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The role of DNP in antigen activation of cellular immune responses Waterfield, John Douglas 1973

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THE ROLE OF DNP IN ANTIGEN ACTIVATION OF CELLULAR IMMUNE RESPONSES  by  JOHN DOUGLAS WATERFIELD B.Sc.  Microbiology  University of B r i t i s h Columbia  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  In the Department of Microbiology  We accept t h i s thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA June 1973  In p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requirements f o r  an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the  L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e  and  study.  I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s for  s c h o l a r l y purposes may  by h i s r e p r e s e n t a t i v e s .  be granted by  permission.  Department of  Microbiology  The U n i v e r s i t y of B r i t i s h Vancouver 8, Canada  Department or  I t i s understood t h a t copying or p u b l i c a t i o n  of t h i s t h e s i s f o r f i n a n c i a l g a i n written  the Head o f my  Columbia  s h a l l not be  allowed without  my  ABSTRACT  In animals immunized with 2,4 dinitrophenyl (DNP) haptenc a r r i e r protein conjugates, no i n v i t r o c e l l u l a r response i s e l i c i t e d by DNP, either alone, or when coupled to a heterologous carrier.  In contrast, animals immunized with haptenic peptide-  c a r r i e r conjugates do mount an i n v i t r o c e l l u l a r response towards the haptenic peptide.  This apparent inconsistency l e d us to  compare the i n vivo and i n v i t r o c e l l u l a r immune responses to a synthetic peptide antigen and i t s DNP derivative to determine the a c t i v a t i o n s p e c i f i c i t y of the c e l l s evoking this response. Guinea pigs were immunized with either the DNP substituted immunogen (DNP-N-10-C) or i t s unsubstituted form (N-10-C) and subsequent  i n vivo or i n v i t r o c e l l u l a r activation was evaluated  for DNP alone, DNP coupled to the homologous determinant, and DNP coupled to heterologous c a r r i e r s . The data suggests that i n DNP-N-10-C immune guinea pigs, DNP substitution opens a new determinant exhibiting, i n antigen reactive c e l l s , a unique s p e c i f i c i t y towards the DNP moiety as well as a portion of the peptide to which i t i s conjugated. However the DNP group by i t s e l f does not have the configurational requirement to evoke c e l l u l a r activation.  I t therefore plays a minor role i n  a c t i v a t i o n of the c e l l u l a r immune response; the major contribution being supplied by the peptide portion of the 'shared' determinant.  iii  TABLE OF CONTENTS Page INTRODUCTION AND LITERATURE REVIEW  1  I.  Clonal Selection Theory of Immunological D i v e r s i t y  1  II.  C e l l Types Involved i n the Immune Response  2  III.  S p e c i f i c i t y of Humoral Antibody and Immunocompetent Bone Marrow Derived C e l l s  3  IV.  S p e c i f i c i t y of Immunocompetent Thymus Derived Cells  V.  D e f i n i t i v e Studies on the Hapten-Carrier  .  5  9  Relationship Using DNP,  MATERIALS AND METHODS I.  .  11  Synthesis, P u r i f i c a t i o n , and Quantitation of the Antigens, Antigenic Determinants, and Peptides  .  .  .  . 11  II.  Immunization of Guinea Pigs  2k  III.  Skin Tests  2k  IV.  Migration I n h i b i t i o n  V.  Lymphocyte Stimulation  VT.  S t a t i s t i c a l Analysis  ' . . . . 25 .  .  .  .  RESULTS AND DISCUSSION  26 28  29  I.  Skin Tests  29  II.  Migration I n h i b i t i o n Factor (MIF) Production  3^-  III.  Lymphocyte Stimulation  kk  IV.  Concluding Discussion  52  APPENDIX  56  LITERATURE CITED  58  LIST OF TABLES  The amino acid composition and the expected molar r a t i o s of the amino acids i n the antigen and peptide determinants  Amino acid degradation values of Beckman standard amino acids The amino acid composition and the expected molar r a t i o s of the amino acids i n the DNP antigens and DNP peptide determinants  The amino acid composition and the expected molar r a t i o s of the amino acids i n the DNP antigen and DNP peptide determinants Skin reactions observed on guinea pigs immunized with N-10-C Skin reactions observed on guinea pigs immunized with N-10-C Skin reactions observed on unimmunized guinea pigs The e f f e c t of the homologous antigen, haptenic peptides, and DNP substituted heterologous compounds on the migration of spleen c e l l s derived from guinea pigs sensitized to DNPN-10-C The e f f e c t of the homologous antigen, haptenic peptides, and DNP substituted heterologous compounds on the migration of spleen c e l l s derived from guinea pigs sensitized to N-10-C  LIST OF TABLES  The effect of the homologous antigen, haptenic peptides, and M P substituted heterologous compounds on the migration of spleen c e l l s derived from unsensitized guinea pigs  -thymidine incorporation i n lymph node c e l l cultures from DNP-N-10-C sensitized guinea pigs i n response to the substituted and unsubstituted forms of the homologous antigen, haptenic . peptides, and M P substituted heterologous compounds  H-thymidine incorporation i n lymph node c e l l cultures from N-10-C sensitized guinea pigs i n response to the substituted and unsubstituted forms of the homologous antigen, haptenic peptides, and M P substituted heterologous compounds  H-thymidine incorporation i n lymph node c e l l cultures from unsensitized (control) guinea pigs i n response to the substituted and unsubstituted forms of the main antigen, haptenic peptides, and M P substituted heterologous compounds previously used  LIST OF FIGURES Figure  1.  Flow diagram of s o l i d phase peptide synthesis  2.  DNP substitution of the N-TRI peptide  3.  Space f i l l i n g model of the NH -heptapeptide (N-HEPTA) before and after DNP substitution  ABBREVIATIONS  N-IO-C  NH -Ala-Tyr -Lys -He -Ala -Asp -Ser -(Gly)  N-HEPTA  NH -Ala-Tyr-Lys -He-Ala-Asp-Ser-COOH  N-TRI  NH -Ala-Tyr-Lys-COOH  -C*COOH 10  2  NH -Ile-Ala-Asp-Ser-COOH  N-TETRA  0  N-PENTA  NH -Ala -Tyr -Lys -He -Ala -COOH  DNP-N-IO-C  NHp -Ala-Tyr -Lys -He -Ala-Asp -Ser -(Gly) -C -COOH 1 1 10 DNP DNP  DNP-N-8-N  NH -Ala-Tyr-Lys-Ile-Ala-Asp-Ser-(Gly) -N**-COOH ^ 1 1 8 DNP DNP  DNP-N-HEPTA  NH -Ala-Tyr-Lys-Ile-Ala-Asp-Ser-COOH 1 1 DNP DNP  DNP-N-TRI  NH -Ala-Tyr-Lys-COOH 1 1 DNP DNP  DNP-N-PENTA  NH -Ala-Tyr-Lys-Ile-Ala-COOH 1 1 DNP DNP  SER  NH^-Ser-Ser-Ser-Ser-COOH  2  k DNP-SER k  *  C  ** N  NH -Ser-Ser-Ser-Ser-COOH 1 DNP Ala-Pro-Va1-Gin-Glu-COOH Ala -Tyr -Lys -He -Ala -Asp -Ser -COOH 1 DNP  ACKNOWLEDGEMENT S  I would l i k e to thank Dr. J u l i a Levy f o r her invaluable encouragement, refreshing suggestions, and constructive of both the research and writing of t h i s thesis.  criticism  I also wish to  thank Dr. Douglas Kilburn f o r h i s h e l p f u l guidance of the research and Mrs. Barbara K e l l y f o r her cooperative contributions to the work.  F i n a l l y , I would l i k e to extend my thanks to my committee,  Dr. J . J . R. Campbell, Dr. J. B. Hudson, Dr. D. G. Kilburn, and Dr. J. Levy, f o r t h e i r suggestions i n editing the f i n a l draft of the thesis.  INTRODUCTION AND  LITERATURE REVIEW  Central to any study of the immune response i s an understanding of how lymphoid c e l l s can detect and respond to the presence of antigen i n t h e i r environment.  Many theories, both i n s t r u c t i o n a l and  s e l e c t i o n a l , have been proposed over the years to account f o r the s p e c i f i c i t y involved i n the immune response ( B r e i n l and Haurowitz, 1930;  Pauling, 1940;  Burnet, 1959  and Jerne, 1971).  At the present  time, the experimental evidence indicates that the s e l e c t i o n a l theories are the most plausible, and hence have become the most widely accepted.  I.  Clonal Selection Theory of Immunological  Diversity  The c l o n a l selection theory of immunity, the basis of a l l s e l e c t i o n a l theories, postulates that lymphoid c e l l s are completely r e s t r i c t e d i n the ranges of antigenic determinants to which they may respond;  r e s t r i c t e d to the extent that one immunocompetent  cell  w i l l distinguish only one antigenic determinant, and hence have only one s p e c i f i c i t y .  This theory also proposes that the determination  of t h i s s p e c i f i c i t y occurs p r i o r to the f i r s t exposure of the c e l l to antigen, probably before immunological maturation. Directoevidence f o r such a r e s t r i c t e d s p e c i f i c i t y has been shown by single c e l l experiments (Nossal et a l , 1964)  and autoradiography  2  (Naor and Sulitzeanu, 19&7 and Humphrey and K e l l e r , 1971). ments employing autoradiography demonstrate  Experi-  that antigen binds  s p e c i f i c a l l y to only a small number of c e l l s , presumably through immunoglobulin-like receptors.  Before entering into a further  discussion of c e l l u l a r s p e c i f i c i t y , i t i s necessary to f i r s t examine the c e l l types involved i n the immune responses.  II.  C e l l Types Involved i n the Immune Response  Immune responses to most antigens involve two c e l l types, macrophages and  lymphocytes.  Macrophages have been shown to be important i n i n v i t r o antibody responses to sheep red blood c e l l s i n mice (Mosier, 1967) > i n v i t r o antibody responses to 1961  and Fishman and Adler, 1967)•  a n  phage i n rabbits and rats Macrophage-associated  ^ i-  n  (Fishman,  antigen  has also been found i n the i n vivo antibody responses to hemocyanins (Unanue and Askonas, 1968), and to heat-aggregated bovine serum albumin (Mitchison, 1969).  However, despite the requirement f o r  macrophages i n most immune responses, i t i s generally thought that t h e i r role i s not i n antigenic s p e c i f i c i t y . Lymphocytes, on the other hand, exhibit a unique  specificity  for antigen, and are e s s e n t i a l i n the development of both c e l l u l a r and humoral immunity.  The lymphocyte population has recently been  further defined as a minimum of two d i s t i n c t c e l l populations, the  3  T c e l l (thymus derived) population, and the B c e l l (Bursa of Fabricius or bone-marrow derived) population.  