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Studies on antigen binding cells involved in cellular immunity to ferredoxin peptides Pearson, Terry W. 1974

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STUDIES ON ANTIGEN BINDING CELLS INVOLVED IN CELLULAR IMMUNITY TO FERREDOXIN PEPTIDES by TERRY WILLIAM PEARSON B.Sc. (Honours Microbiology) The University of British Columbia, 1967 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN THE DEPARTMENT of MICROBIOLOGY WE ACCEPT THIS THESIS AS CONFORMING TO THE REQUIRED STANDARD THE UNIVERSITY OF BRITISH COLUMBIA JANUARY, 1974 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 L i a L i u L f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . 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 c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p urposes may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s un d e r s t o o d t h a t c o p y i n g o r 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 s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f Microbiology The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada i i ABSTRACT Previous studies with conjugates containing the Nh -^terminal and COOH-terminal antigenic determinants of oxidized ferredoxin from C_. pasteurianum indicated a need for at least two determinants to stimulate DNA synthesis in sensitized lymphocytes. This suggested a mechanism involving cell cooperation, a possibility which has been investigated here by selectively inactivating cells binding one or the other of the deter-minants. Cells from immunized guinea pigs were tested in vitro for their capacity to bind antigen or to be stimulated by it before and after "anti-gen suicide" with radioiodinated conjugates containing the Nl^-terminal or COOH-terminal determinants of oxidized ferredoxin. A microculture 3 system for assessing antigen induced stimulation of H-thymidine uptake by lymphocytes was developed for this work. The data show that: 1) Lymphocytes from unimmunized guinea pigs bind both l^-terminal -4 and COOH-terminal determinants at a frequency of about 10 . In immune animals the proportion of antigen binding cells increased about 4-6 fold. The frequency of cells binding the determinants depends markedly on the specific activity of antigens employed. 2) Both T and B lymphocytes bind the antigenic determinants from oxidized ferredoxin. 3) Specific inactivation of cells binding either determinant was achieved by antigen suicide with I-Nh^-terminal or I COOH-terminal s-BSA conjugates. Synergy occurs between the [^-terminal binding cells and COOH-terminal binding cells in the proliferative response of sensi-tized lymph node cells challenged with oxidized ferredoxin in  vitro. Evidence from B cell depletion studies indicates that this is a T cell-T cell interaction. i v TABLE OF CONTENTS Page INTRODUCTION . . . 1 I The Clonal Selection Theory 1 II Antigen Binding Cells . . . . 2 III Functional Significance of Antigen Binding Cells . . . . 4 IV Antigen Receptors on Lymphocytes . . . 6 V Cell Cooperation in Immune Responses 10 VI Cell Mediated Immunity . . . . . . 14 VII Cell Cooperation in Cell Mediated Responses 18 VIII Antigen Suicide 20 IX Thesis Objectives 22 MATERIALS AND METHODS 23 I Peptide Synthesis 23 II Performic Acid Oxidation of Ferredoxin 23 III Immunization Procedures 30 IV Skin Tests 30 V Tissue Culture Medium. . . . . 30 VI Preparation of Cells . . . . 31 VII Migration Inhibition . . . . . . . . . 31 VIII Lymphocyte Culture 32 IX Harvesting of Radioactively Labelled Cultures 32 X Preparation of Succinylated BSA (s-BSA) 35 XI Coupling of Peptides to s-BSA 39 V Table of Contents (Continued) Page XII Amino Acid Analysis 39 XIII Iodination of Antigens 40 XIV Gel Filtration 40 XV Determination of Specific Activity . . . . . . 41 XVI Immunological Activity Testing . . . . . . . . 41 XVII Antigen Suicide. . 42 XVIII Antigen Binding Studies 44 XIX Antigens for Binding Experiments and Autoradiography . . 44 XX Preparation of Gelatin-Coated Slides 45 XXI Autoradiography 45 XXII Processing of Slides 46 XXIII Examination of Slides 46 XXIV Macrophage Autoradiography 46 XXV Cell Separation - Nylon Columns. . . . . . . . . . . . . . 47 XXVI Cell Separation - Anti-IgG Plates. . . . . . . . . . . . 48 XXVII Anti-IgG Autoradiography 49 RESULTS AND DISCUSSION 51 I Skin Tests . . . . . . . . . . . . . . . 51 II Lymphocyte Stimulation . . . 57 (a) Stimulation of Guinea Pig Lymph Node Cells by PHA and ConA. 58 vi Table of Contents (Continued) Page (b) Stimulation of Sensitized Guinea Pig Lymph Node Cells by N ] 0C 63 (c) Stimulation of Sensitized Guinea Pig Lymph Node Cells by OFD . . . . 63 (d) Stimulation of Sensitized Guinea Pig Lymph Node Cells by PPD . . . 63 (e) Stimulation of Sensitized Guinea Pig Lymph Node Cells by KLH . . . 64 (f) Stimulation of Unsensitized Guinea Pig Lymph Node Cells by Test Antigens 64 (g) Reproducibility. . . . . 70 III Peptide Conjugates. . . . . . . . . . . 74 IV Estimation of Coupling. 74 V Immunological Activity Tests . . . . 74 VI Iodination. . . . . . . . . . . 79 VII Antigen Binding Experiments . . . . . . . 81 (a) Uptake of Antigen by Macrophages 81 (b) Uptake of Antigen by Dead Cells 82 (c) Effect of Antigen Concentration 82 (d) Effect of Immunization . . . . . 86 (e) The Effect of B Cell Depletion on Antigen Binding Cells. . 86 VIII Immunological Significance of Antigen Binding Cells . . 91 v i i Table of Contents (Continued) Page (a) Detection of Cell Death by Autoradiography. . . . 91 (b) Lymphocyte Stimulation - Low Dose and High Dose Antigen Suicide . . . . . . . 92 (c) Lymphocyte Stimulation Using B Cell Depleted Populations - High Dose Antigen Suicide . . . . . 95 IX Concluding Discussion. . . . . . . . . . . . 103 X Literature Cited 112 vii i LIST OF TABLES Page I The amino acid composition and expected molar ratios of the amino acids in the synthetic peptides used. . . . 29 II Skin reactions observed on guinea pigs immunized with N1QC 53 III Skin reactions observed on guinea pigs immunized with C ,C 54 mai IV Skin reactions observed on guinea pigs immunized with OFD and KLH. 55 V Skin reactions observed on unimmunized guinea pigs . . . 56 3 VI H-thymidine uptake in guinea pig lymph node cell replicate microcultures dispensed with a constant delivery syringe and processed with the microharvester apparatus 72 3 VII H-thymidine uptake in guinea pig lymph node cells in response to varying concentrations of ConA . . . . . . . 62 3 VIII H-thymidine incorporation in H]QC or OFD/KLH immunized guinea pig lymph node cells in response to varying antigen concentrations . . . . . . . . . . . . . . . . . 65 3 IX H-thymidine incorporation in unimmunized (control) guinea pig lymph node cells in response to antigen preparations . . . . . . . . . . . 69 3 X Antigen induced H-thymidine incorporation in guinea pig lymph node cell cultures from sensitized guinea pigs 71 XI Reproducibility of themicroculture-harvester system: ^H-thymidine incorporation in microcultures stimulated with OFD, KLH and PPD 73 XII Ratios of amino acids in s-BSA and its ^-terminal and COOH-terminal peptide conjugates 76 ix List of Tables (Continued) Page XIII Biological activity of the peptide conjugates and their iodinated derivatives - skin reactions observed on guinea pigs immunized with N^C 77 XIV Inhibition of migration by NH2-terminal and C00H-terminal peptides, s-BSA-peptide conjugates and their iodinated derivatives. 78 XV Numbers of antigen binding macrophages in peritoneal exudates from unsensitized and N-|gC sensitized guinea pigs 84 XVI Antigen binding cells in lymph nodes from NirjC and OFD immunized and unimmunized guinea pigs - detection using low specific activity ^25T_-|aije-|-je(j molecules. . . 88 XVII The effect of cell fractionation on numbers of immuno-globulin positive guinea pig lymph node cells as detected by 125i_anti-IgG autoradiography. . . . . . . . 89 XVIII The effect of B cell depletion on numbers of antigen binding cells in OFD sensitized guinea pig lymph node cells - detection using high specific activity 125i-labelled molecules . . . . . . . . . . . . . 90 XIX Antigen binding lymph node cells from OFD immunized guinea pigs - detection using high specific activity '25i_iabelled molecules before and after allowing antigen suicide 92 3 XX Antigen stimulation of H-thymidine incorporation in cultures of NirjC sensitized guinea pig lymph node cells treated with low doses of high specific activity 125i_Nl0c„ . . . . . . . . . . . . . . . . . 96 3 XXI Antigen stimulation of H-thymidine incorporation in cultures of N-JQC sensitized lymph node cells treated with low doses of high specific activity '"i-CsBSA. . . 97 X List of Tables (Continued) Page 3 XXII Antigen stimulation of H-thymidine incorporation in cultures of Cma"|C sensitized guinea pig lymph node cells treated with low doses of high specific activity 125i_CsBSA , . . . 98 3 XXIII OFD stimulation of H-thymidine incorporation in cultures of OFD sensitized guinea pig lymph node cells treated with high doses of high specific activ-ity 125J_SBSA a n d peptide conjugates. . . . . . . . . . 99 3 XXIV KLH stimulation of H-thymidine incorporation in cultures of KLH sensitized guinea pig lymph node cells treated with high doses of high specific activity 125i_ sBSA and peptide conjugates . . . . . . . . . . . . . . 100 3 XXV OFD stimulation of H-thymidine incorporation in B cell depleted cultures of OFD sensitized guinea pig lymph node cells treated with high doses of high specific activity 125I_SBSA and peptide conjugates. . . 101 3 XXVI KLH stimulation of H-thymidine incorporation in B cell depleted cultures of KLH sensitized guinea pig lymph node cells treated with high doses of high specific activity 125I_SBS/\ and peptide conjugates. . . 102 xi LIST OF FIGURES Page 1 . Flow chart of solid phase peptide synthesis. . . . . . . . 2 4 2 . Amino acid sequence and haptenic determinants of the synthetic immunogen N-jgC . . . . 2 6 3 . Space-filling model of the synthetic immunogen N-jnC. . . . 2 7 4 . Microharvester apparatus -• photograph 3 6 5 . The aspirator and filter units - detail (diagram). . . . . 3 7 6 . The aspirator-washer unit - detail (photograph). . . . . . 3 8 7 . The filter unit - detail showing removal of filter discs (photograph) . . . . . . . . . . . . . . . . . . . . . . . 3 8 8 . Antigen suicide procedure - flow chart . . . . . . . . . . 4 3 9 . PHA stimulation Vs cell number - day two . . . . . . . . . 5 9 1 0 . PHA stimulation Vs cell number - day three . . . . . . . . 6 0 1 1 . PHA stimulation Vs cell number - day four. . . . . . . . . 61 1 2 . N-JQC stimulation Vs cell number. 6 6 1 3 . N^QC stimulation Vs incubation time. . . . . . . . . . . . 6 7 1 4 . PPD stimulation Vs cell number . . . . . . . . . . . . . . 6 8 1 5 . Elution profile of N-JQC iodination mixture . . . . . . . . 8 0 1 6 . The effect of antigen concentration on the antigen binding capacity of N i n C sensitized guinea pig lymphocytes . . . . 8 5 ABBREVIATIONS IgG Immunoglobulin of heavy chain antigenic type y (gamma) IgM Immunoglobulin of heavy chain antigenic type u (mu) DNP-POL Dinitrophenylated polymerized flagellin ABA-Tyr Azobenzenearsonate-L-Tyrosi ne DNA Deoxyribonucleic acid PDG Poly-D-glutamic acid MIF Migration inhibition factor Budr Bromodeoxyuridine GVH Graft versus host MLC Mixed leukocyte culture CML Cell mediated lympholysis N,QC The NH2~terminal haptenic peptide and the COOH-terminal hap-tenic peptide of C_. pasteurianum ferredoxin bridged by 10 glycine residues OFD Oxidized ferredoxin C ,C The symmetrical synthetic molecule formed by bridging two COOH-terminal peptides with a glycine-malonic acid bridge KLH Keyhole limpet hemocyanin R.P.M.I. Medium number 1640 from the Roswell Park Memorial Institute 1640 PBS Phosphate buffered saline* TCA Trichloracetic acid s-BSA Succinylated bovine serum albumin * Saline and phosphate buffered saline of isotonic strength, PBS: NaCl 8 gm/1 KH2P04 0.2 gm/1 KC1 0.2 gm/1 Na2HP04.7H2o 2.17 gm/1 xi i i Abbreviations (Continued) N-sBSA The ^-terminal haptenic peptide covalently linked to suc-cinylated BSA in a ratio of approximately 20:1 (peptide: sBSA) C-sBSA The COOH-terminal haptenic peptide covalently linked to suc-cinylated BSA in a ratio of approximately 22:1 (peptide: sBSA) EDCI l-ethyl-3-(3-dimethylaminopropyl) carbodiimide HC1 FCS Fetal calf serum PPD Purified protein derivative (from Mycobacterium tuberculosis) PHA Phytohemagglutinin (obtained from Phaseolus vulgaris) ConA Concanavalin A xiv ACKNOWLEDGEMENTS I would like to thank Dr. CT. Beer, Dr. J.J.R. Campbell, Dr. J. W. Thomas and Dr. G. Weeks for their time and suggestions as my com-mittee members, Dr. R.E. Falk for his willingness to serve as my external examiner and to Dr. Julia Levy for her time and help as a mem-ber of my committee and for her constant help in many aspects of the work leading to this thesis. Thanks also to Dr. Jim Hudson, Barb Kelly, Dr. Bob Miller, Dr. Chuck Slonecker, Dr. J. Tonzetich, Dr. Nadine Wilson and Dr. Jack Yensen for their consultary contributions in dif-ferent aspects of this work, to Maureen Fairhurst and Jim Richter for technical help, to Dale Krieger for help in preparation of the figures, to Jeanette Davidson and Carol Chapman for their patience and skill in preparation of the final draft, to my fellow graduate students, Dale Gregerson, Fumio Takei, Doug Waterfield and Rob Watson for their help-ful suggestions, criticisms and comradeship during the past few years, and to Dolores Schendel for introducing me to the microculture systems which made this work possible. This thesis would not have been completed without the direction, concern, and friendship of Dr. Doug Kilburn and the support and encouragement of Tom Ellison, lady Anne Malcolm and my family. Thank you very much for keeping the country in the city. 1 INTRODUCTION I Clonal Selection Theory According to Burnet's clonal selection theory (14), immune responses involve the triggering of precommitted clones of antigen sensitive cells and their subsequent development to give a specific humoral or cellular immunity. Central to thistheory are the following points: (1) Individual lymphocytes are precommitted to synthesis of anti-body of a single specificity and are restricted to production of this single molecular species. (2) There are sites present on the cell surface which recognize a given antigen and initiate production of a specific antibody. (3) The antigen recognition site is linked to the antibody it controls, that is, individual antigen reactive cells con-stitutively produce antibody which reacts with antigen and triggers the cell to proliferate and produce large quantities of antibody. (4) The antibody response is due in part to proliferation of the cells producing antibody. That is, the cell specialization is heritable (clonal). Hypotheses regarding generation of diversity of precommitted lym-phocytes be they somatic generation (12,13,14,15,19,36,61,116) or germ-line theories (32,33,54) are compatible with clonal selection. The generation of immune diversity will not be discussed here as the clonal selection theory and the ideas central to this thesis involve the immune 2 system after it has gained diversified competence. II Antigen Binding Cells Naor and Suliztneau (79) originally tested Burnet's hypothesis that lymphoid cells react specifically with antigen. Using autoradiographic techniques they found a small number of mouse spleen cells bound radio-iodinated bovine serum albumin (BSA) in vitro. Byrt and Ada observed that a similar number of cells bound radioiodinated flagellin and hemocyanin (17). Both groups tested antigen binding under conditions that inhibited phagocytosis. As predicted by the clonal selection theory, only a small proportion of lymphoid cells bound any given antigen, Ada (1) has re-viewed the data on radiolabeled antigen binding in lymphoid populations. The major observations are: (1) The binding reaction is usually complete within 60 minutes at 4 C although a time dependence is evident (1). (2) A majority of cells labelled are small lymphocytes although medium lymphocytes, macrophages and plasma cells are occasion-ally labelled (79). (3) Ultrastructural analysis showed that antigen (in this case ferritin) was present on the cell membrane and was bound in a "patchy" fashion on the lymphocyte surface (68). (4) The number of lymphocytes binding any one antigen is a small proportion of the total cell number, usually in the order of 10~^  - 10"^  in unprimed populations (1). (5) In immune animals, the proportion of antigen binding cells increased, usually about ten-fold (80,55,2). 