Prediction of immunodominant helper T cell antigenic sites from the primary sequence

We have used a data base of 23 known immunodominant helper T cell antigenic sites located on 12 proteins to systematically develop an optimized algorithm for predicting T cell antigenic sites. The algorithm is based on the amphipathic helix model in which antigenic sites are postulated to be helices with one face predominantly polar and the opposite face predominantly apolar. Such amphipathic structures can form when the polarity of residues along the sequence varies with a more or less regular period. Hence they can be identified by methods (so called power spectrum procedures) that detect periodic variations in properties of a sequence. The choice of power spectrum procedure, hydrophobicity scale, and model parameters are examined. An algorithm is tested by comparing the predicted amphipathic segments with the locations of the known T cell sites, counting the number of matches, and calculating the probability of getting this number by chance alone. The optimum algorithm, which predicts the largest number of sites with the lowest chance probability, uses the Fauchere-Pliska hydrophobicity scale and a least squares fit of a sinusoid as its power spectrum procedure. By applying this algorithm, 18 of the 23 known sites are identified (75% sensitivity) with a high degree of significance (p less than 0.001). The success of the algorithm supports the hypothesis that stable amphipathic helices are fundamentally important in determining immunodominance. This approach may be of practical value in designing synthetic vaccines aimed at T cell immunity.     

Strong conformational propensities enhance T cell antigenicity

The ability to predict T cell antigenic peptides would have important implications for the development of artificial vaccines. As a first step towards prediction, this report uses a new statistical technique to discover and evaluate peptide properties correlating with T cell antigenicity. This technique employs Monte Carlo computer experiments and is applicable to many problems involving protein or DNA. The technique is used to evaluate the contribution of various peptide properties to helper T cell antigenicity. The properties investigated include amphipathicities (alpha and beta), conformational propensities (alpha, beta, turn and coil), and the correlates of alpha-helices, such as the absence of helix-breakers and the positioning of the residues which stabilize alpha-helical dipoles. We also investigate segmental amphipathicity. (A peptide has this property when it contains at least two disjoint subpeptides, one hydrophobic, one hydrophilic.) Statistical correlations and stratifications assessed independent contributions to T cell antigenicity. The findings presented here have important implications for the manufacture of peptide vaccines. These implications are as follows: if possible, peptide vaccines should probably be those protein segments which have a propensity to form amphipathic alpha-helices, which do not have regions with a propensity to coil conformations, and which have a lysine at their COOH-terminus. The last two observations are of particular use in manufacturing peptides vaccines: they indicate where the synthetic peptides should be terminated. These implications are supported by the findings given below. The significances (p values) support the following statistical generalites about antigenic conformations: most helper T cell antigenic sites are amphipathic alpha-helices; alpha-helical amphipathicity and propensity to an alpha-helical conformation contribute independently to T cell antigenicity; there is evidence that some T cell antigenic sites are beta conformations instead of alpha-helices; T cell antigenic sites avoid random coiled conformations; and T cell antigenic sites are usually not segmentally amphipathic. alpha-Helical amphipathicity was significant, but segmental amphipathicity was not. This has implications for the dimensions of the structure interacting with the hydrophobic portion of an amphipathic T cell antigenic site. Lysines are unusually frequent at the COOH-terminal of T cell antigenic sites, even after accounting for tryptic digests. These lysines can stabilize alpha-helical peptides by a favorable interaction with alpha-helical dipoles.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structural features of T-cell recognition: applications to vaccine design

T lymphocytes, in contrast to antibodies, appear to recognize primarily a limited number of antigenic sites on any given antigenic protein. We find that a single site can so dominate the T-cell repertoire that the presence or absence of a response to one immunodominant site can make the difference between a high responder and a low responder, even though low responders respond to other sites almost as well as high responders. Besides interaction with major histocompatibility complex (MHC) molecules, the mode by which the antigen is processed into fragments for T-cell recognition also determines which sites are seen. The products of natural processing of the protein appear to be larger than the synthetic peptides and contain structures which hinder binding to certain MHC molecules or to the T-cell receptor. A third factor in immunodominance is the intrinsic structure of the antigenic site. We have shown that amphipathic helices have a higher than random chance of being immunodominant, and have developed a computer program to locate such structures in protein amino acid sequences. We prospectively predicted sites in the malaria circumsporozoite protein and found that the four most widely recognized sites in an endemic area of West Africa were all predicted. Similarly, we identified two helper T-cell sites from the HIV (AIDS virus) envelope, and have now shown that immunization with these elicits enhanced antibody responses to the whole envelope when injected into monkeys. These sites are also recognized by human T cells from volunteers who had been immunized with a recombinant vaccinia virus expressing the HIV envelope. Also, because cytotoxic T lymphocytes (CTLS) may play a critical role in defence against AIDS, we have used a recombinant vaccinia virus and transfectants expressing the HIV envelope gene to induce specific CTLS against the HIV envelope. Using synthetic peptides, we were able to identify the first CTL recognition site in the AIDS virus. These results may contribute to the rational design of vaccines.     