T c e l l s are o r i g i n a l l y produced  i n the bone marrow or other hematopoietic tissue, and reside tempora r i l y i n the thymus, before populating the peripheral lymphoid organs (Davies, 1 9 6 8 ) .  B c e l l s also originate from the bone marrow, but  populate the peripheral lymphoid  organs d i r e c t l y .  The T c e l l s are  thought to be mainly responsible f o r e l i c i t i n g the delayed hypers e n s i t i v i t y type or c e l l u l a r reaction ( M i l l e r and M i t c h e l l , while the B c e l l s d i f f e r e n t i a t e  1968),  into plasma c e l l s capable of synthes-  i z i n g immunoglobulin f o r the expression of humoral immunity ( M i l l e r and Osoba, 1 9 6 7 ) .  Both of these c e l l populations appear to exhibit  antigenic s p e c i f i c i t y .  As i t i s the aim of t h i s thesis to r e s t r i c t  the study of s p e c i f i c i t y to the T c e l l population, the s p e c i f i c i t y of the B c e l l population and i t s humoral antibody products w i l l only be l i g h t l y covered.  I I I . S p e c i f i c i t y of Humoral Antibody and Immunocompetent Bone Marrow Derived C e l l s .  Landsteiner ( 1 9 6 2 ) ,  i n studying humoral immunity, demonstrated  that defined chemical groups (haptens), which by themselves were incapable of e l i c i t i n g antibody formation, when coupled to proteins (carriers)  could, upon immunization,  s p e c i f i c anti-hapten antibodies.  result  i n the synthesis of  He also found that chemical modi-  k  f i c a t i o n of the haptens resulted i n a loss of binding a f f i n i t y to the s p e c i f i c antibody.  These r e s u l t s suggested that humoral s p e c i f i c i t y  could exist, i n many cases, to haptenic groups alone.  Since t h i s  early work, haptenic compounds have been used extensively i n studies on T c e l l and B c e l l s p e c i f i c i t y . The s p e c i f i c i t y of B cells'has been studied by various techniques mainly focusing at the c e l l u l a r receptor l e v e l .  In some cases, i t has  been shown that B c e l l s can be s p e c i f i c a l l y inactivated (tolerized) i n vivo by contact with free hapten, preventing the expression of B c e l l function (antibody production). administration of 2,k  Katz et_ a l (1971), found that  dinitrophenyl (DNP)  conjugates of "nonimmunogenic"  amino acid copolymers exerted a suppressive e f f e c t on the capacity of guinea pigs to display anti-DEP antibody responses to with a DNP  conjugate of a strong antigen.  immunization  Moreover, this hapten-  s p e c i f i c unresponsiveness was found to be an i n t e r n a l e f f e c t and not just the blocking of c e l l surf ace.. receptors by the DEP In further experiments  copolymers.  on the s p e c i f i c i t y of c e l l u l a r receptors,  Wigzell and Makela (1970) showed that they could select out populations of B c e l l s with preformed s p e c i f i c i t y receptors towards DEP i n immunized animals as w e l l as i n unimmunized animals. The o v e r a l l r e s u l t s suggest that B c e l l s carry a receptor with a s p e c i f i c i t y that can be directed, i n most circumstances, towards the haptenic determinants on an immunogenic molecule.  These receptors  appear to be of an "immunoglobulin-like" nature, since anti-immuno-  5  g l o b u l i n serum i n h i b i t s antigen binding (Byrt and Ada, 1969;  Dwyer  and Mackay, 1970).  IV.  S p e c i f i c i t y of Immunocompetent Thymus Derived C e l l s .  Hapten-carrier conjugates have been used to study the s p e c i f i c i t y of such T c e l l functions as:  helper c e l l a c t i v i t y , delayed skin hyper-  s e n s i t i v i t y , stimulation of DNA  synthesis i n cultures of sensitized  lymphocytes, macrophage i n h i b i t o r y factor (MIF) production, and c e l l mediated c y t o t o x i c i t y . Paul et_ a l (1970a) found, using 2,4 dinitrophenyl (DNP) pig  guinea  albumin, that the hapten alone could not function as a c a r r i e r f o r  other haptens i n an anti-hapten antibody response. However, the hapten could contribute to the c a r r i e r determinant, and stimulate the formation of c a r r i e r - s p e c i f i c c e l l s capable, of enhancing the antibody response to other haptens presented on the same molecule. In studying the s p e c i f i c i t y of delayed skin reaction using the t r i n i t r o p h e n y l and para toluenesulfonyl haptens, Benacerraf and Levine (1962) found that t h i s s p e c i f i c i t y involved large areas of the sensit i z i n g antigen beside the hapten.  Their results showed that animals  which gave strong delayed reactions to the immunizing  conjugate did  not cross-react to conjugates of the same hapten with a d i f f e r e n t c a r r i e r protein.  These results were v e r i f i e d and extended by G e l l and  S i l v e r s t e i n (1962a,b), using the azobensenesulfonate hapten. Data was  6  presented showing that the s p e c i f i c i t y of the delayed skin reaction to hapten-protein conjugates involved a considerable degree of contribution by the protein c a r r i e r .  They carried t h i s work further  by showing the extent of a hapten contribution to delayed skin reactions as well.  Ortho or para modification of the  meta-azobenzene-  sulfonate hapten was found to have a measurable effect on the skin reactions i n animals immunized to meta-azobenzenesulfonate. Oppenheim et a l (1967), using guinea p i g albumin-orthanilic acid conjugates found that i n v i t r o lymphocyte p r o l i f e r a t i o n i n response to antigen was mainly c a r r i e r s p e c i f i c .  However, they  also noted that the p r o l i f e r a t i v e response was p a r t i a l l y i n h i b i t e d by high concentrations of haptens, indicating a minor hapten contribution to the response.  When ot and 4 DEP-oligolysines were  used as the hapten-carrier conjugates (Schlossman et_ a l , 1969), i t was found that 9 ^ DEP-nona-L-lysine caused l i t t l e or no stimul a t i o n of DEA synthesis by lymphoid c e l l s from guinea pigs  immunized  to 5 i DNP-nona-L-lysine, although the immunizing peptide was an excellent stimulator.  This also indicated a marked c a r r i e r  dependence, but further showed that sensitized c e l l s could d i s criminate among compounds which d i f f e r e d from one another i n the p o s i t i o n of the dinitrophenyl group. When the s p e c i f i c i t y of MIF production was studied, David et a l (1964) demonstrated, using DNP-bovine IgG i n an i n v i t r o system, that the s p e c i f i c i t y was directed against the c a r r i e r .  He l a t e r  7  expanded t h i s work and showed, using the same assay, that sensitized peritoneal exudate c e l l s could discriminate between various oligolysines;  DNP-  a heptamer or larger being required f o r the i n h i b i t i o n  of migration of peritoneal exudate c e l l s (David e t _ a l , 1968). Henney (1970) followed the development of a population of cytotoxic lymphocytes  i n animals immunized with DNP-human IgG and found  that the s p e c i f i c i t y of c y t o l y s i s was markedly c a r r i e r dependent.  51 The degree of c y t o l y s i s by splenic lymphocytes of  Cr l a b e l l e d  mastocytoma c e l l s was only noticeable when the target c e l l s were coupled with the immunizing hapten-carrier conjugate or with the c a r r i e r alone. The findings summarized above indicate that i n certain haptenc a r r i e r systems, the c e l l s p a r t i c i p a t i n g i n the c e l l u l a r immune response can bear a receptor with a complex s p e c i f i c i t y pattern; an antigenic determinant containing elements of both hapten and carrier. I t must be noted that not a l l hapten-carrier systems exhibit t h i s shared s p e c i f i c i t y pattern.  Leskowitz (1963), has shown that  immunization with p-azo-benzenearsonate-poly-L-tyrosine induces a delayed type reaction which i s wholly hapten s p e c i f i c .  Our own work  has shown that s p e c i f i c peptide sequences of larger protein molecules can also be defined as being haptenic (Mitchell et a l , 1970 K e l l y et_ a±_, 1971).  Immunization  and  with the entire protein molecule  induces a delayed type reaction which, l i k e Leskowitz's molecule,  8  i s t o t a l l y hapten s p e c i f i c :  MIF production and delayed skin hyper-  s e n s i t i v i t y being e l i c i t e d by the peptide sequences  themselves  (Waterfield et a l , 1972). I t has thus been w e l l established that certain d i s p a r i t i e s exist i n these hapten-carrier systems as to whether a given hapten w i l l be c a r r i e r dependent or c a r r i e r independent i n e l i c i t i n g a delayed hypersensitive reaction.  The o v e r a l l r e s u l t s previously  discussed have shown that f o r some haptens, such as DNP, part of the c a r r i e r seems to be required to form an antigenic determinant capable of inducing T c e l l function. as peptide sequences,  However, other haptens, such  do not require any s p e c i f i c contribution of  a c a r r i e r molecule. These facts can only leave workers i n the f i e l d with a battery of questions to answer.  Does DNP, when coupled to globular proteins,  create an antigenic determinant near the point of coupling, which i n c e l l s sensitized to t h i s conjugate w i l l show a s p e c i f i c i t y towards both hapten and carrier?  I f so, how many such shared  determinants are formed i n the coupling procedure when globular proteins are used as carriers?  To what extent does the DNP moiety  contribute to the s p e c i f i c i t y of recognition of the combined determinant?  In t h i s determinant, does the haptenic sequence on  the c a r r i e r exist as such before, or was i t newly formed by the coupling procedure? At the present time, i n the study of T c e l l s p e c i f i c i t y these  9  questions  remain unanswered as the main c a r r i e r molecules used have  been either large globular proteins or polymers of amino acids. These compounds have the disadvantage of lacking characterization with regard to the primary structure of the antigenic  (haptenic)  determinants present. We have therefore decided to study the contribution of a t y p i c a l c a r r i e r dependent hapten (DNP)  i n a c t i v a t i n g the c e l l u l a r  immune responses using an immunogenic peptide c a r r i e r whose haptenic sequences have been previously defined. evaluate the r o l e of DNP  This w i l l allow us to  i n these responses when the hapten i s  coupled e x c l u s i v e l y to a known c a r r i e r independent haptenic  V.