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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 British Columbia A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE In the Department of Microbiology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June 1973 In presenting this thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission fo r extensive copying of t h i s thesis for scholarly purposes may be granted by the Head of my Department or by h i s representatives. It i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of M i c r o b i o l o g y  The University of B r i t i s h Columbia Vancouver 8, Canada ABSTRACT In animals immunized with 2,4 dinitrophenyl (DNP) hapten-carrier protein conjugates, no in vitro cellular response is el i c i t e d by DNP, either alone, or when coupled to a heterologous carrier. In contrast, animals immunized with haptenic peptide-carrier conjugates do mount an in vitro cellular response towards the haptenic peptide. This apparent inconsistency led us to compare the in vivo and in vitro cellular immune responses to a synthetic peptide antigen and its DNP derivative to determine the activation specificity of the cells 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 in vivo or in vitro cellular activation was evaluated for DNP alone, DNP coupled to the homologous determinant, and DNP coupled to heterologous carriers. The data suggests that in DNP-N-10-C immune guinea pigs, DNP substitution opens a new determinant exhibiting, in antigen reactive cells, a unique specificity towards the DNP moiety as well as a portion of the peptide to which i t is conjugated. However the DNP group by i t s e l f does not have the configurational requirement to evoke cellular activation. It therefore plays a minor role in activation of the cellular immune response; the major contribution being supplied by the peptide portion of the 'shared' determinant. i i i TABLE OF CONTENTS Page INTRODUCTION AND LITERATURE REVIEW 1 I. Clonal Selection Theory of Immunological Diversity 1 II. Cell Types Involved in the Immune Response 2 III. Specificity of Humoral Antibody and Immunocompetent Bone Marrow Derived Cells 3 IV. Specificity of Immunocompetent Thymus Derived Cells . . 5 V. Definitive Studies on the Hapten-Carrier Relationship Using DNP, 9 MATERIALS AND METHODS 11 I. Synthesis, Purification, and Quantitation of the Antigens, Antigenic Determinants, and Peptides . . . . 11 II. Immunization of Guinea Pigs 2k III. Skin Tests 2k IV. Migration Inhibition ' . . . . 25 V. Lymphocyte Stimulation . . . . 26 VT. S t a t i s t i c a l Analysis 28 RESULTS AND DISCUSSION 29 I. Skin Tests 29 II. Migration Inhibition 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 ratios of the amino acids in the antigen and peptide determinants Amino acid degradation values of Beckman standard amino acids The amino acid composition and the expected molar ratios of the amino acids in the DNP antigens and DNP peptide determinants The amino acid composition and the expected molar ratios of the amino acids in 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 effect of the homologous antigen, haptenic peptides, and DNP substituted heterologous compounds on the migration of spleen cells derived from guinea pigs sensitized to DNP-N-10-C The effect of the homologous antigen, haptenic peptides, and DNP substituted heterologous compounds on the migration of spleen cells derived from guinea pigs sensitized to N-10-C LIST OF TABLES The effect of the homologous antigen, haptenic peptides, and MP substituted heterologous compounds on the migration of spleen cells derived from unsensitized guinea pigs -thymidine incorporation in lymph node c e l l cultures from DNP-N-10-C sensitized guinea pigs in response to the substituted and unsubstituted forms of the homologous antigen, haptenic . peptides, and MP substituted heterologous compounds H-thymidine incorporation in lymph node c e l l cultures from N-10-C sensitized guinea pigs in response to the substituted and unsubstituted forms of the homologous antigen, haptenic peptides, and MP substituted heterologous compounds H-thymidine incorporation in lymph node c e l l cultures from unsensitized (control) guinea pigs in response to the substituted and unsubstituted forms of the main antigen, haptenic peptides, and MP substituted heterologous compounds previously used LIST OF FIGURES Figure 1. Flow diagram of solid 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 N-HEPTA N-TRI N-TETRA N-PENTA DNP-N-IO-C DNP-N-8-N DNP-N-HEPTA DNP-N-TRI DNP-N-PENTA SER k DNP-SER k * C ** N NH -Ala-Tyr -Lys -He -Ala -Asp -Ser -(Gly) -C*COOH 10 NH -Ala-Tyr-Lys -He-Ala-Asp-Ser-COOH NH2-Ala-Tyr-Lys-COOH NH0-Ile-Ala-Asp-Ser-COOH NH2 -Ala -Tyr -Lys -He -Ala -COOH NHp -Ala-Tyr -Lys -He -Ala-Asp -Ser -(Gly) -C -COOH 1 1 10 DNP DNP NH -Ala-Tyr-Lys-Ile-Ala-Asp-Ser-(Gly) -N**-COOH ^ 1 1 8 DNP DNP NH -Ala-Tyr-Lys-Ile-Ala-Asp-Ser-COOH 1 1 DNP DNP NH -Ala-Tyr-Lys-COOH 1 1 DNP DNP NH -Ala-Tyr-Lys-Ile-Ala-COOH 1 1 DNP DNP NH^-Ser-Ser-Ser-Ser-COOH 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 like to thank Dr. Julia Levy for her invaluable encouragement, refreshing suggestions, and constructive criticism of both the research and writing of this thesis. I also wish to thank Dr. Douglas Kilburn for his helpful guidance of the research and Mrs. Barbara Kelly for her cooperative contributions to the work. Finally, I would like to extend my thanks to my committee, Dr. J. J. R. Campbell, Dr. J. B. Hudson, Dr. D. G. Kilburn, and Dr. J. Levy, for their suggestions in editing the f i n a l draft of the thesis. INTRODUCTION AND LITERATURE REVIEW Central to any study of the immune response is an understanding of how lymphoid cells can detect and respond to the presence of antigen in their environment. Many theories, both instructional and selectional, have been proposed over the years to account for the specificity involved in the immune response (Breinl and Haurowitz, 1930; Pauling, 1940; Burnet, 1959 and Jerne, 1971). At the present time, the experimental evidence indicates that the selectional theories are the most plausible, and hence have become the most widely accepted. I. Clonal Selection Theory of Immunological Diversity The clonal selection theory of immunity, the basis of a l l selectional theories, postulates that lymphoid cells are completely restricted in the ranges of antigenic determinants to which they may respond; restricted to the extent that one immunocompetent c e l l w i l l distinguish only one antigenic determinant, and hence have only one specificity. This theory also proposes that the determination of this specificity occurs prior to the f i r s t exposure of the c e l l to antigen, probably before immunological maturation. Directoevidence for such a restricted specificity has been shown by single c e l l experiments (Nossal et al, 1964) and autoradiography 2 (Naor and Sulitzeanu, 19&7 and Humphrey and Keller, 1971). Experi-ments employing autoradiography demonstrate that antigen binds specifically to only a small number of cells, presumably through immunoglobulin-like receptors. Before entering into a further discussion of cellular specificity, i t is necessary to f i r s t examine the c e l l types involved in the immune responses. II. C e l l Types Involved in 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 in in vitro antibody responses to sheep red blood cells in mice (Mosier, 1967) > a n ^ i - n in vitro antibody responses to phage in rabbits and rats (Fishman, 1961 and Fishman and Adler, 1967)• Macrophage-associated antigen has also been found in the in vivo antibody responses to hemocyanins (Unanue and Askonas, 1968), and to heat-aggregated bovine serum albumin (Mitchison, 1969). However, despite the requirement for macrophages in most immune responses, i t is generally thought that their role is not in antigenic specificity. Lymphocytes, on the other hand, exhibit a unique specificity for antigen, and are essential in the development of both cellular and humoral immunity. The lymphocyte population has recently been further defined as a minimum of two distinct 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 cells are originally produced i n the bone marrow or other hematopoietic tissue, and reside tempor-a r i l y in the thymus, before populating the peripheral lymphoid organs (Davies, 1 9 6 8 ). B cells also originate from the bone marrow, but populate the peripheral lymphoid organs directly. The T cells are thought to be mainly responsible for e l i c i t i n g the delayed hyper-sensitivity type or cellular reaction (Miller and Mitchell, 1 9 6 8 ) , while the B cells differentiate into plasma cells capable of synthes-izing immunoglobulin for the expression of humoral immunity (Miller and Osoba, 1 9 6 7 ) . Both of these c e l l populations appear to exhibit antigenic specificity. As i t is the aim of this thesis to restr i c t the study of specificity to the T c e l l population, the specificity 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. III. Specificity of Humoral Antibody and Immunocompetent Bone Marrow Derived Cells. Landsteiner ( 1 9 6 2 ) , in 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, result in the synthesis of specific anti-hapten antibodies. He also found that chemical modi-k fication of the haptens resulted in a loss of binding a f f i n i t y to the specific antibody. These results suggested that humoral specificity could exist, in many cases, to haptenic groups alone. Since this early work, haptenic compounds have been used extensively in studies on T c e l l and B c e l l specificity. The specificity of B cells'has been studied by various techniques mainly focusing at the cellular receptor level. In some cases, i t has been shown that B cells can be specifically inactivated (tolerized) in vivo by contact with free hapten, preventing the expression of B c e l l function (antibody production). Katz et_ a l (1971), found that administration of 2,k dinitrophenyl (DNP) conjugates of "nonimmunogenic" amino acid copolymers exerted a suppressive effect on the capacity of guinea pigs to display anti-DEP antibody responses to immunization with a DNP conjugate of a strong antigen. Moreover, this hapten-specific unresponsiveness was found to be an internal effect and not just the blocking of c e l l surf ace.. receptors by the DEP copolymers. In further experiments on the specificity of cellular receptors, Wigzell and Makela (1970) showed that they could select out populations of B cells with preformed specificity receptors towards DEP in immun-ized animals as well as in unimmunized animals. The overall results suggest that B cells carry a receptor with a specificity that can be directed, in most circumstances, towards the haptenic determinants on an immunogenic molecule. These receptors appear to be of an "immunoglobulin-like" nature, since anti-immuno-5 globulin serum inhibits antigen binding (Byrt and Ada, 1969; Dwyer and Mackay, 1970). IV. Specificity of Immunocompetent Thymus Derived Cells. Hapten-carrier conjugates have been used to study the specificity of such T c e l l functions as: helper c e l l activity, delayed skin hyper-sensitivity, stimulation of DNA synthesis in cultures of sensitized lymphocytes, macrophage inhibitory factor (MIF) production, and c e l l mediated cytotoxicity. Paul et_ a l (1970a) found, using 2,4 dinitrophenyl (DNP) guinea pig albumin, that the hapten alone could not function as a carrier for other haptens in an anti-hapten antibody response. However, the hapten could contribute to the carrier determinant, and stimulate the form-ation of carrier-specific cells capable, of enhancing the antibody response to other haptens presented on the same molecule. In studying the specificity of delayed skin reaction using the trinitrophenyl and para toluenesulfonyl haptens, Benacerraf and Levine (1962) found that this specificity involved large areas of the sensi-tizing 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 different carrier protein. These results were verified and extended by Gell and Silverstein (1962a,b), using the azobensenesulfonate hapten. Data was 6 presented showing that the specificity of the delayed skin reaction to hapten-protein conjugates involved a considerable degree of contribution by the protein carrier. They carried this 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 in animals immunized to meta-azobenzenesulfonate. Oppenheim et a l (1967), using guinea pig albumin-orthanilic acid conjugates found that in vitro lymphocyte proliferation in response to antigen was mainly carrier specific. However, they also noted that the proliferative response was par t i a l l y inhibited 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_ al, 1969), i t was found that 9 ^ DEP-nona-L-lysine caused l i t t l e or no stimu-lation of DEA synthesis by lymphoid cells from guinea pigs immunized to 5 i DNP-nona-L-lysine, although the immunizing peptide was an excellent stimulator. This also indicated a marked carrier dependence, but further showed that sensitized cells could dis-criminate among compounds which differed from one another in the position of the dinitrophenyl group. When the specif i c i t y of MIF production was studied, David et a l (1964) demonstrated, using DNP-bovine IgG in an in vitro system, that the specificity was directed against the carrier. He later 7 expanded this work and showed, using the same assay, that sensitized peritoneal exudate cells could discriminate between various DNP-oligolysines; a heptamer or larger being required for the inhibition of migration of peritoneal exudate cells (David et_al, 1968). Henney (1970) followed the development of a population of cyto-toxic lymphocytes in animals immunized with DNP-human IgG and found that the speci f i c i t y of cytolysis was markedly carrier dependent. 51 The degree of cytolysis by splenic lymphocytes of Cr labelled mastocytoma cells was only noticeable when the target cells were coupled with the immunizing hapten-carrier conjugate or with the carrier alone. The findings summarized above indicate that in certain hapten-carrier systems, the cells participating i n the cellular immune response can bear a receptor with a complex specificity pattern; an antigenic determinant containing elements of both hapten and carrier. It must be noted that not a l l hapten-carrier systems exhibit this shared speci 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 is wholly hapten specific. Our own work has shown that specific peptide sequences of larger protein molecules can also be defined as being haptenic (Mitchell et al, 1970 and Kelly et_ a±_, 1971). Immunization with the entire protein molecule induces a delayed type reaction which, like Leskowitz's molecule, 8 is tota l l y hapten specific: MIF production and delayed skin hyper-sensitivity being e l i c i t e d by the peptide sequences themselves (Waterfield et al, 1972). It has thus been well established that certain disparities exist in these hapten-carrier systems as to whether a given hapten w i l l be carrier dependent or carrier independent in e l i c i t i n g a delayed hypersensitive reaction. The overall results previously discussed have shown that for some haptens, such as DNP, part of the carrier seems to be required to form an antigenic determinant capable of inducing T c e l l function. However, other haptens, such as peptide sequences, do not require any specific contribution of a carrier molecule. These facts can only leave workers in 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 in cells sensitized to this conjugate w i l l show a specificity towards both hapten and carrier? If so, how many such shared determinants are formed in the coupling procedure when globular proteins are used as carriers? To what extent does the DNP moiety contribute to the specificity of recognition of the combined determinant? In this determinant, does the haptenic sequence on the carrier exist as such before, or was i t newly formed by the coupling procedure? At the present time, in the study of T c e l l specificity these 9 questions remain unanswered as the main carrier 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 typical carrier dependent hapten (DNP) in activating the cellular immune responses using an immunogenic peptide carrier whose haptenic sequences have been previously defined. This w i l l allow us to evaluate the role of DNP in these responses when the hapten is coupled exclusively to a known carrier independent haptenic sequence. V.- Definitive Studies on the Hapten-Carrier Relationship Using DNP. Early definitive work in 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 al, 1969). The major antigenic determinants of the O-Fd molecule have since been extensively characterized (Mitchell et al, 1970 and Kelly et al, 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 al, 1972) showed that a' synthetic immunogen could be constructed by solid phase peptide synthesis, consisting of these two haptenic determinants separated by a ten glycine bridge or spacer (N-10-C). This defined immunogen was selected for the study of the role of DNP in activating a cellular 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 yielding 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 cellular immune response in the presence of antigen was measured by the following means - in vivo, delayed skin hypersensitivity and in vitro, MIF production and stimulation of DNA synthesis. These types of cellular activation were evaluated for DEP alone, DEP coupled to the EH 2-ter-minal determinant, and DEP coupled to heterologous carriers. MATERIALS AND METHODS I. Synthesis, Purification, and Quantitation of the Antigens, Antigenic Determinants and Peptides. a. Solid phase peptide synthesis. The immunogen, N-10-C, was synthesized according to the Merrifield (1964) method of solid phase peptide synthesis, with modifications by Stewart and Young (19^9: Fig- l ) • This technique, recently automated in our laboratory by Douglas G-. Hull, involves the use of an insoluble resin, co-polystyrene divinylbenzene (Schwarz Mann Chemical Co.) to which the desired COOH-terminal amino acid (glutamic acid in the case of N-10-C) has already been covalently