Polymorphic HLA-DR7 beta 1 chain residues that are involved in T cell allorecognition

The contributions to allorecognition of polymorphic amino acids in the HLA-DR7 beta 1 chain were analyzed by using mutant DR7 beta 1 chains with single amino acid substitutions at position 4, 11, 13, 25, 30, 37, 57, 60, 67, 70, 71, 74, or 78. Transfectants expressing mutant DR7 molecules were used as stimulators for six DR7-alloreactive T cell clones. The majority of the substitutions had profound effects on the ability of the DR7 molecule to stimulate one or more T cell clones. Nine of the 13 substitutions completely abrogated recognition by at least one clone. The finding that each of the substitutions in the beta-strands in the floor of the peptide binding groove affected T cell allorecognition supports the model of allorecognition in which the complex of a self-peptide bound to a class II molecule is recognized by the TCR. Interestingly, the substitution at position 4, which is predicted to be located outside the peptide binding groove, decreased the ability of the DR7 molecule to stimulate some clones. Each of the DR7-alloreactive T cell clones had a unique reactivity pattern in response to the different mutant molecules, indicating that the TCR of each clone recognized the DR7 molecule differently. Surprisingly, many of the mutant DR7 molecules induced proliferation by one or more clones that was greater than 125% of the proliferation induced by the wild-type DR7 molecule. These data indicate that multiple polymorphic residues, predicted in the class II model to be located in both the beta-strands and alpha-helix of the DR7 beta 1 chain, contribute to allorecognition of the DR7 molecule.     

Functional analysis of DR17(DR3)-restricted mycobacterial T cell epitopes reveals DR17-binding motif and enables the design of allele-specific competitor peptides.

We have previously shown that p3-13 (KTIAY-DEEARR) of the 65-kDa heat shock protein (hsp65) of Mycobacterium tuberculosis and Mycobacterium leprae is selected as an important T cell epitope in HLA-DR17+ individuals, by selectively binding to (a pocket in) DR17 molecules, the major subset of the DR3 specificity. We have now further studied the interaction between p3-13, HLA-DR17 and four different TCR (V beta 5.1, V beta 1, and V beta 4) by using T cell stimulation assays, direct peptide-DR binding assays, and a large panel (n = 240) of single amino acid substitution analogs of p3-13. We find that residues 5(I) and 8(D) of p3-13 are important DR17 binding residues, whereas the residues that interact with the TCR vary slightly for each DR17-restricted clone. By using N- and C-terminal truncated derivatives of p2-20 we defined the minimal peptide length for both HLA-DR17 binding and T cell activation: the minimal peptide that bound to DR17 was seven amino acids long whereas the minimal peptide that activated T cell proliferation was eight amino acids in length. Furthermore, two new DR17-restricted epitopes were identified on hsp70 and hsp18 of M. leprae. Alignment of the critical DR17-binding residues 5(I) and 8(D) of p3-13 with these two novel epitopes and two other DR17-binding peptides revealed the presence of highly conserved amino acids at positions n and n + 3 with I, L, and V at position n and D and E at position n + 3. D and E are particularly likely to interact with the DR17-specific, positively charged pocket that we have defined earlier. Based on these results, a set of single amino acid substituted analogs that failed to activate these T cell clones but still bound specifically to DR17 was defined and tested for their ability to inhibit T cell activation by p3-13 or other DR17-restricted epitopes. Those peptides were able to inhibit the response to p3-13 as well as other DR17-restricted mycobacterial epitopes in an allele-specific manner, and are anticipated to be of potential use for immunotherapeutic and vaccine design strategies.     

