used  in  m a t e r i a l o r medium a l o n e .  l a b o r a t o r y h a d shown t h a t  couple cell  control  for the  T h i s p r o t o c o l was  were f i r s t  pulsed  incubated  later with  washing between a d d i t i o n s .  FdllF  98 Following  a 2 h exposure t o Fd, t h e c e l l s  cultured  without  Suppression in  receiving  were washed and  any f u r t h e r a d d i t i o n s .  o f t h e a n t i - F d Ab r e s p o n s e  a concentration-dependent  maximum o f 6 0 % s u p p r e s s i o n  by F d l l F  manner u s i n g e i t h e r  was  protocol.  was a t t a i n e d u s i n g t h e o r i g i n a l  of Ag p u l s i n g f o l l o w e d by c o - c u l t u r i n g w i t h F d l l F . emphasized that t h e data results  from d i f f e r e n t  curvilinear binding  activity  preparation protocol, Other naive  1979;  suppression investigators  with and  spleen  cells  cells  f o r suppression  (1985).  effectiveness  were c o - c u l t u r e d w i t h (Taniguchi, work,  were p u l s e d  proposed by Hoffmann  would bind t o T c e l l s  response.  (Fig. 12).  100% s u p p r e s s i o n  with  Saito,  and Tada  suppression factor  cells  when  both  was  followed  t o "arm them"  r e p o r t e d by Moser,  pulsing, i s provided  during the suppressed  mutual  and t h e F d l l F  Kauffman,  An a l t e r n a t e e x p l a n a t i o n , f o r t h e  of FdllF  network theory,  and Ag-  By r e v i s i n g t h e  nearly  has been  be  observed  t h e assay  For the present  assay  using the  1 0 0 % was n o t e d  have a c h i e v e d  A  I t should  suppression  The e f f e c t i v e n e s s o f p u l s i n g s p l e e n  Abbas  their  approaching  when t h e s p l e e n  factor  that  The  were r e p r o d u c i b l e .  Kapp e t a l . 1976).  Ag.  that both  A g a n d T s F a t t h e same t i m e  observed by  technique  of FdllF.  between percent  suggested  or Ag-primed  soluble  o f F i g . 10 w a s c o m p i l e d  batches  relationship  achieved  state,  (1980).  When t h e s p l e e n  preventing cells  "plus-minus"  I t was p o s t u l a t e d  monovalent Ag-binding  o f complementary  stimulation,  by t h e  were  specificity  and  factors inhibit  t h e i n d u c t i o n o f an Ab first  exposed t o Ag  99 f o l l o w e d by reached  co-culturing  a maximum a n d  possible  that  "headstart" of F d l l F  towards  the  additional "revised"  d i d not  not.  were  A10F  similar  as  A10F  the  argued  1987;  they  Due  assay,  CTL  (Figs.  a  suppress  the  effects  (Steele,  with FdllF activity  being  in Fig.  experiments, was  themselves s a m e mAb.  In  to  alter  P815  the  w i t h DBA/2 s p l e e n  Chan,  Stammers,  Collectively,  FdllF  were  response  does not  FdllF  not.  while  factors  using the  or  the  cells  Singhai, data  are  Ag-specific.  of F d l l F  was  also  shown i n v i v o .  o f F d - i m m u n e , DBA/2 H-2  t o the wide  seen  t o P815,  CTL  11)  the  whereas A10F  syngeneic  reaction  and  u n r e l a t e d Ag.  the molecules  manner and  lymphocyte  As  t o an  although the  isolated  10  idea that  response  assays,  were b o t h  compared t o c o n t r o l  FdllF.  Th  of suppression,  criss-cross  Chan e t a l . 1988).  responses  levels  response  that  cellular  of a mixed  Biological s e r u m Ab  It i s  suppressive  Ag-specific.  culture  suppressed  does  was  in reciprocal  histoincompatibletargets Levy  given the  "original"  t h e Ab  a concentration-dependent  consistent  as  suppress  in different  magnitude  and  using the  of F d l l F  I t c o u l d be  addition,  on  had  suppression  ( F i g . 