In vitro selection of SV40 T antigen epitope loss variants by site-specific cytotoxic T lymphocyte clones

SV40-transformed cells of C57BL/6 (B6) mouse origin (H-2b) express four distinct predominant antigenic sites, I, II, III, and IV, on SV40 large tumor (T) Ag that are recognized by SV40 T Ag-specific CTL clones. In this study, we selected SV40 T Ag-positive cell lines which had lost one or more of the antigenic sites, by in vitro cocultivation of a SV40-transformed B6 mouse kidney cell line (K-0) with SV40 T Ag site-specific CTL clones, Y-1 (site I specific), Y-2 (site II specific), Y-3 (site III specific), and Y-4 (site IV specific). All of the CTL-resistant cell lines expressed large quantities of cell surface H-2 class I Ag. K-1 cells selected by CTL clone Y-1 lost the expression of antigenic sites I, II, and III, but not site IV. K-2 and K-3 cells selected by CTL clones Y-2 and Y-3, respectively, were found to be negative for sites II and III but expressed sites I and IV. K-4 cells selected by CTL clone Y-4 lost the expression of only site IV. K-1,4 cells (sites I-, II-, III-, IV-) were selected from K-1 cells by cocultivation with CTL clone Y-4, K-2,4 cells (sites I+, II-, III-, IV-) were selected from K-2 cells by CTL clone Y-4. K-3,1 cells (sites I-, II-, III-, IV+) were selected from K-3 cells by CTL clone Y-1, and K-3,1,4 cells (sites I-, II-, III-, IV-) were selected from K-3,1 cells by CTL clone Y-4. From K-4 cells, K-4,1 cells (sites I-, II-, III-, IV-) and K-4,3 cells (sites I+, II-, III-, IV-) were selected by CTL clone Y-1 and Y-3, respectively. The antigenic site loss variant cell lines K-1, K-1,4, K-3,1 K-3,1,4, K-4,1, and K-4,3 synthesized SV40 T Ag molecules of 75, 75, 78, 78, 81, and 88 kDa, respectively. Expression of wild-type SV40 T Ag in the antigenic site loss variants by infection with SV40 or transfection with cloned SV40 DNA restored the CTL recognition sites on the variant cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

PROANAL version 2: multifunctional Program for analysis of multiple protein sequence alignments and for studying the structure-activity relationships in protein families

A new version of the program PROANAL is described. A multiplelinear regression analysis of the protein structure–activityrelationship allows one to investigate the combina–tionsof protein sites and factors influencing the activity. The programalso provides the possibility to seek out protein sites, conservativeor variable in variations of physico–chemical characteristics,and regions with high or low values of these characteristics.PROANAL2 may be useful in the simulation of protein–engineeringexperiments and in the search of a number of protein regionssuch as functional sites, secondary structures, solvent-exposedregions, T– and B–cell antigenic determinants, etc.     

Sequence-Based Antigenic Change Prediction by a Sparse Learning Method Incorporating Co-Evolutionary Information

Rapid identification of influenza antigenic variants will be critical in selecting optimal vaccine candidates and thus a key to developing an effective vaccination program. Recent studies suggest that multiple simultaneous mutations at antigenic sites accumulatively enhance antigenic drift of influenza A viruses. However, pre-existing methods on antigenic variant identification are based on analyses from individual sites. Because the impacts of these co-evolved sites on influenza antigenicity may not be additive, it will be critical to quantify the impact of not only those single mutations but also multiple simultaneous mutations or co-evolved sites. Here, we developed and applied a computational method, AntigenCO, to identify and quantify both single and co-evolutionary sites driving the historical antigenic drifts. AntigenCO achieved an accuracy of up to 90.05% for antigenic variant prediction, significantly outperforming methods based on single sites. AntigenCO can be useful in antigenic variant identification in influenza surveillance.     