Antigen-specific T cells with monogamous or promiscuous restriction patterns are sensitive to different HLA-DR beta chain substitutions

The contributions of the amino acids at 13 polymorphic positions in the HLA-DR7 beta 1 chain to T cell recognition of two antigenic peptides of tetanus toxin (p2 and p30) were assessed using transfectants expressing mutant DR7 beta 1 chains as APC for six toxin-specific T cell clones with two different restriction patterns: monogamous (restricted by DR7 only) or promiscuous (restricted by DR7; DR1; DR2, Dw21; and DR4, Dw4). Each of the 13 substitutions significantly decreased or eliminated the ability of the DR7 molecule to present a peptide to one or more of the T cell clones, but none of the substitutions abolished recognition by all clones. Interestingly, substitutions at positions 4 and 25, which are predicted in the class II model to be located outside the peptide binding groove, decreased the ability of the DR7 molecule to present Ag to some clones but not to others. Each of the four clones specific for the p2 peptide and the two clones specific for peptide p30 had a different reactivity pattern to the panel of DR7 beta 1 mutants, indicating that the TCR of each clone has a different view of the p2/DR7 or p30/DR7 complex. These data emphasize the complexity of the interactions of multiple residues in DR7 beta 1 chains in Ag-specific T cell recognition.     

Analysis of the permissive association of a malaria T cell epitope with DR molecules.

In this study we examined the association of a promiscuous malaria T cell epitope, CS.T3, to different HLA-DR alleles. A large series of singly substituted or truncated variants of CS.T3 was prepared and tested for the ability to be recognised in association with, or to bind to, three distinct HLA-DR alleles (DR1, DRw11, and DRw14(w6)) and three natural variants of HLA-DRw11. We found that although association with the different DR molecules mapped to identical or closely overlapping regions of the peptide, distinct substitutions could drastically influence the capacity of the peptide to interact with one but not another of the three DR molecules tested. Based on analysis of the distribution of residues recognized by T cell clones restricted to the different DR alleles, we suggest that the peptide CS.T3 is not bound, at least for the three DR examined, as an alpha-helix. In addition we tested three subtypes of DRw11 as APC for the CS.T3 analogues and observed that the peptide is most likely bound in the same conformation to the three natural variants of the DRw11 molecule.     

Single amino acid changes in DR and antigen define residues critical for peptide-MHC binding and T cell recognition.

Single amino acid substitutions of Ag and MHC were used to analyze the fine structure of the influenza hemagglutinin (HA)-derived epitope (HA 307-319) recognized in the context of DR7 molecules by a T cell clone. Putative T cell (HA 308, 310, 311, 313, and 316) and DR (HA 309, 312, and 317) contact residues of the Ag were identified by the use of single amino acid-substituted analogs that were tested for their T cell-activating and DR-binding capacities. The peptide-DR7-T cell interaction was further characterized by the use of a panel of 13 site-directed DR7 mutant transfectants analyzed for their capacity to present Ag to T cells, and for their purified mutant DR7 molecules to bind HA 307-319 or its single amino acid-substituted analogs. Eight mutants lost their Ag-presenting function, whereas only one had any decrease in peptide binding. Finally, for three of the mutants it was possible to correct the deleterious effects of mutation by using a particular single amino acid-substituted analog of the peptide molecule. The observed pattern of complementation led to a model that predicts that the Ag assumes an extended conformation, with a turn, in the binding groove, such that the following residues are in close proximity: DR 86-HA 309, DR 71-HA 312, DR 30-HA 314, and 315.     

Fine specificity of murine class II-restricted T cell clones for synthetic peptides of influenza virus hemagglutinin. Heterogeneity of antigen interaction with the T cell and the Ia molecule

We have previously demonstrated diversity in the specificity of murine, H-2k class II-restricted, T cell clones for the hemagglutinin (HA) molecule of H3N2 influenza viruses and have mapped two T cell determinants, defined by synthetic peptides, to residues 48-68 and 118-138 of HA1. In this study we examine the nature of the determinant recognized by six distinct P48-68-specific T cell clones by using a panel of truncated synthetic peptides and substituted peptide analogs. From the peptides tested, the shortest recognized were the decapeptides, P53-62 and P54-63, which suggests that the determinant was formed from the 9 amino acids within the sequence 54-62. Asn54 was critical for recognition since P49-68 (54S) was not recognized by the T cell clones. Furthermore this peptide analog was capable of competing with P48-68 for Ag presentation, thereby suggesting that residue 54 is not involved in Ia interaction and may therefore be important for TCR interaction. Residue substitutions at position 63 also affected T cell recognition, but in a more heterogeneous fashion. Peptide analogs or mutant viruses with a single amino acid substitution at position 63 (Asp to Asn or Tyr) reduced the responses of the T cell clones to variable extents, suggesting that Asp63 may form part of overlapping T cell determinants. However since the truncated peptide P53-62 was weakly recognized, then Asp63 may not form part of the TCR or Ia interaction site, but may affect recognition through a steric or charge effect when substituted by Asn or Tyr. Ag competition experiments with the two unrelated HA peptides, P48-68 and P118-138, recognized by distinct T cell clones in the context of the same restriction element (I-Ak), showed that the peptides did not compete for Ag presentation to the relevant T cell clones, whereas a structural analog of P48-68 was a potent inhibitor. This finding is discussed in relation to the nature of the binding site for peptide Ag on the class II molecule.     