10).  (Table II) supported the  I I showed t h a t  tested  period  i n observed  s u p p r e s s i v e i n t h e Ab  did  to plateau  production before the  difference  activity  Conversely,  in  Ab  protocol  FdllF  was  pulsing  experiments  suppressive  Table  appeared  the percentage  began.  Despite  11,  t h e Ag  with FdllF,  mice were  The suppressed,  a n i m a l s , when c h a l l e n g e d w i t h F d range  i n Ab  responses  of the  and  mice  100 following  their  according  to their  or  PBS  way,  initial  s e r u m Ab  "treatment"  Again,  Ab  because  recorded  as  levels  groups p r i o r  approximately  pretreatment  immunization,  equal  levels of the  and  to  animals  receiving  wide  receive  range  changes  of  either  levels  ranked  FdllF,  factor. having  A10F,  In  this  similar  FdllF,  responses,  i n Ab  were  placed into  numbers o f a n i m a l s  would  individual  the  the  before  A10F,  or  data  was  and  PBS.  after  treatment. It the  was  clear  from  FdllF-treated  administration levels  relatively Ab  levels  close  response 14,  Ag  "no  control  t o Fd  that  response  they  and  can  group  large  probably  animals  more s l o w l y  such  KLH.  of the  were c a p a b l e  of  as  Ab  increased.  The  account  the  for  remained  shown t h a t  a  compared t o the  data  animals  responding  of  anti-Fd  that  Indeed,  identical  levels  factor  the  E a r l i e r work had  develops  a more c o m p l e x Ag  following  by  of Fd  line.  t h e Ab  In contrast,  groups had  change"  that  decreased  immunogenicity  s h o w i n g t h e Ab  indicated  had  o r A10F  response to  o f F i g . 13  challenge.  o f most o f t h e  to the  secondary  PBS  poor  data  animals  and  of the  the  to  readily  the  of F i g . KLH,  to  this  Ag. It  was  challenge the  noted  appeared  experiment  hours  of F d l l F  factor shown).  that  resulted I f an  the  timing  t o be  of Figs.  of  critical 13  treatment.  factor f o r the  and  14,  The  inability  in a failure  to  immune r e s p o n s e  Ag  was  suppress  administration  and  Ag  suppressive effect. given within t o g i v e Ag the  i s determined  by  several  soon  response  In  after  (data  a balance  not  between  101 help  and  suppression,  delivery  of  outcome.  a  In  this  Ag  helped  was  "suppressor"  assay  order  determine  the  A n a l y s i s of  biochemical  FdllF  of  of the  active  analyzed  SN  i n comparison to was  In  addition,  b a n d was  was  initially  considered  to  they In  50k  M  were an  eluted  r  but  minor  to  m a t e r i a l was  filtration  M  r  SN,  (FPLC).  not  in  of  suppression.  cells  to FdllF  and  suppression.  Ag-binding  spent  reducing  FdllF  fractionated FdllF  culture  unique to  recovered. present  controls.  the  Fdll band  the  greater reason,  i t  component.  i n the  low  a  ( F i g . 15).  this  possible FdllF  t o have  showed  Although at  For  at  SN.  A predominant  eluted material  i t was  of  immunoaffinity  SDS-PAGE,  material.  were p r e s e n t  thought  molecule  permitted the  components a l s o p r e s e n t  isolate  The  was  another  because they  initially  attempt  as  by  i n the  i n comparison to  There were o t h e r , 45  of the  control  80k  unique to F d l l  concentrations  of  a p p e a r e d t o be  always present a p r o t e i n at  not  level  m a t e r i a l from F d l l  culture r  i n f l u e n c e the  measurement of the  of  FdllF  number o f bands t h a t  30k  can  time  difficulties  exposure  observed  limited  at  signal  the  f o r the  identification  e l u t e d m a t e r i a l , as  M  by  made p o s s i b l e b e c a u s e B 1 6 G  enrichment The  in vitro  instance, the to  i t i s p o s s i b l e that the  i s also supported  the  Biochemical  The  "helper" or  This  developing  then  range  of  concentrations,  significance.  