bound through i t s free carboxyl group. Sequential coupling of amino acids is carried out by the formation of a peptide bond between the a-carboxyl of the next required t-butyl-oxycarbonyl (t-BOC) amino acid (Schwarz Mann Chemical Co.) and the free amino group of the attached amino acid. The peptide is 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 is achieved Fig. I: Flow diagram of solid phase peptide synthesis C H H 3 H C L - H O A C ; ( C 2 H 5 ) 3 N D E P R O T E C T ; N E U T R A L I Z E ll 2 H R, O i 11 ii C H - C + C O + H - N - C - C - O - C H ( C O P O L Y M E R 3 i 2 l 2vrv C H . i s o b u t y l e n e Boc amino aci,d d i i m i d e H amino a c y l p o l y m e r C O U P L E C H O H R 0 0 H R O I 3 || | \A II | 11 II C H - C - 0 - C - N - C - C - N - C - C - 0 - C H 0 ( 0 / P O L Y M E R 3 I | i C H ^ H Boc p e p t i d e H p o l y m e r H B r - F 3 C C 0 0 H C L E A V E C H H R O H R . O II 2 I I II I I 1 II / — \ C H - C + C O + H N - C - C - N - C - C - O H + B r - C H ( O ) P O L Y M E R 3 | 2 | i ^ \ — / C H H H o i s o b u t y l e n e p e p t i d e * Modified from 'Solid Phase Peptide Synthesis' by John Stewart and Janis Young, p.3 by treatment of the peptide resin with IN HCl in gla c i a l acetic acid (HOAC). The acidic conditions are neutralized by addition of triethylamine (TEA) before subsequent peptide bond formation takes place in 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 resin is washed with organic solvents to prevent build-up of non-reacted amino acids. The amino acids were added in a 4.0 molar excess of the i n i t i a l substitution on the resin. To ensure maximum coupling, the DCC was added in a 6.0 molar excess. Upon completion, the peptide is cleaved from the resin by bubbling anhydrous HBr (Matheson Co.) through a scrubbing vessel, containing 2 0 $ trifluoroacetic acid-80$, anisole, into the suspension of resin in anisole-trifluoro-acetic acid. Finally, the anisole and trifluoroacetic acid are removed by flash evaporation. The cleavage mixture is placed in a round-bottomed flask, which is attached to a flash evaporator (Buchler). A vacuum is 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 is stored for purification. The peptides N-HEPTA, SER^, and N-TRI were synthesized according to the above method, using the appropriate t-BOC amino acyl polymer in each case. The remaining antigens and antigenic determinants, N-8-N, N-PENTA, and N-TETRA were provided by D. G. Hull. b. P u r i f i c a t i o n of the antigens, a n t i g e n i c determinants and peptides. N-10-C was p u r i f i e d by column chromatography on Sephadex G-25 FINE (Pharmacia). The Sephadex was poured i n t o a 2 . 5 cm x 100 cm column (Pharmacia) and e q u i l i b r a t e d w i t h 0.1N TEA - 0 .IN HOAC. The antigen was a p p l i e d to the column i n approximately 2 . 0 ml of the s t a r t i n g b u f f e r , and the column subsequently developed. A f l o w r a t e of 6 mis per hour was maintained, and 4 .0 ml 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 of the peptide was determined by a n a l y s i s of the samples on a Beckman DBG spectrophoto-meter at 2 8 0 and 230 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 pooled, concentrated by f l a s h evaporation, and stored at 4 C f o r amino a c i d a n a l y s i s . The N-HEPTA peptide was p u r i f i e d by column chromatography on Dowex Ag 1X8 (200-400 mesh, Bi o r a d Co.). The prepared r e s i n was poured i n t o a 1 . 6 cm x 70 cm column (Vancouver S c i e n t i f i c Glass) and e q u i l i b r a t e d w i t h 5 . 0 $ p y r i d i n e . The peptide was a p p l i e d t o the column i n approximately 4 . 0 ml of 5 . 0 $ p y r i d i n e . The column was developed w i t h the f o l l o w i n g g radients: 5 . 0 $ p y r i d i n e to 0 . 1 N HOAC - 4 . 0 N p y r i d i n e at 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 at pH 5 . 8 , 1.0 N HOAC - 3 . 8 N p y r i d i n e to 1 0 . 0 N HOAC - 2 . 0 N p y r i d i n e at pH 4.05. A f l o w r a t e of 16 mis per hour was maintained, and 4 . 0 ml 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 for determination of peptide content by the quantitative ninhydrin method of Hirs et a l ( 1 9 5 6 ) . The peptide-containing fractions were pooled, concentrated, and stored at 4 C for amino acid analysis. The N-TRI and SER^ were purified by column chromatography on Sephadex G-15 (Pharmacia). The resin 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 in 5 . 0 ml of this prepara-tion, and the column was developed with the same. A flow rate of 6 . 0 mis per hour was maintained, and 8 . 0 ml fractions were collected. Localization of the peptides were determined by spectral analysis at 280 and 230 A, and the peptide-containing fractions 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: N-10-C, E-8-E, E-HEPTA, SER^, 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, in aqueous solution, was placed in a 1 0 0 . 0 ml round-bottomed flask 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 stirred gently for 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 is present in the peptides (Fig. 2, Fig. 3). However, i t is also possible for 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 significant extent in the substituted prep-arations. d. Purification 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 precipitate, leaving any unreacted 2,4-dinitrobenzenesulfonic acid in solution. The precipitate was centrifuged in a Sorvall GLC-1 at 220 x g for 15 min., and the supernatant discarded. The precipitate 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 optical density of the supernatant at 278 A was less than 0.05. The pellet was then solublized with 1.0$ sodium bicarbonate in d i s t i l l e d water. Fig. 2: DNP substitution of the N-TRI peptide 0 2 ? H 3 -2 |QT + N H 0 - C H - C O O - T Y R O S I N E - N H - C H , 2,4 d i n i t r o b e n z e n e s u l f o n i c a c i d N - T R I pH 10 • O a N ? H 3 °2 N"{0^ ~ N H ~ C H ~ c o ° - T Y R O S I N E - N H - C H g N°2 D N P - N - T R I e. Quantitation of a l l proteins, peptides, and amino acids. After purification of a l l antigens and peptides, an aliquot from each sample was taken up in 6.0 N HCl in a small ampoule, evacuated, sealed, and hydrolyzed at 110 C for 18 hours. The hydrolysates were then washed three times by flash evaporation, and f i n a l l y taken up in pH 2.2 starting buffer for analysis on a Beckman 120 amino acid analyzer. The amino acid composition, and the expected molar concentrations of the antigens and peptides purified are shown in Table I. The expected molar ratios have been corrected for 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 II. 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 ratios of the DNP coupled antigens and peptides are shown in Tables III and IV. Again, the expected molar ratios have been corrected for amino acid degradation caused by hydrolysis. As can be seen by comparing Tables I, III, and IV, the substitution of peptides was at least 90$ of theoretical 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 in subsequent experiments. Table I: The amino acid composition and the expected molar ratios of the amino acids in the antigen and peptide determinants. Amino acid molar ratios (M/M) of synthetic peptide N-10-C N-HEPTA W-TRI Amino acid Expected Found Expected Found Expected Found Aspartic 1 0.91 1 0 . 6 8 Serine .1 1 . 0 2 l 1 . 4 5 Glutamic 2 2.19 Proline 1 1 . 4 0 Glycine 10 1 0 . 2 6 Alanine 3 3.30 2 2 . 0 1 1 1 . 0 0 Valine 1 1 . 2 0 Isoleucine .1 0 . 8 6 1 1 . 0 3 Tyrosine 1 0 . 7 6 1 0 . 8 3 1 0 . 9 0 Lysine 1 0 . 9 L 1 1 . 0 5 1 1 . 2 2 Table II: Amino acid degradation values of Beckman standard amino acids Amino acids * 0 hr 12 hr Time 18 hr 24 hr 48 hr Aspartic .0967 .0989 .0945 .0936 .0903 Threonine .0962 .0949 .0912 .0883 .0823 Serine .1031 .0929 .0873 .0850 .0757 Glutamic .1015 .1000 .0973 .0948 .0916 Proline .0970 .1002 .0944 .0937 .0958 Glycine .0966 .0961 .0937 .0922 .0892 Alanine .0968 .0976 .0949 .0926 .0902 Valine .0947 .0962 .0930 .0910 .0795 Methionine .0959 .0903 .0901 .0886 .0830 Isoleucine .0973 .0970 .0960 .0937 .0899 Leucine .0947 .0963 .0930 .0917 .0879 Tyrosine .0964 .0956 .0918 .0895 .0820 Phenylalanine .0982 .0982 .0954 .0952 .0919 * Beckman amino acid samples (0.1 p.m) were taken and hydrolyzed for 0 hr, 12 hr, 18 hr, 24 hr and 48 hr, to determine the effect of the time of hydrolysis on the degradation of individual 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 theoretical starting value of 0.1 |amoles. Table III: The amino acid composition and the expected molar ratios of the amino acids in the DNP antigens and DNP peptide determinants. Amino acid molar ratios (M/M) of synthetic peptide DNP-N--10-C DNP-N-8-N DNP-N-HEPTA Amino acid Expected Found Expected Found Expected Found Aspartic 1 1 . 0 3 2 2.4 1 0 . 6 3 Serine 1 0 . 8 0 2 2 . 3 1 0 . 8 9 Glutamic 2 2 . 1 0 Proline 1 1 . 2 0 Glycine 10 10.50 8 6 . 6 Alanine 2 2.40 3 3 . 1 1 1 . 0 8 Valine 1 1 . 13 Isoleucine 1 0 . 7 5 2 1 . 8 9 1 0 . 9 3 Tyrosine 1 0 . 8 2 2 0 . 5^ 1 0 . 8 9 Lysine 0 0 0 0 . 