3 Can one equate the binding cells in a virgin population to the pre-cursors of clones as postulated by Burnet? On the basis of numbers alone, no conclusion can be reached. There appear to be too many binding cells -4 4 for a given antigen. At a frequency of 10 , binding cells for only 10 different antigens are allowed. It is argued however that this figure (10 ) includes cross-reacting cells. Also, a hierarchy of binding cells for a given antigen exists. That is, some cells bind antigen at low concentrations and bind progressively more antigen as the concentration increases. Other cells bind antigen only at relatively high concentrations ( 1 ). This observation can be explained by random generation of receptor diversity. As the concentration of antigen increases, there will be more cells that bind antigen due to less stringent specificity requirements. The main question regarding the significance of antigen binding cells concerns their identity with immuno-competent cells. 4 III Functional Significance of Antigen Binding Cells An increasing body of evidence points to the conclusion that at least a portion of the cells binding a given antigen are immunocompetent: (1) Wigzell and Andersson used antigen coated glass bead columns to selectively remove immunocompetent cells from a lymphoid population while reactivity against an unrelated antigen remained intact (137). Recovery of the depleted cells was unsuccessful. (2) Edelman et a l (37, 103, 104), Truffa-Bachi and Wofsy (125) and Wofsy et al (138) achieved specific cell elimination using immunoadsorbant techniques, and recovered the antigen binding cells. (3) Deletion of specific immunocompetent cells was reported by Dutton and Mishell (35) using a "hot-pulse" technique. In vitro incubation of mouse spleen cells stimulated by sheep erythrocytes and given a pulse of high specific activity tritiated thymidine, removed anti sheep erythrocyte antibody forming cells while leaving the response to burro erythrocytes intact. (4) Lymphocytes stimulated with the protein antigen streptokinase-streptodornase varidase (SK-SD) were inactivated using Budr (5-Bromo-2-deoxyuridine) and light (139). Zoschke and Bach found that thymidine uptake was deleted when SK-SD was the stimulating antigen, while t'je response to unrelated antigens was left intact. (5) Diener (31), using cells separated by velocity sedimentation showed that a large proportion of antigen binding cells were induced by antigen to differentiate into larger cells. These 5 larger lymphocytes followed the distribution profile of immuno-competence in a cell population. The significance of the antigen binding cells not stimulated by antigen is uncertain. The relationship between immune competence and antigen binding cells is further complicated by a functional division in the immune system. Introduction of antigen into an animal may stimulate humoral immunity, eel 1-mediated immunity, or both, depending on the nature of the antigen and method of administration. The division in the immune system is attributable to two functionally distinct classes of lymphocytes: B Lymphocytes (Bursa or bone marrow derived) are associated with humoral immunity and are the progenitors of antibody forming cells (18,24). T Lymphocytes (Thymus processed or thymus derived) mediate cellular immunity and also function as "helper" cells in the production of antibody (18 ). Katz and Benacerraff (57) have reviewed the specificity of immune responses attributed to T and B lymphocytes. 6 IV Antigen Receptors on Lymphocytes The recognition of antigen molecules by lymphoid cells is the first step required for initiating the complex events involved in the immune response. The chemical nature of cell surface antigen receptors has recently been under intense investigation in several laboratories. Orig-inally, Sell and Gell (111) observed blast cell formation in cultures treated with antiimmunoglobulin antibody, suggesting that immunoglobulin is present on the cell surface and that interaction with this "receptor" in some way stimulates lymphocytes to proliferate. The presence of immunoglobulin on the surface of B lymphocytes has been demonstrated quite conclusively: (1) The proportion of immunoglobulin positive lymphocytes in various tissues was examined using radioiodinated or fluorescent anti-immunoglobulin antibodies (89,92,128). Spleen, lymph nodes, peripheral blood, and thymus lymphocytes contained 45-60%, 10-30%, 10-25% and less than 1% labelled lymphocytes respectively, figures that correspond to the proportion of B cells in these tissues (93). (2) Stronger evidence that immunoglobulin carrying cells are B cells was found by comparing cells from thymectomized, irradiated, bone marrow reconstituted mice and control untreated mice (130). No immunoglobulin negative lymphocytes were found in the "B re-constituted" animals. (3) Using class specific anti-immunoglobulin antibody, the surface immunoglobulin was shown to be mainly IgM and IgG in the mouse (127) IgM in the rabbit and man, and IgG in the guinea pig (87). 7 The nature of the T cell receptor is sti l l controversial: (1) Raff, using fluorescein coupled anti IgG, could not demon-strate immunoglobulin on the surface of murine lymphocytes possessing the alloantigen Theta (0) (93). (2) Perkins et abusing ultastructural autoradiography (go) and Karnovsky using freeze-etching techniques (56) failed to detect surface immunoglobulin on T lymphocytes, (3) Using lengthy autoradiographic exposure times, Bankhurst et al labelled thymus and thymus derived lymphocytes with radioio-dinated anti-light chain antibody (8). (4) Marchalonis et_ aj_, isolated 7S IgM from the surface of human and murine thymus lymphocytes having iodinated surface proteins by the lactoperoxidase iodination technique (69). Uhr et al , however, were unable to reproduce these results using similar methodology 027). The difficulty in detecting immunoglobulin on T cells has been ex-plained in several ways: (1) Extended exposure times needed for detection of immunoglobulin on T cells in autoradiographic procedures suggests that T lymphocytes possess a much lower density of immunoglobulin than B cells ( 8 ). (2) Lack of T cell labelling with anti-immunoglobulin antibody reflects relative accessibility between T and B lymphocyte surface immunoglobulin (69). (3) The T cell receptor may not be a conventional immunoglobulin 8 Mitchison has proposed the term IgX (75) and Nossal IgT (83). (4) Feldmann (40) has recently shown the existence of an Ig-antigen complex which is shed from T cells. Cone et al_. (21) showed identity of the Ig in this complex with 7S IgM, using lacto-peroxidase iodination techniques. Receptor shedding may cause difficulty in surface detection. Although a paucity of evidence exists showing T cell surface immuno-globulin directly, the experiments of Marchalonis (69), Feldmann (40) and Cone et al_. (21), indicate that immunoglobulin is present on T cell surfaces. Since the most convincing evidence was gained using the extremely sensitive surface iodination technique, perhaps other labelling techniques, which depend on using anti-immunoglobulin antibody, are inadequate. Demonstration of surface Ig doesn't necessarily assign it a role in the immune response. However, a number of studies indicate that immuno-globulin is, in fact, the antigen receptor on both T and B lymphocytes: (1) Radiolabeled antigen binding reactions are inhibited by anti-immunoglobulin antibodies (2, 134). (2) The in vitro humoral response to several antigens is inhibited by anti-light chain sera (63). (3) In vivo immune response to several antigens is inhibited in mice reconstituted with anti-u treated spleen cells (134). (4) Rosette formation in T cell enriched populations is inhibited by anti u and anti light chain sera ( 63). 9 (5) Graft versus host reactions are inhibited by anti-light chain antisera (70). (6) Specific removal of T lymphocytes using "antigen-suicide" techniques is inhibited by anti-immunoglobulin sera (9, 100). Functional inhibition studies implicating immunoglobulin as T cell antigen receptor are reviewed by Simonsen (23). The precise events that lead to lymphocyte triggering after antigen binding to cell receptors are largely unknown. Lipid-protein surface membrane components undergo conformational alteration after antigen-receptor interaction and it is hypothesized that these allosteric changes are involved in lymphocyte activation (27). There appears to be a re-lationship between the localization of antigen and the activation of lymphocytes, by the mobilization of surface receptors to form patches or caps (56,95,30). 10 V Cell Cooperation in Immune Responses The elucidation of the mechanism of lymphocyte stimulation by antigens is complicated by the apparent interaction of T and B lympho-cytes. Studies on this cooperative process have been concerned almost entirely with production of humoral antibody, with the following obser-vations emerging: (1) Both thymus cells and bone marrow-derived cells are required to restore immune capacity to an irradiated animal (18,24). (2) In hapten-carrier experiments, both thymus-derived cells primed to an immunogenic carrier and bone marrow cells primed to hapten are required for secondary anti hapten response to the hapten-carrier conjugate (76,77). (3) Antigen specificity exists in both thymus-derived and bone-marrow-derived populations (9,76,77). (4) In some instances, intact living thymus cells and bone marrow cells are needed for antibody production (24), while in others some antigens are thymus independent and stimulate B cells directly (72). (5) Both thymus-derived and bone marrow-derived cells have a radiosensitive component in their restorative capacity in cell cooperation (24). (6) Both hapten and carrier moieties must be present on the same molecule in order to obtain an anti hapten response upon antigen challenge (76,77). 11 Several theoretical models have been proposed to account for these data: Two Cell Hypotheses (1) The "Local Concentration" Hypothesis. Mitchison (75) suggested that initially the T cells react with antigenic determinants present on the carrier molecule, localizing haptenic determinants for present-ation to the hapten-reactive B cell via an "antigen bridge" between the cells. The T cell fulfills a passive role acting as a helper cell presenting the antigen to the B cell in the required concen-tration or spatial arrangement. (2) The "Cross-Linking" Hypothesis. Bretcher and Cohn (11) postulated that union between B cells and antigen is necessary for activation but that cross-linking of antigen is required. An antibody to a different determinant on the antigen must react with the antigen adsorbed to the B cell. The antigen specific receptors, either on the T cells or after secretion may cross-link the antigen molecules on the B cells. (3) The "Factor" Hypothesis. Dutton et al^  (34) suggested that both T and B cells bind antigen. After antigen binding, T cells become activated and secrete a non-specific stimulus to the B cells, which become competent to respond to antigen. Schimpel and Wecker, using allogeneic T cell stimulation, found non-specific soluble T cell factors which enhanced the B cell response to any antigen capable of stimulating them (106). Feldmann^lT^u^stantiated this report using syngeneic mouse cells in vitro. Now-specific T cell 12 factor was produced providing a strong T cell response was generated, Recent work by Feldmannand his colleagues favors a 3 cell collaborative system: (1) Using metabolic inhibitors, it was found that virgin T cells must divide before becoming functional helper cells (40). (2) Cell co-operation occurred with T cells separated from B cells by a cell impermeable nucleopore membrane (39). (3) Adherent cells were required for antibody response to hapten-protein antigens that require T and B lymphocyte co-operation. Response to a T cell-independent antigen (DNP-POL) was not diminished by removal of adherent cells (41). (4) A specific "collaborative factor" was produced by activated T cells, and became macrophage bound. These macrophages retained sufficient collaborative factor to trigger B cells into antibody production (40). (5) Treatment of collaborating macrophages with anti kappa chain or anti u chain antisera abrogated the antibody response (40). The last observation led to the view that T cells had produced an IgM-like receptor molecule, which when complexed to antigen became liberated from the T cell surface and attached to macrophages. The monomeric surface IgM found on human and mouse thymocytes by Marchalonis:et al_ (69) was compared to isolated collaborative factor (21). Complete identity was found. The tendency of T and B cell surface immunoglobulin to bind to macrophages was then investigated by Cone et al_., using surface radio-13 iodination techniques (21). T cell derived IgM product bound to mac-rophages while B cell surface immunoglobulin did not. Three Cell Hypotheses (1) T Cell-Macrophage-B Cell Feldmann (40) formulated the following model: Activated thymus cells release IgM-like molecules complexed with the inducing antigen. This shed complex binds to the macrophage surface and a concentrated depot of antigenic determinants is formed. The macrophages present the deter-minants to B cells which are then triggered. (2) Macrophage-T Cell-B Cell An alternative three cell model was proposed by Katz and Unanue (58). Antigen is initially bound by macrophages which present the anti-gen to T cells. The T cells are then activated and in turn trigger B cells. The three cell models are particularly attractive in that they suggest mechanisms governing control of the antibody response, that is, regulation by macrophage mediation. Regulation of humoral antibody production using a three cell model has been reviewed by Feldmann and Nossal (41). 14 VI Cell Mediated Immunity. As has been discussed in the previous section, the activation of B lymphocytes proceeds by a complex series of events probably involving, for most antigens, the collaboration of T and B c e l l s , and macrophages. Like B lymphocytes, T cells bind antigen (101), demonstrate functional specificity ( 9 ) , and are effector c e l l s . Is i t l i k e l y then that T cell activation is analagous to B cell activation? Studies involving in vitro T cell function (M.I.F. production, H-TdR uptake, and cytotoxicity) have shown that: (1) Upon activation by antigen, T lymphocytes differentiate to become blast cells (126). (2) In the presence of the sensitizing antigen, T cells secrete a variety of non-specific factors (lymphokines) such as migration inhibition factors, chemotactic factors, cytotoxic factors, and mitogenic factors, which appear to play a role in c e l l -mediated immunity ( ). (3) In the presence of the sensitizing antigen, T cells differentiate into cytotoxic effector cells (50), Hapten-carrier experiments on delayed hypersensitivity reactions suggest that specificity in MIF production(25), lymphocyte proliferation (84), and antigen induced cytolysis (50), is carrier dependent. These data, obtained with structurally undefined DNP-protein conjugates have led to the almost dogmatic view that cells reacting in cell-mediated responses bear a receptor with a complex specificity pattern, containing elements of both hapten and carrier (115), that there is a requirement for 15 a larger functional antigenic determinant than that required for inter-action with B cells or circulating antibody (42) and that the recognition is not as specific as in humoral immunity. Many results obtained using defined low molecular weight antigens are in direct contradiction to the studies involving complex undefined hapten-carrier systems. The mono-valent molecule azobenzenearsonate-L-tyrosine (ABA-Tyr) demonstrates positive skin tests (62,81,82), and stimulates lymphocytes into cell division in vitro (4,5,16). The carboxy-terminal dodecapeptide of Bovine glucagon also stimulates in vitro cell division in sensitized animals. Several workers suggest that T cell recognition, although extremely sensitive, may be less specific than that demonstrated in antibody pro-duction: and Marshall-Clarke (1) Hoffman and Kappler (53) and Playfair (91) demonstrated cross-reactivity of helper T cells to erythrocyte antigens which exhibited no serological cross-reactivity. (2) Thompson £ l .al. (123) described cross reactions between lysozyme and carboxymethylated lysozyme in cell mediated immunity. Previously, Gerwing and Thompson showed no serological cross-reactivity between lysozyme and its carboxymethylated derivative (43). (3) Cross-reactivity in cell mediated immunity was shown by Parish (86) using flagellin and acetoacetylated flagellin in rats and and Ada in mice by Cooper (22) using flagella from two strains of Salmonella that show no serological cross-reactivity. 