i n i t s n a t i v e form,  B16G-  using high performance  m a t e r i a l was  r e s o l v e d as  gel a  single  102 peak,  but  unexpectedly,  at  a M  greater than  r  of p r e v i o u s l y reported T c e l l - d e r i v e d not  exceeded  FdllF  had  affinity  145k  (Beaman e t  aggregated enrichment  or  intrinsic  there  are  nature  explanations  c o l u m n p r o t o c o l may  of  TsF  for  of  for aggregation.  The  have been  compounded by  f u r t h e r concentration of the  w h i c h was  required f o r high performance  activity.  In  comprised a separate  also  identified  This  active  reducing 30k  M  i n the  fractions  experiment,  vicinity  m a t e r i a l from  SDS-PAGE,  a pair  of bands  p r o t e i n s was  binding  activity  comparison Despite  the  filtration, Ag-binding experiments.  the and  thought  because  to the  at  other  failure  high M  t o have  of the  low  of  45k  the  M  .  little  could  sample,  Ag-binding was  peak. when a n a l y z e d  l a r g e amounts o f b o t h  about  This  activity  r  fractions,  One  and  filtration.  suppressive  individual  contained  conditions.  that possessed  of t h i s  p r o t e i n s d e s c r i b e d above.  r  contained these  of  Besides  denature  under very  p e a k was  has  solution,  upon r e c o v e r y  gel  that  immunoaffinity  material to  acidic  the  peak  80k  and  fractions  However,  the  significance  concentrations  by  also  role  for  of  Ag-  observed,  in  p r o t e i n bands.  to  determine  correlation suppressive  of the activity  the 80k was  native M and  30k  of F d l l F  r  M  r  confirmed  r  have  TsF  P i e r c e 1983).  i n aqueous  M  either  aggregate  The  native  molecules  Aggregation and  have caused F d l l F  The  e x p l a n a t i o n was  conditions used  (Webb, K a p p ,  hydrophobic  other  One  gel filtration.  been p r e v i o u s l y r e p o r t e d the  Ag-binding  a l . 1984).  under the  200k.  proteins with  by  gel  with  these  103 The  F d l l F - e n r i c h e d m a t e r i a l was a l s o f r a c t i o n a t e d  preparative  using  SDS-PAGE, run under non-reducing c o n d i t i o n s .  This  same t e c h n i q u e was used s u c c e s s f u l l y f o r both A10F (Chan e t a l . 1988)  and GAT-TsF  Fig. in  (Sorensen, P i e r c e ,  20, heavy bands at 80k M f r a c t i o n 4.  r  and Webb 1983).  were r e c o v e r e d from both samples  No A g - b i n d i n g a c t i v i t y was a s s o c i a t e d with these  or any f r a c t i o n s i n t h e v i c i n i t y ;  i e . f r a c t i o n s 1 t o 11 ( f i g . 19)  from e i t h e r F d l l - o r DClO-derived m a t e r i a l . 80k  M  materials  r  As shown i n  To determine i f the  were d i f f e r e n t by some other p h y s i c a l parameter,  b e s i d e s M , they were a n a l y z e d by two-dimensional g e l r  electrophoresis the  and observed t o be i d e n t i c a l ,  l i m i t s of  technique.  However, 30k M in  within  r  bands, unique t o F d l l  f r a c t i o n s 12 t o 14.  binding  material,  T h i s was c o i n c i d e n t  were  with t h e f i r s t Ag-  a c t i v i t y peak of the F d l l - d e r i v e d m a t e r i a l .  