2 7 0 0 Table IV: The amino acid composition and the expected molar r a t i of the amino acids in the DNP antigen and DNP peptide determinants. Amino acid molar ratio (M/M) of antigen and peptides DNP-LYSOZYME DNP-N-PENTA DNP-N-TRI Amino acid Expected Found Expected Found Expected Found Aspartic 2 1 2 3 . 0 Threonine 7 8 . 0 Serine 10 l l . 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 -1 1 . 2 1 0 0 1 0 . 8 2 1 0 . 8 7 1 0 . 9 3 0 0 . 0 0 0 0 The DNP substituted amino acid was quantitated using the extinction coefficients described by Sanger ( 1 9 4 9 ) . A 20 |_imolar solution of DNP serine in 1% sodium bicarbonate has an absorbance at 350 A of 0 . 3 1 2 . Beer's law is obeyed in solutions of DNP amino acids for concentrations less than 50 Mmoles per ml. II. Immunization of Guinea Pigs. Albino guinea pigs, weighing approximately 300 grams were immunized with 250 |j.grams DNP-N-10-C in phosphate buffered saline pH 7 - 0 (PBS-0.15 M NaCl, 0 . 0 0 2 M NaPO^ buffer, 0 . 0 2 $ sodium azide) emulsed in an equal volume of complete Freund's adjuvant (Difco). The injection series was given in a total volume of 1 . 0 ml as follows: 0 . 4 ml intramuscularly into the shoulder muscles, 0 . 4 ml intramuscularly into the gluteal muscles of the hind legs, and 0 . 2 ml intraperitoneally. The animals were l e f t for approximately three weeks before skin tests and other immunological procedures were carried out. III. Skin Tests. Skin tests were carried out by injecting 0 . 2 ml of test antigens and peptides in PBS intradermally into the shaven and depillated (with Nair) flanks of the test animals. The concentrations of the peptides used were derived according to the maximum degree of solubility of each particular DNP peptide in 1 $ sodium bicarbonate. The results were read after two hours (immediate reactions) and after 2k hours (delayed reactions) from the time of injection. An erythema of greater than 0 . 6 cm was considered a positive reaction. IV. Migration Inhibition. The migration inhibition 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 aseptically removed, and placed in a petri dish ( 1 0 0 mm diameter, Falcon Plastics) containing PBS plus 5 . 0 $ heat-inactivated f e t a l calf serum (Gibco). Clumps of cells 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 cells were then packed by centrifugation at 220 x g for 10 minutes in a Sorvall GLC-1 and washed once with Eagles MEM (North American Biological, see appendix) containing 15$ heat-inactivated f e t a l calf serum plus 100 units of p e n i c i l l i n and 100 (ig of strepto-mycin per ml. This combination w i l l hereafter be designated as Eagles MEM. The cells were then resuspended in Eagles MEM to give a 2 0 $ c e l l suspension. Capillary tubes ( 0 . 8 to 1 . 0 mm in diameter and 75 nun long, Fisher) were f i l l e d with the c e l l suspension and sealed at one end with miniseal (Dade Co.). The tubes were then centrifuged at 220 x g for 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 in each chamber. The chambers were f i l l e d with Eagles MEM containing various concentrations of the test antigens and peptides. The chambers were then sealed, and incubated for 24 hours at 37 C in a CO^ enriched atmosphere. Migration was measured as the rectanglular area encompassing the furthest extent of c e l l travel, using a calibrated stage on a Wild M40 microscope. The results were expressed as percent of migration of spleen cells in comparison to the migration of cells in the control chambers containing no antigen or peptide. V. Lymphocyte Stimulation. Lymphocyte stimulation was carried out according to a modi-fication of the technique described by Dutton and Eady ( 1 9 6 4 ) . Guinea pigs were exsanguinated, and the popliteal, inguinal, and mesenteric lymph nodes were removed and made into a single c e l l suspension as previously described for the spleen. The cells were then packed by centrifugation at 220 x g for 15 minutes, and washed once with RPMI-1640 (Gibco, see appendix) containing 15$ heat-inactivated f e t a l calf serum plus 100 units of p e n i c i l l i n 27 and 100 ug of streptomycin per ml. In future, this combination w i l l be designated as l6k0. The cells were taken up in 1640 and counted. The c e l l concentration was then adjusted to a total of 5 x 10^ cells per ml with l6k0. Only viable cells, as determined by trypan blue exclusion, were counted on an eosinophyl haemo-cytometer (Spiers-Levy). A viable count of greater than 9 5 $ was mandatory for use of the cells in the stimulation test. The test antigens and peptides were set up in quadruplicate in a microtitre plate (Limbro Chemical) with 5 x 10^ viable cells in a 0.1 ml volume per microtitre 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 well in a total volume of 0.05 ml. Finally, each well was topped up with 0.1 ml of l6k0 media. The microtitre plate was then incubated at 37 C in a CO^ enriched atmosphere. After three days, 1.0 \iCi of H-thymidine (specific activity 26 Ci per mmole, (New England Nuclear) was added to each microtitre well. The cells were harvested 18 hours later by a microharvesting method developed by T. Pearson from a model of the Hartsman micro-precipitator. This technique involved suction removal of the cells from each microtitre well onto a 1 .3 cm diameter glass fibre 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$ meth-anol and 41.0 ml of Liquifluor (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 725) Average counts per minute were recorded from 1 minute counts on each sample. The results were expressed as a ratio of stimulation of lymph node cells in 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 inhibition 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 3 6 0 / 6 7 computer. The results were expressed as a T probability with values of less than 0.01 being considered as significant. 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 in 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 injecting the preparations in 0.2 ml PBS intradermally into the depillated flanks of the animals. The develop-ment of an erythema greater than 6 mm in diameter after 2 hours indicated the presence of circulating antibody to the challenging antigen (immediate reaction). The persistence of an erythema after 24 hours indicated that an in vivo cellular 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 significant delayed response. Unfortunately, this cannot be inter-preted as an in vivo difference in cellular recognition since no delayed response was e l i c i t e d by either peptide in the N-10-C immune animals (Table VI). These results are probably a direct reflection of the concentrations of the peptides used, which were considerably Skin reactions observed on guinea pigs immunized with MP-N-10-C. Challenging Test dose Skin reactions a antigen umoles per ml . ... , -, -, ° immediate delayed (2hr) (24hr) DNP-N-10-C 0.125 8/11 10/11 DEP-N-HEPTA 0 . 0 5 0/11 6 /n N-TETRA 0 . 0 5 0/11 3/H DNP-BSA 0 . 1 2 5 0/11. 3/7 Saline control 0 . 2 ml 0/11 0/11 Figures represent the ratio of the number of animals showing positive reactions ( 6 mm) to the number of animals tested. 31 Table VI: Skin reactions observed on guinea pigs immunized with N-10-C. Challenging Antigen Test dose [jmoles per ml Skin reactions a immediate (2hr) delayed ( 2 U h r ) DNP-N-10-C 0.125 DNP-N-HEPTA 0 . 0 5 N-TETRA 0 . 0 5 DNP-BSA 0 . 1 2 5 Saline control 0 . 2 ml 5/10 0 / 1 0 0/10 0 / 8 0/10 7/10 1/10 2/10 3 / 8 0/10 Figures represent the ratio of the number of animals showing positive reactions (6 mm) to the number of animals tested. lower than the concentrations of the original immunogen. The concen-tration range in these tests was widely restricted due to the hydro-phobic nature of the peptides after DNP substitution. It should also be noted at this time that, although the NH 2-terminal heptapeptide (N-HEPTA) was used for DNP substitution, the complete haptenic sequence of the moleculeris contained in the tetrapeptide (N-TETRA - unpublished observations). DNP, when conjugated to BSA, produced a marginal reaction in DNP-N-10-C immune animals. However this effect was also noted in N-10-C immune animals, indicating that the response was. probably a non-specific one. These results imply that the DNP moiety, although an integral part of the DNP-N-10-C immunogen, cannot cause an in vivo cellular response without the aid 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 alter the determinant to such a significant extent that in vivo cellular activation and humoral anti-body binding is prevented in animals which are sensitized to the unconjugated form. As mentioned before, the DNP-N-HEPTA and N-TETRA fa i l e d to give any significant response, and the DNP-BSA conjugate gave only a marginal non-specific one. The specificity of both the immediate and delayed reactions is shown by the failure of a l l the preparations tested to cause an erythema in control guinea pigs (Table VII). 