Human T cells recognize polymorphic and non-polymorphic regions of the Plasmodium falciparum circumsporozoite protein.

In order to characterize T cell epitopes in the Plasmodium falciparum circumsporozoite (CS) protein sequence, we isolated T cell clones, from non-immune donors, which reacted with synthetic peptides corresponding to two predicted CS protein T cell epitopes. Peptide CS.T3 (corresponding to a non-polymorphic region of the CS protein, residues 378-398) was recognized in association with either DR2 or DRw9 restriction elements. T cell clones recognizing CS.T3 also reacted with the sporozoite-derived CS protein. Peptide CS.T2 corresponds to a polymorphic region (residues 325-341) of the CS protein. Unlike the CS.T3-specific clones, the CS.T2-specific clones did not recognize the CS protein. Since the CS.T2 peptide includes residues which are polymorphic in different P. falciparum isolates, we investigated whether these residues were critical for recognition of the peptide. We show here that a single amino acid substitution at a position of the CS protein which shows genetic polymorphism affects recognition of the sequence by human T cells. The implications of these data for malaria vaccine development are discussed.     

The pigeon cytochrome c-specific T cell response of low responder mice. I. Identification of antigenic determinants on fragment 1 to 65

An examination of the proliferative response to pigeon cytochrome c fragments 1 to 65 and 1 to 80 by T cells from mice that are low responders to the native molecule revealed that some of the strains could respond to antigenic determinants on these fragments. T cell clones derived from B10.A(3R) and B10.A(4R) mice were used to characterize the antigenic determinants on fragment 1 to 65. All of the clones recognized syngeneic A beta:A alpha Ia molecules as their restriction element. Three B10.A(3R) clones and six B10.A(4R) clones recognized fragment 39 to 65. Another four B10.A(4R) clones responded to fragment 1 to 38. By stimulating with a series of cytochrome c fragments from different species, as well as a synthetic peptide, it was possible to localize the antigenic determinant(s) recognized by the B10.A(3R) clones to residues 45 to 58. Each clone showed a unique pattern of responsiveness to the various fragments, suggesting a diversity of T cell receptors specific for the same peptide. One B10.A(3R) clone could be stimulated by many of the 1 to 65 fragments in association with allogeneic B10.SM presenting cells and by tuna fragment 1 to 65 in association with B10.M presenting cells, although the rank order of potency for several of the fragments was different than that observed with syngeneic antigen-presenting cells. In addition, the clone was poorly reactive to a synthetic peptide containing a conservative substitution, serine for threonine, at position 49. The implications of these results for subsite dissection (agretope and epitope) of the antigenic determinant recognized by this clone are discussed.     

Mutations in the third, but not the first or second, hypervariable regions of DR(beta 1*0101) eliminate DR1-restricted recognition of a pertussis toxin peptide.