16 Several possibilities exist to explain these observations: (1) Differences between T and B cells are a reflection of sensi-tivity although the receptors may be the same. A lower affin-ity requirement for the stimulation of T cells is suggested by Parish (86). (2) Apparent differences in sensitivity of T and B cells may re-flect the difference in sensitivity of detection of function although they possess identical requirements for stimulation. (3) There are determinants that are specific for T cells and stimulate T cells but not B cells. In the case of cross-reactions, it would be assumed that common T cell determinants were present. This last possibility, that there is a functional dissociation of determinants for T and B cells is supported by the finding that certain antigens induce cell-mediated reactions rather than humoral responses (66,126). Further evidence has been found using synthetic and natural antigens: (1) Senyk et aj_., using the natural polypeptide glucagon, found antibody specificity primarily for the ami no-terminal hepta-1 decapeptide, while only intact glucagon and its carboxy-terminal dodecapeptide stimulated cell division in sensitized cells in culture (112,113). (2) Using the synthetic conjugates ABA-poly-D-glutamic acid and ABA-DNP, Alkan et aj . (4) found that cellular immunity was directed against the ABA group (carrier) while antibody was produced to the DNP and PDG (hapten). 17 It is interesting, however, that when ABA is joined to a protein moiety i t s role is reversed and i t acts as a hapten (108), The C-terminal undecapeptide of glucagon acts in an analogous manner (113). Work with haptenic peptides from tobacco mosaic virus protein (118) and from oxidized ferredoxin (135) has shown that: There is no direct correlation between delayed skin reactions and lymphocyte stimulation. Haptenic peptides, when not bound to a carrier, e l i c i t skin reactions and MIF in immune animals but do not stimulate thymidine uptake by lymphocytes. Hence, carrier specificity cannot be a requirement for the production of MIF. This is contrary to David and Schlossman (27) who showed that only immunogenic DNP-oligolysines eli c i t e d MIF production. From these observations, i t appears that for T c e l l s , activation may occur without subsequent proliferation: (1) Bloom (10) found that production of antigen induced MIF is dissociated from DNA synthesis and is associated with a non-dividing c e l l . (2) Rocklin (96) using Budr-light inactivation of dividing c e l l s , found production of MIF was unaffected by removal of lympho-cyte proliferation in response to antigen. These data suggest that either one population of T cells responds differentially to different antigenic stimulus or that two functionally separate populations exist. 1 8 VII Cell Cooperation in Cell Mediated Responses Lonai and Feldman (65,66) reported synergism between T and B cells in in vitro xenograft reaction, implicating lymphocyte interactions in cell mediated immunity. However, using column purified T cells, .Goldstein et_ al_. demonstrated complete autonomy of T-cells for sensitization (45) and cytotoxic effector function (46). The possibility that T-T interac-tion occurs is supported by several lines of investigation: (1) Raff and Cantor (94) have demonstrated the presence of T cell subpopulations in the mouse. (2) T-T synergism in GVH reactions has been claimed by ? Asofsky (6) and Tigelaar and Asofsky (124). (3) The results of Schendel et^  al_. (105) that mixed leukocyte cul-ture (MLC) activation is required for the cell mediated lympho-cysis (CML) reaction and that the cells responding in MLC are separable from those mediating CML by adsorption on specific monolayers (7) imply that cell-cell cooperation is involved in the CML reaction. There is no direct evidence for either T-B or T-T interaction in cell-mediated immunity to soluble protein antigens. A two cell collabor-ation is suggested, however, by the work of Waterfield et^  al_. (135) and Levy et al. (64) using chemically defined peptides: (1) Only divalent or polyvalent antigens (more than one antigenic determinant per molecule) are immunogenic. (2) Only immunogenic molecules stimulate thymidine incorporation in lymphocytes from sensitized animals. Haptenic peptides do not. In some situations however, molecules containing a single antigenic determinant to which the cells are sensitized are sufficient for stimulation of thymidine uptake. 19 These results suggest some sort of bridging mechanism for T cell activation and the work in this thesis was designed to further test this hypothesis. Mitchell and The antigens synthesized byALevy et aJL contain the amino-terminal heptapeptide and the carboxy-terminal pentapeptide which have been identified as the antigenic determinants of the ferredoxin molecule (73). These peptides can be synthetically linked to form immunogenic analogs of the natural protein. For the major part of this investiga-tion, the peptide N^QC (a synthetic analogue of oxidized ferredoxin from £. pasteurianum) has been used since the two chemically distinct antigenic determinants would be postulated to react with at least two different populations of lymphocytes. Cell cooperative models proposed for the production of humoral antibodies postulate the existence of dif-ferent lymphocyte populations which interact with separate antigenic determinants on the eliciting antigen. If an analogous situation exists in cell-mediated immunity, then the divalent ferredoxin peptides can be used to look at cell interaction in delayed hypersensitivity reactions using the following approach: (1) Removal or inactivation of cells reacting with either anti-genic determinant should abrogate responses requiring cell cooperation. (2) Mixing a cell population functionally depleted in cells reacting to only one antigenic determinant with one depleted in cells reacting to the other determinant should restore cell cooperative responses. 20 Specific inactivation of cells binding a single antigenic deter-minant was attempted using radioactive antigen suicide in this thesis. VIII "Antigen Suicide" Radiolabelled antigen binding cells were first examined for immuno-competence by Ada and Byrt using an "antigen-suicide" technique ( 3). Spleen cells from unimmunized mice were incubated with polymerised flag-125 ellin labelled to high specific activity with Iodine. After removal of unbound antigen, treated cells were stored overnight to allow radia-tion death of cells binding antigen. Cell populations pretreated with radiolabel1ed flagellin failed to react to a subsequent challenge with the antigen. Response to unrelated antigens remained intact, demonstra-ting specific clonal deletion. It has long been known that lymphocytes display marked sensitivity to ionizing radiations. With this in mind, Ada and Byrt explained their 125 results in the following manner: Antigen binding cells specific for I labelled flagellin adsorbed the antigen onto their surface. During iso-tope decay, electrons of relatively weak energy are emitted and are absorbed a few microns from their source. Cells binding significant amounts of antigen receive relatively high doses of ionizing radiation and are inactivated. Other cells are left unharmed. Evidence supporting specific clonal elimination of antigen binding lymphocytes by antigen suicide is derived from several sources: (1) Specific removal of antibody response to hemocyanin in both immune and non-immune animals was achieved by Unanue (132) 21 125 using I hemocyanin suicide in vitro. 125 (2) Humphrey and Keller (55) used the synthetic antigen I-TIGAL in vivo and selectively inhibited the response to that antigen. Antigen suicide techniques are adaptable to studying hapten-carrier function and c e l l cooperation in the immune response: (1) Using an adoptive transfer system, Basten et a_l_ showed that specificity of the antibody response to fowl gamma globulin and horse erythrocytes was dictated by both T and B lymphocytes ( 9). (2) Roelantsand Askonas were able to test helper cell function in an adoptive transfer system using DNP-ovalbumin and DNP-hemocyanin. They specifically removed helper function for 125 anti-DNP response using high specific a c t i v i t y , I-hemocyanin (100). (3) Specific inactivation of DNP hapten-binding cells using i r r a d i -ation suicide was reported recently by Golan and Borel (44). On a theoretical basis, specific antigen suicide has the potential to functionally impair cells binding a single antigenic determinant and thus may be one of the most powerful techniques for investigating aspects of molecular recognition and cell-cooperation in a functionally hetero-genous lymphocyte population. 22 Thesis Objectives. To develop a microculture system for measuring mitogen and antigen induced stimulation of uptake of radioactive isotopes by lymphocytes. To examine antigen binding cells for ferredoxin peptides using auto-radiography and cell separation techniques. To develop an "antigen suicide" technique to specifically inactivate cells reacting with a single antigenic determinant. To test for cell-cell interaction in the eel 1-mediated immune res-ponse to ferredoxin peptides using functional inactivation of cell populations by "antigen suicide" techniques. 23 MATERIALS AND METHODS I Peptide Synthesis In the solid phase method of peptide synthesis, the peptide being synthesized is covalently attached to an insoluble copolystyrene-divinyl benzene resin and this is insolubilized or in "solid phase". Reagents used in the synthesis, along with unattached amino acids remain soluble and can easily be washed from the resin. The necessity for purification procedures at intermediate steps in the synthesis is thus eliminated. The reaction sequence is as follows: (Figure 1). (1) Chloromethyl groups on the resin react with the salt of a t-butyloxycarbonyl (t-Boc) amino acid to form an ester linkage with the carboxyl of the first amino acid. (2) The t-Boc group protecting the amino group of the attached amino acid is removed with anhydrous trifluoracetic acid to allow it to react with the carboxyl group of the incoming protected amino acid. (3) The coupling reaction is carried out by dicyclohexylcarbodii-mide and involves formation of a peptide bond between the amino group of the coupled amino acid and the carboxyl group of the incoming amino acid, its amino group being protected by t-Boc. The deprotection, addition of new protected amino acid, and coupling steps are repeated until the peptide sequence is complete. (4) The completed peptide is cleaved from the resin by a nucleo-philic displacement reaction using HBr gas. The terminal t-Boc group is removed at the same time by an elimination reaction. 24 Figure 1. Flow Diagram of Solid Phase Peptide Synthesis ( l ) (2) ( 3 ) (4) CH O H R.O 1 « J II i 11 II CH - C -O-C-N-C-C-O-CH d 1 i 2 CH 3 H o OLYMER Anhydrous Trifluoracetic Acid DEPROTECT 1 NEUTRALIZE M H 2 « I^O CH 3-C + C 0 2 + H-N-C-C-O-CH / o > CHg 2 > ' POLYMER isobutylene t-BOC amino acid CH, amino acyl polymer diimide COUPLE • - n H R 2 ° H R l ° C HA - ? - 0 - C - N - C - C - N - C - C - 0 - C H <(o^>POLYMER H X  3 , CH, i H t-BOC peptide polymer HBr CLEAVE H R o H R o 1 i II i i 1 II CO + H - N - C - C - N - C - C - O H a i , H H B r ~ C H 0 / o V OLYMER peptide 25 The following peptides, synthesized by the solid phase method of Merrifield (71) with the modifications suggested by Stewart and Young (119), were used: (1) NF -^terminal haptenic heptapeptide from the oxidized ferredoxin molecule of Clostridium pasteurianum (Figure 2). Sequence: NH2-Ala-Tyr-Lys-Ile-Ala-Asp-Ser-COOH Designated: N (2) COOH-terminal haptenic pentapeptide from the same molecule (Figure 2). Sequence: NH2-Ala-Pro-Val-Gln-Glu-C00H Designated: C (3) The two haptenic peptides bridged by ten amino acids (Figure 2). Sequence: NH2-Ala-Tyr-Lys-Ile-Ala-Asp-Ser-(Gly)10-Ala-Pro-Val-Gl .rr-Glu-COOH Designated: N-JQC (4) A symmetrical molecule consisting of two carboxyterminal pentapep-tides bridged by one malonic acid and ten glycine residues. Sequence: COOH-Glu-Gln-Val-Pro-Ala-Mal-(Gly)10-Ala-Pro-Val-Glh-Glu-COOH Designated: C-jC (A space-filling model of Nir)C is shown in Figure 3.) Figure 2. Amino acid sequence and haptenic determinants of the synthetic immunogen (N-10-C). N H ^ - A l a - T y r - L y s - I l e - A l a - A s p - S e r - ( G l Y I Q ) - A l a - P r o - V a l - G i n - Glu-COOH ro 28 An automated synthesizer built by Mr. Doug Hull in Dr. Levy's laboratory was used in all syntheses. T-Boc amino acids were purchased from Sigma Chemical Company, or from Schwarz-Mann, and the chloromethyl-ated resin from Bio-Rad. Peptides were cleaved from the resin using HBr. Tyrosine containing peptides were cleaved with HBr scrubbed with anisole. Peptides were purified by gel filtration on Sephadex G-15 and G-25 columns. After acid hydrolysis, representative aliquots were analyzed for amino acid content on a Beckman Model 120 amino acid ana-lyzer. Expected and observed ratios of amino acids of the synthesized peptides are presented in Table I. Synthesis and purification from Levy et a l (64). II Performic Acid Oxidation of Ferredoxin. Ferredoxin from Clostridium pasteurianum was purchased from Sigma Chemical Co. (F3250 Type 1), and its performic acid oxidized derivative (OFD) was prepared by the method of Tanaka et a l (121) in the following manner: 30 mg Ferredoxin was dissolved in 4.0 ml distilled H,>0 and 2.0 ml 15% TCA +0.1 ml 2-mercaptoethanol added. The mixture was allowed to stand for 1 hour at 4 C. The precipitate was collected by centrifu-gation and washed consecutively with 5.0 ml dH20, 5 ml 95% ethanol and 5.0 ml ether. The final precipitate was dried under vacuum, dis-solved in 2.0 ml concentrated formic acid and 4.0 ml performic acid (prepared from 9.0 ml formic acid + 1.0 ml 30% H202) was added. The oxidation was allowed to proceed for 4 hours at -10 C. The oxidized ferredoxin was lyophilized, dissolved in 10.0 ml distilled H20 and quantitated by amino acid analysis. Table I. The amino acid composition and expected molar ratios of the amino acids in the synthetic peptides used in this study. Peptide sequences are given on page 25. Amino acid molar ratios of synthetic peptides (M/M) Amino acid Expected Found Expected C 2 Found N Expected C 3 10L Found CmalC Expected 4 Found Aspartic 1.0 1.16 1.0 0.91 Serine 1.0 0.94 1.0 1.02 Glutamic 2.0 2.02 2.0 2.19 4.0 4.30 Proline 1.0 1.03 1.0 1.40 2.0 2.20 Glycine 10.0 10.26 10.0 9.50 Alanine 2.0 2.05 1.0 1.00 3.0 3.30 2.0 1.90 Valine 1.0 , 0.94 1.0 1.20 2.0 2.07 Isoleucine 1.0 0.98 1.0 0.36 Tyrosine 1.0 0.85 1.0 0.76 Lysine 1.0 1.50 1.0 0.94 Synthesized by T. Pearson Synthesized by D. Waterfield 4 Synthesized by D. Hull Synthesized by D. Hull 30 III Immunization Procedures. Random bred male guinea pigs weighing 500-800 g were immunized with N-JQC or OFD in sterile saline and 50% by volume of complete Freund's adjuvant (Difco) in a total volume of 1.0 ml. Each guinea pig received 250 yg total, in the following manner: 0.2 ml intramus-cularly into the shoulder muscles and 0.2ml intraperitoneally. Guinea pigs were immunized with C^-jC in an identical manner. In several experiments, animals were immunized with 250 yg keyhole limpet hemo-cyanin (KLH) mixed with the OFD dose. All animals were skin tested 11-14 days after initial sensitization. IV Skin Tests. Hair was removed from the flanks of the test animals by shaving followed by treatment with a depillating agent. Test antigens (50 -100 yg in 0.1 ml sterile saline) were injected intradermally along with a saline control. Results were read at 3 hours (immediate reac-tion) and 24 hours (delayed reaction) from the time of challenge. Erythema of greater than 0.6 cm was considered positive, as saline controls were always less than 0.6 cm in diameter. V Tissue Culture Medium. Guinea pig cells were cultured in R.P.M.I. 1640 (Gibco) contain-ing 15% heat inactivated (56°C, 30 min) fetal calf serum, 100 units penicillin and 100 yg streptomycin per ml. Medium components were made up separately and combined after sterilization by millipore 31 filtration. The pH was then adjusted to 7.1 - 7.2 with IN HC1. This medium is termed complete medium. Incomplete medium contained no fetal calf serum. VI Preparation of Cells. Guinea pigs were sacrificed by a sharp blow on the head and were exsanguinated by heart bleeding. The animals were shaved and soaked with 70% ethanol. Mesenteric, inguinal, popliteal and axil-lary lymph nodes were removed aseptically, trimmed of fat, and teased gently apart in phosphate buffered saline, containing 5% FCS, using curved forceps. Cell clumps were broken up by expulsion through a tuberculin syringe with a number 26 needle. The cells were washed once by centrifugation, resuspended in 1.0 ml PBS and centri-fuged through a discontinuous fetal calf serum gradient (100%, 50%, 25% FCS in PBS) to remove debris. Spleen cell suspensions were pre-pared in an identical manner. Viable cell numbers were determined by trypan blue exclusion. All procedures were carried out at room temperature in a laminar flow air hood. VII Migration Inhibition. Migration inhibition was performed as a modification of the method described by David et al_ (26). Capillary tubes were filled with a 20% spleen cell suspension and plugged with modelling clay, They were centrifuged at 170 x g_ for 5 minutes, cut just below the cell-fluid interface and placed on the bottom of Mackaness type 32 chambers. Measurement of migration in the presence and absence of antigen was performed by determining the rectangular area encompas-sing the furthest extent of confluent macrophage travel, using a calibrated microscope stage. All tests were performed in duplicate and two capillaries used in each test. VIII Lymphocyte Culture. A miniaturized system of lymphocyte culture and stimulation was developed. The method is derived from the mixed leukocyte culture (MLC) test of Hartzman et aj_. (48). Lymph node cells in a total vol-ume of 0.25ml were placed in the wells of flat bottomed microtitre plates (LINBRO ISFB 96-TC) covered with sterile plastic lids (LINBRO #55), and incubated in a C02 enriched humidified atmosphere. Cell concentration, antigen and mitogen concentration, and length of incu-bation were varied in order to optimize culture conditions. 