M. Z. Atassi

Genetic control and intersite influences on the immune response to sperm whale myoglobin.

Determination of the precise antigenic structure of sperm-whale myoglobin (Mb) has enabled us to focus our attention on the molecular and cellular factors that control and regulate the immune responses to the protein antigen. Our studies have shown that the immune responses to sperm-whale Mb are controlled by genes in the I region of the major histocompatibility complex (H-2) of mice. More importantly, the responses to the synthetic antigenic sites are each under separate genetic control. The recognition of the antigenic sites by antibodies is independent of the immunized species and of the time the antisera are obtained after the initial immunization (from nine days up to a year). The same sites are recognized by antisera raised in rabbit, goat, pig, cat, chicken and outbred and inbred mice. The same sites recognized by mouse B-cells are also recognized by mouse T-cells. No meaningful genetic control of antibody affinity was observed. Autoimmune antibody and T-lymphocyte proliferative responses were readily generated by immunizing an animal with self-Mb. With mouse Mb, the autoimmune T-lymphocyte response was under genetic control and mapped with the I-A and the H-2D end of the H-2 gene complex. In other recent studies we have shown, using several Mb variants, that the binding capacity of an antigenic site is fully accounted for by substitutions in the antigenic sites (actual contact residues) and in residues close (within 7.A) to the sites (i.e. environmental residues). The overall response to Mb is regulated by inter-site influences which can either be of a cooperative (help) nature or of a suppressive nature. Finally, genetic control of the responses to individual antigenic sites on a protein is not only determined by the genetic constitution of the host but also by the chemical properties of the individual sites. The H-2 subregions mapping the responses to given antigenic sites can also recognize other sites, which were previously unrecognizable in a homologous protein, if the chemical properties of these sites are suitably altered.

T‐LYMPHOCYTE RECOGNITION OF SPERM‐WHALE MYOGLOBIN. SPECIFICITY OF T‐CELL RECOGNITION FOLLOWING NEONATAL TOLERANCE WITH EITHER MYOGLOBIN OR SYNTHETIC PEPTIDES OF AN ANTIGENIC SITE

Recently, in investigating the responses of T‐cell from high responder mice that were primed with myoglobin (Mb) or with synthetic peptides containing antigenic site 5 and increasing in length stepwise by increments of two residues, we observed that T‐cell recognition was highly dependent on conformation. In the present studies, tolerization experiments were carried out to further investigate this finding. Neonatal mice (BALB/cByJ) were either tolerized with Mb or with synthetic peptides of Mb containing antigenic site 5. Tolerization with Mb and subsequent immunization with Mb gave T‐cells that did not proliferate in vitro to Mb or any of the peptides. T‐cells from mice that were tolerized with a truncated peptide 139‐153 (having deletions at Tyr‐151 and Ala‐144) and subsequently immunized with Mb proliferated in vitro to Mb and to peptides 132‐153, 135‐153 and 143‐153. T‐cells from mice that were tolerized with native Mb and subsequently immunized with peptides (which are unfolded in solution) did not proliferate in vitro to Mb, but responded well to the peptides. Conversely, tolerization with peptides had no effect on the recognition of, and the response to, native Mb by the T‐cells, whereas the response to the peptides was completely removed. It was thus concluded that the recognition of protein antigens (or at least of Mb) by T‐cells is (like the recognition by antibody) dependent on the conformation of the antigen.

T-lymphocyte recognition of sperm-whale myoglobin. Responses of T-cells from mouse strains primed with synthetic peptide carrying antigenic site 5 and relation to antigen presentation.

In the preceding communication of this series, the fine specificity requirements for T‐cell recognition of one of the Mb antigenic sites (antigenic site 5) were examined. Seven synthetic peptides containing antigenic site 5 and progressively increasing in length to the left by increments of two residues up to 22 residues in length were studied with regard to their ability to stimulate T‐cell proliferation of Mb‐primed T‐cells from high responder H‐2d and H‐2s mouse strains. Unexpectedly, it was found that, unlike smaller and longer peptides, some intermediate‐sized peptides failed to stimulate T‐cell proliferation even though they contained the full antigenic site. This indicated that lack of proliferative stimulating activity by a peptide does not necessarily imply absence of an antigenic site in the peptide. The results enabled us to conclude that T‐cell recognition of native proteins (or at least of Mb) is dependent on protein conformation. In the present work, we have examined the ability of peptide‐primed T‐cells from H‐2d and H‐2s mouse strains to proliferate to Mb. Immunization with peptide 145‐151 (antigenic site 5) afforded T‐cells that did not proliferate in vitro to Mb or any of the synthetic peptides 145‐153, 143‐153, 141‐153, 139‐153, 137‐153, 135‐153 or 132‐153. When mice were primed with either peptide 143‐153 (11 residues) or peptide 132‐153 (22 residues), The T‐cells obtained did not respond to Mb but responded in each case very well to peptides 132‐153, 135‐153, 137‐153 and gave lower, but significant, response to the shorter peptides 145‐153 and 143‐153. Intermediate‐sized peptides did not stimulate T‐cell proliferations and neither did truncated peptides that had deletions at Tyr‐151 and Ala‐144. These findings underscore the fine specificity of the T‐cell and indicate a high level of conformational dependency for T‐cell recognition. It is also concluded that macrophage recognition and presentation of protein antigen must involve the intact native protein and it is probably not related to the processing and fragmenting of the antigen by macrophage. The latter activity is more related to the role of macrophage in clearance.