-  D e f i n i t i v e Studies on the Hapten-Carrier  sequence.  Relationship Using  DNP.  Early d e f i n i t i v e work i n elucidating the antigenic nature of certain proteins established native ferredoxin, from Clostridium pasteurianum, and i t s performic  acid oxidized derivative (O-Fd) to  be the molecule of choice (Nitz ejt a l , 1969).  The major antigenic  determinants of the O-Fd molecule have since been extensively characterized ( M i t c h e l l et a l , 1970  and K e l l y et a l , 1971).  They  were found to constitute the NH,_,-terminal heptapeptide (N) of the molecule, having the sequence NH^-Ala-Tyr-Lys-He-Ala-Asp-Ser-COOH and the COOH-terminal tetrapeptide (C) with the amino acid sequence NH  -pro-Val-Gin-Glu-COOH.  Later studies (Levy et a l , 1972)  showed  that a' synthetic immunogen could be constructed by s o l i d phase peptide synthesis, consisting of these two haptenic determinants separated by a ten glycine bridge or spacer (N-10-C). This defined immunogen was DNP  selected f o r the study of the role of  i n activating a c e l l u l a r immune response, as the DNP  substituted  form would consist of elements of both hapten dependent and hapten independent determinants.  DEP couples only to the  NH^-terminal  determinant y i e l d i n g a defined immunologically bivalent hapten-carrier conjugate (DEP-E-10-C).  Guinea pigs were immunized with either DEP-N-  10-C or E-10-C i n complete Freund's adjuvant.  The c e l l u l a r immune  response i n the presence of antigen was measured by the following means -  i n vivo, delayed skin h y p e r s e n s i t i v i t y and i n v i t r o ,  production and stimulation of DNA  synthesis.  MIF  These types of c e l l u l a r  a c t i v a t i o n were evaluated f o r DEP alone, DEP coupled to the EH -ter2  minal determinant, and DEP coupled to heterologous  carriers.  MATERIALS AND METHODS  I.  Synthesis, P u r i f i c a t i o n , and Quantitation of the Antigens, Antigenic Determinants and Peptides.  a.  Solid phase peptide synthesis.  The immunogen, N-10-C, was synthesized according to the  (1964) method of s o l i d phase peptide synthesis, with  Merrifield  modifications by Stewart and Young  (19^9: Fig- l ) • This technique,  recently automated i n our laboratory by Douglas G-. Hull, involves the use of an insoluble resin, co-polystyrene (Schwarz  divinylbenzene  Mann Chemical Co.) to which the desired COOH-terminal  amino acid (glutamic acid i n the case of N-10-C) has already been covalently bound through i t s free carboxyl group.  Sequential  coupling of amino acids i s carried out by the formation of a peptide bond between the a-carboxyl of the next required t - b u t y l oxycarbonyl  (t-BOC) amino acid (Schwarz Mann Chemical Co.)  free amino group of the attached amino acid.  and the  The peptide i s then  elongated by sequential removal of the t-BOC group from the attached amino acid and coupling of the next t-BOC amino acid, so that chain elongation progresses from the COOH-terminal to the NHg-terminal of the desired peptide.  Removal of the t-BOC group  which blocks the free amino end on the growing chain i s achieved  Fig.  I:  Flow diagram of s o l i d phase peptide synthesis  C H  H  3  HCL-HOAC;  (C H ) N 2  D E P R O T E C T  CH  +CO  i  3  l  2  CH.  amino  Boc amino  CH I  CH  3-  I  3  O  H  || |  R  0  \A  aci,d  0  (COPOLYMER  acyl  polymer COUPLE  diimide  H  II  |  R O 11 II  C - 0 - C - N - C - C - N - C - C - 0 - C H | H  CH^ Boc  i H  peptide  0  ( 0 /  3  CH  H  R  O  H  R.  II  I  I  II  I  I  2  - C + C O  3  |  CH  o  isobutylene  *  1  O  L  Y  M  E  R  C L E A V E  O II  + H N - C - C - N - C - C - O H | i H H  2  P  polymer HBr-F CC00H  C H  R A L I Z E  2vrv  H  isobutylene  3  ; N E U T  H R, O i 11 ii + H - N - C - C - O - C H  2  ll - C  5  + B r - C H  / — \  ^  (O)POLYMER \—/  peptide  Modified from 'Solid Phase Peptide Synthesis' by John Stewart Janis Young, p.3  and  by treatment of the peptide r e s i n with IN HCl i n g l a c i a l acetic acid (HOAC).  The a c i d i c conditions are neutralized by addition  of triethylamine (TEA) before subsequent peptide bond formation takes place i n the presence of the next t-BOC amino acid and d i cyclohexylcarbodiimide (DCC).  The only exception to this technique  involves active esters of t-BOC amino acids which require no coupling reagents.  After each step, the r e s i n i s washed with  organic solvents to prevent build-up of non-reacted amino acids. The amino acids were added i n a 4 . 0 molar excess of the i n i t i a l substitution on the resin. was  To ensure maximum coupling, the DCC  added i n a 6.0 molar excess.  Upon completion, the peptide  i s cleaved from the r e s i n by bubbling anhydrous HBr  (Matheson Co.)  through a scrubbing vessel, containing 2 0 $ t r i f l u o r o a c e t i c acid80$, anisole, into the suspension of r e s i n i n a n i s o l e - t r i f l u o r o acetic acid.  F i n a l l y , the anisole and t r i f l u o r o a c e t i c acid are  removed by f l a s h evaporation.  The cleavage mixture i s placed i n  a round-bottomed flask, which i s attached to a f l a s h evaporator (Buchler).  A vacuum i s applied, and the anisole and t r i f l u o r o -  acetic acid evaporate from the mixture.  After several washes  with d i s t i l l e d water, the peptide i s stored f o r p u r i f i c a t i o n . The peptides N-HEPTA, SER^,  and N-TRI were synthesized  according to the above method, using the appropriate t-BOC amino a c y l polymer i n each case.  The remaining antigens and antigenic  determinants, N-8-N, N-PENTA, and N-TETRA were provided by D. G. H u l l .  b.  P u r i f i c a t i o n o f the antigens, antigenic  determinants  and p e p t i d e s .  N-10-C was p u r i f i e d b y column chromatography on Sephadex G-25 FINE ( P h a r m a c i a ) .  The Sephadex was poured i n t o a 2 . 5 cm x 1 0 0 cm  column (Pharmacia) and e q u i l i b r a t e d w i t h 0.1N T E A - 0 . I N HOAC. The a n t i g e n was a p p l i e d t o t h e column i n a p p r o x i m a t e l y 2 . 0 m l o f t h e s t a r t i n g b u f f e r , and t h e column s u b s e q u e n t l y developed.  A flow rate  o f 6 m i s p e r hour was m a i n t a i n e d , and 4 . 0 m l samples were c o l l e c t e d on an LKB f r a c t i o n c o l l e c t o r .  L o c a l i z a t i o n o f t h e p e p t i d e was  d e t e r m i n e d b y a n a l y s i s o f t h e samples on a Beckman DBG s p e c t r o p h o t o meter a t 2 8 0 and 2 3 0 A.  The p e p t i d e - c o n t a i n i n g f r a c t i o n s were  p o o l e d , c o n c e n t r a t e d b y f l a s h e v a p o r a t i o n , and s t o r e d a t 4 C f o r amino a c i d  analysis.  The N-HEPTA p e p t i d e was p u r i f i e d b y column chromatography on Dowex Ag 1 X 8 (200-400 mesh, B i o r a d Co.).  The p r e p a r e d r e s i n was  poured i n t o a 1 . 6 cm x 7 0 cm column (Vancouver S c i e n t i f i c G l a s s ) and equilibrated with 5.0$pyridine.  The p e p t i d e was a p p l i e d t o t h e  column i n a p p r o x i m a t e l y 4 . 0 m l o f 5 . 0 $ p y r i d i n e . developed w i t h the f o l l o w i n g g r a d i e n t s :  The column was  5.0$pyridine to 0 . 1 N  HOAC - 4 . 0 N p y r i d i n e a t pH 6.4, 0 . 1 N HOAC - 4.0 N p y r i d i n e t o l . O N HOAC - 3 . 8 N p y r i d i n e a t pH 5 . 8 , 1.0 N HOAC - 3 . 8 N p y r i d i n e t o 1 0 . 0 N HOAC - 2 . 0 N p y r i d i n e a t pH 4.05.  A f l o w r a t e o f 16 mis p e r  hour was m a i n t a i n e d , and 4 . 0 m l f r a c t i o n s were c o l l e c t e d on a LKB  fraction collector.  A 0 . 1 ml aliquot was taken from each sample  f o r determination of peptide content by the quantitative ninhydrin method of Hirs et a l ( 1 9 5 6 ) .  The peptide-containing f r a c t i o n s were  pooled, concentrated, and stored at 4 C f o r amino acid analysis. The N-TRI and SER^ were p u r i f i e d by column chromatography on Sephadex G-15  (Pharmacia).  The r e s i n was poured into a 5 . 0 cm  x 100 cm column (Kontes), equilibrated with 0.1 H TEA  - 0.1 I HOAC.  The peptides were applied to the column i n 5 . 0 ml of t h i s preparat i o n , and the column was  developed with the same. A flow rate of  6 . 0 mis per hour was maintained, and 8 . 0 ml f r a c t i o n s were collected.  L o c a l i z a t i o n of the peptides were determined  by  s p e c t r a l analysis at 2 8 0 and 2 3 0 A, and the peptide-containing f r a c t i o n s were subsequently pooled, concentrated, and stored at 4 C.  c.  DEP substitution of proteins, peptides, and amino acids.  A l l the proteins, peptides, and amino acids: E-HEPTA, SER^,  N-10-C, E-8-E,  N-PEETA, E-TRI, E-TETRA, Lysozyme, Bovine serum  albumin (BSA), and serine, were coupled with 2 , 4 dinitrophenyl (DEP) by the following procedure. Each peptide preparation, i n aqueous solution, was placed i n a 1 0 0 . 0 ml round-bottomed f l a s k with a 1 0 . 0 molar excess of 2,4-dinitrobenzenesulfonic acid (Eastman).  The pH  was  adjusted to approximately pH 10 with 6.0 H NaOH, and the reaction mixture was s t i r r e d gently f o r 2 4 hours.  The 2 , 4 dinitrobenzene-  sulfonic acid couples mainly to the pt-NHg-terminals of the peptide sequences, and to the €.-NHg of lysine, i f lysine i s present i n the peptides (Fig. 2, F i g . 3). However, i t i s also possible f o r the DNP to couple to the tyrosine residues under prolonged reaction conditions.  Amino acid analysis showed that tyrosine had not  coupled with DNP to a s i g n i f i c a n t extent i n the substituted preparations.  d.  P u r i f i c a t i o n of DNP antigens, peptides, and amino acids.  Upon completion of coupling, the pH of each peptide suspension was lowered to pH 4.0 with 6.0 N HCl.  This caused the  DNP substituted compounds to p r e c i p i t a t e , leaving any unreacted 2,4-dinitrobenzenesulfonic acid i n solution.  