We have examined the role of 12 polymorphic residues of the beta-chain of the HLA-DR1 class II molecule in T cell recognition of an epitope of pertussis toxin. Murine L cell transfectants expressing wild-type or mutant DR1 molecules (containing single amino acid substitutions in DR(beta 1*0101)) were used as APC in proliferation assays involving nine DR1-restricted T cell clones specific for peptide 30-42 of pertussis toxin. Four different patterns of recognition of the mutants were found among the pertussis-specific clones. Residues in the third hypervariable region (HVR) of DR(beta 1*0101) are critically important for all the T cell clones; amino acid substitutions at positions 70 and 74 abrogated recognition by all of the T cell clones, and substitutions at positions 67 and 71 eliminated recognition by most of the clones. In contrast, most single amino acid substitutions in the first and second HVR, predicted to be located in the floor of the peptide binding groove, had little or no effect on the proliferative responses of these clones. However, the involvement of beta-chain first and second HVR residues was demonstrated by the inability of transfectants expressing wild-type DR(beta 1*0404) (DR4Dw14) or DR(beta 1*1402) (DR6Dw16) to present peptide to these clones. These beta-chains have completely different first and second HVR compared with DR(alpha,beta 1*0101) although the third HVR are identical. These results illustrate the functional importance of third HVR residues of DR(beta 1*0101) and allow definition of the molecular interactions of the DR1 molecule with the 30-42 peptide.     

Fine specificity of T cell recognition of the same peptide in association with different I-A molecules

The fine specificity of the response of T cell clones derived from B10.BR and B10.S congenic mouse strains restricted by I-Ak and I-As molecules, respectively, and which recognize the same 17 amino acid sequence (102-118) of myoglobin, has been investigated and compared with that of T cell clones specific for the same peptide with I-A.d The critical amino acid residues within the 102-118 region of myoglobin required for stimulation of I-Ak-and I-As-restricted T cell clones specific for this determinant were compared using a panel of synthetic peptide analogs. Residues 109, 113, and 116 were critical for stimulation of clones from both haplotypes, although the precise fine specificity varied, even among clones using the same restriction molecule. Residues 109 and 116 are also critical for stimulation of myoglobin-specific I-Ad-restricted clones (Berkower, I., L. A. Matis, G.K. Buckenmeyer, F.R. N. Gurd, D. L. Longo, and J. A. Berzofsky. J. Immunol. 132:1370, 1984). There was also considerable overlap in the size of the minimal determinant necessary for full activity: 106-118 for B10.BR and B10.D2 (Cease, K. B., I. Berkower, J. York-Jolley, and J. A. Berzofsky. J. Exp. Med. 164:1779, 1986) clones and 102-117 for B10.S clones. Despite this similarity in fine specificity, T cell clones were genetically restricted and could not be stimulated with the 102-118 peptide presented by Ia molecules of other haplotypes that could also present this epitope to syngeneic clones. These results suggest that binding of an immunogenic peptide to class II molecules is not sufficient to ensure recognition by a given T cell antigen receptor specific for the peptide, but do not indicate whether the major histocompatibility complex molecules interact directly with the T cell antigen receptor or induce a different recognizable conformation of the peptide.     

T cell receptor junctional regions and the MHC molecule affect the recognition of antigenic peptides by T cell clones.

Nine independent pigeon cytochrome c-specific T cell clones were analyzed by using a panel of antigenic peptide analogs presented in association with three allelic IE-encoded MHC glycoproteins. Eight of the T cell clones expressed a TCR composed of a unique alpha- and beta-chain amino acid sequence, and concordantly, each of these T cell clones exhibited a unique Ag specificity. This was true for several clones which differed only in TCR V-J junctional regions. Interestingly, for a given clone, the response to some of the peptide analogs depended to a large extent on the allelic form of the presenting MHC molecule. A simple interpretation of these data would suggest that certain positions of the peptide Ag are most important for Ag-MHC molecule interactions, and that these specific interactions can influence the antigenic epitope recognized by the TCR. We suggest that an antigenic peptide binds to an MHC glycoprotein in a distinct way, but may retain a measure of flexibility.     

Mycobacterium leprae-specific T cells from a tuberculoid leprosy patient suppress HLA-DR3-restricted T cell responses to an immunodominant epitope on 65-kDa hsp of mycobacteria.