A single  band was observed i n f r a c t i o n 12, whereas two o r t h r e e seen i n f r a c t i o n s 13 and 14. repeated, u s i n g unique 30k M  r  detected  When p r e p a r a t i v e  bands were  SDS-PAGE was  10 times t h e amount of s t a r t i n g sample, t h e  material  from the f r a c t i o n s was r e c o v e r e d and  a n a l y z e d by two-dimensional g e l e l e c t r o p h o r e s i s .  In s p i t e of the  numerous c o r r e s p o n d i n g spots shared between t h e c o n t r o l and F d l l F material, there in  were some unique p o l y p e p t i d e s  p a r t i c u l a r ) i n the F d l l F  (and one major one  sample at around t h e 30k M  r  position.  However, these two-dimensional g e l r e s u l t s a l s o demonstrated how heterogeneous the r e c o v e r e d 30k M It  was t h e r e f o r e  r  m a t e r i a l was (data not shown).  shown t h a t an unique 30k M  r  p r o t e i n with  104 associated Ag-binding derived  SN.  This moiety  proteolysis  yielding  with Ag-binding the of  30k  M  both Ag-binding  and  activity  M  one  material  r  denatured  SDS  gel  TsF  r e p o r t e d by  ( F i g . 19)  susceptible binding  with  either  "identical" was  than is  one  an  additional  80k  spotting  or  45k  that  ( f i g . 18)  pattern  seen  two-dimensional  the  rule  out  region"  the  that  of the  between the  TsF,  molecule.  of the  non-reducing  a 24k  M  one M  protein  component  protein associates  r  80k  actually the  M  r  Indeed,  material,  that  and  B16G unique  Fdll  FdllF  of  that  banding  FdllF. more  material  i s specific  material  the  ( f i g . 21)  consist  "unique"  i s that  was  molecule.  polypeptide.  r  The  Ag-  r  i t i s a component p a r t o f  and  30k  The  (1982)  gel electrophoresis  that  control  no  suppressor  with the  e l o n g a t e d s p o t s may  possibility  30k M  capable  partially  into  nonspecific  r  was  i t was  Cantor  )  that  recovered  TsF  M  was  i n the  and  I t degraded M  that  14k  breakdown p r o d u c t .  McVay-Boudreau,  a 45k  than  Since  the bottom  i s composed o f more t h a n  Another  position  expected.  be  either  Fdll-  some  (less  protein  only a p a r t of the  m o l e c u l a r s p e c i e s and  hidden.  "constant Mr  the  that  the  possibility  demonstrated  recovered from  may  and  formally  Conceivably,  ever  It i s possible  a n a l y z e d by  does not  was  to proteolysis.  molecule  unknown.  alternative  suppressive activity.  Fresno,  Whether F d l l F is  An  could conclude  activity  have undergone  part of a larger  or constituted  Ag-binding  may  recovered from  s m a l l e r peptide fragments  was  suppressive  c o u l d be  itself  activity.  molecule  r  activity  for a  at the  would not  80k be  105 Two fig.  bands 18.  An  t h e B16G lower  M  was  but  component  r  as  unique  Fdll  i s frequently  always  i t s r e c o v e r y as  clearly  proteins  in  recovered  present  compared w i t h the  composed o f a then  heavily  yielded Ag-binding ( f i g . 20,  from  due  from  to  30k  M  the  fractions  of both  the  eluted  30k  13  45k  assuming  M  chain,  the  protein  r  r  the  is  recovered  30k  fractionation  contained several polypeptide 14).  45k  material,  There  proteins.  p r e p a r a t i v e SDS-PAGE  and  expected  column.  gels) that with other  be  equivalent  imunoaffinity  that  and  and  components would  contaminated  fractions  30k  quantities,  the non-reducing  i n the  two  (from two-dimensional  was  quantities  the  i n equimolar  evidence  present  identified  i t i s not  of the p r o t e i n s  Additionally,  bands  M  actually  observed  protein  r  45k  a heterodimer,  recovery clear  were  r  shown).  