The p r e c i p i t a t e was  centrifuged i n a S o r v a l l GLC-1 at 220 x g f o r 15 min., and the supernatant discarded.  The p r e c i p i t a t e was then washed with  d i s t i l l e d water adjusted to pH 4.0 with 6.0 N HCl, and subsequently repelleted.  This procedure was repeated u n t i l the o p t i c a l density  of the supernatant at 278 A was less than 0.05.  The p e l l e t was  then solublized with 1.0$ sodium bicarbonate i n d i s t i l l e d water.  Fig.  2:  DNP  substitution of the N-TRI peptide  ? 3H  0  2  2 |QT+NH  - C H - C O O - T Y R O S I N E - N H - C H ,  0  N - T R I  2,4  dinitrobenzenesulfonic  acid pH  10  •O N a  ? 3 H  °2 "{0^~ N  N  °2  N  H  ~  C  H  ~  c  o  °  -  TYROSINE-NH-CHg D N P - N - T R I  e.  Quantitation of a l l proteins, peptides, and amino acids.  After p u r i f i c a t i o n of a l l antigens and peptides, an aliquot from each sample was taken up i n 6.0 evacuated,  N HCl i n a small ampoule,  sealed, and hydrolyzed at 110 C f o r 18 hours.  The  hydrolysates were then washed three times by f l a s h evaporation, and f i n a l l y taken up i n pH 2.2  s t a r t i n g buffer f o r analysis on a  Beckman 120 amino acid analyzer.  The amino acid composition,  and the expected molar concentrations of the antigens and peptides p u r i f i e d are shown i n Table I.  The expected molar r a t i o s have  been corrected f o r the degradation of the amino acids caused by the 18 hour hydrolysis.  Degradation values on Beckman amino  acid standards from 0 to 48 hours are shown on Table I I . Upon coupling with DNP,  a l l antigens were again quantitated  and the degree of substitution evaluated by amino acid analysis. The amino acid composition, and the expected molar r a t i o s of the DNP coupled antigens and peptides are shown i n Tables I I I and IV.  Again, the expected molar r a t i o s have been corrected f o r  amino acid degradation caused by hydrolysis. As can be seen by comparing Tables I, I I I , and IV, the substitution of peptides was  at least 90$ of t h e o r e t i c a l  DNP  values, not taking into consideration a 5$ allowable machine error.  Thus, the DNP peptides were evaluated as pure and homo-  genous enough to use i n subsequent  experiments.  Table I:  The amino acid composition and the expected molar r a t i o s of the amino acids i n the antigen and peptide determinants.  Amino acid molar r a t i o s (M/M)  N-10-C Amino acid  Expected  of synthetic peptide  N-HEPTA Found  Expected  W-TRI  Found  Aspartic  1  0.91  1  0.68  Serine  .1  1.02  l  1.45  Glutamic  2  2.19  Proline  1  1.40  Glycine  10  10.26  Alanine  3.30  2  2.01  Valine  3 1  Isoleucine  .1  0.86  1  1.03  Tyrosine  1  0.76  1  Lysine  1  0.9L  1  Expected  Found  1  1.00  0.83  1  0.90  1.05  1  1.22  1.20  Table I I :  Amino acid degradation values of Beckman standard amino acids  Time Amino acids * Aspartic Threonine Serine Glutamic Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine  *  0 hr 12 hr 18 hr  24 hr  48 hr  .0967 .0989 .0945 .0936 .0903 .0962 .0949 .0912 .0883 .0823 .1031 .0929 .0873 .0850 .0757 .1015 .1000 .0973 .0948 .0916 .0970 .1002 .0944 .0937 .0958 .0966 .0961 .0937 .0922 .0892 .0968 .0976 .0949 .0926 .0902 .0947 .0962 .0930 .0910 .0795 .0959 .0903 .0901 .0886 .0830 .0973 .0970 .0960 .0937 .0899 .0947 .0963 .0930 .0917 .0879 .0964 .0956 .0918 .0895 .0820 .0982 .0982 .0954 .0952 .0919  Beckman amino acid samples (0.1  p.m) were taken and hydrolyzed  f o r 0 hr, 12 hr, 18 hr, 24 hr and 48 hr, to determine the effect of the time of hydrolysis on the degradation of i n d i v i d u a l amino acids.  At the designated times, a sample was quantitated on a  Beckman amino acid analyzer.  The results are expressed as the  umolar content l e f t undegraded from the t h e o r e t i c a l s t a r t i n g value of 0.1 |amoles.  Table I I I :  The amino acid composition and the expected molar r a t i o s of the amino acids i n the DNP antigens and DNP peptide determinants.  Amino acid molar r a t i o s (M/M) of synthetic peptide  DNP-N--10-C Amino acid  Expected  DNP-N-8-N  Found  Expected  Found  DNP-N-HEPTA Expected  Found  Aspartic  1  1.03  2  2.4  1  0.63  Serine  1  0.80  2  2.3  1  0.89  Glutamic  2  2.10  Proline  1  1.20  Glycine  10  10.50  8  6.6  Alanine  2  2.40  3  3.1  1  1.08  Valine  1  1.13  Isoleucine  1  0.75  2  1.89  1  0.93  Tyrosine  1  0.82  2  0 . 5^  1  0.89  Lysine  0  0  0  0.27  0  0  Table IV:  The amino acid composition and the expected molar r a t i of the amino acids i n the DNP antigen and DNP peptide determinants.  Amino acid molar r a t i o (M/M) of antigen and peptides DNP-LYSOZYME Amino acid Aspartic Threonine Serine  Expected  Found  21  23.0  7  8.0  10  ll. 8  Glutamic  5  6.8  Proline  2  2.9  Glycine  12  12  Alanine  12  .12  Valine  6  6  Isoleucine  6  5-0  Leucine  8  8.1  Tyrosine  3  3.0  Phenylalanine  3  3.0  Lysine  0  1.6  Arginine  l  -  DNP-N-PENTA Expected  Found  1  1.21  1  0.82  DNP-N-TRI Expected  Found  0  0  1  0.87  1  0.93  0  0.00  0  0  The DNP substituted  amino acid was quantitated using the  extinction c o e f f i c i e n t s described by Sanger ( 1 9 4 9 ) .  A 2 0 |_imolar  solution of DNP serine i n 1% sodium bicarbonate has an absorbance at 350 A of 0 . 3 1 2 .  Beer's law i s obeyed i n solutions of DNP  amino acids f o r concentrations less than 50 Mmoles per ml.  II.  Immunization of Guinea Pigs.  Albino guinea pigs, weighing approximately 3 0 0 grams were immunized with 2 5 0 |j.grams DNP-N-10-C i n phosphate buffered  saline  pH 7 - 0 (PBS-0.15 M NaCl, 0 . 0 0 2 M NaPO^ buffer, 0 . 0 2 $ sodium azide) emulsed i n an equal volume of complete Freund's adjuvant (Difco). 1.0  The i n j e c t i o n series was given i n a t o t a l volume of  ml as follows:  0 . 4 ml intramuscularly  muscles, 0 . 4 ml intramuscularly  into the shoulder  into the g l u t e a l muscles of the  hind legs, and 0 . 2 ml intraperitoneally.  The animals were l e f t  f o r approximately three weeks before skin tests and other immunological procedures were c a r r i e d out.  I I I . Skin Tests.  Skin tests were c a r r i e d out by i n j e c t i n g 0 . 2 ml of test antigens and peptides i n PBS intradermally  into the shaven and  d e p i l l a t e d (with Nair) flanks of the test animals.  The  concentrations of the peptides used were derived according to the maximum degree of s o l u b i l i t y of each p a r t i c u l a r DNP peptide in 1 $ sodium bicarbonate.  The r e s u l t s were read after two hours  (immediate reactions) and after 2k hours (delayed reactions) from the time of i n j e c t i o n .  An erythema of greater than 0 . 6 cm  was considered a p o s i t i v e reaction.  IV.  Migration Inhibition.  The migration i n h i b i t i o n test was carried out according to a modification of the technique described by David et a l ( 1 9 6 4 ) . Guinea pigs were exsanguinated,  and spleens were a s e p t i c a l l y  removed, and placed i n a p e t r i dish ( 1 0 0 mm diameter,  Falcon  P l a s t i c s ) containing PBS plus 5 . 0 $ heat-inactivated f e t a l c a l f serum (Gibco).  Clumps of c e l l s were teased out of the spleen  and broken into a single c e l l suspension by repeatedly forcing them through a 1 ml tuberculin syringe (Fisher). The c e l l s were then packed by centrifugation at 2 2 0 x g f o r 10 minutes i n a S o r v a l l GLC-1 and washed once with Eagles MEM (North American B i o l o g i c a l , see appendix) containing 1 5 $ heat-inactivated f e t a l c a l f serum plus 1 0 0 units of p e n i c i l l i n and 1 0 0 (ig of streptomycin per ml. This combination  w i l l hereafter be designated as  Eagles MEM. The c e l l s were then resuspended i n Eagles MEM to give a 2 0 $ c e l l suspension.  C a p i l l a r y tubes ( 0 . 8 to 1 . 0 mm i n  diameter and 75 nun long, Fisher) were f i l l e d with the c e l l and sealed at one end with miniseal (Dade Co.).  suspension  The tubes were then  centrifuged at 220 x g f o r 5 minutes, and cut just below the c e l l f l u i d interface.  They were placed at the bottom of MacKaness type  chambers, two i n each chamber. MEM  The chambers were f i l l e d with Eagles  containing various concentrations of the test antigens and  peptides.  The chambers were then sealed, and incubated f o r 24 hours  at 37 C i n a CO^ enriched atmosphere.  Migration was measured as the  rectanglular area encompassing the furthest extent of c e l l t r a v e l , using a c a l i b r a t e d stage on a Wild M40 microscope.  The results were  expressed as percent of migration of spleen c e l l s i n comparison to the migration of c e l l s i n the control chambers containing no antigen or peptide.  V.  Lymphocyte Stimulation.  Lymphocyte stimulation was  carried out according to a modi-  f i c a t i o n of the technique described by Dutton and Eady ( 1 9 6 4 ) . Guinea pigs were exsanguinated,  and the p o p l i t e a l , inguinal,  and  mesenteric lymph nodes were removed and made into a single c e l l suspension as previously described f o r the spleen.  The c e l l s were  then packed by centrifugation at 220 x g f o r 15 minutes, and washed once with RPMI-1640 (Gibco, see appendix) containing 1 5 $ heat-inactivated f e t a l c a l f serum plus 100 units of p e n i c i l l i n  27  and 100 ug of streptomycin per ml. w i l l be designated as l6k0. counted.  In future, t h i s combination  The c e l l s were taken up i n 1640 and  The c e l l concentration was then adjusted to a t o t a l of  5 x 10^ c e l l s per ml with l6k0.  Only viable c e l l s , as determined  by trypan blue exclusion, were counted on an eosinophyl haemocytometer (Spiers-Levy).  A v i a b l e count of greater than 9 5 $ was  mandatory f o r use of the c e l l s i n the stimulation test.  The test  antigens and peptides were set up i n quadruplicate i n a m i c r o t i t r e plate (Limbro Chemical) with 5 x 10^ viable c e l l s i n a 0.1 ml volume per m i c r o t i t r e well.  The antigens and peptides, at an  i n i t i a l concentration of 0.05 M-m per ml of 1640, were added to each w e l l i n a t o t a l volume of 0.05 ml. topped up with 0.1 ml of l6k0 media.  