1 micro-3 curie H-thymidine (specific activity 2 Ci per mmole-NEN) was added to each microtitre culture well 16 - 18 hours prior to harvesting. All materials were dispensed using automatic repeating syringe dispen-sers (Hamilton Co. #PB-600-l). Triplicate cultures were run in most experiments, quadruplicate or sextaplate in others. IX Harvesting of Radioactively Labelled Cultures. Lymphocyte stimulation was studied using the incorporation of tritiated thymidine into DNA. Due to the technical burden of har-vesting multiple samples and the length of time required to precipitate 33 each microtitre culture separately, a machine which performs simultan-eous precipitation of several microtitre cultures was built. The apparatus was re-designed from the prototype observed by the author in Dr. Fritz Bach's laboratory in Madison, Wisconsin. A similar model is discussed by Hartzman et al. (49). Harvesting of lymphocyte microcultures was achieved quickly and efficiently using a machine built in this department. After appropri-ate incubation of lymphocyte microcultures, tritiated thymidine is added to the culture and 16 hours later the well contents are aspirated onto glass fibre filters. The filters are then washed, placed in glass vials and dried in an oven at 100 C for 30 minutes. Scintillation fluid was added (60% toluene, 40% methanol, and 41 ml NEN Liquifluor in 1.0 liter) and the samples counted for one minute on a Nuclear Chicago scintillation counter (Model 725). Using the microprecipitator appara-tus, one row of microtitre plate wells (12 cultures) can be harvested in about one minute. The microprecipitator consists of four main parts: (1) a reservoir-valve control unit, (2) an aspirator-washer unit, (3) a filter unit, (4) a vacuum unit (Figure 4 ). The reservoir-valve control unit consists of a glass bottle, reser-voir (containing saline or distilled water) connected to the washer unit by tygon tubing. The flow of wash fluid from the reservoir is con-trolled by a microswitch activated solenoid valve. Flow is by gravity feed. It is possible to add TCA and methanol washes by the addition of 34 two extra microswitch-solenoid combinations. The aspirator-washer unit (Figure 5,6) functions to remove the cul-tures from the microtitre wells, to wash the wells free of cells and to aspirate the cultures and washings onto glass fibre filters where they are washed free of unincorporated isotope. The tubing from the reser-voir allows the appropriate wash fluid to flow into the hollow acrylic handle. The fluid flows down the outside of 12 pieces of stainless steel tubing which project into the microtitre wells. Microtitre plates are fixed in place by a spring-loaded acrylic wedge attached to the base plate. The stainless steel tubes are connected to 12 lengths of tubing which carry the well contents and wash fluid to the wells in the filter unit. The filter unit (Figure 5, 7) holds the glass fibre filter strips (Reeve Angel) to allow application of suction and removal of filtrate. An acrylic baseplate contains 12 porous polypropylene discs upon which the filter strips are placed. The 12 polyethylene tubes from the aspir-ator-washer unit f it into 12 holes in the top piece of the filter unit. A filter strip is placed on the base plate and the top piece is clamped firmly down using a manual quick-release clamp. An "o" ring at the bot-tom of each top-piece cylinder prevents vacuum leakage, leakage from one aspirating channel to the next and also "punches out" glass fibre discs from the filter strip. The vacuum unit consists of an electric vacuum pump connected via two liquid traps to an exhaust port on the base of the filter unit. All 35 filtrates are collected in the traps and are disposed of as radioactive waste. A normal operating sequence is as follows: The top unit is clamped in place over a filter strip and the vacuum turned on. The contents of the microtitre wells are aspirated onto the filters and the wells washed with water by activating the solenoid, allowing water to flow into the wells. The wells are scraped free of adherent cells with the plastic tube tips. After 15 - 20 seconds of constant washing, the clamp is re-leased, the filter peeled back and the filter discs remaining are removed after turning off the vacuum. Originally, cells were washed with saline, TCA and then methanol. Washing with water gives identical results. The aspirator and filter units can be easily dissassembled and back-ground radioactivity removed by overnight soaking in "Count off" fluid (NEN). Background counts are usually in the order of 10 - 20 cpm/filter disc. Providing there is adequate washing, no contamination is found from one filter strip to the next. X Preparation of Succinylated BSA (s-BSA). Bovine serum albumin (200 mg, twice crystallized, Miles Research Laboratories, Kankakee, 111.) in 3.0 ml distilled water, was placed in pencil dialysis tubing (Visking, Union Carbide) and dialyzed exhaustively against 0.1 N NaCl to remove contaminating ions. The pH was adjusted to 7.5 with N NaOH and 500 mg succinic anhydride added gradually with stir-ring over a one hour period. pH was maintained at 7.5 with 5.0 N NaOH. Stirring was continued for one more hour and reactants then removed by exhaustive dialysis at 4 C against distilled water. Figure 4. Microharvester Apparatus. 1. Reservoir-Valve Control Unit a) Reservoir b) Solenoid Valve Microswitch c) Solenoid Valve 3. Filter Unit f) Top Plate g) Filter Holder h) Microtitre Plate i) Clamp 2. Aspirator-Washer Unit d) Aspirating Needles e) Tygon Tubing Vacuum Unit j) Vacuum Traps k) Vacuum Pump Figure 5. The aspirator and filter units. Figure 6 . The Aspirator-Washer Unit - Detail a) Tygon tubing from Reservoir b) Acrylic handle c) Stainless steel aspirator needles - Tygon tipped d) Tygon tubing - to f i l t e r unit Figure 7 . The F i l t e r Unit - Detail showing Removal of F i l t e r Discs e) Base plate f) Top plate showing rubber "0" rings g) F i l t e r holder h) Clamp i) Microtitre plate 39 XI Coupling of Peptides to s-BSA. The Nh -^terminal heptapeptide and the COOH-terminal pentapep-tide of the ferredoxin molecule were conjugated to s-BSA via a water soluble carbodiimide reaction. This procedure was based on the method for modification of carboxyl groups in proteins of Hoare and Koshland (51). 1 Ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl (EDCI) was obtained from OTT Chemical Company. In a total volume of 1.0 ml of water, 10 mg of s-BSA and 10 mg of the peptide were mixed. The pH was adjusted to 4.75 with 0..IN HCl and 0.25 ml of DH20 contain-ing 100 mg EDCI was added. The reaction was allowed to proceed for 2 hours with constant stirring and the pH maintained at 4.75 with IN HCl during this time. A further 100 mg EDCI was added to complete the reaction. Reactants were removed by dialysis at 4°C against 0.001NHC1 (4 x 1000 ml over 48 hr). The conjugates were solubilized by dropwise addition of 5N NaOH to pH 12.0. After 3 - 12 hr, the pH was adjusted to 7.0 with slow addition of IN HCl. Succinylated BSA was used to ensure that coupling occurred exclusively through the free amino groups of the peptides. XII Amino Acid Analysis. Protein concentrations were determined by the method of Lowry et al_ (67), and approximately 0.02 ymoles of each conjugate acid hydrolyzed for analysis (6N HCl, 18 hr, 105°C in vacuo). The hydro-lysates were dried in a flash evaporator, washed free of acid with distilled water and quantitated by amino acid analysis on a Beckman 40 Model 120 amino acid analyzer by the method of Spackman et al_ (117). The degree of coupling of peptides was estimated by calculating differences in key amino acids between the conjugates and s-BSA; these being indicative of the amount of peptide present in each con-jugate. XIIIIodination of Antigens. Iodination was performed in microvolumes by a modification of the procedure of Hunter and Greenwood (47). 0.02 Millicuries of 125 carrier free Iodine as Mai (0.01ml in 0.IN NaOH, New England Nuclear) was mixed with 2 - 10 ug protein (0.02ml in 0.4Mphosphate buffer, pH 7.5) and chloramine T (sodium salt of N-chloro-p-toluene sulfonamide, Eastman Kodak - 200 yg in 0.02ml) was added. Iodination was allowed to proceed 1 - 5 minutes and the reaction then termina-ted by addition of sodium metabisulfite (200 yg in 0.02ml). Non-Radioactive KI was then added (0.05ml of 1% solution) and the reactants removed by gel filtration on Sephadex G-15 or by dialysis against a continu-ous flow of saline. All manipulations were carried out in a fume hood and reagents added with micro pipettes inserted into specially 127 adapted tuberculin syringes. Cold iodinated ( Iodine) antigens used as controls, were prepared in an identical manner. XIV Gel Filtration. A 30 x 1 cm column was packed to a height of 10 cm with Sephadex G-15. The column was equilibrated with 0.04 M phosphate buffer pH 7.5 41 and then treated with 5.0 ml heat inactivated fetal calf serum (FCS) in order to inhibit non-specific adsorption of protein. A drop of FCS was added to each sample prior to application to the column. Eluting buffer was 0.04 M phosphate pH 7.5 and the flow rate was adjusted to 12 drops per minute. Fractions of 8 drops per tube were collected by hand and tubes read using a Geiger-Muller counter (Nuclear Chicago). Peak tubes were pooled and adjusted to physiological osmolarity with NaCl. XV Determination of Specific Activity. All iodinated peptides and proteins were quantifated after adjusting for loss on the column or on the dialysis membranes, Specific activity was calculated by converting CPM to DPM after counting on an Isocap 300 scintillation counter (Iodine channel is 80% efficient). Antigens were frozen and specific activity cal-125 culated on the basis of a 60.2 day half-life for the I isotope. XVI Immunological Activity Testing All synthesized peptides, s-BSA conjugates and their iodinated derivatives were tested for immunological function both in vivo by using skin tests and in vitro by migration inhibition. Denaturation of proteins can occur in the presence of chlora-mine-T used in Iodination procedures (85). Iodinated antigens were 127 thus tested for activity by skin testing with I labelled proteins 125 and assuming that analogous I labelled proteins would react in an identical manner. 42 XV11 Antigen Suicide. Radioactive "antigen suicide" was carried out as in Figure 8, and is a modification of the procedure of Ada et al. ( 3 ). Guinea pig spleen or lymph node cells were incubated with antigens labelled to a 125 7 high specific activity with I. Cells (2 x 10 in 0.5 ml complete 1640 + 0.015 M sodium azide) were placed in 10 ml disposable plastic test tubes (Fisher #2001) and cooled to 4°C. Antigens (100 ng - 2 yg in 0.2 ml saline) were cooled to 4°C and mixed with the cells. The total volume was made up to 2.0 ml with complete 1640 + azide medium and the mixture was incubated for 1 hour at 4°C. Incubation was at 4°C in the presence of azide in order to inhibit uptake of anti-gen by macrophages. The cells were then centrifuged from the mixture (750 x g_, 5 minutes), the supernate removed by aspiration, and the cells resuspended in the same medium containing azide (1.0 ml). Un-bound antigen was removed by centrifuging the cells through three 1.0 ml discontinuous fetal calf serum gradients (100%, 75%, 50%, 25% in PBS). The cell pellet was then resuspended in 0.2 ml PBS + 15% FCS and stored at 4°C for 24 hours to allow radiation death. Cells were resuspended in 1.0 ml PBS and centrifuged through a discontinuous fetal calf serum gradient to remove unbound antigen and cell debris. The washed cells were resuspended in complete 1640 medium, incubated at 37°C for 2-3 hours, counted and prepared for lymphocyte stimulation, MIF tests and autoradiography. Control cells were treated in an iden-127 tical fashion but with saline or I labelled antigens. 43 Figure 8. Antigen Suicide Procedure - Flow Chart Lymph node cells (spleen cells) 125 127 I Antigens I Antigens (Control) 4UC 1 hour Centrifuge through FCS gradients 3x I Resuspend cells in 0.5 ml PBS + 15% FCS Refrigerate at 4°C 24 hours to allow irradiation death .1 Incubate at 37 C 2 - 3 hours Lymphocyte stimulation Centrifuge through FCS gradients Resuspend in med ium, count viable cells Autoradiography MIF (spleen cells) 44 XVIII Antigen Binding Studies. Varying concentrations of iodinated antigens were incubated with 2 x IO'7 guinea pig lymph node cells in a total volume of 0.2 ml in complete medium 1640 + 0.015 M sodium azide at 4 C for 1 hour. The cells were then freed of unbound antigen by centrifugation through FCS gradients (X3), resuspended in 0.2 ml complete medium 1640, precipita-ted with 15% TCA (0.8 ml) and t r i p l i c a t e samples pipetted onto glass fibre f i l t e r s . The f i l t e r s were then washed with PBS and methanol, dried and counted in an Isocap 300 s c i n t i l l a t i o n counter. A range of antigen concentration was tested in order to ascertain saturation curves for use in antigen suicide experiments. XIX Antigens for Binding Experiments and Autoradiography. 125 Proteins were labelled to low specific activity with Iodine by a chloramine-T method modified from Hunter and Greenwood (47). One mCi 1 2 5Iodine as Nal (N.E.N. - NEZ-033) was mixed with 100 yg protein (0.1 ml in 0.4 M phosphate buffer pH 7.5). Chloramine T was added (0.02 ml of 20 yg/ml in phosphate buffer pH 7.5) and after one minute the re-action was terminated with the addition of 0.02 ml sodium metabisulfite (200 yg/ml in 4 M phosphate buffer, pH 7.5). Non radioactive KI was then added (0.05 Ml of 1% solution), the volume made up to 0.3 ml with FCS and the reactants removed by gel f i l t r a t i o n on Sephadex G-15. Specific ac t i v i t i e s ranged between 0.1 and 5.0 yCi/yg protein. 