FdllF  be  M  unique  amount o f  forming to  45k  eluates,  (data not If  at  M  r  Therefore  equimolar  c o m p o n e n t s may  obscured  by  other  actually  be  contaminating  proteins. The the  known s t r u c t u r e s  1984) and  i d e a of a h e t e r o d i m e r i c form  and  mAb.  the At  TCR  of  that  present,  t h e h e t e r o d i m e r i c TCR Saito  et  suggested TCR.  a l . 1987;  Loh  similarities  Zheng e t  s e c r e t e d by  r e p o r t e d TsF have been  molecules:  i s i n keeping  (eg. S a i t o  and  c h a r a c t e r i z e d by  i t i s unknown i f F d l l F a/15  et a l . 1987).  o r 876 Recent  hybridoma  cloning t o any  et  a l .  of  1986;  r e p o r t s have  d e s c r i b e d an A g - b i n d i n g that  gene  (Brenner  with  Taniguchi  i s related  between hybridoma-derived  a l . (1988)  a Th  of F d l l F  combined w i t h the  TsF  and  the  a/15  molecule I - J  +  chain  of  106 an  inducer  and  TsF t o give  Moorhead  with  t w o mAb  TCR fi c h a i n of  (1988)  CD3-expressing  activity.  Fairchild,  characterized a DNP-specific  specific genes.  suppressive  f o r the products  o f Vjj8,  TsF t h a t  a family  Ts^ hybridomas TsF.  correlated with  I n a d d i t i o n , a mAb  specific  chain,  immunoprecipitated  a p r o t e i n o f t h e same M  a  chain,  from t h e hybridoma  cells.  1988)).  able  factors)  hand,  i t i s known t h a t  t o b i n d Ag i n t h e absence  suggests  that  although  surface Ts  (45k) as t h e  o f MHC  i s co-  (Clevers  et a l .  (and t h e i r  from t h e a/fi TCR-bearing  differ  r  f o r t h e TCR  ( T h e CD3 m o l e c u l e ,  t h e a / f i TCR o n t h e T c e l l  On t h e o t h e r  soluble  of murine  the a b i l i t y to  a  with  reacted  K u c h r o o e t a l . (1988) s h o w e d t h a t s e l e c t i o n  b i n d Ag and produce  expressed  Kubo,  cell  by  being  (Webb e t a l . 1 9 8 9 ) .  t h e Ts r e c e p t o r  may b e s i m i l a r ,  This  i t i s not  t o t h e a / f i TCR.  identical  Summary  The  T s F - s p e c i f i c mAb,  soluble the  Ag  at least  a  Although  f o r the study  and i s o l a t i o n  of  systems. tool  i n t h e development  In several respects,  a continuous  Fdll.  FdllF,  r a i s e d f o r t h e c h a r a c t e r i z a t i o n o f a P815-  was a c r i t i c a l  hybridoma.  for  by t h e Ts h y b r i d o m a ,  TsF, i t has been u s e f u l  i n other B16G  as  secreted  Ab was o r i g i n a l l y  specific TsF  mediator  B16G, h a s b e e n u s e d t o i s o l a t e  source  four  years  f o rFdllF. with  this  cell  of the Fdll  line  Firstly, Fdll  respect  was a g o o d remained  to the production  of  choice stable FdllF.  107 Secondly,  the exposure 2+  influx  event cell  ions, dye  as measured u s i n g  ( S t e e l e , Chu,  secreted  receptor  form  studies  Ag-specific antiserum, (North  the  intracellular  suggest  molecule. anti-p30,  the interaction  i s unknown.  the presence Lastly,  was  e t a l . 1988).  and Levy  (Imboden e t a l . 1 9 8 6 ) .  of a receptor do  a calcium-sensitive  Chan, N o r t h ,  has been a s s o c i a t e d w i t h surface  flux  t o Fd induces  .  o f Ca  fluorescent  of F d l l  using  shown  membrane-associated moiety,  Whether F d l l F the  i s a  calcium  membrane-associated,  FACS a n a l y s i s , t h e r a b b i t  to bind  These data  This  of ligand with i t s  However,  of a  1987).  