An induced fit hypothesis for antigen recognition by T lymphocytes: a role for specific antigen retention structures on antigen-presenting cells

The nature of T lymphocyte recognition of foreign antigens is not known, despite recent advances in elucidating the cellular structures that may be involved in the specific interactions. The central difficulty in this process is that T cells respond to foreign antigen only in the context of major histocompatibility complex (MHC) antigens expressed by another antigen-presenting cell. In addition, T cells that interact with class II MHC antigens do not bind foreign protein antigens in their native form, but seem to recognize only proteolytic peptide fragments as the relevant antigen. The simplest explanation for these observations is that the class II MHC antigens themselves bind antigenic peptides to form the appropriate determinant that interacts with the antigen-specific T cell receptor. However, to date no such antigenic complex has been found with MHC antigens despite rigorous attempts at their demonstration. One alternative explanation described here is that there is no preexisting foreign antigen-MHC antigen complex prior to interaction with T cells, and it is the T cells that cause the two moieties to become associated for recognition by a single antigen-specific T cell receptor. Central to this mechanism is that foreign antigenic peptides must be associated with specific antigen retention structures (SARS) expressed by antigen-presenting cells which retain and protect the peptide on the cell surface. These SARS, upon interaction with T cell membrane moieties, would subsequently associate with MHC antigens. A hypothesis to describe this mechanism is developed to account for published observations of antigen processing by antigen-presenting cells and T cell antigen recognition, and makes several predictions that are experimentally testable. This mechanism is also generally applicable to other cellular interactions in which soluble peptide mediators may become associated with surface components of one cell type, and this newly formed complex is in turn recognized by a receptor on a second cell type to deliver functional signals.     

Recognition of hepatitis B surface antigen by human T lymphocytes. Proliferative and cytotoxic responses to a major antigenic determinant defined by synthetic peptides

The antigenic sites for human T lymphocytes on hepatitis B surface Ag (HBsAg) were studied by using synthetic oligopeptides. T cell lines of the helper/inducer class, which were isolated from hepatitis B vaccine recipients, were found to react strongly and in an Ag-specific way with peptides corresponding to a sequence of 10 to 30 amino acids near the amino terminus of the HBsAg molecule. Cells with surface expression of the antigenic determinant contained in these synthetic peptides induced both proliferative and cytotoxic responses in the hepatitis B-specific T cells. The results indicate that amino acid residues 24-27 of HBsAg could be directly involved in this T cell determinant. Inhibition studies with mAb to MHC class II Ag and target cells from various HLA-typed individuals suggest that some T cell responses to this determinant of HBsAg might be restricted by the DPw4 molecule. However, the possibility exists that more than one of the MHC class II molecules could be involved as restricting elements of T cell responses to this synthetic peptide. In vivo experiments with synthetic peptides such as those described here are needed to demonstrate the possibility of enhancing HBsAg immune responses in some individuals.     

Fine mapping two distinct antigenic sites on simian virus 40 (SV40) T antigen reactive with SV40-specific cytotoxic T-cell clones by using SV40 deletion mutants.

The existence of two distinct antigenic sites at the surface of simian virus 40 (SV40)-transformed H-2b cells has been previously demonstrated (A. E. Campbell, L. F. Foley, and S. S. Tevethia, J. Immunol. 130:490-492, 1983) by using two independently isolated SV40-specific cytotoxic T-lymphocyte (CTL) clones, K11 and K19. We identified amino acids in the amino-terminal half of SV40 T antigen that are essential for the recognition of antigenic sites by these CTL clones by using H-2b cells transformed by mutants that produce T antigen truncated from the amino-terminal or carboxy-terminal end or carrying overlapping internal deletions in the amino-terminal regions of SV40 T antigen. The results show that CTL clone K11 failed to recognize and lyse target cells missing SV40 T-antigen amino acids 189 to 211, whereas CTL clone K19 lysed these cells. The cell lines missing SV40 T-antigen amino acids 220 to 223 and 220 to 228 were not lysed by CTL clone K19 but were susceptible to lysis by CTL clone K11. Two other cell lines missing amino acids 189 to 223 and 189 to 228 of SV40 T antigen were not lysed by either of the CTL clones but were lysed by SV40-specific bulk-culture CTL if sufficient amounts of relevant restriction elements were expressed at the cell surface. The SV40 T-antigen amino acids critical for the recognition of an antigenic site by CTL clone K11 were identified to be 193 to 211; 220 to 223 were identified as critical for recognition by CTL clone K19. The deletion of these amino acids from the T antigen resulted in the loss of antigenic sites specific for CTL clones K11 and K19.     