45 XX Preparation of Gelatin-Coated Slides. Glass slides were washed in chromic acid for 1 hour, rinsed in tap water 10 minutes and washed with several liters of distilled water. The slides were then immersed for 2 minutes into a gelatin solution pre-cooled to 21°C (3.75 gm bacteriological gelatin in 750 ml distilled water). After dipping, excess gelatin was drained off and slides were dried at 37°C for 2 hours. Slides were stored at 4°C in sealed black boxes and discarded if more than 4 weeks old. XXI Autoradiography. Immune or non-immune lymph node cells were layered onto 100% FCS and clumps allowed to settle. The uppermost layer of cells was cen-trifuged and resuspended in complete 1640 +0.015 M Sodium azide. The cells (1 x 107 in 0.2 ml) were cooled to 4°C and then 1 yg 1 2 5 I anti-gen was added. The total volume was made up to 0.5 ml and the mixture kept at 4°C for 60 minutes with periodic shaking. Unbound antigen was removed by centrifugation of the mixture through five discontinuous FCS gradients. Cells were resuspended in 0.2 ml 50% FCS (in PBS) and painted onto gelatin coated slides using a fine paint brush. The slides were then fixed in methanol for 30 minutes, washed in water, dried, and dipped in Nuclear Track emulsion (Kodak NTB2). The slides were dried for 1 hour in a vertical position and stored for three days - one week at 4°C in sealed black boxes prior to developing. 46 Autoradiography using high specific activity antigens was per-formed directly on cells taken from antigen suicided cell preparations, XXII Processing of Slides. Autoradiograph slides were developed by immersion into DEKTOL developer for 2 minutes, distilled water for 30 seconds and Kodak fixer for 5 minutes. Softening of the emulsion was prevented by pre-cooling all solutions to 17°C. Developed slides were washed in cold running water for 30 minutes, air dried, and stained for 5 minutes with 2% Giemsa in N phosphate buffer pH 7.0. XXIII Examination of Slides. Cells were examined under oil immersion using a light microscope. Labelled cells were scored positive i f they had a definite lymphoid morphology and greater than 10 grains overlying them or immediately adjacent. Between 10,000 and 20,000 cells were examined in each experi-ment when using low specific activity antigens and 5,000 cells when using high specific activity preparations. XXIV Macrophage Autoradiography. Peritoneal exudate cells were collected by washing the peritoneal cavity of guinea pigs with sterile PBS sixteen hours after intraperi-toneal injection of 20.0 ml of sterile mineral o i l . The cells in the washings were packed by centrifugation at 200 x £ for 10 minutes and then washed 2x with PBS. The cells were resuspended in RPMI 1640 47 complete medium and adjusted to 1.5 x 10 viable macrophages/ml. 3 x 10 cells were incubated with iodinated antigens (1 yg ) in a total volume of 0.3 ml. Cells were freed of unbound antigen by centrifuga-tion through three FCS gradients and were then smeared on gelatin coated slides and processed for autoradiography as previously des-cribed. Cells were scored positive if they had a definite macrophage morphology and greater than 10 grains overlying them, One thousand cells were counted in each experiment. XXV Cell Separation-Nylon Columns Guinea pig lymph node cell populations were fractionated by their differential adherence to nylon fibres in the following manner: Nylon fibre inserts were removed from LP-1 Leuko-Pak leukocyte filters (Fenwal Laboratories - Morton Grove, Illinois) and were soaked in changes of distilled water for 1 week at 4°C and for 24 hours at 37°C, The nylon was dried in an oven at 100°C for 2 hours and 0.6 g of dry fibre was packed into 5.0 ml syringe barrels fitted with poly propy-lene disc supports. The nylon 'columns' were sterilized by autoclav-ing. The columns were washed with complete 1640 medium (care being taken to remove all air bubbles) then filled with complete medium and incubated for 30 minutes at 37°C. The medium was run to the level 7 o of the nylon fibre and 5 x 10 - 1 x 10° cells in 1.0 ml complete 1640 were gently layered on with a Pasteur pipette. The cells were run into the column and washed in with 1.0 ml complete medium. The column 48 was topped up with 2.0 ml medium and incubated for 30 minutes at 37 C in a C02 enriched, humidified atmosphere. Cells were eluted at 1 drop per second, with the addition of medium until the effluent became clear (approximately 5.0 mis). Viable cell numbers were determined by trypan blue exclusion and aliquots then prepared for lymphocyte stimulation, antigen suicide experiments and autoradiography. From Eisen et al (38). XXVI Cell Separation - Anti IgG Plates Anti IgG plate separation of cells was performed in collaboration with Fumio Takei using a method developed by Mr. Takei in this labora-tory. Essentially, the following procedure was followed: Sheep or rabbit anti guinea pig IgG was purified by specific immunoadsorbance to Sepharose 4 B beads coupled with DEAE purified guinea pig IgG via cyano-gen bromide activation. Ten gauge nylon strip (Polypenco 4") was cut to fit the bottom of 120 x 30 mm polystyrene petri dishes (Falcon). The nylon discs were partially hydrolyzed (3.0 N HC1/37 C/2hr), washed with distilled water and then glued onto the bottom of the petri plates with polystyrene solution in toluene. The plate bottoms were washed with distilled water and 5.0 ml of 2.5% glutaraldehyde in 0.2 M sodium bicarbonate buffer (pH 9.4) was added to each plate and incubation at room tempera-ture allowed for 30 minutes. The plates were washed with distilled water to remove unattached glutaraldehyde. 1.0 ml purified anti guinea pig IgG antibody (0.5 mg/ml) in bor-ate buffered saline was added to each plate and the plates were 49 incubated at 4 C for 24 hours. Radioiodinated antibody was added as a tracer to determine the extent of coupling. After incubation, the plates were washed with PBS and stored with PBS containing 15% inactiva-ted FCS and 5.0 mg/ml sodium azide. Free aldehyde groups remaining on the nylon sheet couple to FCS in this step. 2.0 ml of lymph node cell suspension (2.5 x 10^  cells/ml in PBS + 15% FCS + 0.015 M NaNg) were added to each IgG plate and the plates were incubated 30 minutes at 4 C with periodic agitation. The plates were then washed three times with 3.0 ml PBS containing 15% FCS and the effluent cells were recovered and washed 3X with PBS + 15% FCS. Viable cell numbers were determined by trypan blue exclusion and aliquots pre-pared for lymphocyte stimulation, antigen suicide experiments and auto-radiography. XXVII Anti-IgG Autoradiography Unfractionated (U) cells and fractionated (E or effluent) cells were examined for the presence of surface immunoglobulin using anti-IgG autoradiography by the following procedure: 1 x 10^  cells in 0.2 ml PBS + 0.015 M sodium azide + 15% FCS were incubated at 4 C for 1 125 hour in the presence of 7.0 yg I labelled sheep anti guinea pig IgG (specific activity 0.36MCi/yg). The purified sheep anti guinea pig IgG was prepared by Fumio Takei in this laboratory and was shown to be homogenous by immunodiffusion. The cells were washed free of unbound anti IgG by centrifugation through three discontinuous fetal calf serum gradients and slides prepared and developed as previously described. Immunoglobulin positive cells were identified as those cells with lymphocytic morphology and having 10 or more grains over lying or associated with them. A minimum of 500 cells were counted for each cell preparation. (Procedure modified from Ada (1~). 51 RESULTS AND DISCUSSION I Skin Tests. The ability of OFD, N 1 Q C , PPD and KLH to elicit immediate or delayed skin reactions was tested in sensitized and unsensitized guinea pigs. The sensitized animals had been immunized 1 0 - 1 4 days earlier with OFD (or N -JQC), and KLH in complete Freund's adjuvant. Antigen preparations were injected in 0 . 1 ml saline intradermally into the flanks of the animals. An erythema of greater than 6 mm was considered a positive skin reaction as saline controls were always of lesser diam-eter. Development of a positive reaction after 3 hours indicated the presence of circulating antibody to the antigen (immediate reaction), Skin erythema existing after 24 hours indicated that a cell-mediated reaction to the antigen had occurred (delayed reaction), Animals giving positive delayed reactions were used in subsequent experiments requiring immune animals. Skin test negative guinea pigs were boosted and skin tested 1 0 - 1 4 days later. In N-JQC immunized animals, 5 0 yg N -JQC, ^mal^ anc* P P D e^c'*'tec' only delayed reactions indicating the absence of circulating antibody to these antigens (Table II). Several animals failed to respond to N-JQC and Cm ,C, an observation reflected later in the degree of lymphocyte ma i stimulation found with these guinea pigs. In C ^ C immunized animals, delayed responses were found in all cases (Table III). Animals immunized with both OFD and KLH gave positive delayed 52 skin reactions with all antigens tested (Table IV). KLH gave both immediate and delayed reactions indicating that both humoral and cellu-lar immunity were induced. Large areas of erythema (up to 50 mm diameter) found in delayed responses to KLH indicated that a high degree of cellular immunity had been induced. Specificity of the skin reactions was demonstrated by the absence of erythema after skin testing in unimmunized (control) guinea pigs (Table V). 53 Table II: Skin reactions observed on guinea pigs immunized with N i nC Challenging Test dose Skin Reactions Average Diameter antigen yg per 0.1 ml Immediate Delayed of Erythema (mm) (3 hours) (24 hours) (24 hours) N1QC 50 0/14 10/14 10 C m a l C 50 0/14 9/14 10 PPD 50 0/14 14/14 15 Saline Control 0.1 ml 0/14 0/14 2 Figures represent the ratio of the number of animals showing positive reactions (> 6mm) to the number of animals tested). 54 Table III: Skin reactions observed on guinea pigs immunized with C m g 1 C Challenging antigen Test dose yg per 0.1 ml Skin Reactions Immediate Delayed (3 hours) (24 hours) Average Diameter of Erythema (mm) N10C CmalC PPD Saline Control 50 50 50 0.1 ml 0/4 0/4 0/4 0/4 4/4 4/4 4/4 0/4 10 9 18 3 Figures represent the ratio of the number of animals showing positive reactions (> 6mm) to the number of animals tested, 55 Table IV: Skin reactions observed on guinea pigs immunized with OFD and KLH Challenging Test dose Skin Reactions  antigen yg per 0.1 ml Immediate Average Delayed Average (3 hours) Diameter (24 hours) Diameter of of Erythema Erythema (mm) (mm) N10C 50 0/10 8/10 9 OFD 50 0/18 14/18 10 PPD ^ 50 0/18 16/18 15 KLH 50 10/18 25 16/18 23 Saline Control 0.1 ml 0/18 0/18 3 Figures represent the ratio of the number of animals showing positive reactions (> 6mm) to the number of animals tested. 56 Table V : Skin reactions observed on unimmunized guinea pigs Challenging Test dose Skin Reactions Average Diameter antigen pg per 0.1 ml Immediate Delayed of Erythema (mm) (3 hours) (24 hours) N10C 50 0/4 0/4 2 CmalC 50 0/4 0/4 2 OFD 50 0/2 0/2 2 PPD 50 0/4 0/4 5 KLH 50 0/2 0/2 5 Saline 0.1 ml 0/4 0/4 2 Figures represent the ratio of the number of animals showing positive reactions (> 6mm) to the number of animals tested. 57 II Lymphocyte Stimulation Lymphocyte transformation has been assessed by measuring the up-take of tritiated thymidine into the DNA of lymphocytes undergoing blastogenesis. The non-specific activation of lymphocytes by mitogens or the specific activation of sensitized cells by antigens, results in an increased degree of DNA synthesis prior to cell division. The degree of DNA synthesis (and thus degree of stimulation) can be quantitated 3 by comparing the amount of H-thymidine incorporated into the DNA of lymphocyte cultures containing antigen or mitogen to that incorporated into the DNA of cell cultures without added antigen or mitogen. Experiments were carried out to determine optimal culture condi-tions for the stimulation of guinea pig lymph node cells using phyto-hemagglutinin, concanavalin A, and the protein antigens OFD, N^QC, PPD and KLH, employing a microculture system and a semi-automatic multiple sample harvester. 58 (a) Stimulation of Guinea Pig Lymph Node Cells by PHA and ConA. Phytohemagglutinin (PHA) and Concanavalin A (ConA) induce blast-like transformation in T lymphocytes (120) and were thus chosen to investi-gate optimal parameters for the microculture and stimulation of T lympho-cytes. As the incubation time increased, maximum PHA response was found in cultures containing lower cell density, ie. at day two, maximum PHA 5 response was obtained with 5 x 10 cells/well (Figure 9) at day 3 with 2.5 x 105 cells/well (Figure 10)and at day 4 with 1 x 105 cells/well (Figure 11). Culture wells with added PHA became quite acid after 24 hours incu-5 6 bation especially with high cell numbers (5 x 10 - 1 x 10 /well), sug-gesting that in these cultures, conditions soon became toxic. This 5 would explain the rapid decrease in lymphocyte response with 5 x 10 cells/well between day 2 and 3. Cultures containing lower cell density reach peak stimulation much later as there are fewer responding cells and the cultures do not become excessively acid until after lengthy 5 incubation. PHA stimulation using 5 x 10 cells/well was chosen as a measure of T cell viability in subsequent experiments. i The response of guinea pig lymph node cells to varying concentra-tions of ConA is shown in Table VII. A final ConA concentration of 4.0 yg /ml was used in all further tests. 225h 2 0 0 H 150L ^5 10 1 2.5 5 Cell no. x 10" Figure 9 . PHA Stimulation Vs Cell Number. Stimulation represents the ratio of the average H-thymidine incorporation in triplicate cultures with PHA (1/100) present to the average 3H thymidine incorporation in triplicate con-trol cultures without PHA. Cultures were pulsed with H thymidine at 48 hours and har-vested 16 hours later. Results from 2 guinea pigs are shown. lire 10- PHA Stimulation Vs Cell Number. Stimulation: as defined in Figure 9 . 3 Cultures were pulsed with H-thymidine at 72 hours and harvested 16 hours later. Results from 2 guinea pigs are shown. 61 Cell no. x 10 ^  Figure 11. PHA Stimulation Vs Cell Number. Stimulation: as defined in Figure 9. 3 Cultures were pulsed with H thymidine at 96 hours and harvested 16 hours later. Results from 2 guinea pigs are shown. 62 Table VII: H-thymidine uptake in guinea pig lymph node cells in response to varying concentrations of ConA Final ConA Concentration (ug /ml) CPM (± standard deviation)^ 0.0 863 (± 176) 1.0 761 (± 86) 2.0 9,061 (± 261) 4.0 25,092 (± 1302) 8.0 24,961 (± 1313) 20.0 8,042 (± 651) 40.0 4,691 (± 402) Average CPM and standard deviation were calculated from quadruplicate cultures of guinea pig lymph node cells (5 x 105/well), pulsed with 3 H-thymidine after 48 hours incubation and harvested 16 hours later. 63 (b) St imulation of Sensitized Guinea Pig Lymph Node Cells by N^QC The stimulation of N-JQC and OFD immunized guinea pig lymph node cells by N ^ C diluted in RPMI 1640 was tested at final concentrations varying from 12 - 96 yg /ml (Table VIII) A final N-jQC concentration of 24 yg /ml was used in all subsequent experiments. The effect of vary-ing cell concentrations and incubation period on the N-JQC response is shown in Figure 12 S 13 respectively. Maximum response was obtained 5 using 5 x 10 cells/well and a four-day incubation period. (c) Stimulation of Sensitized Guinea Pig Lymph Node Cells by OFD. Stimulation of OFD immune lymph node cells by varying concentra-tions of oxidized ferredoxin is shown in Table VIII A four day incuba-5 tion period (96 hours) and a cell concentration of 5 x 10 cells/well were used in all tests. The optimum response was obtained with a final OFD concentration of .0008 y moles (4.8 yg ) per ml, and this concen-tration was used in all subsequent experiments. (d) Stimulation of Sensitized Guinea Pig Lymph Node Cells by PPD. All animals were immunized with antigens emulsified in complete Freund's adjuvant (CFA), therefore purified protein derivative (PPD) stimulated cells from immunized animals. (Freund's adjuvant consists in part of M. tuberculosis, the bacterium from which PPD is derived). Optimal stimulation was achieved with a final concentration of 10 yg PPD per ml (Table VIII) As with N 1 Q C and OFD, a cell concentration of 5 5 x 10 /well yielded maximum stimulation of thymidine uptake (Figure 14). 64 (e) Stimulation of Sensitized Guinea Pig Lymph Node Cells by KLH. Of the antigens tested in this study, keyhole limpet hemocyanin (KLH) induced the greatest thymidine uptake. Varying concentrations 5 of KLH were tested at day four using 5 x 10 cells/well (Table VIII). Maximum response was obtained with a final concentration of 20 yg: KLH per ml. (f) Stimulation of Unimmunized (Control) Guinea Pig Lymphocytes by Test Antigens.  