also  to the Fdll suggest  possibly FdllF,  hybridoma  the presence  expressed  by  of a  Fdll  hybridoma. The p r o p e r t i e s o f F d l l F other in  reported  vitro  TsF.  a 30k M  consistent with  I t bound Ag,  and i n v i v o ,  composition,  were  r  had suppressive  and has as a p a r t Ag-binding  the Ag-binding  several  important  determinants not  interest  areas,  of FdllF.  such  However,  one c h a i n .  Further  establish  the molecular  both  molecular  FdllF  that  i f a n y , t o t h e known  suppressor for activity  were  FdllF i s  i s actually studies  of FdllF TCR  that  further investigation.  biochemical  composition  of  restrictions  characterized t o warrant  more t h a n  by  the characterization  as e x p r e s s i o n  that  activity  I t i s acknowledged  i t i s concluded  i s the p o s s i b i l i t y  relationship,  w o r k was  (eg. I-J) and g e n e t i c  addressed.  sufficiently  molecule  of i t s  shown  chain.  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Analysis of antigen-specific, Ig-restricted cell-free material made b y I - J L y - 1 c e l l s ( L y - 1 T s i F ) t h a t i n d u c e s L y - 2 cells t o e x p r e s s s u p p r e s s i v e a c t i v i t y . E u r . J . Immunol. 11:905-12. +  Y a m a u c h i , K., D. M u r p h y , H. C a n t o r , a n d R. K. G e r s h o n . 1 9 8 1 b . A n a l y s i s o f an a n t i g e n - s j j e c i f i c , H - 2 - r e s t r i c t e d c e l l - f r e e p r o d u c t ( s ) made b y " I - J Ly-2 c e l l s (Ly-2TsF) t h a t suppresses Ly-2 c e l l - d e p l e t e d spleen c e l l a c t i v i t y . E u r .J . Immunol. 11:913-18. Y e e , G. K., J . G. L e v y , M. L . K r i p k e , a n d S. E . U l l r i c h . 1 9 8 9 . The r o l e o f s u p p r e s s o r f a c t o r s i n t h e r e g u l a t i o n o f immune responses by u l t r a v i o l e t radiation-induced s u p p r e s s o r Tlymphocytes. I I I . I s o l a t i o n o f a suppressor f a c t o r with t h e B16G m o n o c l o n a l a n t i b o d y . C e l l . Immunol, ( i n p r e s s ) . Z h e n g , H., M. B o y e r , A. F o t e d a r , B. S i n g h , a n d D. R. G r e e n . 1 9 8 8 . An a n t i g e n - s p e c i f i c h e l p e r T c e l l h y b r i d o m a p r o d u c e s an antigen-specific suppressor inducer molecule with i d e n t i c a l a n t i g e n i c f i n e s p e c i f i c i t y . J . Immunol. 140:1351-58.  117 APPENDIX THE C O M P O S I T I O N S Bicarbonate  OF B U F F E R S AND  SOLUTIONS  buffer  1.59 g N a C 0 2.93 g N a H C 0 0.2 g N a N i n 1 1 o f d i s t i l l e d w a t e r , a d j u s t t o p H 9.6 w i t h H C 1 ; s t o r e a t 4°C ( V o l l e r , B i d w e l l , a n d B a r t l e t t 1 9 7 6 ) . 2  3  3  3  Diethanolamine  buffer  97 m l d i e t h a n o l a m i n e 0.1 g M g C l . 6 H 0 0.2 g N a N i n 8 0 0 m l o f d i s t i l l e d w a t e r , a d j u s t t o p H 9.8 w i t h H C 1 ; a d d d i s t i l l e d w a t e r t o 1 1; s t o r e a t 4°C ( V o l l e r , B i d w e l l , a n d B a r t l e t t 1976) . 2  2  3  Phosphate b u f f e r e d  s a l i n e (PBS)  0.137 M N a C l 2 7 . 0 mM K C 1 7 1 . 9 mM N a H P 0 . 1 2 H 0 1 4 . 7 mM K H P 0 2  4  2  2  4  Phosphate buffered PBS 0.05%  Tween 20  saturated  s a l i n e c o n t a i n i n g Tween 20 ( P B S - T w e e n )  (v/v) ( V o l l e r ,  (NH ) SQ 4  2  B i d w e l l , and B a r t l e t t  1976).  