F i n a l l y , each w e l l was  The m i c r o t i t r e plate was  then incubated at 3 7 C i n a CO^ enriched atmosphere. days, 1.0 \iCi of H-thymidine  After three  ( s p e c i f i c a c t i v i t y 2 6 C i per mmole,  (New England Nuclear) was added to each m i c r o t i t r e well.  The  c e l l s were harvested 1 8 hours l a t e r by a microharvesting method developed by T. Pearson from a model of the Hartsman microprecipitator.  This technique involved suction removal of the  c e l l s from each m i c r o t i t r e well onto a 1 . 3 cm diameter glass f i b r e f i l t e r (Reave Angel).  The f i l t e r s were then dried and  mixed with 4.0 ml of s c i n t i l l a t i o n f l u i d ( 6 0 $ toluene, 40$ methanol and 41.0 ml of L i q u i f l u o r (New England Nuclear) per l i t r e ) and counted on a Nuclear-Chicago s c i n t i l l a t i o n counter (model 7 2 5 )  Average counts per minute were recorded from 1 minute counts on each sample.  The results were expressed as a r a t i o of stimulation  of lymph node c e l l s i n comparison to the stimulation of the control wells containing no antigen or peptide.  VI.  S t a t i s t i c a l Analysis.  Average migration i n h i b i t i o n and lymphocyte stimulation, standard deviation, and Student's t test were performed on a l l data using a triangular regression package (TRIP) program on an IBM 360/67  computer.  The r e s u l t s were expressed as a T p r o b a b i l i t y  with values of less than 0 . 0 1 being considered as s i g n i f i c a n t .  RESULTS AND DISCUSSION  I.  Skin Tests  The a b i l i t y of various DNP conjugated and unconjugated compounds to e l i c i t either immediate or delayed skin reactions was tested i n sensitized and unsensitized (control) animals.  The sensitized animals  had previously been immunized with either DNP-N-10-C or N-10-C. The tests were performed by i n j e c t i n g the preparations  i n 0.2 ml PBS  intradermally into the d e p i l l a t e d flanks of the animals.  The develop-  ment of an erythema greater than 6 mm i n diameter after 2 hours indicated the presence of c i r c u l a t i n g antibody to the challenging antigen (immediate reaction).  The persistence of an erythema after  24 hours indicated that an i n vivo c e l l u l a r reaction to the antigen (delayed reaction) had occurred. In the DNP-N-10-C immune animals i t was found that 0.125 |j.m of DNP-N-10-C e l i c i t e d both immediate and delayed reactions (Table V). However, neither of the haptenic peptides, DNP-N-HEPTA or N-TETRA, gave immediate reactions, and only the DNP-N-HEPTA produced a s i g n i f i c a n t delayed response.  Unfortunately,  t h i s cannot be i n t e r -  preted as an i n vivo difference i n c e l l u l a r recognition since no delayed response was e l i c i t e d by either peptide i n the N-10-C immune animals (Table VI). of the concentrations  These r e s u l t s are probably a d i r e c t r e f l e c t i o n of the peptides used, which were considerably  Skin reactions observed on guinea pigs immunized with MP-N-10-C.  Challenging antigen °  Test dose umoles per ml  Skin reactions . ... , -, -, immediate delayed (2hr) (24hr) a  0.125  8/11  0.05  0/11  N-TETRA  0.05  0/11  3/H  DNP-BSA  0.125  3/7  Saline c o n t r o l  0 . 2 ml  0/11.  DNP-N-10-C DEP-N-HEPTA  10/11  0/11  Figures represent the r a t i o of the number of animals showing p o s i t i v e reactions ( 6 mm) to the number of animals tested.  6/n  0/11  31  Table VI:  Skin reactions observed on guinea pigs with N-10-C.  Challenging Antigen  Test dose [jmoles per ml  immunized  Skin reactions immediate (2hr)  a  delayed (2Uhr)  DNP-N-10-C  0.125  5/10  7/10  DNP-N-HEPTA  0.05  0/10  1/10  N-TETRA  0.05  0/10  2/10  DNP-BSA  0.125  0/8  3/8  Saline c o n t r o l  0 . 2 ml  0/10  0/10  Figures represent the r a t i o of the number of animals showing p o s i t i v e reactions (6 mm) to the number of animals tested.  lower than the concentrations of the o r i g i n a l immunogen.  The concen-  t r a t i o n range i n these tests was widely r e s t r i c t e d due to the hydrophobic nature of the peptides after DNP substitution.  I t should also  be noted at t h i s time that, although the NH -terminal heptapeptide 2  (N-HEPTA) was used f o r DNP substitution, the complete haptenic sequence of the moleculeris contained i n the tetrapeptide (N-TETRA - unpublished observations).  DNP, when conjugated to BSA, produced a marginal  reaction i n DNP-N-10-C immune animals.  However t h i s effect was also  noted i n N-10-C immune animals, i n d i c a t i n g that the response was. probably a non-specific one. These results imply that the DNP moiety, although an i n t e g r a l part of the DNP-N-10-C immunogen, cannot cause an i n vivo c e l l u l a r response without the a i d of the homologous antigen. In the N-10-C immune animals i t was found that DNP-N-10-C again e l i c i t e d both immediate and delayed reactions, although to a lesser extent.  This suggested that substitution of the NHg-terminal deter-  minant of N-10-C with DNP does not a l t e r the determinant to such a s i g n i f i c a n t extent that i n vivo c e l l u l a r a c t i v a t i o n and humoral a n t i body binding i s prevented i n animals which are sensitized to the unconjugated form.  As mentioned before, the DNP-N-HEPTA and N-TETRA  f a i l e d to give any s i g n i f i c a n t response, and the DNP-BSA conjugate gave only a marginal non-specific one. The s p e c i f i c i t y of both the immediate and delayed reactions i s shown by the f a i l u r e of a l l the preparations tested to cause an erythema i n control guinea pigs (Table VII).  33  Table VII:  Skin reactions observed on unimmunized  Challenging Antigen  Test dose pinoles per ml  guinea pigs.  Skin reactions  a  immediate (2hr)  delayed (24hr)  DNP-N-10-C  0.125  0/4  0/4  DNP-N-HEPTA  0.05  0/4  0/4  N-TETRA  0.05  0/4  1/4  DNP-BSA  0.125  1/4  0/4  0/4  0/4  Saline control  0.2 ml  Figures represent the r a t i o of the number of animals showing p o s i t i v e reactions (6 mm) to the number of animals tested.  As the delayed skin reactions could not be used to e s t a b l i s h a d i f f e r e n t i a l response between the DEP substituted determinant and the unsubstituted determinant  (N-HEPTA)  (E-TETRA), i t was necessary to carry  out more quantitative tests f o r delayed hypersensitivity.  II.  Migration I n h i b i t i o n Factor (MIF) Production.  The spleen c e l l s of animals s e n s i t i z e d to either N-10-C or DEP-E10-C were tested f o r t h e i r a b i l i t y to produce MIF, i n the presence of E-10-C or i t s DEP derivative, an assortment of DEP substituted or unsubstituted peptides forming a portion of or a l l o f the  EH^-terminal  hapten, as w e l l as i n the presence of some DEP substituted heterologous compounds.  Tests were also carried out using combinations of peptides,  i n order to determine whether or not blocking of MIF production could be achieved.  The t e s t involves antigen s p e c i f i c a c t i v a t i o n of sensi-  t i z e d c e l l s i n v i t r o to produce MIF, a non-specific compound thought to be e l i c i t e d by sensitized.T c e l l s i n the presence of s p e c i f i c antigen, which i s capable of preventing the migration of c e l l s (macrophages) from c a p i l l a r y tubes (David et a l , 1 9 6 4 ) .  The r e s u l t s of these tests  are presented i n Tables VIII and IX.  a.  The Response of DEP-E-10-C Sensitized Cells to the Test Antigens.  In the c e l l s taken from animals s e n s i t i z e d to DEP-N-10-C, i t  Table VIII:  The e f f e c t of the homologous antigen, haptenic peptides, and DNP substituted heterologous compounds on the migration of spleen c e l l s derived from guinea pigs s e n s i t i z e d to DNP-N-10-C.  Antigen  Concentration i n chambers (l-imoles/ml)  Migration $  toprobability^  a  no. of animals  DNP-N-10-C  0.025  ^3.3 ± l U . 9  7  N-10-C  0.025  69. 4 + 20.1  6  DNP-N-HEPTA  0.05  I6.7 +  7  N-TETRA  0.05  DNP-LYSOZYME  0.025  DNP-SER 4 DNP  5-7 + 16.1 55.6  7  .001  .000  .000 .001 .000 .000  7  .167  0.05  86.7 + 27.3 87. h + 23.8  7  .934  0.05  90.6 + 28.1  7  .hl3  DNP + N-HEPTA  0.05  + 12.9  7  .000  DNP-N-TRI + N-TETRA  0.05 each  88.4 + 23.1  4  .080  4  .330  each  51.4  DNP-N-TRI  0.05  DNP-N-PENTA  0.05  102.3 + 13.0 78.6 + 15.5  4  .003  N-PENTA  0.05  77.1  + 12.6  4  .002  Eagles MEM  1 ml  100  7  These figures f o r percent migration represent the averaged percent migration on chambers containing antigen, peptide, or heterologous protein compared to control chambers incubated without any of these preparations. The t p r o b a b i l i t y represents the r e s u l t s of a .Student's t t e s t c a r r i e d out on the i n d i v i d u a l migration values f o r each antigen as compared with the values f o r the media controls. The dashed t p r o b a b i l i t i e s represents analysis carried out amongst similar antigens. A t p r o b a b i l i t y of less than 0 . 0 1 can be considered as s t a t i s t i c a l l y significant.  Table IX:  The e f f e c t of the homologous antigen, haptenic peptides, and DEP substituted heterologous compounds on the migration of spleen c e l l s derived from guinea pigs sensitized to N-10-C.  Antigen  DEP-E-10-C  Concentration i n chambers (nmoles/ml)  0.025  Migration  t probability  a  ' $  67.9  no. of animals  + 10.8  E-10-C  0.025  68.4 + 10.4  8  DEP-E-HEPTA  0.05  69.4 + 13.1  6  E-TETRA .  0.05  71.3  +  i  .000  7  14.3  8  .778  .000 .000  .764  .000  DEP -LY SOZYME  0.025  86.1  24.8  8  .010  DEP-SEE 4  0.05  8  .001  DEP  0.05  84.2 + 1 3 . 1 + 15.6 96.9  7  .303  DEP + N-HEPTA  0 . 0 5 each  8  .000  DEP-E-TRI + N-TETRA  0 . 0 5 each  2  .009  DEP-E-TRI  0.05  + 11.5 69.4 + 18.3 + 107.2 3.79  2  .371  DEP-E-PEETA  0.05  85.6 + 13.6  2  .193  N-PEETA  0.05  78.9  + 24.7  2  .130  Eagles MEM  1 ml  100  70.5  8  These figures f o r percent migration represent the averaged percent migration i n chambers containing antigen, peptide, or heterologous protein compared to control chambers incubated without any of these preparations. The t p r o b a b i l i t y represents the results of a student's t t e s t c a r r i e d out on the i n d i v i d u a l migration values f o r each antigen as compared with the values f o r the media controls. The dashed t p r o b a b i l i t i e s represents analysis c a r r i e d out amongst similar antigens. A t p r o b a b i l i t y of less than 0 . 