The specificity of the antigen preparations used for stimulation is shown by experiments performed with cells from unimmunized guinea pigs. PPD was the only antigen causing significant stimulation (Table IX ) which could be attributed to its reported mitogenic properties for B cells (120). PHA response was used as a positive control in these experiments. 65 Table VIII. H thymidine incorporation in N1QC or OFD/KLH immunized guinea pig lymph node cells in response to varying anti-gen concentrations Sensitizing Antigen Challenging Antigen CPM ± standard (yg /ml final concentration) deviation2 N10C 96 yg 402 + 69 48 yg 481 + 82 24 yg 488 + 64 12 ng 211 + 61 0 yg 206 + 41 PPD 40 yg 302 + 61 20 yg 841 + 112 10 yg 876 + 102 2 yg 318 + 56 0 yg 326 + 48 OFD/KLH OFD 19.2 yg • 488 + 102 9.6 yg 511 + 31 4.8 yg 509 + 48 2.4 yg 221 + 61 0 yg 246 + 86 100 yg 206 + 06 40 yg 408 + 68 20 yg 2486 + 202 10 yg 1681 + 142 0 yg 302 + 21 Antigens were all injected in CFA resulting in a parallel sensitization to PPD. Calculated from triplicate cultures of guinea pig lymph node cells (5 5 3 x 10 cells/well) pulsed with H-thymidine at 96 hours and harvested at 112 hours. Figure 12. N 10 2.5 5 Cell no. x 1 0 " 5 C Stimulation Vs Cell Number. Stimulation: as defined in Figure 9 3 10 Cultures were pulsed with harvested 16 hours later, are shown. H thymidine at 96 hours and Results from 3 guinea pigs 72 96 120 H o u r s Figure 13. N,nC Stimulation Vs Incubation Time. Stimulation represents the ratio of the average H-thymidine incorporation in triplicate cultures con-incorporation in triplicate control cultures without Cultures containing 5 x 10 cells/well were pulsed with 3H thymidine at 3 different time intervals as indicated on the abcissa. Cells were harvested 16 hours later. Results from 3 guinea pigs are shown. 3 taining average thymidine 1 2J5 5 10 Cell no. x10~5 Figure 14. PPD Stimulation Vs Cell Number. 3 Stimulation represents the ratio of the average H-thymidine incorporation in triplicate cultures containing PPD (10 ugm/ ml) to the average thymidine incorporation in triplicate control cultures without PPD. 3 Cultures were pulsed with H thymidine at 96 hours and har-vested 16 hours later. Results from 3 guinea pigs are shown. 69 Table IX: H-thymidine incorporation in unimmunized (control) guinea pig lymph node cells in response to antigen preparations used in lymphocyte stimulation experiments Challenging Antigen (yg /ml) Final Concentration Average^  Stimulation No- of Animals Tested N10C 1.0U0.06 2 PPD 2.06±0.31 2 OFD 0.98±0.09 2 KLH 0.96±0.10 2 PHA 68.04±8.90 2 Figures were calculated from triplicate cultures of guinea pig lymph node cells (5 x 10 /well) pulsed with H-thymidine at 96 hours and 3 harvested at 112 hours. The ratio of H-thymidme incorporation in 3 cultures with antigen present to H incorporation in cultures con-taining no antigen is averaged from experiments with both animals. 70 Stimulation results compiled from experiments with several animals are given in Table X Results are presented as the average stimulation ratio plus or minus the standard deviation. KLH consistently induced the greatest thymidine uptake in sensitized animals. PPD also gave good stimulation in all animals tested. The response to N Q^C was not consistently above 2-fold and depended markedly on the preparation used. For this reason, OFD was used for immunization and stimulation in all later experiments. (g) Reproducibility Reproducibility using the microharvester apparatus was tested by labelling, pooling and redistributing PHA and ConA stimulated and unstimulated guinea pig lymph node cells into microtitre wells before harvesting. Results are shown in Table VI. The average percent stan-dard deviation between replicates ranged between 3.5% - 12.5% in un-stimulated (control) cultures and between 0.5% - 5.2% in ConA and PHA stimulated cultures respectively. Other reports indicate that an error of 3.5% - 4.5% Standard deviation) is added simply by dispensing MLC samples with Hamilton syringes (49). Maximum error introduced by the labelling and harvesting procedures therefore is in the order of 9.0% average standard deviation. Reproducibility of the microculture-harvester system is shown in a compilation of data from actual experiments (Table XI), 71 Table X : Antigen induced H-thymidine incorporation in guinea pig lymph node cell cultures from sensitized guinea pigs Immunizing Antigen Stimulating Antigen Final Antigen Concentration Average^  Stimulation No. of Animals Tested N10C N10C .01 ymoles/ml 2.3U0.91 10 PPD 10 yg /ml 3.88±1.00 10 CmalC N10C .01 ymoles/ml 1.48±0.23 5 PPD 10 yg /ml 3.38+1.06 5 C nC mai .01 ymoles/ml 2.38±0.51 5 OFD, KLH OFD .0008 ymoles/ml 2.95±1.13 10 PPD 10 yg /ml 4.09±1.54 10 KLH 20 yg /ml 8.55+4.65 10 Figures for average stimulation are derived from the ratio of H-thymidine incorporation in triplicate cultures with antigen present to those without for each animal. 72 Table VI: H-thymidine uptake in guinea pig lymph node cell replicate microcultures dispensed with a constant delivery syringe and processed with the microharvester apparatus Sample Number Control (CPM) PHA1 (CPM) Sample Number Control (CPM) ConA2 (CPM) 1 1102 29,163 1 762 36,421 2 1412 28,204 2 871 37,402 3 976 31,216 3 804 37,116 4 1111 31,001 4 811 36,902 5 1042 34,696 5 901 35,111 6 1007 28,921 6 799 37,911 7 963 27,041 7 789 37,004 8 915 30,263 8 851 36,991 9 1181 30,211 9 802 37,100 10 1411 29,402 10 807 37,102 11 1002 27,402 11 820 38,009 12 1316 27,826 12 819 37,931 1119±140 29,612±1554 820±29 37,083±192 Average percent standard Control 12.5 deviation: PHA Stimulated 5.2 Average percent standard Control deviation: PHA Stimulated PHA (1/100) stimulated and unstimulated (control) guinea pig lymph node 3 cells labelled with H-thymidine after 48 hours in culture, dispensed into microtitre plates (5 x 10 cells/well) in 0.25 ml volumes using a Hamilton constant delivery syringe. ConA (4 yg /ml) stimulated and unstimulated (control) guinea pig lymph node cells treated as in 1. 73 Table XI • Reproducibility of the microculture-harvester system: 3 1 H-thymidine incorporation by microcultures stimulated with OFD, KLH and PPD. Average CPM (± Standard Deviation) Control OFD KLH PPD 1 402 ( 2.4) 929 ( 0.2) 4057 ( 0.1) 961 ( 1.0) 2 226 ( 2.5) 435 (16.5) 442 (29.1) 3298 (11.5) 3 826 (22.0) 21985 ( 7.5) 17334 (10.6) 44758 ( 8.6) 4 1900 (10.2) 8361 (12.8) 10548 ( 9.5) 6378 (13.3) 5 491 ( 8.6) 1213 (24.0) 1971 ( 8.2) 1998 (12.1) Average percent 9.1% 12.2% 14.4% 9.3% standard deviation 1 5 Lymph node cells from OFD/KLH immunized guinea pigs (5 x 10 mononuclear cells per well dispensed with a Hamilton constant delivery repeating 3 syringe) pulsed with H-thymidine after 96 hours incubation and harves-ted 16 hours later with the microharvester apparatus. 2 Each experiment was performed with cells from a different guinea pig. 3 CPM ± percent standard deviation calculated from triplicate cultures from individual guinea pigs. Experiment 74 III Peptide Conjugates The Nl^-terminal or the COOH-terminal haptenic peptides of ferre-doxin were covalently linked to succinylated BSA (s-BSA) producing the conjugates termed N-sBSA and C-sBSA. Because of the probability of mul-tiple binding of such molecules which contain approximately 20 peptides per molecule of s-BSA their avidity should be much greater than that of the peptides alone making their use in autoradiography and antigen sui-cide experiments preferable. The number of tyrosine residues present 125 in the BSA molecule also enables labelling with Iodine to a high specific activity, especially important in achieving sensitivity in auto-radiography and effectiveness in antigen suicide experiments. Iodination of the tyrosine in BSA is essential in C-sBSA conjugates as the COOH-terminal peptide contains no tyrosine. IV Estimation of Coupling The degree of coupling of peptides was estimated by calculating differences in key amino acids between the conjugates and s-BSA (Table XII). The N-sBSA conjugate was estimated to contain 20 NH2-terminal pep-tides per molecule and the C-sBSA conjugate 22 COOH-terminal peptides per molecule. V Biological Activity Tests Biological activity of s-BSA and its peptide conjugates was tested in vivo by skin testing and in vitro by production of MIF. The ability of the different preparations to elicit delayed skin reactions in N i nC immunized guinea pigs is shown in Table XIII.Inhibition of migration of sensitized cells induced by the peptide conjugates is shown in Table XIV. All preparations containing either Nh -^terminal or COOH-terminal peptides gave positive skin reactions and elicited MIF indi-cating that the conjugates retained biological activity. The specifi-city of these reactions is demonstrated by the negative reactions seen using unconjugated s-BSA alone. Table XII. Ratios of amino acids in s-BSA and its Nh -^terminal and COOH-terminal peptide conjugates. Amino Acid s-BSA N-sBSA C-sBSA Asp 0.88 2.392 1.04 Thr 0.41 0.49 0.56 Ser 0.33 0.40 0.40 Glu 1.34 1.31 2.30 Pro 0.23 0.19 0.97 Gly 0.35 0.29 0.33 Ala 0.74 2.84 1.32 Val 0.56 0.58 1.13 Met 0.06 0.09 0.03 lieu 0.20 1.25 0.25 Leu 1.00 1.00 1.00 Tyr 0.32 1.30 0.19 Phe 0.43 0.43 0.53 Calculated using leucine as 1.0 in each analysis. Figures underlined correspond to the amino acids found in the NH terminal or COOH-terminal peptides. 77 Table XIII. Biological activity of the peptide conjugates and their iodinated derivatives - skin reactions observed on guinea pigs immunized with N i nC. Challenging Test Dose Delayed Skin Reaction1 Diameter Preparation (yg /0.1 ml) (24 hours) of Erythema (mm) N1QC 50 2/2 9 NH2-terminal 50 2/2 10 COOH-terminal 50 2/2 9 s-BSA 50 0/2 3 N-sBSA 50 2/2 12 C-sBSA . 5 0 2/2 10 1 2 7 I -N C 50 2/2 10 1 2 7I-sBSA 50 0/2 5 127I-NsBSA 50 2/2 10 127I-CsBSA 50 2/2 13 Saline (Control) 0.1 ml 0/4 4 Figures represent the ratio of the number of animals showing positive reactions (> 6 mm) to the number of animals tested. 78 Table XIV. Migration inhibition by N^-terminal and COOH-terminal peptides, sBSA-peptide conjugates, and their iodinated 2 derivatives. Test Preparation Test Dose Chamber Concentration Percent ? Migration + Standard Deviation ^-terminal 0.05 ymole/ml 67% t 9.1 COOH-terminal 0.05 ymole/ml 72 + 4.8 s-BSA 150 yg /ml 106 t 19.4 N-sBSA 150 yg. /ml 60 + 6.9 C-sBSA 150 yg /ml 74 i 13.1 127 '^'i-Ns-BSA 150 yg/ml 72 + 12.1. 1 ?7 '^'i-CsBSA 150 yg /ml 71 + 9 - 1 Medium (Control) - 100 The migration of spleen cells from N-JQC immunized guinea pigs measured after 18 hours incubation at 37 C . 127 I-labelled conjugates were tested with the assumption that analogous 125 I-labelled proteins would react identically. Percent migration measured in chambers containing peptides, s-BSA conjugates or their iodinated derivatives compared to control chambers containing culture medium alone. Measurement of migration was per-formed by determining the rectangular area encompassing the furthest extent of confluent macrophage travel, using a calibrated microscope stage. The average area of two migration patterns from each animal was calculated. 79 VI Iodination Iodinated proteins were freed from unreacted Iodine by gel fi ltra-tion. The elution profile of an N-JQC Iodination mixture is shown in Figure 15. The labelled N^QC was frontally eluted as a single well 125 defined peak while unbound I was included in the gel and eluted much 125 later. Between 30% - 50% of the I label was associated with the first peak, depending on the nature and quantity of the protein iodina-ted. Elution profiles of the sBSA conjugates were identical although 125 proportionally more I was associated with the protein peak. Experi-ments using Iodinated molecules as tracers showed that less than 100 ng protein was adsorbed to the Sephadex beads providing the column was washed with FCS prior to sample application. Specific activities (cal-culated after correcting for column loss) ranged from 100 - 500j*Ci per yg for N-|QC and from 100 yCi- 1 mCi perug for s-BSA and its peptide conjugates. Iodination of proteins for autoradiography was performed using high concentrations of protein in the reaction mixture (50 - 100 yg /0.1 ml). Specific activities of proteins labelled in this manner ranged from 0.4 to 1.0 yCiper yg, 127 I-labelled proteins were collected by pooling tubes correspond-125 ing with peak tubes from elution profiles of analogous I reaction mixtures. All Iodinated preparations were tested for biological 125 activity with skin tests and MIF tests using I-labelled proteins 125 assuming that the analagous I-labelled molecules would react identi-cally. Results from these tests indicated that the Iodination procedure did not significantly alter the biological activity of the peptide determinants (Table XIII, XIV). 5 10 15 20 25 30 35 Tube no. Figure 1 5 . E lut ion P r o f i l e of N-JQC Iodination Mixture. P r io r to sample app l i ca t i on , the column ( G - 1 5 Sephadex 1 cm x 1 0 cm) was equ i l ibrated with 0 . 0 1 M phosphate buffer pH 7 . 5 and 5 . 0 ml inact ivated FCS. E lut ing buffer was 0 . 0 1 M phosphate pH 7 . 5 adjusted to 1 2 drops per minute. Eight drop f ract ions were co l lec ted by hand. 81 VII Antigen Binding Experiments The potential of lymphocytes to bind antigen can best be tested in  vitro using a cell suspension in which every cell is exposed to the same antigen concentration. This approach was first explored by Naor and Suliztneau (79) who wished to test a tenet of Burnet's Clonal Selection Hypothesis which proposed that only a small proportion of an animal's total lymphocyte population would possess receptors for any one antigen (or antigenic determinant). Autoradiographic examination of cells exposed in vitro to radioactively labelled antigen revealed only a small proportion of cells binding antigen. The number of antigen binding cells observed is influenced by various parameters however and thus asserts a need for concern about specificity and interpretation (Reviewed by Ada, 1 ). In the work reported here, antigen binding experiments were designed to determine the maximum number of cells specifically binding a given antigen or antigenic determinant while minimizing interference from non-specific uptake of labelled antigen. The following parameters were considered in all antigen binding studies: (a) Uptake of Antigen by Macrophages Cell suspensions usually contain macrophages and polymorphonuclear leukocytes which may take up antigens non-specifically, Cohn (20) reported that phagocytosis by macrophages is inhibited by 15 mM sodium azide while the binding of antigen to lymphocytes remains unaffected. 125 Macrophage uptake of I"N-|nC a n c ' t n e iodinated peptide conjugates was 82 tested both in the presence and absence of sodium azide. The results are shown in Table XV. A high proportion of macrophages from both immune and non-immune animals bound all iodinated proteins tested when incubated in complete medium at 37 C. This uptake was inhibited by incubation at 4 C in the presence of sodium azide (15 mM). Azide was included as a medium component in all subsequent antigen binding experi-ments. (b) Uptake of Antigen by Dead Cells. Damaged cells, cell debris, and isolated nuclei may bind labelled antigen thus making it difficult to determine numbers of antigen binding cells. In order to minimize interference from non-specific uptake of antigen, lymphoid tissues were teased gently apart in the presence of 5% fetal calf serum and the cell suspensions layered onto 100% fetal calf serum. Clumps of aggregated cells and cell debris were allowed to set-tle out and were discarded. Dead cells and debris were also removed by centrifugation of the cell suspensions through discontinuous fetal calf serum gradients. Cells were counted positive only i f they were completely intact with a well defined periphery, evenly stained, possessed a defi-nite lymphoid morphology and had 10 or more grains overlying or associa-ted with them. Emulsion which gave more than 30 background grains per microscope field was discarded. (c) Effect of Antigen Concentration. If antigen receptors are generated by a random process (as proposed 83 in the Clonal Selection Theory) there will be an increasing number of receptors available for binding antigen as the degree of complementar-ity (between receptors and antigenic determinants) decreases. The fre-quency distribution of grain counts at a given antigen concentration supports this view as a marked variation in the number of antigen molecules bound per cell is seen. Ada ( 1 ) examined the heterogeneity of antigen binding to murine spleen cells using varying concentrations of hemocyanin. Between antigen concentrations of 50 - 1500 ng/ml the number of antigen binding cells increased almost proportionately with the increase in antigen concentration. At a concentration higher than 1500 ng/ml the number of binding cells did not increase. 