The polar tuberculoid type (TT) of leprosy, characterized by high T cell reactivity to Mycobacterium leprae, is associated with HLA-DR3. Surprisingly, DR3-restricted low T cell responsiveness to M. leprae was found in HLA-DR3-positive TT leprosy patients. This low responsiveness was specifically induced by M. leprae but not by M. tuberculosis and was seen only in patients and not in healthy controls. We studied this patient-specific, M. leprae-induced, DR3-restricted low T cell responsiveness in depth in one representative HLA-DR3-positive TT leprosy patient by using T cell clones. From this patient two types of T cell clones were obtained: one type was cross-reactive with M. tuberculosis and recognized an immunodominant epitope (amino acids 3 to 13) on the 65-kDa heat shock protein (hsp) the other type was M. leprae specific and reacted to a protein other than the 65-kDa one. To examine whether these M. leprae-specific T cell clones were responsible for the DR3-restricted low responsiveness to M. leprae, we tested them for the ability to suppress the proliferation of the DR3-restricted, 65-kDa, hsp-reactive clones. The DR3-restricted, M. leprae-specific T cells completely suppressed the proliferative responses of DR3-restricted, cross-reactive T cell clones to the 65-kDa hsp from the same patient as well as from other individuals. Also, DR3-restricted responses to an irrelevant Ag were suppressed by the M. leprae-specific T cell clones. However, no suppression of non-DR3-restricted T cell responses was seen. Although the mechanism must still be elucidated, this M. leprae-induced, DR3-restricted immunosuppression may at least partly explain the observed DR3-associated low T cell responsiveness in TT leprosy patients.     

Functional analysis of the interaction of the antigen-specific T cell receptor with its ligands

Increasing the number of antigen-specific T cell clones in a T cell proliferation assay resulted in a shift in the antigen dose-response curves toward higher amounts of antigen (i.e., more antigen was required to achieve a given degree of stimulation). The antigen dose-response curve shifts were found to reflect the competition that occurred between the antigen-specific T cell receptors for their ligand, a combination of antigen and Ia molecule. This observation made it possible to determine whether the difference in the potency with which several synthetic cytochrome c analogs could stimulate one cytochrome c-specific T cell clone was due to a difference in the avidity of the antigen-specific receptors on the T cell clone for the different Ia molecule-antigen combinations. It was demonstrated that a single amino acid substitution at position 103 (which greatly diminished the potency of the analog) did not significantly alter the avidity of the T cell antigen-specific receptor for its ligand. In contrast, a substitution at position 99 (which resulted in a comparable decrease in potency) caused a dramatic loss of avidity. These results are consistent with the previous designation of residue 99 as one site on the antigen that contacts the T cell antigen-specific receptor, and of residue 103 as one part of the antigen that contacts the Ia molecule.     

Autoimmune T cells: immune recognition of normal and variant peptide epitopes and peptide-based therapy

Experimental autoimmune encephalomyelitis (EAE) results from T helper (TH) cell recognition of myelin basic protein (MBP). We have characterized TH cell reactivity in B10.PL and PL/J (H-2u) mice to 39 N-terminal MBP peptide derivatives of different lengths and with individual amino acid substitutions. The peptide determinant of murine MBP can be divided into a minimal stimulatory core region (residues 1-6) and a tail region (residues 7-20) that alters the structure of the core region to affect both T cell recognition and MHC binding. Core recognition by B10.PL and PL/J mice is highly similar but in one case strain dependent. Peptide analogs that do not stimulate MBP-specific TH cells but bind to the I-Au molecule competitively inhibit T cell reactivity to MBP in vitro and prevent the induction of EAE in vivo.     

Nonrandom selection of T cell specificities in anti-HLA-DR responses. Sequence motifs of the responder HLA-DR allele influence T cell recruitment