0 1 can be considered as s t a t i s t i c a l l y significant.  b  appeared that the homologous antigen was s i g n i f i c a n t l y more active immunologically i n v i t r o than was i t s unsubstituted counterpart. This apparent s p e c i f i c effect of DNP-substitution was supported by the observation that DNP-N-HEPTA caused considerably more i n h i b i t i o n than did the N-TETRA peptide, which has been shown previously to contain the immunologically active amino acids of the N-hapten (see Abbreviations f o r comparison between DNP-N-HEPTA and N-TETRA).  I t should be  mentioned at t h i s time that the concentrations of the peptides have been increased i n r e l a t i o n to that of the immunogen, as t h e o r e t i c a l l y , according to the c l o n a l selection theory, a reduced e f f e c t would be seen using the peptides, since only one clone of c e l l s i s activated i n these tests; the clone responding to the NH^-terminal.  When the  DNP-N-10-C antigen i s used, the clone reacting to the COOH-terminal haptenic peptide i s also activated, causing a s i g n i f i c a n t l y greater i n h i b i t i o n of migration than that caused by single peptides. The o v e r a l l r e s u l t s suggest that the antigenic s p e c i f i c i t y of reactive c e l l s i n DNP-N-10-C immune animals exists towards both the DNP-moiety and the N-HEPTA haptenic peptide when presented i n a conjugated form. NHg-terminal of  The degree to which the N-TETRA peptide and the N-10-C  activates the DNP-N-10-C reactive c e l l s i s  probably an i n d i c a t i o n of the degree of c r o s s - r e a c t i v i t y existing between the substituted and unsubstituted forms i n t h i s assay.  DNP,  by i t s e l f , or conjugated to heterologous c a r r i e r s (SER^ and Lysozyme), w i l l not cause a c t i v a t i o n of c e l l s to produce MIF.  These r e s u l t s  agree with those of workers using other hapten-carrier systems (David et a l , 1 9 6 4 ) and imply that there i s a molecular c o n f i g u r a t i o n a l requirement  f o r c e l l u l a r activation, the DNP hapten not f u l f i l l i n g necessary to activate the c e l l s (David et a l 1.96k,  the requirement and Carpenter  and Brandriss,  196k).  The data, however, could also be explained by the p o s s i b i l i t y that i n coupling DNP onto the N-HEPTA peptide, an a d d i t i o n a l haptenic determinant  other than the o r i g i n a l N-TETRA has been created i n the  N-HEPTA sequence, hence producing an enhanced i n v i t r o activation. To distinguish these two p o s s i b i l i t i e s , a p a r t i a l requirement f o r DNP i n the s p e c i f i c i t y receptors, or two d i s t i n c t antigenic determinants, DNP-N-TRI and DNP-N-PENTA were synthesized.  N-TRI consists  of the three NH -terminal amino acids of the N-HEPTA. and does not 2  contain any sequences of the N-TETRA haptenic peptide.  The N-PENTA,  however, i s a bridge peptide, consisting of the f i v e NH^-terminal amino acids of the N-HEPTA peptide, and contains only two amino acids of the N-TETRA (see Abbreviations).  As can be seen from Table VIII,  DNP-N-TRI does not seem to be able to cause MIF production.  The DNP-  N-PENTA and N-PENTA however appear to cause marginal a c t i v a t i o n of the c e l l s , i n d i c a t i n g that no extra determinant has been created i n the N-HEPTA peptide. The above data shows that the DNP groups substituted onto either N-10-C or N-HEPTA enhance the production of MIF i n c e l l s from animals  sensitized to DNP-N-10-C i n comparison to the e f f e c t  produced by t h e i r unsubstituted counterparts. This implies a s p e c i f i c role of DNP i n the recognition of the antigens by the sensitized c e l l s However, the tests involving DNP-N-TRI and DNP-N-PENTA and t h e i r unsubstituted counterparts indicate that no extra determinant has been created, since no a c t i v a t i o n was observed by the tripeptide, and only marginal a c t i v a t i o n was caused by either the N-PENTA or the DNP-N-PENTA.  As the peptapeptide overlaps with the N-TETRA sequence,  i t would appear that activation mainly r e s u l t s from the presence of the immunologically active amino acids i n t h i s sequence rather than to the DNP moiety.  b.  The Response of N-10-C Sensitized Cells to the Test Antigens.  Migration of spleen c e l l s from N-10-C immunized guinea pigs was measured i n the presence of these preparations to test the proposed s t r u c t u r a l model of s p e c i f i c i t y .  These experiments were designed i n  part to determine i f substitution of the antigen and haptenic peptides with DNP caused any conformational changes i n the molecules,  prevent-  ing recognition of the N-TETRA haptenic sequence as i t existed i n the o r i g i n a l N-HEPTA peptide. As can be seen from the results i n Table IX, there i s no s i g n i f i c a n t difference i n the i n h i b i t i o n caused by DNP-N-10-C and that caused by N-10-C.  This i s further v e r i f i e d by the results of  40  the DNP-N-HEPTA and N-TETRA haptens, i n d i c a t i n g that DNP s u b s t i t u t i o n of the immunogen or peptide does not prevent t h e i r subsequent i n v i t r o recognition by c e l l s previously s e n s i t i z e d to the unsubstituted form. The DNP-TRI, DNP-N-PENTA, and N-PENTA peptides i n h i b i t i o n of migration,  show no s i g n i f i c a n t  although i t i s possible that i f larger groups  of animals had been used, marginal a c t i v a t i o n might have been demonstrable with N-PENTA.  The data does indicate however, that the N-TETRA,  as previously mentioned, contains the immunologically active amino acid sequence of the NHg-terminal antigenic determinant of N-10-C.  The DNP  molecule, when coupled to heterologous c a r r i e r s , d i d not cause s i g n i f icant a c t i v a t i o n of c e l l s .  This would be expected because the T c e l l s  were not immunized to the DNP substituted immunogen and the heterologous c a r r i e r s i n no way cross-react to i t . The observation that with N-10-C s e n s i t i z e d c e l l s b a s i c a l l y no differences i n MIF production were obtained  i n the presence of DNP sub-  s t i t u t e d or unsubstituted antigens shows that the DNP moiety does not obstruct c e l l u l a r recognition of the antigen.  The f a c t that s i g n i f i c a n t  differences i n MIF production were observed under comparable circumstances i n DNP-N-10-C s e n s i t i z e d c e l l s supports the p o s s i b i l i t y of some kind of s p e c i f i c role of the DNP groups i n c e l l recognition.  Since  apparently no extra antigenic determinant was created by DNP substitution (see above), i t i s possible that DNP under these circumstances functions by expanding the number of antigen sensitive c e l l s r e c r u i t e d during immunization.  c.  Receptor Blocking Experiments.  The nature of the s p e c i f i c surface receptors on T c e l l s a mystery at t h i s time.  There i s data, somewhat tenuous  remains  and not  unequivocally accepted, which imply that surface immunoglobulin  acts  as the recognition s i t e f o r antigen. (Basten et a l , 1 9 7 1 and Roelants and Askonas, 1 9 7 1 ) .  However, there-'.is an equal weight of data unable  to support t h i s p o s s i b i l i t y (Uhr and V i t e t t a , 1 9 7 3 ) .  That there i s  s p e c i f i c recognition of antigen i s not disputed, but whether or not a c t i v a t i o n i s comparable to B c e l l a c t i v a t i o n requirements i s not clear. Since i t i s d i f f i c u l t to v i s u a l i z e antigen recognition by T c e l l s as something other than analogous to immunoglobulin  binding with antigen,  the following argument was made regarding the function of DNP  sub-  s t i t u t i o n on the N-hapten, to j u s t i f y the experimental system to be presented.  i.  Since the ' s p e c i f i c i t y ' sequence of the W-hapten i s  contained i n the N-TETRA, i t i s possible that these amino acids are compulsory f o r recognition at the T c e l l surface.  ii.  In the DNP-N-HEPTA peptide, both DNP  substitutions  are located i n the N-TRI portion of the peptide and do not overlap with the N-TETRA.  iii.  I f the ' s p e c i f i c i t y ' sequence must enter a  molecular crypt i n the recognition s i t e i n order to activate sensitized T c e l l s , i t i s possible that such an i n t e r a c t i o n might be blocked i n the presence of amino acid sequences forming the 'outer region' of the recognition s i t e .  To investigate t h i s p o s s i b i l i t y , c e l l s from animals  sensitized  to DNP-N-10-C and N-10-C were measured f o r MIF production i n the presence of either N-HEPTA or N-TETRA, when these peptides were used alone or mixed with DNP or DNP-N-TRI. Tables VIII and IX.  The r e s u l t s are tabulated i n  The data obtained from DNP-N-10-C sensitized  c e l l s showed very c l e a r l y that while DNP on i t s own d i d not i n h i b i t MIF production i n the presence of antigenic peptides, DNP-N-TRI was able to completely abrogate the response of these c e l l s to the N-TETPA. That t h i s e f f e c t was s p e c i f i c was demonstrated by the equivalent experiment i n N-10-C sensitized c e l l s , where MIF production e l i c i t e d by N-TETRA. was the same either i n the absence or presence of DNP-N-TRI. This data indicates a number of p o s s i b i l i t i e s :  i.  The surface recognition s i t e s on T c e l l s may  involve a mechanism similar to that seen i n immunog l o b u l i n ligand  formation, i n that entry of the  antigenic determinant may be involved.  into a ' r e c i p r o c a l ' f i t ' crypt  ii.  DNP on i t s own does not appear to be capable  of blocking the i n t e r a c t i o n of N-HEPTA with c e l l s sensitized to DNP-N-10-C or N-10-C.  iii.  DNP-N-TEI, which contains both DNP substitutions  and the three amino acids d i s t a l to the ' s p e c i f i c i t y ' sequence of the N-HEPTA peptide, i s capable of abrogating the response of DNP-N-10-C sensitized c e l l s to the N-TETRA, but not the response of N-10-C sensitized c e l l s to the same peptide.  