125 The effect of increasing concentration of I labelled N^QC and the BSA conjugates on binding of labelled antigen by N-|QC immune lympho-cytes is shown inFigurel6. Maximum binding was achieved with antigen concentrations of 1.0 yg /0.2 ml (2 x 10^  cells) and higher for all preparations tested. This concentration (1 yg /0.2 ml) was used in sub-sequent experiments requiring high levels of radiation (autoradiography and antigen suicide). Since s-BSA and its peptide conjugates were labelled to approximately the same specific activity, the increased 1 2 5 1 2 5 1 2 5 counts seen with I-N-sBSA and '"iC-sBSA over IsBSA incubation mixtures reflects the specificity of the binding. The low counts 125 observed in *-N-|QC incubation mixtures may be due to the low specific 125 activity of this antigen. Alternatively, I N-JQC which is monovalent for the NHL-terminal and COOH-terminal determinants may be bound less avidly than the polyvalent peptide-sBSA conjugates. 34 Table XV . Number of antigen binding macrophages in peritoneal exudates from unsensitized and N-JQC sensitized guinea pigs. Treatment 1 2 5I-labelled 1 Protein Labelled Cells Unimmunized 2 per 103 Immunized Medium + 0.15 M azide 1 hour at 0°C NioC NsBSA CsBSA 0 1 0 1 1 1 Medium only 1 hour at 37°C NioC NsBSA CsBSA 502 511 312 471 516 418 Medium only 16 hours at 37°C NIOC NsBSA CsBSA 900 920 817 802 891 916 Specific activities were: N1QC 0.8 f-^ Ci/^ g, NsBSA 1.4 ^Ci/jtg and CsBSA 125 1.0 /J-Ci/^g- 1.0 yg I-protein was used in each incubation mixture. Slides were exposed for 7 days. Cells were scored positive if they possessed a definite macrophage mor-phology and had 10 or more grains overlying or associated with them. 1000 cells were examined in each test. 85 5.0 eo I O s o 110 100 1000 2000 Nanograms of Antigen Figure 16. The effect of antigen concentration on the antigen bind-ing capacity of N-JQC sensitized guinea pig lymphocytes. 125 Varying concentrations of I labelled proteins were incubated with 1 x 10' lymphocytes in a total volume of 0.2 ml and the number of bound C P M determined after 1 hour at 4 C . 125 125 I-sBSA (1.0 uci/ug). A- V I-N10C (0.4MCi/Kq I-CsBSA(1.0 ^Ci/yg) V25I-NsBSA(l .0 Ci/^g 86 (d) Effect of Immunization The numbers of cells specifically binding N-^C and the NHg-terminal and COOH-terminal peptide determinants were determined by auto-radiography using lymph node cells from immunized and unimmunized guinea pigs. The results are presented in Table X V I . A four to five fold increase in the number of antigen binding cells was seen in both N-JQC and OFD immunized animals. The specificity of the binding reaction was 125 indicated by the absence of cells binding I-sBSA. Approximately equal numbers of lymphocytes were labelled with N-jQC and the peptide conjugates, a somewhat disconcerting observation as it was expected that the number of cells binding N J Q C would equal the sum of the numbers binding each determinant. This result parallels the findings of the batch scintillation counting experiment where cells incubated with I -N-JQC bound relatively low counts in comparison with those incubated in the presence of the sBSA conjugates. Low specific activity or low avidity for cell receptors may explain this observation. The labelled cells were small to medium sized lymphocytes although labelled macrophages and dead cells were occasionally seen. A few lymphocytes with grain counts higher than background were not scored positive because of the arbitrary limit of a minimum of 10 grains per positive lymphocyte. (e) The Effect of B Cell Depletion on Antigen Binding Cells Unfractionated (U) cells and fractionated (E or eluted) cells from nylon fibre columns and anti-IgG plates were examined for the presence 87 of surface immunoglobulin using anti-IgG autoradiography. B cells were equated with immunoglobulin positive cells and were identified as those cells with intact lymphocytic morphology having 10 or more grains overlying or associated with them. The numbers of immuno-globulin positive cells found in unfractionated and fractionated pop-ulations are presented in Table XVII . The effect of B cell depletion on numbers of cells binding antigenic determinants of OFD is shown in Table XVIII. 88 Table XVI. Antigen binding cells in lymph nodes from N^C and OFD immunized and unimmunized guinea pigs - detection using 125 low specific activity I-labelled molecules. 1 2 5I-labelled preparation' 2 4 Antigen Binding Cells per 10 Lymphocytes Unimmunized N ^ Q C Immune OFD Immune s-BSA 0 1.0 1.0 N-sBSA i .o • 5.0 6.0 C-sBSA 1.0 5.0 4.0 N 10 C 1.0 4.0 5.0 Specific activities of the iodinated molecules ranged from 0.4 - 1.0 uCi per yg. 1.0 yg was used in each reaction mixture. 4 10 cells were counted on each autoradiograph slide. Slides were exposed for 7 days. TableXVII- The effect of cell fractionation on numbers of immunoglobulin positive guinea pig lymph 125 2 node cells detected by I-anti IgG autoradiography. Experiment Treatment Percent Immunoglobulin Percent Number Cell Yield Positive2 Cells/500 Positive Cells 1 none 100 204 40.8 nylon fibre 22.0 13 2.6 2 none 100 224 44.8 nylon fibre 20.2 12 2.4 3 none 100 220 44.0 nylon fibre 16.0 8 1.6 4 none 100 229 45.8 anti IgG plate 59.0 42 8.4 5 none 100 211 42.2 anti IgG plate 54.0 71 14.2 Specific activity of the I-labelled sheep anti-guinea pig IgG was 0.36 yPVy 9 7 . 0 ug was incubated with 10^  cells in each experiment. Cells were identified as immunoglobulin positive if they possessed intact lymphoid morphology and had 10 or more grains overlying them or immediately adjacent. 500 cells were counted on each slide. Tab! e XV111. The effect of B cell depletion on the number of antigen binding cells in OFD sensitized 125 guinea pig lymph node cells - detection using high specific activity I-labelled molecules. Experiment Cells Percent 1 2 5I-labelled ABC4/5 x 103 Lymphocytes Number Ig Bearing Preparation3 Cells2 Grain Number - (10-20) (21-30) (> 30) Total 1 Unseparated 40.8 sBSA 3 2 0 5 NsBSA 12 4 0 16 CsBSA 15 3 0 18 Eluted 2.6 sBSA 2 1 0 3 NsBSA 14 1 0 15 CsBSA 16 1 0 17 2 Unseparated 44.8 s-BSA 0 1 1 2 N-sBSA 18 4 . 0 22 C-sBSA 14 2 2 18 Eluted 2.4 s-BSA 1 1 0 2 N-sBSA 14 5 0 19 C-sBSA 14 2 0 16 B cells were depleted using nylon fibre columns. Detected by anti-IgG autoradiography. Specific activities ranged between 196 - 300 yCi/yg. 3 5x10 cells were counted on each autoradiograph slide. Slides were exposed for 7 days. 91 VIII Immunological Significance of Antigen Binding Cells (a) Detection of Cell Death by Autoradiography The functional significance of antigen binding cells was investi-gated using the radioactive antigen suicide technique in an attempt to inactivate lymphocytes binding the antigen N^C or either the Nh^ -terminal or COOH-terminal antigenic determinants. The antigen suicide technique depends on the marked sensitivity of lymphocytes to ionizing radiations (55 ). If the radiation from bound antigen leads to death of the cell, the outcome might be detec-table by autoradiography. The number of cells killed by radiation 125 125 from bound I-r^-terminal and I-COOH-terminal antigenic determin-ants was estimated by comparing autoradiograph slides before and after antigen suicide (Table XIX). A marked decrease in the number of anti-gen binding cells was seen after antigen suicide. The greatest decrease was seen in cells with higher grain numbers, a result compati-ble with the idea that cells binding most antigen would be most easily killed. Cells inactivated but not killed would stil l be observed as antigen binding cells, likewise with cells not lysed by the radiation. Shedding of hot antigen may account for these observations although it is unlikely under the conditions used. (b) Lymphocyte Stimulation - Low Dose Antigen Suicide The functional activity of cell populations (before and after anti-gen suicide) was tested in vitro using antigen induced lymphocyte stimu-lation as an index of cellular immunity. Initial experiments used low doses of 1 2 5I-labelled N i nC (100 ng/5 x 107 cells) with the object of Tab!eXIX. Antigen binding lymph node cells from OFD immunized guinea pigs - detection using high 125 specific activity I-labelled molecules before and after antigen suicide. Experiment Number 1 2 5I-labelled Preparation^ Treatment Cells 2 3 3 Antigen Binding Cells /5 x 10 Lymphocytes Grain Number - (10-20) (21-30) (> 30) Total sBSA N-sBSA C-sBSA Slides made at zero time 0 18 14 1 4 2 1 0 2 2 22 18 sBSA Stored 2 0 1 3 N-sBSA 24 hours 8 0 0 8 C-sBSA at 4 C 3 0 0 3 2 sBSA Slides 3 2 0 5 N-sBSA made at 12 4 0 16 C-sBSA zero 15 3 0 18 time sBSA Stored 0 0 0 0 N-sBSA 24 hours 4 1 0 5 C-sBSA at 4 C 7 0 0 7 Specific activities of iodinated molecules ranged" from 196-300 yCi/ufl. Cells were spread onto slides immediately after labelling or after allowing radiation death by incubating for 24 hours at 4 C-3 5 x 10 cells were counted on each autoradiograph slide. Slides were exposed for 7 days before developing. 93 eliminating only highly avid antigen binding cells and minimizing non-specific cell inactivation. The results are shown in Table XX. After suicide, the average proliferative response to N-jQC was 32.7% of the untreated control response. The decreased N-JQC response was highly significant as shown by T test (p < 0.01). Inactivation was specific since the decrease in the PPD response was not statistically signifi-cant (p > 0.10). If the in vitro proliferative response to OFD involves the co-operation of cells directed to each of the antigenic determinants, then the inactivation of cells binding either determinant should eliminate the response. To test this, specific inactivation of cells binding the COOH-terminal determinant was attempted using low doses of 125I-CsBSA (Tables XXI and XXII). Thymidine uptake induced by N 1 Q C and Cma-|C was decreased significantly (p << 0.005) in both C -jC and N^QC sensitized cells indicating that COOH-terminal binding cells func-tion in the stimulation response to these antigens and are inactivated by the iodinated COOH-terminal s-BSA conjugate. The lack of signifi-cant decrease in response to PPD (p > 0.10) demonstrates the specificity of killing with 125I-CsBSA. 7 1 ?R High doses (1.0 ug /2 x 10 lymphocytes) of I-labelled antigens were used in an attempt to further decrease the stimulation response 125 in all subsequent experiments. I-sBSA treated cells were included as an additional control, all stimulation ratios were based on these cells treated with non-specific radiation. Specific reduction of the OFD response (Table XX111) was found 94 using both I-NsBSA and I-CsBSA (p « 0.005 and P « 0.01 respec-tively) while responses to the control antigen KLH (TableXXIV ) (P = 0.10 and 0.025 respectively) were not significantly reduced. Cell mixing experiments were also performed in order to test cell-cell interaction, ie. mixing of a cell population suicided with the l^-terminal determinant with a population suicided with the COOH-terminal determinant should restore the response to control levels if cell synergy occurs. If no synergy occurs, the stimulation value obtained by mixing the two populations is expected to be the average of the sum of the contribution of two populations. This expected stimulation ratio was compared with the observed values in all cell mixing experiments. Chi-square analysis of expected and observed stimulation ratios in mixed populations showed that the OFD response was significantly higher (p << 0.005) than would be expected in the absence of synergy (Table XXIII), The failure to restore the OFD res-ponse fully may be due to decreased numbers of active l^-terminal and COOH-terminal binding lymphocytes in the suicided cell preparations brought about by non-specific irradiation from the bound antigen. The observed KLH responses were significantly lower than would be expected (p « 0.005), (Table XXIV). This indicates inhibition of response rather than enhancement as seen with OFD stimulation in mixed popula-tions. The reason for this inhibition is unclear although the reduced KLH response may reflect poor culture conditions, perhaps due to toxic products contributed by each suicided population. 95 (c) Lymphocyte Stimulation Using B Cell Depleted Populations - High Dose Antigen Suicide  The effect of antigen suicide on lymphocyte stimulation in B cell depleted populations is shown in Table XXV (OFD stimulation) and Table XXVI (KLH stimulation). Specific depression of the OFD response was achieved with 125I-NsBSA and 125I-CsBSA (p « 0.005 in both cases) while the response to KLH was not significantly reduced (p > 0.10 in both cases). Cell mixing experiments again indicated synergy between the two populations in the response to OFD (Table XXV )• The differ-ences in observed and expected values for KLH stimulation were again statistically significant but were lower in 3 out of 5 experiments (Table XXVI). Incubation of cell populations in serum-free medium after antigen 125 suicide resulted in a decrease in I counts bound to the cells, Counts remaining after this incubation usually were in the order of 10% of the total counts before incubation in the serum-free medium (approximately 100-200 cpm/5 x 105 cells). Table XX . Antigen stimulation of H-thymidine incorporation in cultures of N1QC sensitized guinea pig lymph node cells treated with low doses1 of high specific activity 1 2 5 I - N i n C . Experiment Number Stimulating Antigen N10C PPD Treatment of Cells Untreated2 1 2 5 I - N 1 Q C 3 % Untreated 1 2 5 I -N C % 1 1.28 0.0 1.31 96.7 2 2.07 55.1 1.60 96.6 3 2.11 43.2 2.10 71.82 Average: 32.7 ± 28.9% 88.4 ± 14 P Values: P < 0.01 P > 0.10 Antigen dose used was 100 ng/5 x 10° lymphocytes. The specific activity of the '"i-N^C prepara-tions ranged from 100-400 uCi/ug Ratio of 3H-thymidine incorporation in triplicate cultures containing antigen to those without antigen. PPD (10 yg /ml) and N-]gC (24 yg ./ml) were used for stimulation. Average percent of control antigen response over background after treating cells with high specific activity ^ 2 5Tgc . ie. stimulation ratio suicided cells -1.0 ^ IQQO^ stimulation ratio untreated cells -1.0 Table XXI. Antigen stimulation of H-thymidine incorporation in cultures of N^C sensitized lymph node cells 1 125 treated with low doses of high specific activity I-CsBSA. Experiment Number Antigen (Stimulation) - N10C Cma lC PPD Treatment of Cells Untreated I2 1 2 5I-CsBSA3 % Untreated 125I-CsBSA % Untreated 125I-CsBSA % 1 3.36 47.0 1.36 0.0 - -2 1.28 0.0 - 1.31 225 3 2.07 40.1 - 1.60 96.6 4 4.95 54.6 - 7.86 112.7 5 2.10 14.5 1.32 0.0 2.73 124.8 Average: 31.2 ± 22.; 7% 0.0% 139.7 ± 57.9% P Values • P « 0.005 P > 0.10 Antigen dose used for suicides was 100 ng/5 x 10 lymphocytes. Specific activity of the I-CsBSA preparations ranged from 100 - 500 uc/ug. Ratio of 3H-thymidine incorporation in triplicate cultures containing antigen to those without antigen. Antigen concentrations used were: N-jnC 24 yg /ml, Cma-|C 24 yg ./ml and PPD 10 yg /ml. Average percent of control antigen response over background after treating cells with high specific activity 125I-CsBSA as defined in Table XX . Table XXII. Antigen stimulation of H-thymidine incorporation in cultures of C^-jC sensitized guinea 1 1 ?5 pig lymph node cells treated with low doses of high specific activity I-CsBSA. Experiment Number Stimulating Antigen - Qna lC PPD Treatment of Cells - Untreated 125 1 "l-CsBSA % Untreated 125 '"I-CsBSA % 1 2.25 30.4 1.54 68.5 2 3.24 40.2 1.63 68.2 3 1.92 48.9 3.56 26.8 Average: 39.8 ± 9.1 3% 54.5 ± 23.9 P Values: P « 0.005 P » 0.025 l s 2, 3, as in Table XXI. Table XXIII. OFD stimulation of H-thymidine incorporation in cultures of OFD sensitized guinea pig lymph node 1 12^  cells treated with high doses of high specific activity I-sBSA and peptide conjugates. Experiment Treatment Mixed^ N and C Number o f C e l l s : " None2 1 2 5 3 '"i-sBSA" 3 1 2 5 4 '"i-NsBSA^ 1 2 5 5 ' " i -CsBSA 3 Expected Observed 1 1 . 8 7 1 0 0 % 7 5 . 8 % 1 0 8 % 9 1 . 9 % 6 8 . 9 % 2 2 . 0 9 1 0 0 0 . 0 2 5 . 7 2 5 . 7 3 9 . 5 1 3 2 . 3 1 1 0 0 1 5 . 3 6 9 . 5 4 2 . 4 5 . 3 9 4 1 . 9 3 4 5 . 1 8 3 . 3 1 3 5 . 7 1 0 9 . 5 1 9 0 . 4 5 1 . 6 1 1 1 1 . 4 6 0 . 3 - 1 1 . 8 3 5 . 9 6 9 . 1 6 1 . 8 9 8 9 . 9 6 3 . 8 6 5 . 0 6 4 . 4 7 0 . 0 7 5 . 8 6 8 9 . 9 9 0 . 6 9 0 . 4 9 0 . 5 1 6 7 . 2 8 4 . 4 0 1 1 1 . 5 5 8 . 8 7 4 . 7 6 6 . 7 7 7 . 