33 Table VII: Skin reactions observed on unimmunized guinea pigs. Challenging Antigen Test dose pinoles per ml Skin reactions a immediate (2hr) delayed (24hr) DNP-N-10-C 0.125 DNP-N-HEPTA 0.05 N-TETRA 0.05 DNP-BSA 0.125 Saline control 0.2 ml 0/4 0/4 0/4 1/4 0/4 0/4 0/4 1/4 0/4 0/4 Figures represent the ratio of the number of animals showing positive reactions (6 mm) to the number of animals tested. As the delayed skin reactions could not be used to establish a differential response between the DEP substituted determinant (N-HEPTA) and the unsubstituted determinant (E-TETRA), i t was necessary to carry out more quantitative tests for delayed hypersensitivity. II. Migration Inhibition Factor (MIF) Production. The spleen cells of animals sensitized to either N-10-C or DEP-E-10-C were tested for their a b i l i t y to produce MIF, in the presence of E-10-C or i t s DEP derivative, an assortment of DEP substituted or un-substituted peptides forming a portion of or a l l o f the EH^-terminal hapten, as well as in the presence of some DEP substituted heterologous compounds. Tests were also carried out using combinations of peptides, in order to determine whether or not blocking of MIF production could be achieved. The test involves antigen specific activation of sensi-tized cells in vitro to produce MIF, a non-specific compound thought to be e l i c i t e d by sensitized.T cells in the presence of specific antigen, which is capable of preventing the migration of cells (macrophages) from capillary tubes (David et al, 1 9 6 4 ). The results of these tests are presented in Tables VIII and IX. a. The Response of DEP-E-10-C Sensitized Cells to the Test Antigens. In the cells taken from animals sensitized to DEP-N-10-C, i t Table VIII: The effect of the homologous antigen, haptenic peptides, and DNP substituted heterologous compounds on the migration of spleen cells derived from guinea pigs sensitized to DNP-N-10-C. Antigen Concentration Migration a toprobability^ in chambers (l-imoles/ml) $ no. of animals DNP-N-10-C 0 . 0 2 5 ^3.3 ± l U . 9 7 . 0 0 0 6 9 . 4 . 0 0 1 N-10-C 0 . 0 2 5 + 2 0 . 1 6 . 0 0 1 DNP-N-HEPTA 0.05 I 6 . 7 + 5-7 7 . 0 0 0 + 1 6 . 1 . 0 0 0 N-TETRA 0.05 5 5 . 6 7 . 0 0 0 DNP-LYSOZYME 0 . 0 2 5 8 6 . 7 + 2 7 . 3 7 . 1 6 7 DNP-SER 4 0.05 8 7 . h + 2 3 . 8 7 . 9 3 4 DNP 0.05 9 0 . 6 + 2 8 . 1 7 .hl3 DNP + N-HEPTA 0.05 each 5 1 . 4 + 1 2 . 9 7 . 0 0 0 DNP-N-TRI + N-TETRA 0.05 each 8 8 . 4 + 2 3 . 1 4 . 0 8 0 DNP-N-TRI 0.05 1 0 2 . 3 + 1 3 . 0 4 . 3 3 0 DNP-N-PENTA 0.05 7 8 . 6 + 1 5 . 5 4 . 0 0 3 N-PENTA 0.05 7 7 . 1 + 1 2 . 6 4 . 0 0 2 Eagles MEM 1 ml 100 7 These figures for 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 probability represents the results of a .Student's t test carried out on the individual migration values for each antigen as compared with the values for the media controls. The dashed t probabilities represents analysis carried out amongst similar antigens. A t probability 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 effect of the homologous antigen, haptenic peptides, and DEP substituted heterologous compounds on the migration of spleen cells derived from guinea pigs sensitized to N-10-C. Antigen Concentration Migration a t probability b in chambers ' (nmoles/ml) $ no. of animals DEP-E-10-C 0 . 0 2 5 6 7 . 9 + 1 0 . 8 7 . 0 0 0 E-10-C 0 . 0 2 5 6 8 . 4 + 1 0 . 4 8 . 7 7 8 . 0 0 0 DEP-E-HEPTA 0 . 0 5 6 9 . 4 + 1 3 . 1 6 . 7 6 4 . 0 0 0 E-TETRA . 0 . 0 5 7 1 . 3 + 14.3 8 . 0 0 0 DEP -LY SOZYME 0 . 0 2 5 8 6 . 1 i 2 4 . 8 8 . 0 1 0 DEP-SEE 4 0 . 0 5 84 .2 + 1 3 . 1 8 . 0 0 1 DEP 0 . 0 5 9 6 . 9 + 1 5 . 6 7 . 3 0 3 DEP + N-HEPTA 0 . 0 5 each 7 0 . 5 + 1 1 . 5 8 . 0 0 0 DEP-E-TRI + N-TETRA 0 . 0 5 each 6 9 . 4 + 1 8 . 3 2 . 0 0 9 DEP-E-TRI 0 . 0 5 1 0 7 . 2 + 3 . 7 9 2 . 3 7 1 DEP-E-PEETA 0 . 0 5 8 5 . 6 + 1 3 . 6 2 . 1 9 3 N-PEETA 0 . 0 5 7 8 . 9 + 2 4 . 7 2 . 1 3 0 Eagles MEM 1 ml 100 8 These figures for percent migration represent the averaged percent migration in chambers containing antigen, peptide, or heterologous protein compared to control chambers incubated without any of these preparations. The t probability represents the results of a student's t test carried out on the individual migration values for each antigen as compared with the values for the media controls. The dashed t probabilities represents analysis carried out amongst similar antigens. A t probability of less than 0 . 0 1 can be considered as s t a t i s t i c a l l y significant. appeared that the homologous antigen was significantly more active immunologically in vitro than was i t s unsubstituted counterpart. This apparent specific effect of DNP-substitution was supported by the observation that DNP-N-HEPTA caused considerably more inhibition than did the N-TETRA peptide, which has been shown previously to contain the immunologically active amino acids of the N-hapten (see Abbrevi-ations for comparison between DNP-N-HEPTA and N-TETRA). It should be mentioned at this time that the concentrations of the peptides have been increased in relation to that of the immunogen, as theoretically, according to the clonal selection theory, a reduced effect would be seen using the peptides, since only one clone of cells is activated in these tests; the clone responding to the NH^-terminal. When the DNP-N-10-C antigen is used, the clone reacting to the COOH-terminal haptenic peptide is also activated, causing a significantly greater inhibition of migration than that caused by single peptides. The overall results suggest that the antigenic specificity of reactive cells in DNP-N-10-C immune animals exists towards both the DNP-moiety and the N-HEPTA haptenic peptide when presented in a conjugated form. The degree to which the N-TETRA peptide and the NHg-terminal of N-10-C activates the DNP-N-10-C reactive cells is probably an indication of the degree of cross-reactivity existing between the substituted and unsubstituted forms in this assay. DNP, by i t s e l f , or conjugated to heterologous carriers (SER^ and Lysozyme), w i l l not cause activation of cells to produce MIF. These results agree with those of workers using other hapten-carrier systems (David et al, 1964) and imply that there is a molecular configurational requirement for cellular activation, the DNP hapten not f u l f i l l i n g the requirement necessary to activate the cells (David et a l 1.96k, and Carpenter and Brandriss, 196k). The data, however, could also be explained by the possibility that in coupling DNP onto the N-HEPTA peptide, an additional haptenic determinant other than the original N-TETRA has been created in the N-HEPTA sequence, hence producing an enhanced in vitro activation. To distinguish these two possib i l i t i e s , a pa r t i a l requirement for DNP in the specificity receptors, or two distinct antigenic deter-minants, DNP-N-TRI and DNP-N-PENTA were synthesized. N-TRI consists of the three NH 2-terminal amino acids of the N-HEPTA. and does not contain any sequences of the N-TETRA haptenic peptide. The N-PENTA, however, is a bridge peptide, consisting of the five 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 activation of the cells, indicating that no extra determinant has been created in 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 in cells from animals sensitized to DNP-N-10-C in comparison to the effect produced by their unsubstituted counterparts. This implies a specific role of DNP in the recognition of the antigens by the sensitized cells However, the tests involving DNP-N-TRI and DNP-N-PENTA and their unsubstituted counterparts indicate that no extra determinant has been created, since no activation was observed by the tripeptide, and only marginal activation 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 results from the presence of the immunologically active amino acids in this sequence rather than to the DNP moiety. b. The Response of N-10-C Sensitized Cells to the Test Antigens. Migration of spleen cells from N-10-C immunized guinea pigs was measured in the presence of these preparations to test the proposed structural model of specificity. These experiments were designed in part to determine i f substitution of the antigen and haptenic peptides with DNP caused any conformational changes in the molecules, prevent-ing recognition of the N-TETRA haptenic sequence as i t existed in the original N-HEPTA peptide. As can be seen from the results in Table IX, there is no significant difference in the inhibition caused by DNP-N-10-C and that caused by N-10-C. This i s further verified by the results of 40 the DNP-N-HEPTA and N-TETRA haptens, indicating that DNP substitution of the immunogen or peptide does not prevent their subsequent in vitro recognition by cells previously sensitized to the unsubstituted form. The DNP-TRI, DNP-N-PENTA, and N-PENTA peptides show no significant inhibition of migration, although i t is possible that i f larger groups of animals had been used, marginal activation might have been demon-strable 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 carriers, did not cause signif-icant activation of cells. This would be expected because the T cells were not immunized to the DNP substituted immunogen and the hetero-logous carriers in no way cross-react to i t . The observation that with N-10-C sensitized cells basically no differences in MIF production were obtained in