Molecular mapping of a histocompatibility-restricted immunodominant T cell epitope with synthetic and natural peptides: implications for T cell antigenic structure

We have prepared synthetic and natural peptides that have allowed delineation of a major antigenic site of myoglobin recognized by histocompatibility (I-Ed)-restricted T cell clones. The smallest peptide capable of stimulating T cell proliferation consisted of residues 136-146. Residues Glu 136, Lys 140, and Lys 145 were essential for antigenicity, whereas Lys 133 and Tyr 146 added potency but were not required for antigenicity. The periodicity of these residues suggests that folding the peptide into its native alpha-helical structure may be essential for antigenicity either by forming a hydrophilic binding site for the T cell receptor or by participating in antigen presentation. The same folding could also produce a hydrophobic site on the opposite side of the alpha-helix that could participate in hydrophobic interactions. The extrapolation of these findings to other known peptide antigens suggests that this tendency to form an amphipathic alpha-helix may be a general property of antigenic sites recognized by T cells, perhaps due to a different functional role of each type of site in eliciting T cell responses.     

Immunodominant sites of human T cell lymphotropic virus type 1 envelope protein for murine helper T cells

HTLV-I (human T cell lymphotropic virus type 1) is the retrovirus causally related to adult T cell leukemia/lymphoma and is also associated with a neurological disorder, tropical spastic paraparesis, or HTLV-I-associated myelopathy. The development of these two different diseases among HTLV-I-infected individuals may depend in part on differences in their T cell immunity associated with a difference of HLA phenotype. Peptides corresponding to 17 sites in the HTLV-I envelope protein were tested for their antigenicity for lymph node cells from B10.BR, B10.D2, B10.A(5R), and B10.HTT congenic mice, representing four independent MHC haplotypes, immunized with the native envelope protein. Ten of the 17 tested sites were predicted to be amphipathic alpha-helical sites and all of them were found to be antigenic for at least one of the four MHC congenic strains of mice. Three of the 17 sites were amphipathic 3(10)-helical sites and four sites were predicted to be non-helical sites: none of the 3(10)-helical sites were antigenic and only one of four non-predicted sites was found to be immunodominant. Furthermore, three potent immunodominant peptides, V1E1 (342-363), V1E8/SP4a (191-209), and V1E10 (141-156) were also shown to be immunogenic; i.e., these peptides could be used to immunize mice to elicit proliferative responses of lymph node cells to the native HTLV-I envelope protein. Furthermore, these three peptides were able to prime animals for an enhanced antibody response to the native protein. Because this priming followed the same Ir gene control as the proliferative response, it probably reflects the ability of these peptides to prime helper T cells. The localization of immunodominant sites in HTLV-I envelope protein in mice may be useful for finding antigenic and immunogenic sites in humans, for developing a peptide vaccine for the virus, and possibly for aiding in prognosis for the development of different disease manifestations of HTLV-I infection.     

Functionally distinct agretopic and epitopic sites. Analysis of the dominant T cell determinant of moth and pigeon cytochromes c with the use of synthetic peptide antigens