T‐ LYMPHOCYTE RECOGNITION OF SPERM‐WHALE MYOGLOBIN. RECOGNITION OF SYNTHETIC PEPTIDES CARRYING ANTIGENIC SITE 5 BY MYOGLOBIN‐PRIMED T‐CELLS

Previous studies from this laboratory have resulted in the determination of the antigenic structure of sperm‐whale myoglobin (Mb). In the present work, we have investigated the fine specificity requirements for T‐cell recognition of one of the Mb antigenic sites (antigenic site 5). The antigenic site (peptide 145‐153) and seven progressively longer peptides, increasing in length stepwise by two residues at a time, up to 22 residues in length (peptide 132‐153), were synthesized. In addition, four truncated peptides were synthesized with intentional deletions at Tyr‐ 151 and Ala‐ 144. The T‐cell recognition of these purified synthetic peptides was examined here in detail in three strains of mice (BALB/cByJ, B10.D2/n and SJL/J). Mb‐primed mice afforded T‐cells which proliferated to smaller peptides (two or four residues longer than the site; i.e. peptides 145‐153 and 143‐153) and more so to the longer peptides 135‐153 and 132‐153 and to Mb. No response was obtained to the truncated peptides, thus underscoring the fine specificity T‐cells. No response was obtained also to intermediate‐sized peptides. The latter result, due to an unfavourable mode of folding, suggested a conformational dependency in T‐lymphocyte recognition.

GENETIC CONTROL OF THE IMMUNE RESPONSE TO MYOGLOBIN: XVI. CONTROL OF ANTIBODIES WITH PRESELECTED SPECIFICITIES FOLLOWING IMMUNIZATION WITH FREE SYNTHETIC PEPTIDES REPRESENTING THE ANTIGENIC SITES OR SURFACE NON‐IMMUNOGENIC LOCATIONS IN THE PROTEIN

Previous studies in this laboratory have resulted in the determination of the antigenic structure of myoglobin. The present work was carried out to investigate the genetic control of the murine antibody response to myoglobin following immunization with free (i.e., not coupled to a carrier) synthetic antigenic sites or other peptides corresponding to surface regions of myoglobin that are not immunogenic when the native molecule is the immunizing antigen. Synthetic peptides corresponding to antigenic site 1 (peptide 15‐22), site 2 (peptide 56‐62), site 3 (peptide 94‐100), site 4 (peptide 113‐120), site 5 (peptide 145‐151) and two surface regions, peptide 1‐6 and peptide 121‐127, were injected in complete Freunds adjuvant in different strains of mice. Serum antibodies specific for myoglobin were subsequently obtained and were measured by means of a radioimmune plate binding assay in which Mb was used as the solid phase antigen. It was found that the genetic control of the antibody response to myoglobin following immunization with the free synthetic peptides was different from the genetic control obtained following immunization with native myoglobin. The significance of this finding is discussed.

GENETIC CONTROL OF THE IMMUNE RESPONSE TO HAEMOGLOBIN: IV. Ly‐1+ T‐CELLS AND APPROPRIATE NON‐H‐2 GENES ARE REQUIRED FOR IN VITRO RESPONSES TO α ‐ AND β‐SUBUNITS OF HUMAN ADULT HAEMOGLOBIN

Experiments were conducted to determine if non‐H‐2 gene effects could be demonstrated in mice which had been primed to either the α‐subunit or β‐subunit of human haemoglobin. It was found that C3H.SW (H‐2b) and Balb/c (H‐2d) mice are low responder mice to α‐chain of a haemoglobin when compared to H‐2 identical B10 (H‐2b) and B10.D2(H‐2d) mice. B120.S and A.SW (both H‐2s) are responsive to β‐chain challenge while Balb/c mice are low responders in contrast to high responder B10.D2 mice. Ly‐1+ cells were demonstrated to be required (by cell depletion experiments) for an in vitro T‐cell proliferative response to either subunit. In these experiments, Ly‐2+ cells were not of crucial importance.

Myoglobin-reactive T cell clones.

Myoglobin has been used for a considerable period of time as a “model” antigen for immunological studies. Since the sequence and crystal structure of myoglobin from sperm whale skeletal muscle are well known, myoglobin can be studied in great detail as an antigen. The immunological studies that have been done with Mb to date have provided useful information bearing on a number of important questions. We have used myoglobin as a model antigen to study via the new and powerful techniques made possible by T cell cloning. Since the T cell clones which we utilize are of the helper/inducer subset (TH, see below) and therefore occupy a central role in vivo in controlling immune responsiveness, these techniques should provide both specific and generalized information relating to the immune responses to complex protein antigens.