the presence of DNP sub-stituted or unsubstituted antigens shows that the DNP moiety does not obstruct cellular recognition of the antigen. The fact that significant differences in MIF production were observed under comparable circum-stances in DNP-N-10-C sensitized cells supports the possi b i l i t y of some kind of specific role of the DNP groups in c e l l recognition. Since apparently no extra antigenic determinant was created by DNP substitution (see above), i t is possible that DNP under these circumstances functions by expanding the number of antigen sensitive cells recruited during immunization. c. Receptor Blocking Experiments. The nature of the specific surface receptors on T cells remains a mystery at this time. There is data, somewhat tenuous and not unequivocally accepted, which imply that surface immunoglobulin acts as the recognition site for antigen. (Basten et al, 1971 and Roelants and Askonas, 1 9 7 1 ) . However, there-'.is an equal weight of data unable to support this po s s i b i l i t y (Uhr and Vitetta, 1 9 7 3 ) . That there is specific recognition of antigen is not disputed, but whether or not activation is comparable to B c e l l activation requirements is not clear. Since i t is d i f f i c u l t to visualize antigen recognition by T cells as something other than analogous to immunoglobulin binding with antigen, the following argument was made regarding the function of DNP sub-stitution on the N-hapten, to justify the experimental system to be presented. i . Since the 'specificity' sequence of the W-hapten is contained in the N-TETRA, i t is possible that these amino acids are compulsory for recognition at the T c e l l surface. i i . In the DNP-N-HEPTA peptide, both DNP substitutions are located in the N-TRI portion of the peptide and do not overlap with the N-TETRA. i i i . If the 'specificity' sequence must enter a molecular crypt in the recognition site in order to activate sensitized T cells, i t is possible that such an interaction might be blocked in the presence of amino acid sequences forming the 'outer region' of the recognition site. To investigate this possibility, cells from animals sensitized to DNP-N-10-C and N-10-C were measured for MIF production in the presence of either N-HEPTA or N-TETRA, when these peptides were used alone or mixed with DNP or DNP-N-TRI. The results are tabulated in Tables VIII and IX. The data obtained from DNP-N-10-C sensitized cells showed very clearly that while DNP on i t s own did not inhibit MIF production in the presence of antigenic peptides, DNP-N-TRI was able to completely abrogate the response of these cells to the N-TETPA. That this effect was specific was demonstrated by the equivalent experiment in N-10-C sensitized cells, where MIF production e l i c i t e d by N-TETRA. was the same either in the absence or presence of DNP-N-TRI. This data indicates a number of possi b i l i t i e s : i . The surface recognition sites on T cells may involve a mechanism similar to that seen in immuno-globulin ligand formation, in that entry of the antigenic determinant into a 'reciprocal'fit' crypt may be involved. i i . DNP on i t s own does not appear to be capable of blocking the interaction of N-HEPTA with cells sensitized to DNP-N-10-C or N-10-C. i i i . DNP-N-TEI, which contains both DNP substitutions and the three amino acids d i s t a l to the 'specificity' sequence of the N-HEPTA peptide, is capable of abrogating the response of DNP-N-10-C sensitized cells to the N-TETRA, but not the response of N-10-C sensitized cells to the same peptide. This implies a specific participation on the part of the DNP-N-TRI in the recognition site on T cells. It would thus appear that while DNP alone cannot block this reaction, the amino acids in the immediate v i c i n i t y of the DNP can, suggesting again a specific role of this v i c i n i t y of the DNP-N-HEPTA sequence in c e l l recognition. Since blocking by DNP-N-TRI was not observed in N-10-C sensitized cells, i t would appear that the sensitized cells in this population do not specifically recognize this sequence. d. The Response of Unsensitized Cells to the Test Antigen. The spec i f i c i t y of the various test preparations used in the previous set of experiments is established by their performance in c e l l cultures from unimmunized animals. As can be seen from Table X, none of these preparations cause any significant inhibition of migra-tion of cells from unsensitized control guinea pigs. This also indicates that none of the results found in the immunized animals were a direct result of cytotoxicity caused by any of the preparations used. III. Lymphocyte Stimulation. The lymph node cells of animals sensitized to either N-10-C or DNP-N-10-C were tested for their a b i l i t y to in i t i a t e DNA synthesis in 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 in the presence of some DNP substituted heterologous compounds. The test involves specific antigen activation of sensitized cells, causing them to increase the rate of DNA synthesis prior to entry into the division cycle. This degree of synthesis can be quantitatively measured by comparing the amount of ^ H-thymidine label incorporated into the DNA of cells found in c e l l cultures containing antigen to that incorporated into the DNA of c e l l cultures containing no antigen (Dutton and Eady, 1 9 6 4 ). The results of these tests are presented in Tables XI and XII. Table X: The effect of the homologous antigen, haptenic peptides, and DNP substituted heterologous compounds on the migration of spleen cells derived from unsensitized guinea pigs. Antigen Concentration Migration a trprobability* 3 in chambers (fjmoles/ml) $ no. of animals DNP-N-10-C 0 . 0 2 5 N-10-C 0 . 0 2 5 DNP-N-HEPTA 0 . 0 5 N-TETRA 0 . 0 5 DNP -LYSOZYME 0 . 0 2 5 DNP-SEE 4 0 . 0 5 DNP 0 . 0 5 DNP + N-HEPTA 0 . 0 5 each DNP-N-TRI + N-TETRA 0 . 0 5 each DNP-N-TRI 0 . 0 5 DNP-N-PENTA 0 . 0 5 N-PENTA 0 . 0 5 Eagles MEM 1 ml 9 7 . 2 + 2 6 . 1 6 . 6 9 1 . 2 9 3 9 7 . 0 + 1 8 . 2 7 . 0 6 7 9 2 . 2 9 8 . 7 + + 3 0 . 9 1 8 . 7 5 7 . 4 2 5 . 2 3 9 . 502 1 0 0 . 3 + 2 4 . 4 6 . 4 2 1 1 0 2 . 7 + 2 8 . 5 7 . 1 6 4 99.h + 2 5 . 3 7 . 5 8 0 9 8 . 3 + 2 5 . 4 7 . 7 1 9 8 8 . 6 + 1 1 . 5 2 . 4 2 0 9 3 . 5 + 1 1 . 2 2 . 2 2 1 9 5 . 8 + 9 - 3 2 . 3 1 0 8 8 . 1 + 6 . 2 2 . 0 6 0 100 7 These figures for percent migration represent the averaged percent migration in chambers containing antigen, peptide, or heterologous protein compared to control chambers incubated without any of these preparations. The t probability represents the results of a Student's t test carried out on the individual migration values for each antigen as compared with the values for the media controls. The dashed t probabilities represents analysis carried out amongst similar antigens. A t probability of less than 0 . 0 1 can be considered as s t a t i s t i c a l l y significant. 46 Table XI: %-thymidine incorporation in lymph node c e l l cultures from DNP-E-10-C sensitized guinea pigs in response to the substituted and unsubstituted forms of the homologous antigen, haptenic peptides, and DEP substituted hetero-logous compounds. Antigen Concentration Average a Wo. of t probability (|jmoles/ml) stimulation animals DEP-N-10-C 0.05 2 .27 + 1.35 6 . 6 2 2 .000 N-10-C 0.05 2.35 + 1.42 7 .000 DEP-N-8-N 0 . 0 5 .1.45 + 0.83 7 . 0 2 9 DEP-N-HEPTA 0 . 0 5 1.41 + 1 . 0 7 5 .025 DEP-LYSOZYME 0 . 0 5 1.12 + 0.57 6 . 0 2 7 DEP-SER 4 0 . 0 5 0 . 9 0 + 0.3^ 5 .241 DEP 0 . 0 5 0 . 8 0 + 0.32 3 .058 N-TETRA 0 . 0 5 1.39 + 0.95 7 . 0 2 3 PPD 50 ug/ml 1.95 + 0 . 6 7 5 .000 RPMI l640 0 . 0 5 ml 1.00 7 These figures for the average stimulation are derived from the ratio of 3H-thymidine incorporation in cultures with the test preparations present to the 3jj_thymidine incorporation in control cultures from the same guinea pig without these preparations. The results are based on quadruplicate sets of data from tissues taken from DNP-N-10-C sensitized guinea pigs. The t probability represents the results of a Student's t test carried out on the individual stimulation values for each antigen as compared with the values for the media controls. The dashed t probabilities 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 significant. 1 Table XII: -thymidine incorporation in lymph node c e l l cultures from N-10-C sensitized guinea pigs in response to the substituted and unsubstituted forms of the homologous antigen, haptenic peptides, and DEP substituted hetero-logous compounds. Antigen Concentration Average a No. of t probability (|jmoles/ml) stimulation animals DNP-N-10-C 0.05 1 . 8 8 + O.67 8 -- . 6 9 6 - 0 0 0 N-10-C 0 . 0 5 1 . 9 3 + 1 . 0 0 7 -— . 0 0 1 DNP-N-8-N 0 . 0 5 0 . 9 3 + 0 . 5 5 8 . 4 5 4 DNP-N-HEPTA 0 . 0 5 1 . 0 6 + 0 . 7 7 5 . 5 2 3 DNP-LYSOZYME 0 . 0 5 0 . 8 6 + 0 . 2 6 8 . 0 2 3 DNP-SER 4 0 . 0 5 1.24 + 0 . 7 3 7 . 1 8 3 DNP 0 . 0 5 1 . 2 4 + 0 . 9 1 7 . 0 6 2 N-TETRA 0 . 0 5 1.40 1 . 2 5 8 . 1 3 6 PPD 50 p-g/rnl 2 . 5 2 + 1 . 7 0 8 . 0 0 0 RPMI l 6 4 0 0 . 