This implies a s p e c i f i c p a r t i c i p a t i o n on the part of the DNP-N-TRI i n the recognition s i t e on T c e l l s .  I t would thus appear that while DNP  alone cannot block t h i s reaction, the amino acids i n the immediate v i c i n i t y of the DNP can, suggesting again a s p e c i f i c r o l e of t h i s v i c i n i t y of the DNP-N-HEPTA sequence i n c e l l recognition.  Since  blocking by DNP-N-TRI was not observed i n N-10-C sensitized c e l l s , i t would appear that the sensitized c e l l s i n this population do not s p e c i f i c a l l y recognize t h i s sequence.  d.  The Response of Unsensitized C e l l s to the Test Antigen.  The s p e c i f i c i t y of the various test preparations used i n the previous set of experiments i s established by t h e i r performance i n  c e l l cultures from unimmunized animals. none of these preparations  As can be seen from Table X,  cause any s i g n i f i c a n t i n h i b i t i o n of migra-  t i o n of c e l l s from unsensitized control guinea pigs.  This also  indicates that none of the r e s u l t s found i n the immunized animals were a d i r e c t r e s u l t of c y t o t o x i c i t y caused by any of the preparations used.  I I I . Lymphocyte  The  Stimulation.  lymph node c e l l s of animals s e n s i t i z e d to either N-10-C or  DNP-N-10-C were tested f o r t h e i r a b i l i t y to i n i t i a t e DNA synthesis i n the presence of N-10-C or i t s DNP derivative, an assortment of DNP substituted or unsubstituted  peptides forming a portion of or  a l l of the NH^-terminal hapten, as well as i n the presence of some DNP substituted heterologous compounds.  The t e s t involves  specific  antigen a c t i v a t i o n of s e n s i t i z e d cells, causing them to increase the rate of DNA synthesis p r i o r to entry into the d i v i s i o n cycle.  This  degree of synthesis can be q u a n t i t a t i v e l y measured by comparing the amount of ^H-thymidine l a b e l incorporated  into the DNA of c e l l s  found i n c e l l cultures containing antigen to that incorporated the DNA of c e l l cultures containing no antigen 1964). XII.  into  (Dutton and Eady,  The r e s u l t s of these tests are presented i n Tables XI and  Table X:  The e f f e c t of the homologous antigen, haptenic peptides, and DNP substituted heterologous compounds on the migration of spleen c e l l s derived from unsensitized guinea pigs.  Antigen  Concentration i n chambers (fjmoles/ml)  Migration  trprobability*  a  $  no. of animals  DNP-N-10-C  0.025  97.2  + 26.1  6  N-10-C  0.025  97.0 + 18.2  7  DNP-N-HEPTA  0.05  5  N-TETRA  0.05  92.2 + 30.9 + 18.7  7  DNP -LYSOZYME  0.025  100.3 + 24.4  6  .421  DNP-SEE 4  0.05  102.7  + 28.5  7  .164  25.3  7  .580  98.7  99.h  +  .691  .425  .293 .067 .239  . 502  DNP  0.05  DNP + N-HEPTA  0.05  each  98.3 + 25.4  7  .719  DNP-N-TRI + N-TETRA  0.05  each  88.6 + 11.5  2  .420  DNP-N-TRI  0.05  9 3 . 5 + 11.2  2  .221  DNP-N-PENTA  0.05  95.8 +  9-3  2  .310  N-PENTA  0.05  88.1 +  6.2  2  .060  Eagles MEM  1 ml  100  7  These figures f o r percent migration represent the averaged percent migration i n chambers containing antigen, peptide, or heterologous protein compared to control chambers incubated without any of these preparations. The t p r o b a b i l i t y represents the r e s u l t s of a Student's t t e s t c a r r i e d out on the i n d i v i d u a l migration values f o r each antigen as compared with the values f o r the media controls. The dashed t p r o b a b i l i t i e s represents analysis c a r r i e d out amongst similar antigens. A t p r o b a b i l i t y of less than 0 . 0 1 can be considered as s t a t i s t i c a l l y significant.  3  46  Table XI:  %-thymidine incorporation i n lymph node c e l l cultures from DNP-E-10-C sensitized guinea pigs i n response to the substituted and unsubstituted forms of the homologous antigen, haptenic peptides, and DEP substituted heterologous compounds.  Antigen  Concentration (|jmoles/ml)  DEP-N-10-C  0.05  N-10-C  0.05  DEP-N-8-N  0.05  DEP-N-HEPTA  0.05  DEP-LYSOZYME  0.05  DEP-SER 4  0.05  DEP  0.05  Average stimulation a  + 1.35 2.35 + 1.42 .1.45 + 0.83 2.27  1.41 + 1 . 0 7 1.12 + 0.57 0 . 9 0 + 0.3^ 0 . 8 0 + 0.32  N-TETRA  0.05  PPD  50 ug/ml  1.39 + 0.95 1.95 + 0 . 6 7  RPMI l640  0.05  1.00  ml  Wo. of t probability animals  6  .622  .000  7  .000  7  .029  5  .025  6  .027  5  .241  3  .058  7  .023  5  .000  7  These figures f o r the average stimulation are derived from the r a t i o of 3H-thymidine incorporation i n cultures with the test preparations present to the 3jj_thymidine incorporation i n c o n t r o l cultures from the same guinea p i g without these preparations. The results are based on quadruplicate sets of data from tissues taken from DNP-N-10-C s e n s i t i z e d guinea pigs. The t p r o b a b i l i t y represents the r e s u l t s of a Student's t test c a r r i e d out on the i n d i v i d u a l stimulation values f o r each antigen as compared with the values f o r the media controls. The dashed t p r o b a b i l i t i e s represent analysis carried out amongst similar antigens. A t value of less than 0.01 can be considered as s t a t i s t i c a l l y s i g n i f i c a n t .  1  Table XII:  -thymidine incorporation i n lymph node c e l l cultures from N-10-C s e n s i t i z e d guinea pigs i n response to the substituted and unsubstituted forms of the homologous antigen, haptenic peptides, and DEP substituted heterologous compounds.  Antigen  Concentration (|jmoles/ml)  Average stimulation a  No. of t probability animals  1.88  + O.67  8  0.05  1.93  + 1.00  7  DNP-N-8-N  0.05  8  .454  DNP-N-HEPTA  0.05  0.93 + 0.55 1.06 + 0.77  5  .523  DNP-LYSOZYME  0.05  0.86 + 0.26  8  .023  DNP-SER 4  0.05  1.24  7  .183  DNP  0.05  1.24  + 0.73 + 0.91  7  .062  N-TETRA  0.05  1.40  1.25  8  .136  + 1.70  8  .000  DNP-N-10-C N-10-C  0.05  PPD  50 p-g/rnl  2.52  RPMI l 6 4 0  0.05  100  ml  -.696 -—  .001 0 0 0  8  These f i g u r e s f o r the average stimulation are derived from the r a t i o of 3jj-thymidine incorporation i n cultures with the t e s t preparations present to the 3jj-thymidine incorporation i n control cultures from the same guinea pig without these preparations. The r e s u l t s are based on quadruplicate sets of data from tissues taken from N-10-C s e n s i t i z e d guinea pigs. The t p r o b a b i l i t y represents the r e s u l t s of a Student's t t e s t c a r r i e d out on the i n d i v i d u a l stimulation values f o r each antigen as compared with the values f o r the media controls. The dashed t p r o b a b i l i t i e s represent analysis c a r r i e d out amongst s i m i l a r antigens. A t value of less than 0 . 0 1 can be considered as s t a t i s t i c a l l y s i g n i f i c a n t .  a.  The Response of DNP-N-10-C Sensitized C e l l s to the Test Antigens.  The r e s u l t s , presented i n Table XI, show that there i s no difference i n the degree of stimulation caused by either the subs t i t u t e d or the unsubstituted  form of the immunogen.  Also, DNP,  when coupled to a larger c a r r i e r (Lysozyme), f a i l e d to show any stimulation.  This indicates again that DNP, i n i t s e l f ,  does not  have the configurational requirement to e f f e c t c e l l a c t i v a t i o n . These findings are i n agreement with those of other workers i n the field  (Schlossman et a l , 1 9 6 9 and Paul et a l , 1 9 7 0 ) .  r e s u l t s do not explain the observation  However the  that DNP-N-10-C and N-10-C  do not show a d i f f e r e n t i a l response i n the degree of stimulation i n DNP-N-10-C immune lymph node c e l l s . The other haptenic test preparations  did not cause any  s i g n i f i c a n t degree of stimulation i n these experiments.  In general  t h i s was to be expected, since i t has previously been shown that there i s a molecular size requirement needed to activate c e l l s into the d i v i s i o n cycle.  Most of the peptides tested e x i s t below  t h i s l e v e l (Waterfield et a l , 1 9 7 2 ) .  But, i t should have been  expected that DNP-N-8-N would have stimulated obviously has not.  i n t h i s system, as i t  K e l l y et_ a l ( 1 9 7 2 ) , have shown that N-8-N does  stimulate i n N-10-C immune animals.  This suggests that the prep-  aration of DNP-N-8-N used i n these experiments could have been i n some way i n h i b i t o r y to c e l l d i v i s i o n .  It should be noted that the posit ive  control, PPD,  i n these tests  stimulated adequately, since the animals were immunized with a preparat i o n containing Freund's complete adjuvant: an/adjuvant consisting, i n part of M. tuberculosis, the bacterium from which PPD i s derived. The data presented here shows that c e l l s activated to d i v i s i o n do not recognize the differences between the substituted and unsubstituted forms of the antigen. who found that DNP  I t also confirms the observations of other workers  coupled onto heterologous c a r r i e r s does not have the  configurational requirements to cause c e l l activation.  These findings  are not i n complete agreement with those obtained using the MIF  assay;  where the substituted and unsubstituted forms of the antigen showed a quantitative difference i n c e l l u l a r activation.  However, these apparent  differences can possibly be attributed to d i f f e r e n t degrees of sensit i v i t y of the test methods employed and w i l l be discussed i n greater detail later.  b.  The Response of N-10-C Sensitized Cells to the Test Antigens.  Since no d i s t i n c t i o n was exhibited i n DNP-N-10-C immune animals between the substituted and unsubstituted forms of the immunogen, experiments using N-10-C immunized guinea pigs were carried out to see i f DNP  substitution would block the recognition of the N-TETRA deter-  minants i n the N-10-C molecule. As can be seen from Table XII, there i s no s i g n i f i c a n t difference  i n the amount of stimulation caused by DNP-N-10-C or N-10-C. indicates that DNP  This  substitution of the immunogen does not prevent i t s  subsequent i n v i t r o recognition by c e l l s previously sensitized to the unsubstituted form. The remaining data represents the further controls on these experiments.  