Genetic Control of the Immune Response to Hemoglobin and its Subunits

Human adult hemoglobin (Hb) was selected as a model oligomeric protein for investigations into Ir gene function. Separate genetic control of the α- and β-subunits of Hb was demonstrated. The relevant Ir genes were mapped to the I-A subregion and in particular to the Aβ gene for the α-chain of Hb. H-2b and H-2d mice are responders to the α-subunit and H-2d, H-2f and H-2s mice are responsive to the β-subunit. The subunits are not cross-reactive in T-cell assays. Non-H-2 linked genes are also important in Hb-specific immunity. There is a requirement for Ly-1+ T-cells for optimal responses in vitro.

Evidence for cellular but not serologic cross-reactivity between keyhole limpet hemocyanin and sperm-whale myoglobin

Abstract The addition of keyhole limpet hemocyanin (KLH) to cultures of rabbit lymph node cells (LNC) primed with KLH and sperm-whale myoglobin (Mb) induced the synthesis of antibody to Mb as well as to KLH. Several mechanisms for this heterologous induction were considered. It was established that KLH does not nonspecifically activate rabbit T or B lymphocytes. It was also shown that KLH and Mb do not cross-react serologically by several sensitive and specific criteria. Therefore, it was surmised that heterologous induction of Mb antibody synthesis by KLH was due to cellular cross-reactivity between these proteins. Rabbits were primed by the injection of Mb-complete Freunds adjuvant (CFA), alum-Mb, or alum-KLH, and their LNC challenged with KLH, Mb, and synthetic antigenic sites of Mb. These experiments yielded much and diverse evidence for cellular cross-reactivity between KLH and Mb, and especially between KLH and the Mb peptides: KLH plus Mb-primed LNC evoked enhanced anti-KLH and anti-Mb syntheses. KLH plus KLH-sensitized LNC resulted in a lowered anti-Mb antibody response. Mb added to Mb-educated LNC either enhanced or inhibited the anti-KLH antibody response, depending on whether the priming adjuvant was CFA or alum. The addition of Mb to KLH-primed cells enhanced or inhibited the ensuing anti-Mb antibody synthesis; KLH did not affect or inhibit anti-KLH antibody synthesis. Addition of synthetic Mb antigenic sites to Mb-sensitized LNC elevated or suppressed anti-KLH antibody production, depending on the length of time between priming and in vitro challenge. A mixture of KLH and Mb peptide lowered the anti-Mb antibody response of Mb-educated LNC compared to KLH alone. A combination of KLH and Mb peptide also reduced the anti-KLH antibody synthesis of KLH-primed cells compared to KLH per se. The addition of KLH to Mb-sensitized LNC enhanced their uptake of tritiated thymidine, and their transport of tritiated cyclic AMP and protein synthesis. Added Mb induced the synthesis of protein and nonspecific IgG by KLH-primed LNC; Mb peptides evoked protein synthesis by these cells. It is postulated that cross-reactivity at the T-cell level is responsible for the induction of Mb antibody synthesis by adding KLH to either Mb-primed or KLH/Mb-primed LNC. The implications of these findings with respect to cellular and humoral immunity are discussed.

IN VITRO RESPONSES OF MYOGLOBIN-PRIMED LYMPH NODE CELLS TO MYOGLOBIN AND MYOGLOBIN SYNTHETIC ANTIGENIC PEPTIDES

The ultimate objective of the newer immunology is to understand various immune responses and reactions in cellular and molecular terms. The relevant molecules include various antigenic determinants, receptors and antigen-binding molecules on lymphocytic surfaces, molecules produced and secreted by lymphocytes, and lymphocytic surface molecules that recognize and are triggered by helper and/or regulatory molecules produced by other cells (Cold Spring Harbor Symposia, 1976). These cellular and molecular mechanisms have been illuminated by studies with hapten-protein and synthetic peptide systems in inbred strains of mice. Beginning in the early 1970’s Goodman utilized glucagon, Benjamini Tobacco mosaic virus protein and then Levy ferredoxin to obtain much new information about the relationships between antigenic structure of these proteins and their peptides and the capacity of these molecules to induce various immune reactions. We assumed that many questions about such relationships could be approached incisively with peptides derived from proteins of known molecular and antigenic structure. It was also hoped that the complexity of immune responses would be reduced by studying the results of adding single peptides to lymphocytes; this was based on the assumption that some of this complexity was due to the response of different clones of T and/or B lymphocytes to the different antigenic determinants on a protein. We proposed to utilize small peptides approximating in size one antigenic determinant, i.e., a tetrapeptide (Schechter et al., 1966) because such peptides would not effect the cross-linkage of receptors considered necessary for lymphocytic activation (Fanger et al., 1970).