0 5 ml 100 8 These figures for the average stimulation are derived from the ratio of 3jj-thymidine incorporation in cultures with the test preparations present to the 3jj-thymidine incorporation in control cultures from the same guinea pig without these preparations. The results are based on quadruplicate sets of data from tissues taken from N-10-C sensitized guinea pigs. The t probability represents the results of a Student's t test carried out on the individual stimulation values for each antigen as compared with the values for the media controls. The dashed t probabilities represent analysis carried out amongst similar 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 significant. a. The Response of DNP-N-10-C Sensitized Cells to the Test Antigens. The results, presented in Table XI, show that there is no difference in the degree of stimulation caused by either the sub-stituted or the unsubstituted form of the immunogen. Also, DNP, when coupled to a larger carrier (Lysozyme), f a i l e d to show any stimulation. This indicates again that DNP, in i t s e l f , does not have the configurational requirement to effect c e l l activation. These findings are in agreement with those of other workers in the f i e l d (Schlossman et al, 1969 and Paul et al, 1 9 7 0 ) . However the results do not explain the observation that DNP-N-10-C and N-10-C do not show a differential response in the degree of stimulation in DNP-N-10-C immune lymph node cells. The other haptenic test preparations did not cause any significant degree of stimulation in these experiments. In general this was to be expected, since i t has previously been shown that there is a molecular size requirement needed to activate cells into the division cycle. Most of the peptides tested exist below this level (Waterfield et al, 1 9 7 2 ) . But, i t should have been expected that DNP-N-8-N would have stimulated in this system, as i t obviously has not. Kelly et_ a l ( 1 9 7 2 ) , have shown that N-8-N does stimulate in N-10-C immune animals. This suggests that the prep-aration of DNP-N-8-N used in these experiments could have been in some way inhibitory to c e l l division. It should be noted that the posit ive control, PPD, in these tests stimulated adequately, since the animals were immunized with a prepara-tion containing Freund's complete adjuvant: an/adjuvant consisting, in part of M. tuberculosis, the bacterium from which PPD is derived. The data presented here shows that cells activated to division do not recognize the differences between the substituted and unsubstituted forms of the antigen. It also confirms the observations of other workers who found that DNP coupled onto heterologous carriers does not have the configurational requirements to cause c e l l activation. These findings are not in complete agreement with those obtained using the MIF assay; where the substituted and unsubstituted forms of the antigen showed a quantitative difference in cellular activation. However, these apparent differences can possibly be attributed to different degrees of sensi-t i v i t y of the test methods employed and w i l l be discussed in greater detail later. b. The Response of N-10-C Sensitized Cells to the Test Antigens. Since no distinction was exhibited in 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 in the N-10-C molecule. As can be seen from Table XII, there is no significant difference in the amount of stimulation caused by DNP-N-10-C or N-10-C. This indicates that DNP substitution of the immunogen does not prevent i t s subsequent in vitro recognition by cells previously sensitized to the unsubstituted form. The remaining data represents the further controls on these experiments. As before, DNP when coupled to Lysozyme does not cause any stimulation. The smaller peptides also do not cause any stimu-lation for, as has been previously mentioned, they exist below the molecular size requirement necessary for peptide activation of c e l l division. Stimulation with the positive control antigen, PPD, indicates that the cells used in these experiments were viable and could be stimulated by a non-cross reacting antigen. c. The Response of Unsensitized Cells to the Test Antigens. The specificity of the various test preparations used in the experiments employing DNP-N-10-C and N-10-C immunized animals is well illustrated by similar experiments in unimmunized animals. As can be seen in Table XIII, none of the peptides cause any significant cellular activation in the form of c e l l division in unsensitized control guinea pigs. 51 Table XIII: si -thymidine incorporation in lymph node c e l l cultures from unsensitized (control) guinea pigs in response to the substituted and unsubstituted forms of the main antigen, haptenic peptides, and DNP substituted hetero-logous compounds previously used. Antigen Concentration Average a No. of t probability (umoles/ml) stimulation animals DNP-N'-IO-C 0.05 O .98 + 0 . 4 3 6 — . 3 0 9 . 8 5 7 N-10-C 0 . 0 5 0 . 8 4 + 0.3k 7 . 0 8 2 DNP-N-8-N 0 . 0 5 0 . 8 7 + 0 . 3 8 7 .400 DNP-N-HEPTA 0 . 0 5 0 . 8 7 + 0.41 5 . 6 5 8 DNP-LYSOZYME 0 . 0 5 0 . 9 4 + 0 . 3 7 6 . 7 5 3 DNP-SER 4 0 . 0 5 1 . 2 2 + 0.41 7 . 0 5 6 DNP 0 . 0 5 1 . 0 3 + 0 . 2 8 5 . 4 8 6 N-TETRA 0 . 0 5 O .96 + 0 . 3 9 5 • 771 PPD 50 |ig/ml 1 . 5 6 + 0 . 6 8 6 . 0 0 0 RPMI l 6 4 0 0 . 0 5 ml 100 7 These figures for the average stimulation are derived from the ratio of 3H-thymidine incorporation in cultures with the test preparations present to the 3 H -thymidine incorporation in control cultures from the same guinea pig without these preparations. The results are based on quadruplicate sets of data from tissues taken from unsensitized guinea pigs. The t probability represents the results of a Student's t test carried out on the individual stimulation values for each antigen as compared with the values for the media controls. The dashed t probabilities represent analysis carried out amongst similar 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 significant. IV. Concluding Discussion. The aim of this thesis has been to establish the contribution of the DNP molecule in the specificity of activation of cellular immune responses both in vivo and in vitro in animals immunized with a DNP carrier protein conjugate whose antigenic sequences have been determined. The in vivo results showed that DNP, when coupled to a hetero-logous carrier, did not have the configurational requirement to effect a delayed hypersensitive reaction. The substituted immunogen DNP-N-10-C was required to e l i c i t such a reaction. It was also noted that DNP substitution of N-10-C did not block the in vivo recognition of the haptenic N-TETRA sequence in animals immunized to the un-substituted form. The in vitro results, as assayed by MIF production and lymphocyte stimulation, have shown certain inconsistencies. The specificity requirements for cells activated into MIF production in DNP-N-10-C animals seem to be mainly directed towards a 'shared' determinant, consisting partly of the DNP moiety and partly of the N-HEPTA peptide, since DNP substituted antigens were more effective in e l i c i t i n g MIF production than were their unsubstituted counterparts. However, the specifi c i t y requirements of cells sensitized to N-10-C appear to be directed towards the N-TETRA peptide in the NH^-terminal determinant, and are satisfied by either the substituted or unsubstituted form of the NH2-terminal determinant. Cells activated into a state of division by the substituted and unsubstituted antigen do not appear to be able to distinguish these specificity differences. Both in vitro assays are consistent in the fact that DEP, by itself, or coupled to heterologous carrier's, can not activate cells to produce MIF or to synthesize DEA. The apparent differences in the a b i l i t y of both in vitro delayed hypersensitivity reactions to distinguish DEP substituted and unsub-stituted challenging antigens can possibly be explained by any of a number of observations. 1. There appear to be different molecular size requirements for activation of MIF and lymphocyte stimulation and thereby possibly a dissociation of specificity requirements. Small peptides of four amino acids in length (E-TETRA) can produce a significant inhibition of migration in the MIF assay. However i t requires a much larger peptide of 22 amino acids in the case of E-10-C to ini t i a t e DEA synthesis in activated cells. It would appear then, that MIF production can be activated much more easily and is possibly a more sensitive assay. This basis of sensitivity of various requirements of activation involved in these two assays could explain the dis-crepancy. 