The self-restriction of Ag-specific T cell responses is interpreted as the result of a positive selection of the individual's T cell specificities for their compatibility with self-MHC molecules. If the T cell receptor (TCR) specificities in any given individual have an affinity for syngeneic MHC molecules, it is unclear how they interact with allogeneic MHC structures. To approach this question, we analyzed 123 alloreactive HLA-DR4 Dw4 or Dw14 specific T cell clones that were generated from responder/stimulator combinations with defined disparities in the HLA-DR beta 1-chain. Sets of T cell clones were established from three different HLA-Dw4+ responders and compared for their fine specificities. The majority of HLA-DR4 Dw14 specific T cell clones co-recognized HLA-DR1 Dw1+ (33 to 36% of all T cell clones) or HLA-DRw14 Dw16+ (26 to 33%) stimulators, both of which share very similar sequences in the third hypervariable region of the HLA-DR beta 1-chain with the HLA-DR4 alleles Dw4 and Dw14. These data suggest that sequence and structural similarities in the alpha-helical portions of the HLA-DR molecule impose a strong bias on the recognition of allotargets. The second haplotype of the responder did not appear to affect the typical fingerprint of T cell recognition except for the deletion of self-reactive TCR specificities. Nonrandom usage of TCR specificities in anti-HLA-DR responses was also found for HLA-DRw11/DRw13+ and HLA-DRw11/DR7+ T cell donors who did not share any obvious polymorphic sequence stretches with the allostimulators HLA-DR4 Dw4 or Dw14. T cell clones from an HLA-DRw11/DRw13+ responder functionally resembled the TCR specificities derived from the HLA-DR4 Dw4+ donors. T cell clones derived from an HLA-DRw11/DR7+ individual were characterized by a distinct cross-reactivity pattern preferring HLA-DRw13 Dw19+ (50 to 60%) and HLA-DR3+ (43 to 57%) stimulator cells. These findings suggest that the responder HLA-DR alleles influence the structural constraints in the recognition of allo-HLA-DR molecules in closely related and in completely disparate responder/stimulator combinations.     

An influenza A virus-specific and HLA-DRw8-restricted T cell clone cross-reacting with a transcomplementation product of the HLA-DR2 and DR4 haplotypes

The clone TA10 is a T3+ T4+ T8- proliferative and cytolytic human T cell clone. This clone has been shown to be specific for the hemagglutinin of influenza A Texas virus and restricted by an HLA class II molecule associated with the DRw8-Dw8.1 phenotype. Here we show that TA10 and all of its subclones can also react with eight HLA-DRw8 negative, Epstein-Barr virus (EBV)-transformed cell lines or phytohemagglutinin blasts in the absence of influenza antigens. All of these cell lines are HLA-DR2/DR4 with a classic DR2 long haplotype. The only nonreactive HLA-DR2/DR4 cell line observed bears a DR2 short haplotype. Only heterozygous HLA-DR2/DR4 but not parental DR2 or DR4 EBV-transformed cell lines can be recognized by TA10, indicating that the cross-reacting determinant is a transcomplementation product between HLA-DR2 and HLA-DR4 haplotypes. DR-specific, but not DQ- or DP-specific monoclonal antibodies, inhibit in the proliferation assay and in the chromium release test both the DRw8-Dw8.1-restricted and the anti-DR2/DR4 reactions. These results show that HLA-DR-restricted, anti-viral human T cell clone can evidence cross-reactivity for allospecific class II molecules of the major histocompatibility complex, and human CTL can recognize transcomplementation products of class II HLA genes. In addition, the results suggest that a beta-chain coded for by an HLA-DR gene and associated with an alpha-chain coded for by a still unidentified but possibly HLA-DQ gene constitute this functional transcomplementation product.     

Class II-restricted T-cell clones to a synthetic peptide of influenza virus hemagglutinin differ in their fine specificities and in the ability to respond to virus.

Fifteen T-cell clones were derived from BALB/c or DBA/2 mice immunized with a synthetic peptide corresponding to the C-terminal 24 residues (residues 305 to 328) of the HA1 chain of H3 subtype influenza virus hemagglutinin. All of the clones proliferated when the peptide was presented in association with I-Ed. By using shorter homologs, it was shown that the T-cell response was focused predominantly on the region at the N-terminal end of the peptide encompassed by residues 306 to 319. Individual clones recognizing this region differed in their absolute requirements for residues at the extremities of the site and also in their patterns of efficiency of recognition of shorter homologs. One particular clone defined another site of T-cell recognition within residues 314 to 328. The response of the clones to peptide analogs identified certain residues within the sites that were critical for recognition, with the substitution Gln-311----Ser having a differential effect on clones responding to the N-terminal site. Only one of the clones responded well to influenza virus itself. This clone also required relatively low concentrations of the parent peptide for optimum stimulation and was suppressed by higher concentrations. The data demonstrate striking heterogeneity in the T-cell response even to a short synthetic peptide, with different T-cell clones recognizing slightly different but overlapping areas of the molecule.     