The dominant T cell determinant on moth and pigeon cytochromes c in B10.A (E beta k:E alpha k) mice is located in the C-terminal portion of the protein, contained within residues 93-103 or 93-104. Thirty-seven antigen analogs, containing single amino acid substitutions at positions 98, 99, 101, 102, 103, and 104, were synthesized. The effects of the substitutions on in vitro antigenicity and in vivo immunogenicity were determined. Functional assays with T cell clones identified residues 99, 101, 102, and 103 as critical, based on their effect on antigenic potency. Peptides containing substitutions at residues 99, 101, and 102 were capable of eliciting unique clones upon immunization of B10.A mice. This was consistent with the identification of these residues as part of the epitope, the site on the antigen that interacts with the T cell receptor. Immunization with peptides substituted at residue 103, however, failed to elicit clones with unique specificity for the immunogen. When these peptides were tested for their ability to stimulate the T cell clones with antigen-presenting cells from B10.A(5R) mice expressing the E beta b:E alpha k Ia molecule, a consistent change in the relative antigenic potency was observed with 50% of the peptides. The effect of the Ia molecule on the antigenic potency ruled out the possibility that residue 103 nonspecifically affected antigen uptake or processing and identified residue 103 as part of the agretope, the site that interacts with the Ia molecule. The locations of the agretope and the epitope on this antigenic determinant appear to be fixed, even in the presence of large numbers of amino acid substitutions. However, some substitutions were found to affect both the agretope and the epitope, placing limits on the functional independence of the two sites. The results are discussed in terms of the trimolecular complex model of T cell activation and the implications of these data for antigen-Ia molecule interactions.     

Human T cell differentiation antigens and correlation of their expression with various markers of T cell maturation.

Two sets of differentiation antigens are demonstrated on human T cells by using 11 heterologous anti-human antisera raised against various normal and malignant T cells. The two antigenic determinants from the first set of differentiation antigens are expressed only on thymus cells and on T lymphoblasts, whereas the two antigenic determinants from the second set are expressed on blood T cells, Sezary cells, T.CLL cells, and thymus cells. Four T cell phenotypes are thus defined; two phenotypes are expressed only by T lymphoblasts, whereas the other two phenotypes are expressed both by normal and malignant T cells. Moreover, a clear-cut relationship exists between the four T cell antigenic phenotypes and two other markers of T cell differentiation: terminal deoxynucleotidyl transferase and peanut agglutinin. Two phenotypes are linked with the presence of TdT, one phenotype is linked with the affinity for PNA, and the fourth phenotype is correlated with the absence of both markers.     

Brief antigenic stimulation generates effector CD8 T cells with low cytotoxic activity and high IL-2 production

It is currently believed that a brief antigenic stimulation is sufficient to induce CD8 T cells to complete their differentiation program, become effector T cells, and subsequently generate memory. Because this concept was derived from studies in which only a single effector function was analyzed (either IFN-gamma production or target cell lysis), we wondered whether monitoring for multiple effector functions might reveal novel characteristics of effector CD8 T cells elicited by brief or prolonged Ag exposure. Using an in vitro system to generate effector T cells and an in vivo adoptive transfer model to track donor CD8 T cells, we found that the differentiation programs acquired by CD8 T cells after brief or prolonged antigenic stimulation were different. Although the frequencies of IFN-gamma and TNF-alpha producers were comparable for both effector CD8 T cell populations, there were major differences in cytotoxic potential and IL-2 production. Whereas prolonged (>24 h) Ag exposure stimulated effector CD8 T cells with high cytotoxic activity and low IL-2 production, brief (<24 h) stimulation generated effector CD8 T cells with low cytotoxic activity and high IL-2 production. The latter effector T cells rapidly converted into central memory-like CD8 T cells, exhibited long-term survival in adoptively transferred hosts, and gave robust recall responses upon Ag challenge. These data suggest that not all functions of effector CD8 T cells are equally inherited after brief or prolonged antigenic stimulation.     

Peptide epitope mapping in vaccine development: Introduction

Protection from infectious disease by the host immune response requires specific molecular recognition of unique antigenic determinants of a given pathogen. An epitope is an antigenic determinant which: 1) specifically stimulates the immune response (either B or T cell mediated); and 2) is acted upon by the products of these protective mechanisms. In B cell immunity, antibodies produced from stimulation by specific epitopes recognize and bind to these same antigenic structures. Identification of protective epitopes is extremely valuable to successful vaccine development. In order to be protective these antibodies must, in addition to recognition and binding, interfere with some vital step in pathogenesis such as adherence or toxin action. Protein B cell epitopes are frequently composed of the side chains (R-groups) of the amino acids found at solvent-exposed surfaces. These epitopes are classified as continuous (also linear or sequential) if composed of a single antibody-recognizing element located at a single locus of the primary structure. They are discontinuous (or assembled) if more than one physically separated entity is involved. T cell epitopes are peptides on the surface of antigen-presenting cells (macrophages, dendritic cells, and B cells) that are bound to major histocompatibility proteins; the T cell recognizes this peptide-MHC complex. Received 12 August 1996/ Accepted in revised form 03 November 1996     