As before, DNP  any stimulation.  when coupled to Lysozyme does not cause  The smaller peptides  also do not cause any stimu-  l a t i o n f o r , as has been previously mentioned, they e x i s t below the molecular size requirement necessary f o r peptide a c t i v a t i o n of c e l l division.  Stimulation with the p o s i t i v e control antigen, PPD,  indicates  that the c e l l s used i n these experiments were v i a b l e and could be stimulated by a non-cross reacting antigen.  c.  The Response of Unsensitized C e l l s to the Test Antigens.  The s p e c i f i c i t y of the various test preparations used i n the experiments employing DNP-N-10-C and N-10-C immunized animals i s well i l l u s t r a t e d by s i m i l a r experiments i n unimmunized animals. be seen i n Table XIII, none of the peptides  cause any  As  can  significant  c e l l u l a r a c t i v a t i o n i n the form of c e l l d i v i s i o n i n unsensitized control guinea pigs.  51  Table XIII:  si -thymidine incorporation i n lymph node c e l l cultures from unsensitized (control) guinea pigs i n response to the substituted and unsubstituted forms of the main antigen, haptenic peptides, and DNP substituted heterologous compounds previously used.  Antigen  Concentration (umoles/ml)  Average stimulation a  No. of t probability animals  DNP-N'-IO-C  0.05  O.98  + 0.43  N-10-C  0.05  0.84  + 0.3k  7  .082  DNP-N-8-N  0.05  0.87  7  .400  DNP-N-HEPTA  0.05  0.87  + 0.38 + 0.41  5  .658  DNP-LYSOZYME  0.05  0.94  6  .753  DNP-SER 4  0.05  1.22  + 0.37 + 0.41  7  .056  DNP  0.05  1.03  + 0.28  5  .486  N-TETRA  0.05  O.96  +  0.39  5  • 771  + 0.68  6  .000  PPD  50 |ig/ml  1.56  RPMI l 6 4 0  0 . 0 5 ml  100  6  —  .309  .857  7  These figures f o r the average stimulation are derived from the r a t i o of 3H-thymidine incorporation i n cultures with the test preparations present to the 3 -thymidine incorporation i n control cultures from the same guinea p i g without these preparations. The r e s u l t s are based on quadruplicate sets of data from tissues taken from unsensitized guinea pigs. H  The t p r o b a b i l i t y represents the r e s u l t s of a Student's t t e s t c a r r i e d out on the i n d i v i d u a l stimulation values f o r each antigen as compared with the values f o r the media controls. The dashed t p r o b a b i l i t i e s represent analysis c a r r i e d out amongst s i m i l a r antigens. A t value of less than 0 . 0 1 can be considered as s t a t i s t i c a l l y s i g n i f i c a n t .  IV.  Concluding Discussion.  The aim of this thesis has been to establish the contribution of the DNP molecule i n the s p e c i f i c i t y of activation of c e l l u l a r immune responses both i n vivo and i n v i t r o i n animals immunized with a DNP c a r r i e r protein conjugate whose antigenic sequences have been determined. The i n vivo results showed that DNP, when coupled to a heterologous c a r r i e r , d i d not have the configurational requirement to e f f e c t a delayed hypersensitive reaction.  The substituted immunogen DNP-  N-10-C was required to e l i c i t such a reaction.  I t was also noted  that DNP substitution of N-10-C d i d not block the i n vivo recognition of the haptenic N-TETRA sequence i n animals immunized to the unsubstituted form. The i n v i t r o results, as assayed by MIF production and lymphocyte stimulation, have shown certain inconsistencies.  The s p e c i f i c i t y  requirements f o r c e l l s activated into MIF production i n DNP-N-10-C animals seem to be mainly directed towards a 'shared' determinant, consisting p a r t l y of the DNP moiety and p a r t l y of the N-HEPTA peptide, since DNP substituted antigens were more e f f e c t i v e i n e l i c i t i n g MIF production than were t h e i r unsubstituted counterparts.  However, the  s p e c i f i c i t y requirements of c e l l s sensitized to N-10-C appear to be directed towards the N-TETRA peptide i n the NH^-terminal determinant, and are s a t i s f i e d by either the substituted or unsubstituted form  of the NH -terminal determinant. 2  C e l l s activated into a state of  d i v i s i o n by the substituted and unsubstituted antigen do not appear to be able to d i s t i n g u i s h these s p e c i f i c i t y differences.  Both i n  v i t r o assays are consistent i n the f a c t that DEP, by i t s e l f , or coupled to heterologous carrier's, can not activate c e l l s to produce MIF to synthesize The  DEA.  apparent differences i n the a b i l i t y of both i n v i t r o  h y p e r s e n s i t i v i t y reactions to d i s t i n g u i s h DEP s t i t u t e d challenging antigens number of  1.  or  delayed  substituted and unsub-  can possibly be explained by any of a  observations.  There appear to be d i f f e r e n t molecular size requirements f o r  a c t i v a t i o n of MIF  and lymphocyte stimulation and thereby possibly a  d i s s o c i a t i o n of s p e c i f i c i t y requirements.  Small peptides of four  amino acids i n length (E-TETRA) can produce a s i g n i f i c a n t of migration  i n the MIF  assay.  inhibition  However i t requires a much larger  peptide of 22 amino acids i n the case of E-10-C to i n i t i a t e synthesis i n activated c e l l s . production  I t would appear then, that  DEA  MIF  can be activated much more e a s i l y and i s possibly a more  sensitive assay.  This basis of s e n s i t i v i t y of various requirements  of a c t i v a t i o n involved i n these two assays could explain the d i s crepancy.  2.  Work reported by Paul et a l ( 1 9 6 8 )  showed a heterogeneity  of  responsiveness on the part of lymphocytes i n the i n v i t r o DNA synthetic response that has not yet been shown with MIF production. preted this as a heterogeneity  i n the a f f i n i t y of antigen  receptors possessed by d i f f e r e n t i n d i v i d u a l lymphocytes. heterogeneity  They i n t e r binding I f such  of a f f i n i t y existed, a d i f f e r e n t i a l stimulation response  between substituted and unsubstituted forms of the immunogen could quite conceivably be overshadowed by the extensive occurring.  cross-reactions  Thus, even i f a 'shared' determinant encompassed the  s p e c i f i c i t y of reactive c e l l s i n DNP-N-10-C immune animals, the  hetero-  geneity of a f f i n i t y could be such that N-10-C could produce an equivalent e f f e c t .  Unfortunately,  such a hypothesis would require lymphocyte  receptors of more uniform a f f i n i t y f o r the a c t i v a t i o n of MIF and no experimental evidence to confirm this has been presented to date.  3.  F i n a l l y , i t has been postulated (Rocklin, 1 9 7 3 ) that MIF production  and lymphocyte stimulation can be dissociated from one another on the basis that there are d i s t i n c t c e l l populations response;  involved i n each  the c e l l population entering the d i v i s i o n cycle being  d i f f e r e n t from the one producing MIF.  In summary, on the basis of the MIF assay, i t can be stated that an extended determinant appears to be formed when DNP i s coupled onto a haptenic  sequence previously shown to be c a r r i e r independent.  After immunization with this DNP substituted complex, antigen  sensitive c e l l s , showing a c e l l u l a r s p e c i f i c i t y towards both the DNP moiety and a portion of the previous haptenic peptide, can be activated upon challenge.  The DNP group, by i t s e l f or when coupled to heterologous  carriers, does not have the configurational requirement to effect c e l l u l a r activation, while the unsubstituted haptenic peptide can by itself  e l i c i t such a response,  i n d i c a t i n g a considerable degree of  c r o s s - r e a c t i v i t y between the substituted and unsubstituted forms. This implies that, although DNP i s an i n t e g r a l part of the new determinant, i t plays a minor role i n actual a c t i v a t i o n of c e l l s to e l i c i t a delayed hypersensitive reaction. In terms of other DNP-carrier systems i n use at the present time, these results can be interpreted as an a r t i f i c i a l formation of many separate determinants on globular proteins, most of which, upon immunization, w i l l express a c e l l u l a r s p e c i f i c i t y towards a combined or 'shared' determinant.  Therefore, any anti-hapten-carrier delayed  h y p e r s e n s i t i v i t y response w i l l i n f a c t be a heterogeneous a n t i - c a r r i e r response which w i l l depend on the number of haptenic sequences i n the carrier.  APPENDIX  1.  Composition of Eagles Minimum E s s e n t i a l Medium (MEM)  mg/l 8,000.0  NaCl  400.0  KC1 Ng^PO^. 2 H 0 2  MgSO^.7H 0 2  KH  2  P0  CaCl  140.0  2  1, 0 0 0 . 0  Glucose 2  100.0 60.0  1|  MgCl .CH  60.0  2  NaHCO^ L-arginine L-cystine  100.0 55.0 105.0 24.0  mg/l L-methionine  15.0  L-phenylalanine  32.0  L-threonine  48.0  L-tryptophan  10.0  L-tyrosine  36.0  Valine  46.0  Choline C l .  1.00  F o l i c acid  1.00  i - inositol  2.00  Nicotinamide  1.00  D-Ca.pantothenate  1. 0 0  Pyridoxol HCl  1.00  L-Glutamine  292.0  L-histidine  31.0  Riboflavin  0.10  L-isoleucine  52.5  Thiamine-HCl  1.00  L-leucine  52.4  phenol red  L-lysine  58.0  NaHCO^  10.00 2,200.0  57  Composition of RPMI 1 6 4 0  2.  mg/l Ca(W0 ) .4H 0 3  2  2  Glucose  f100.0 2,000.0  mg/l L-phenylalanine  15.0  L-proline  20.0  MgSO^.7H 0  100.0  L-serine  30.0  KC1  400.0  L-threonine  20.0  2  5.0  Na HP0^.7H 0  1, 5 1 2 . 0  L-tryptophane  NaCl  6,000.0  L-tyrosine  20.0 20.0  2  2  L-arginine  200.0  L-valine  L-asparagine  500.0  Biotin  0.2  L-aspartic  20.0  Vitamin B-^  0.005  L-cystine  50.0  D-Ca-pantothenat e  0.25  L-glutamic acid  20.0  Choline C l  3.0  300.0  F o l i c acid  1.0  1.0  i-inositol  35.0  L-glutamine Glutathione Glycine  •  2  10.0  Nicotinamide  1.0  L-histidine  15.0  P.A.B.A.  1.0  L-hydroxyproline  20.0  Pyridoxin - HCl  1.0  L-isoleucine  50.0  Riboflavin  0.2  L-leucine  50. 0'-  Thiamine - HCl  1.0  L-lysine HCl  40.0  Phenol red  5-0  L-methionine  15.0 "  NaHC0  3  2,000.0  LITERATURE CITED  1971.  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