5 9 2 . 0 2 8 0 . 4 4 2 . 7 3 1 . 7 3 7 . 2 4 6 . 3 1 0 3 . 7 1 9 5 . 9 5 6 . 1 6 5 . 8 6 1 . 0 7 1 . 5 Average: 9 2 . 4 ± 1 9 . , 1 5 4 . 6 ± 2 8 . 6 6 7 . 8 ± 3 8 . 0 6 2 . 5 ± 2 7 . 8 8 0 . 6 ± 5 6 . 3 P Values: P « 0 . 0 0 5 P « 0 . 0 1 P « 0 . 0 0 5 (Chi square analysis) Antigen dose used was 1.0 yg 12 x 10' lymphocytes. Specific activities of labelled preparations ranged from 2 0 0 - 1 0 0 0 uCi/u-g Ratio of ^-thymidine incorporation in triplicate cultures containing OFD (4.8 yg /ml) to those without OFD. Average percent of control OFD response over background after treating cells with high specific activity 1 2 5 I -sBSA. 1 2 5 Average percent of I-sBSA control response over background after treating cells with high specific activity 1 2 5 I _ N S B S A . 1 2 5 As in 4 except cells were treated with high specific activity I-CsBSA. Percentage of the 1 2 5 I -sBSA control OFD response of a mixture of 1 2 5I-NsBSA and 1 2 5I-CsBSA treated cel ls . The expected value is calculated as the average of the sum of the contribution of the two populations i f no synergy occurs. TableXXIV . KLH stimulation of H-thymidine incorporation in cultures of KLH sensitized guinea pig lymph node 1 1 ?5 cells treated with high doses of high specific activity I-sBSA and peptide conjugates. Experiment Number Treatment « of Cells:- None^  1 2 5I-sBSA 3 125 4 '"i-NsBSA4 1 2 5I-CsBSA5 Mixed6 Expected N and C Observed 1 9.63 8.24- 83.8% 92.4% 128.7% 110.5% 95.9% 2 1.92 1.92-100.0 176.0 147.8 161.9 147.8 3 10.61 10.61-100.0 49.0 83.4 66.1 39.5 4 14.60 10.67- 70.7 63.2 81.0 72.1 63.5 5 10.02 9.02- 88.9 170.8 102.1 136.4 103.6 6 9.06 9.10-100.5 99.0 115.5 107.2 121.1 7 11.93 11.90- 99.7 89.9 88.0 88.9 62.7 8 3.35 3.12- 90.21 91.0 92.5 91.7 96.2 9 2.59 1.58- 36.4 112.0 124,1 118.0 189.6 10 3.77 3.11- 76.1 95.3 95.7 95.4 94.3 Average: 84.6% 103.8% 105.9% 104.8% 101.4% P Values: P = 0.10 P = 0.025 P < 0.005 (Chi square analysis) As in Table XXIII. 'As in Table except the stimulating antigen was KLH (20 ug./Ml). Table XXV- OFD stimulation of H-thymidine incorporation in B cell depleted cultures of OFD sensitized guinea 1 125 pig lymph node cells treated with high doses of high specific activity I-sBSA and peptide conjugates. Experiment Number Percent** B Cells Treatment ? of Cells:- None 125 3 '"I-sBSA"3 125 4 1 0I-NsBSA* 1 2 5I-CsBSA5 Mixed** Expected N and C Observed 1 2.6 1.80 108.7 50.6% 19.5 % 35.0 % 28.7 % 2 2.4 2.07 100.0 0.0 (119.6f* (119.6)* (165.4)* 3 1.6 3.36 95.8 50.4 50.0 50.2 62.4 4 8.4 2.26 86.5 48.6 29.4 38.9 88.0 5 14.2 3.11 95.3 34.3 39.3 36.8 51.2 Average: 97.3 ± 8.0 36.6 ± 21 .6 34.6 ± 13 .0 40.2 ± 6.9 57.5±24.6 P Values: P « 0.005 P « 0.005 P « 0.005 (Chi square analysis) As in TableXXIII. Cells in experiment 1-3 were depleted of B cells by passage through nylon fibre columns, and in experiments 4-5 by adherence to anti-IgG plates. 125 As detected by I anti-IgG autoradiography. Not included in Chi square analysis. Not included in average. Table XXVI- KLH stimulation of H-thymidine incorporation in B cell depleted cultures of KLH sensitized 1 125 guinea pig lymph node cells treated with high doses of high specific activity I-sBSA and peptide conjugates. Experiment Number g Percent B Cells Treatment of Cells:-2 None 1 2 5I-sBSA 3 125I-NsBSA4 1 2 5I-CsBSA 5 Mixed6 Expected N and C Observed 1 2.6% 2.68 95.8% 100.0 % 99.3% 99.7 % 93.2 % 2 2.4 3.39 67.3 104.9 117.3 (111.1)* (18.0)* 3 1.6 7.02 109.8 66.7 66.7 66.7 75.0 4 8.4 8.49 71.0 57.33 56.3 56.8 70.3 5 14.2 7.11 98.5 101.5 98.1 99.8 98.3 Average: 88.5+18.4 86.0 ± 22.3 87.5 ± 25.2 86.8 ± 23.6 84.2 ± 13.6 P Values: p » 0.10 p » 0.10 p < 0.05 1-6 7-8 * As in TableXXIIexcept KLH (20 yg /ml) was the stimulating antigen. As in Table XXV. Not included in Chi square analysis. 103 IX Concluding Discussion The binding of antigen is believed to be the initial step in the complex sequence of events leading to an immune response specific for the inducing antigen and has led many investigators to examine and de-fine the different parameters affecting the antigen binding reaction. A major shortcoming of most of these investigations is the failure to con-sider the concept that the antigens used possess several, if not many, different antigenic determinants. Presumably these antigens can react with cells possessing specific receptors for any one of the antigenic determinants. The number of cells specifically binding a particular antigenic determinant would therefore be much less than the number bind-ing an antigen containing multiple determinants, an idea which helps explain the observed high frequency of antigen binding cells in virgin populations. While attempts to determine the frequency of antigen bind-ing cells for a single antigen have been successful, a reliable estimate of functional binding cells is hampered by cross-reactivity and binding of determinants on the antigen which do not invoke an immune response. Studies on the diversity of receptors on cell surfaces are also made dif-ficult by employing antigen binding experiments using multideterminant antigens. The aim of this thesis was to examine cells binding the antigenic determinants of C_. pasteurianum oxidized ferredoxin and to study their functional contribution in the eel 1-mediated immune response to the ferredoxin molecule and its synthetic analog N-JQC. It was hoped that binding studies using highly defined antigenic determinants would allow 104 greater definition of the significance of antigen binding cells than has been previously available using undefined antigens. Antigen Binding Macrophages Much recent work has shown that macrophage bound antigen elicits cell-mediated immunity(102,114129). It is clear that macrophages do not determine the specificity of the immune response and there is evidence that the extent to which these cells are needed depends upon the nature of the antigen employed (131). Recent work by Rosenthal and Shevach (102, H4)demonstrated that the recognition of soluble protein antigens by guinea pig T lymphocytes requires the presentation of antigen on histo-compatible macrophages, and that this interaction between macrophage and T lymphocyte can be blocked by alloantisera, implicating the existence of a specific associative event between macrophage bound antigen and T lymphocytes. The in vitro results in this thesis showed that macrophages from immune and non-immune guinea pigs bound radiolabelled Nh -^terminal and COOH-terminal antigenic determinants from the oxidized ferredoxin mole-cule. The binding was energy dependent and non-specific, as up to 92% of macrophages became labelled with the iodinated peptide conjugates, Further work involving macrophage removal is necessary to determine the functional involvement of macrophages in the cellular response to OFD. Antigen Binding Lymphocytes Lymphocytes binding the peptide determinants were found in both 1 0 5 immunized and unimmunized guinea pigs. Since all binding experiments were performed under conditions inhibiting pinocytosis, i t seems likely that the binding of determinants was due to the energy-independent asso-ciation of the determinants with specific receptors on the cell surface. -4 The frequency of cells binding a given determinant was 10 in unimmu-nized animals and increased 4 - 6 fold in O F D and N-JQC immunized guinea pigs. This frequency is rather higher than expected although it must be considered that the system used relatively high concentrations of labelled conjugates and that high avidity binding would be expected with multideterminant molecules. No significant binding differences between the Nh -^terminal and COOH - terminal determinants were found. The absence of cells binding radiolabelled s-BSA supports the view that specific binding was being observed. The use of high specific activity radiolabeled molecules in antigen binding studies revealed an important observation, The frequency of deter-minant binding lymphocytes increased several fold when high specific activity molecules (100 - 300 yCi/yg) were used, thereby detecting anti-gen binding cells that would have gone undetected using standard techniques. Antigen Binding T and B Lymphocytes Specific T and B lymphocytes can be inactivated by highly radioactive antigen indicating that both T and B cells have antigen specific receptors (9,100). Thus labelled cells observed by autoradiography after incuba-tion of lymphocytes with radioactive antigen of high specific activity should represent a mixture of specific T and B cells. These T and B 106 subpopulations of lymphocytes can be distinguished by anti-immunoglobu-lin antisera; only B cells bind sufficient antibody to be detected by low specific activity I anti-immunoglobulin autoradiography (21). The presence of surface immunoglobulin on guinea pig lymphocytes also leads to their specific retention on anti-IgG coupled nylon plates (Fumio Takei - unpublished data). The effluent population from these plates are enriched in Ig-negative cells and the majority of Ig-bearing cells are removed. Similarly, but perhaps with less specificity, Ig-bearing cells are removed from a population of cells by passage through nylon fibre columns (38). Unfractionated cells and cell populations depleted of B cells using anti-Ig plates and nylon columns both contained antigen binding cells specific for the NH -^terminal and COOH-terminal determinants, at least when measured using high specific activity anti-gens. The observation that the B-cell depleted population contains anti-gen binding cells suggests that these cells are antigen binding T lympho-cytes. Several alternative explanations exist: 1) The antigen binding cells are B cells which escaped retention on the anti-IgG plates or nylon fibre columns. This is unlikely as 50% of the antigen binding B lymphocyte population would have to elute consistently in several experiments. Two classes of B cells may exist thus allowing selective retention of one of these B cell classes, although the B cell content varied markedly from experiment to experiment and makes this explana-tion difficult to believe. 2) Recently, it was shown by Webb and Cooper (l36)that cytophilic 107 IgM antibody can adsorb to the surface of T cells. Thus i t is possible that the lymphoid cells in an immune animal can bind antigen through acquired antibody. The failure to detect anti-body to OFD in immunized guinea pigs (135), and the use of extensively washed cells in the work described here argues against binding via passively acquired antibody on T cells. It is possible, however, that acquired antibody on T cells is responsible for antigen binding as only very few molecules of antibody would be required. If, however, this were the case9 it would not invalidate the observations of this work indicat-ing the T cell function can be depressed specifically by inac-tivating binding cells. 3) The cells binding the antigenic determinants are T lymphocytes. This is the most likely explanation and is supported by the decreased T cell stimulation after specific antigen suicide on the B cell depleted populations. Most reports of failure to detect T cells binding soluble antigen have involved the use of low specific activity radiolabeled molecules. Successful detection of antigen binding murine T lymphocytes was reported by Roelants (101) using high specific activity iodinated molecules and recently by Kelly et al_ (60) using the ferredoxin peptides coupled to poly D glutamic acid. It therefore appears that detection of antigen binding T cells depends upon the sensitivity of the assay system used, either because of small amounts of antigen bound by T Lymphocytes or because of the transient nature of antigen binding, problems seemingly 108 overcome by the use of highly radioactive or multideterminant molecules with high binding avidity. Lymphocyte stimulation results obtained with suicided cell popula-tions indicated that specific inactivation of cells responding to OFD and N^ QC was achieved. The responses to the unrelated antigens KLH and PPD were not significantly reduced although some lowering of stimulation was seen in several experiments, possibly due to irradiation. The results 125 from I-sBSA treated cells are consistent with this idea. Successful 125 antigen suicide was achieved with *~^io^ a n c ' w ^ t ' 1 k°* n a n t i9 e m ' c determinants, although the [^-terminal conjugate was slightly more ef-fective. The reason for this is not clear; perhaps slightly greater binding affinity of the ^-terminal accounts for the difference. Alternatively, the radiation from the NH2-terminal determinant may be 125 more effective as the I label is part of the determinant itself whereas the COOH-terminal contains no tyrosine and the radioiodine is carried only in the s-BSA molecule. Several problems were encountered in the suicide procedure: 1) Abrogation of the stimulation response was complete in only a few experiments. Autoradiography showed that a proportion of the determinant binding cells remained after antigen suicide. These cells may have responded to antigenic stimulation. Since the lymphocyte stimulation procedure utilizes sensitized lymph-ocytes, numbers of responding cells may be at different stages in the cell cycle and may escape inactivation either by binding insufficient antigen or by not binding antigen at al l . 109 Progenitor development may occur over the period of assay, relatively unaffected by antigen suicide, and may respond to stimulation. Complete specific abrogation of antibody responses has been reported by several investigators (9, 132) although in a l l cases antigen suicide was performed on virgin (unsensitized) cel l populations using adoptive transfer systems in inbred mice. A similar approach could be used in the work reported here using OFD (and synthetic peptide analogues) with inbred guinea pigs, an attack which was not carried out due to unavailability of these animals. 3 Scin t i l l a t i o n counting of H-thymidine incorporated into the DNA of responding lymphocytes was hindered by the low energy 125 gamma emissions from bound I-labelled molecules. This prob-lem was partially solved by incubating the suicided cells in serum-free medium for 3 hours prior to washing and culturing. Cell surface proteins are shed during this incubation (127) and 125 most of the bound I-labelled antigen was removed. Interfer-125 ence from remaining I was negated by subtracting counts 3 from replicate cultures that had not been pulsed with H-thymi-dine. Standard deviations between replicate samples were large, however, probably due to the high specific activity of the iodinated molecules. This may account for the great varia-tion in the suicide results. Ideally, the DNA should be purified and counted, a procedure which was not followed here because of the technical burden involved in processing large no numbers of cell cultures. Cell Cooperation If lymphocytes are restricted to reacting with a single specificity of antigenic determinant, then the work of Waterfield ejt al_ (135) and Levy et aj_ (64) suggests a two cell collaboration in the cell mediated response to the soluble protein antigen, OFD. The work in this thesis 125 was designed to test this suggestion. Mixing of I-NsBSA treated cul-125 tures with I-CsBSA treated cultures resulted in a partial restoration of the stimulation response to OFD suggesting synergy between cells of the suicided cultures. Mixing experiments performed with B cell depleted cultures indicated that the observed synergy was between T lymphocytes. The response to KLH was not increased significantly in the same mixed populations, demonstrating that the observed synergy was specific for OFD stimulation. There are several possible explanations for these observa-tions: 1) The increased stimulation may be due to priming of cells by residual (bound) N-sBSA and C-sBSA. Mixing of these cultures would result in a greater response to OFD than the cultures con-taining cells treated with only one determinant. This is an unlikely possibility, however, as the amount of bound peptide conjugate is insufficient to cause stimulation. The culture period is also too short to allow primary sensitization to the peptide conjugates. 2) The synergy is due to specific interaction between cells binding I l l the Nh -^terminal and COOH-terminal determinants, the mixed cul-tures containing viable cells of both types. This second explanation is exciting as it is the first indication of T cell interaction to a soluble protein antigen. 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