4  900 g ( N H ) S 0 a r e added t o 1 1 o f d i s t i l l e d water; t h i s i s heated u n t i l (NH ) S0 i s dissolved completely; the hot s o l u t i o n i s t h e n q u i c k l y f i l t e r e d u s i n g Whatman No.1 p a p e r ; t h e s o l u t i o n i s c o o l e d a t room t e m p e r a t u r e t o a l l o w f o r c r y s t a l f o r m a t i o n ; a t t h i s t i m e t h e s o l u t i o n i s a d j u s t e d t o p H 7.4 u s i n g N H O H ( M i s h e l l and S h i i g i 1980). 4  2  4  4  2  4  4  SDS-PAGE 1) S t o c k  Solutions solutions  118 a) 1 0 %  APS  0.05 g A P S 500 u l d i s t i l l e d w a t e r , b) 30%  acrylamide  (30%  prepare T,  3.3%  daily C,  see below)  29 g a c r y l a m i d e (Bio-Rad) 1 g bis-acrylamide (Bio-Rad) i n 100 m l o f d i s t i l l e d w a t e r ; t h i s s o l u t i o n i s f i l t e r e d (0.45 um) s t o r e d a t 4°C, a n d p r o t e c t e d f r o m l i g h t . Note: % T= ( ( g a c r y l a m i d e + g b i s - a c r y l a m i d e ) -r t o t a l v o l u m e ) x 100% % C= ( g b i s - a c r y l a m i d e -f ( g a c r y l a m i d e + g b i s - a c r y l a m i d e ) ) x 100% c) 2 5 % a c r y l a m i d e  ( 2 5 % T,  2.5%  C)  25 g a c r y l a m i d e 0.65 g bis-acrylamide i n 100 m l o f d i s t i l l e d w a t e r ; t h i s s o l u t i o n s t o r e d a t 4°C, a n d p r o t e c t e d f r o m l i g h t . d) s t a c k i n g  (upper)  0.625 M T r i s 0 . 5 % SDS a d j u s t t o pH  (Trizma, 6.8  e) s e p a r a t i n g 1.87 M T r i s 0 . 5 % SDS a d j u s t t o pH f) r u n n i n g 0.25 1.92 1.0%  gel buffer  using  (lower)  8.8  buffer  M Tris M glycine SDS  base, HC1;  (5 x )  T-1503,  HC1;  Sigma)  store at  gel buffer  using  i s filtered  4°C.  (5 x )  store at  4°C.  (10 x )  (G 7 1 2 6 ,  2) f o r 15 m l o f a 1 0 % 6 ml 25% a c r y l a m i d e 3 ml lower g e l b u f f e r 6 ml HPLC-pure water  Sigma)  (% T)  separating gel solution:  (0.45  um)  119 mix w i t h o u t i n t r o d u c i n g 70 u l 1 0 % A P S 7 u l TEMED 3) f o r 7.5 m l o f a 4%  a i r ; t o polymerize,  (% T) s t a c k i n g  gel solution:  1 ml 30% acrylamide 1.5 m l u p p e r g e l b u f f e r 5 ml HPLC-pure water mix w i t h o u t i n t r o d u c i n g a i r ; t o p o l y m e r i z e , 100 u l 1 0 % A P S 10 u l TEMED IEF  add:  add:  solutions  1) S t o c k  solutions  a) 1 0 % C H A P S 0.5 g C H A P S i n 5 m l d i s t i l l e d b) s a m p l e 9.8 3% 2% 100  water.  buffer  M urea CHAPS ampholines mM DTT  c) o v e r l a y  buffer  8 M urea 3% C H A P S 2% a m p h o l i n e s 100 mM DTT sample and o v e r l a y d) 3 0 % a c r y l a m i d e  buffers solution  are aliquoted  and stored  ( 3 0 % T, 2 . 7 % C)  29 g acrylamide 0.8 g bis-acrylamide i n 100 m l o f d i s t i l l e d w a t e r ; t h i s s o l u t i o n s t o r e d a t 4°C, a n d p r o t e c t e d f r o m light.  e)  anolyte  a t -70'C.  i s filtered  ( 0 . 4 5 urn)  120 20 mM NaOH, prepare  f r e s h , degas b e f o r e use.  f) c a t h o l y t e 10 mM H o P 0 , prepare 4  f r e s h , degas b e f o r e use.  2) f o r 5 ml of IEF g e l s o l u t i o n : 2.75 g urea 0.65 ml 30% acrylamide 1 ml 10% CHAPS 0.85 ml HPLC-pure water 0.36 ml pH 3-10 ampholines to polymerize, add: 5 u l 10% APS 3.5 u l TEMED IEF s o l u t i o n s were adapted from t h e method o f Bravo (1984) with the e x c e p t i o n o f the CHAPS and 30% acrylamide s o l u t i o n . The use of CHAPS i n p l a c e o f T r i t o n X-100 was o r i g i n a l l y d e s c r i b e d by Perdew, Schaup, and S e l i v o n c h i c k (1983). The composition of the 30% acrylamide s o l u t i o n was d e s c r i b e d i n t h e Bio-Rad t e c h n i c a l b u l l e t i n 1144.  

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