Central role of thrombospondin-1 in the activation and clonal expansion of inflammatory T cells

Thrombospondin-1 (TSP) is a transiently expressed matricellular protein known to promote chemotaxis of leukocytes to inflammatory sites. However, TSP and its receptor CD36 are abundantly expressed in chronically inflamed tissues such as the rheumatoid synovium. Here, we show that TSP provides the costimulatory signal that is necessary for the activation of autoreactive T cells. Data presented reveal that TSP-mediated costimulation is achieved through its independent interaction with CD36 on APCs and with CD47 on T cells. We propose that a CD47-TSP-CD36 trimolecular complex is a novel costimulatory pathway that significantly decreases the threshold of T cell activation. Consistent with the paradigm that lesions in rheumatoid synovitis are sites of antigenic recognition, the characteristic focal expression of TSP on APCs such as macrophages and fibroblast-like synoviocytes suggest a central role of TSP in the expansion of tissue-infiltrating T cells.     

A hypothesis on the biological role of ABH,lewis and P blood group determinant structures in glycosphingolipids and glycoproteins

A hypothesis is presented that glycosphingolipids of circulating erythrocytes are membrane-packing substances providing for an energetically cheap carbohydrate protective coat at the cell surface. The glycosphingolipids should cover the membrane surface not occupied by functional glycoproteins. This role is envisaged for the globo series of glycosphingolipids which are P^k and P antigens of human blood. Glycosphingolipids of the neolacto series terminated with non-informative A, B, H. Lewis, P₁ antigenic structures as well as with sialic acid residues should serve the same purpose. These carbohydrate structures may be also used for conferring biological inertness on otherwise functionally active carbohydrate structures and provide protection for circulatory and membrane glycoproteins from proteolysis, denaturation and recognition of potentially antigenic sites of protein moieties by the immunosurveillance system of the body. At the external body surface the same carbohydrate structures may protect cells from the action of pathogenic microorganisms and other environmental factors. The roles of the above mentioned carbohydrate sequences on glycosphingolipids and glycoproteins in the development, tumorigenesis and evolution of blood group polymorphism are discussed.Abbreviations GP glycoprotein - GSL glycosphingolipid - GC glycoconjugate     

A method of calculatiing immunochemical cross-reactions between homologous proteins

The effects of amino acid substitutions within the antigenic sites, within the residues close to these sites and within other parts of the molecule on the cross-reaction with the antisera to homologous protein were considered. The method for calculus the values of cross-reactions, based on using primary structures data, X-ray coordinate of one of the homologues and the locations of the antigenic sites is proposed. The values obtained by this method for the cross-reactions of ten myoglobins from various species with antisperm-whale myoglobin sera have a good correlation with the known experimental data. The possibility of using the method to make the location of protein antigenic sites more precise is discussed.     

Genetic regulation of the immune response to hepatitis B surface antigen (HBsAg). VI. T cell fine specificity

In the companion paper it was demonstrated that the T cell proliferative response to HBsAg was controlled by I region genes as was previously shown for in vivo anti-HBs production. In this paper, the structural requirements for T cell recognition of HBsAg were compared with B cell (antibody) recognition of HBsAg. Secondly, we attempted to map determinants on HBsAg required for activation of HBsAg-primed T cells, and we examined the influence of I region genotype on the observed T cell antigenic fine specificity. The results of these studies indicate clear differences between T cell and B cell recognition of HBsAg. T cell activation required significantly less native structure as compared with antibody binding to HBsAg. Reduced and alkylated HBsAg, the subunit polypeptide P25, tryptic fragments of P25, and synthetic peptide analogues of HBsAg were all capable of eliciting a T cell proliferative response, whereas these "denatured" forms of the antigen bind anti-HBs marginally or not at all. Furthermore, the results suggest that T cell recognition sites on HBsAg do not necessarily overlap with B cell recognition sites. Examination of T cell fine specificity in a series of H-2 congenic strains, with the use of HBsAg, P25, tryptic fragments of P25, and synthetic peptides, revealed multiple T cell recognition sites on HBsAg, and the particular site(s) recognized is dependent on the H-2 genotype of the responding strain. Finally, preliminary results indicate that the specificity of human, HBsAg-primed T cells appear to be variable among individuals.