2 . Work reported by Paul et a l ( 1 9 6 8 ) showed a heterogeneity of responsiveness on the part of lymphocytes in the in vitro DNA synthetic response that has not yet been shown with MIF production. They inter-preted this as a heterogeneity in the a f f i n i t y of antigen binding receptors possessed by different individual lymphocytes. If such heterogeneity of a f f i n i t y existed, a differential stimulation response between substituted and unsubstituted forms of the immunogen could quite conceivably be overshadowed by the extensive cross-reactions occurring. Thus, even i f a 'shared' determinant encompassed the specifi c i t y of reactive cells in 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 equiv-alent effect. Unfortunately, such a hypothesis would require lymphocyte receptors of more uniform a f f i n i t y for the activation of MIF and no experimental evidence to confirm this has been presented to date. 3 . Finally, i t has been postulated (Rocklin, 1973) that MIF production and lymphocyte stimulation can be dissociated from one another on the basis that there are distinct c e l l populations involved in each response; the c e l l population entering the division cycle being different 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 is coupled onto a haptenic sequence previously shown to be carrier independent. After immunization with this DNP substituted complex, antigen sensitive cells, showing a cellular specificity 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 cellular activation, while the unsubstituted haptenic peptide can by i t s e l f e l i c i t such a response, indicating a considerable degree of cross-reactivity between the substituted and unsubstituted forms. This implies that, although DNP is an integral part of the new determinant, i t plays a minor role in actual activation of cells to e l i c i t a delayed hypersensitive reaction. In terms of other DNP-carrier systems in 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 immuniz-ation, w i l l express a cellular specificity towards a combined or 'shared' determinant. Therefore, any anti-hapten-carrier delayed hypersensitivity response w i l l in fact be a heterogeneous anti-carrier response which w i l l depend on the number of haptenic sequences in the carrier. APPENDIX 1. Composition of Eagles Minimum Essential Medium (MEM) mg/l mg/l NaCl 8 , 0 0 0 . 0 L-methionine 1 5 . 0 KC1 400.0 L-phenylalanine 3 2 . 0 Ng^PO^. 2 H 2 0 6 0 . 0 L-threonine 4 8 . 0 MgSO^.7H20 1 0 0 . 0 L-tryptophan 1 0 . 0 KH 2 P 0 1| 6 0 . 0 L-tyrosine 3 6 . 0 CaCl 2 140.0 Valine 4 6 . 0 Glucose 1, 0 0 0 . 0 Choline Cl. 1 . 0 0 MgCl2.CH2 1 0 0 . 0 Folic acid 1 . 0 0 NaHCO^ 5 5 . 0 i - i n o s i t o l 2 . 0 0 L-arginine 1 0 5 . 0 Nicotinamide 1 . 0 0 L-cystine 24 .0 D-Ca.pantothenate 1. 00 L-Glutamine 2 9 2 . 0 Pyridoxol HCl 1 . 0 0 L-histidine 3 1 . 0 Riboflavin 0 . 1 0 L-isoleucine 5 2 . 5 Thiamine-HCl 1 . 0 0 L-leucine 5 2 . 4 phenol red 1 0 . 0 0 L-lysine 5 8 . 0 NaHCO^ 2 , 2 0 0 . 0 57 2. Composition of RPMI 1640 mg/l mg/l Ca(W03)2.4H20 f100.0 L-phenylalanine 15.0 Glucose 2,000.0 L-proline 20.0 MgSO^.7H20 100.0 L-serine 30.0 KC1 400.0 L-threonine 20.0 Na2HP0^.7H20 1, 512 .0 L-tryptophane 5.0 NaCl 6,000.0 L-tyrosine 20.0 L-arginine 200.0 L-valine 20.0 L-asparagine 500.0 Biotin 0.2 L-aspartic 20.0 Vitamin B-^ 2 0 . 0 0 5 L-cystine 50.0 D-Ca-pantothenat e 0 .25 L-glutamic acid 20.0 Choline Cl 3.0 L-glutamine 300.0 Folic acid 1.0 Glutathione 1.0 i- i n o s i t o l 35.0 Glycine • 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 1 5 . 0 " NaHC03 2,000.0 LITERATURE CITED Basten, A., Miller, J.F.A.P., Warner, N. L., and Pye, J. 1 9 7 1 . Specific inactivation of thymus-derived (T)- and non-thymus-derived (B)-lymphocytes by 1 2 5 i labelled antigen. Nature New Biology 231 : 104. Benacerraf, B. and Levine, B.B. 1 9 6 2 . 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Fishman, M., and Adler, F.L. 19^7- The role of macrophage-RNA in the immune response. Cold Spr. Harb. Symp. quant. Biol. 3 2 : 3 4 3 . 1 5 . Cell, P.G.H. and Silverstein, A.M. 1 9 6 2 a . Delayed hypersensi-t i v i t y to hapten-protein conjugates. I. The effect of carrier protein and site of attachment to hapten. J. Exp. Med. 115: 1037-6o 1 6 . Henney, C.S. 1970. A cytolytic system for the in vitro detection of cell-mediated immunity to soluble antigen. J. Immunology 105: 9 1 9 . 17. Hirs, C.M.W., Moorse, S., and Stein, W.H. 1 9 5 6 . Peptides obtained by tryptic hydrolysis of performic acid oxidized ribonuclease. J. Biol. Chem. 219_: 6 2 3 . 1 8 . Humphrey, J.M. and Keller, M.V. 1 9 7 0 . Developmental aspects of Antibody formation and structure, ( j . Sterzl and H. Riha eds.) Proc. Symp. Prague Czech. Acad. Sci. Prague and Academic Press, N.Y. 1 9 . Jerne, N.K. 1 9 7 L The somatic generation of immune recognition. Eur. J. Immunology 1: 1. 2 0 . Katz, D.H., Davie, J.M., Paul, W.E. and Benacerraf, B. 1971 . Carrier function in anti-hapten antibody responses. IV. Experimental conditions for the induction of hapten specific tolerance or for the stimulation of anti-hapten anamnestic responses by non-immunogenic hapten-polypeptide conjugates. J. Exp. Med. 134: 2 0 1 . 2 1 . Kelly, B. and Levy, J.G. 1971 . Immunological studies on the major haptenic peptides from performic acid oxidized ferre-doxin from Clostridium pasteurianum. Biochemistry 10: 17&3. 2 2 . Kelly, B., Levy, J.G. and Hull, D. 1 9 7 3 . Cellular and humoral immune responses in guinea pigs and rabbits to chemically defined synthetic peptides. Submitted to Eur. J. Immunology. 2 3 . Landsteiner, K. 1 9 6 2 . The specificity of serological reactions. Revised edition. Dover Publications, N.Y. 6 i 2 4 . Leskowitz, S. 1 9 6 3 . Immunochemical study of antigenic speci f i c i t y in delayed hypersensitivity. II. Delayed hypersensitivity to polytyrosine-azobenzenearsonate and i t s suppression by haptens. J. Exp. Med. 117: 9 0 9 . 25. Levy, J.G., Hull, D., Kelly, B., Kilburn, D.G., and Teather, R.M. 1 9 7 2 . The cellular immune response to synthetic peptides containing sequences known to be haptenic in performic acid--oxidized ferredoxin from Clostridium pasteurianum. Cell. Immunology. 5_: 8 7 . 2 6 . Merrifield, R.B. 1 9 6 4 . Solid-phase peptide synthesis. III. An improved synthesis of bradykinin. Biochemistry 3 : 1 3 8 5 . 2 7 . Miller, J.F.A.P. and Osoba, D. 1 9 6 7 . Current concepts of the immunological function of the thymus. Physiol. Rev. 47: 437 . 2 8 . Miller, J.F.A.P. and Mitchell, J.F. 1 9 5 8 . Cell to c e l l interaction in the immune response. I. Hemolysin-forming cells in neo-natally thymectomized mice reconstituted with thymus or thoracic duct lymphocytes. J. Exp. Med. 128: 8 0 1 . 2 9 . Mitchell, B., Levy, J.G., and Nitz, R.M. 1 9 7 0 . Synthesis of haptenic- C-terminal octapeptides of two cross-reacting bacterial ferredoxin molecules. Biochemistry 9: 1839-3 0 . Mitchison, N.A. 1 9 6 9 . The immunogenic capacity of antigen taken up by peritoneal exudate cells. Immunology 16: 1. 3 1 . Mosier, D.E. 1967. A requirement for two c e l l types for antibody formation in vitro. Science 158: 1575 . 62 3 2 . Naor, D. and Sulitzeanu, D. I 9 6 7 . Binding of radioiodinated bovine serum albumin to mouse spleen cells. Nature 214: 6 8 7 . 33- Nitz, R.M., Mitchell, B., Gerwing, J., and Christensen, J. 1 9 6 9 . Studies on the antigenicity of bacterial ferredoxins. I. The effect of modification of cysteinyl residues on the antigenicity of Clostridium pasteurianum ferredoxin. J. Immunology 103: 319-3 4 . Nossal, G.J. V., Szenberg, A., Ada, G. L., and Austin, CM. 1964. Single c e l l studies on l 8 s antibody production. J. Exp. Med. 119: 4 8 5 . 3 5 . Oppenheim, J.J., Wolstencroft, R.A., and Gell, P.G.H. 1967. Delayed hypersensitivity in the guinea pig to a protein-hapten conjugate and it s relationship to in vitro transform-ation of lymph node, spleen, thymus and peripheral blood lymphocytes. Immunology 12: 8 9 . 3 6 . Paul, W.E., Katz, D.G., Goidl, E.A., and Benacerraf, B. 1 9 7 0 a . Carrier function in anti-hapten immune responses. II. Specific properties of carrier cells capable of enhancing anti-hapten antibody responses. J. Exp. Med. 132: 2 8 3 . 37- Paul, W.E., Siskind, G..W., and Benacerraf, B. 1 9 6 8 . Specificity of cellular immune responses. Antigen concentration dependence of stimulation of DNA. synthesis in vitro by specifically sensitized cells, as an expression of the binding characteristics of cellular antibody. J. Exp. Med. 127: 25. 63 3 8 . Paul, W.E., Stupp, Y., Siskind, G.W. , and Benacerraf, B. 1 9 7 0 b . Immunogenicity of £ DNP-oligo-L-lysyl peptides. Developmental Aspects of Antibody Formation and Structure, ( j . Sterzl and H. Riha eds.). Proc. Symp. Prague Czech. Acad. Sci., Prague and Academic Press, N.Y. 3 9 ' Pauling, L. 1 9 4 0 . A theory of the structure and process of formation of antibodies. J. Amer. Chem. Soc. 6 2 : 2 6 4 3 . 4 0 . Rocklin, R.E. 1 9 7 3 . Production of migration inhibitory factor by non-dividing lymphocytes. J. Immunology 110: 6 7 4 . 4 . 1 . Roelants, S.E. and Askonas, B.A. 1 9 7 L C e l l co-operation in antibody induction. The susceptibility of helper cells to specific lethal radioactive antigen. Eur. J. Immunol. 1: 151 . 4 2 . Sanger, J. 1 9 4 9 . The terminal peptides of insulin. Biochem. J. 45: 5 6 3 . 4 3 . Schlossman, S.F., Herman, J., and Yaron, A. 1 9 6 9 . Antigen recognition: in vitro studies on the specificity of the cellular immune response. J. Exp. Med. 130: 1 0 3 1 . 4 4 . Silverstein, A.M., and Gell, P.G.H. 1962b. Delayed hypersensitivity to hapten-protein conjugates. II. Anti-hapten specificity and the heterogeneity of the delayed response. J. Exp. Med. 115: 1 0 5 3 . 4 5 . 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