Genetic restriction and fine specificity of human T cell clones reactive with rabies virus

Rabies virus-specific T cell clones isolated from a human vaccine recipient were studied for their fine specificity and genetic restriction using synthetic peptides of the viral Ag and mouse fibroblasts transfected with human MHC genes. Two clones were found to react with an epitope present in the rabies glycoprotein, which was presented by the HLA-DR7 molecule. Other T cell clones recognized synthetic epitopes corresponding to the rabies nucleoprotein in association with the HLA-DR7 or HLA-DQw3 molecule, and one clone responded to the viral nucleocapsid Ag in the presence of HLA-DPw4. T cell clones that exhibited different cross-reactivity patterns among several virus strains were found to recognize closely situated epitopes (within 15 amino acid residues), which were presented in the context of the same MHC molecule. The lack of recognition of a particular virus strain by a T cell clone was attributable in some cases to amino acid variations of the Ag that appear to affect the T cell's receptor for Ag specificity and not the ability of that epitope to associate with the corresponding MHC molecule. Comparisons of the T cell cross-reactivity patterns with various rabies and rabies-related viruses, the fine antigenic specificity, and MHC restriction may aid in understanding the role of individual amino acid variations among virus strains in the induction of cross-protective immunity.     

Mapping of distinct serologic and T cell recognition epitopes on an HLA-DR beta-chain.

A new DR beta-chain allele is defined that is identical to the previously described DR6b molecule except for the first hyperpolymorphic region, where the new allele displays the same polymorphisms found on DR8 and DR12 genes. Two distinct epitopes have been mapped on this new allele. The polymorphism in common with DRw8 and DRw12 is recognized by mAb GS313-9D11. However, alloreactive T cell clones specific for DR6b cells (Dw9) recognize this allele, whereas Dw8-specific T cell clones do not. The mAb determinant maps to the first beta-sheet and probably involves a polymorphic residue lying outside the helix. The binding of mAb 9D11 to this region does not interfere with TCR binding. Alloreactive T cell recognition is associated with polymorphisms located predominantly on the alpha-helical portion of the molecule.     

A systematic molecular analysis of the T cell-stimulating antigens from Mycobacterium leprae with T cell clones of leprosy patients. Identification of a novel M. leprae HSP 70 fragment by M. leprae-specific T cells

Both protective immunity and immunopathology induced by mycobacteria are dependent on Ag-specific, CD4+ MHC class II-restricted T lymphocytes. The identification of Ag recognized by T cells is fundamental to the understanding of protective and pathologic immunity as well as to the design of effective immunoprophylaxis and immunotherapy strategies. Although some T cell clones are known to respond to recombinant mycobacterial heat shock proteins (hsp) like hsp3 65, the specificity of most T cells has remained unknown. We therefore have undertaken a specificity analysis of 48 well defined Mycobacterium leprae- and/or Mycobacterium tuberculosis-reactive (Th-1-like) T cell clones. Most clones (n = 44) were derived from different leprosy patients, and the remainder from one healthy control. Their HLA restriction molecules were DR2, DR3, DR4, DR5, DR7, DQ, or DP. T cell clones were stimulated with large numbers (n = 20 to 40) of mycobacterial SDS-PAGE-separated fractions bound to nitrocellulose. Each clone recognized a single fraction or peak with a particular Mr range. Some of the clones (n = 7) recognized the fraction that contained the hsp 65 as confirmed with the recombinant Ag. Most clones (n = 41), however, responded to Ag other than the hsp 65. Nine clones responded to a 67- to 80-kDa fraction. Five of them responded also to an ATP-purified, 70-kDa M. leprae protein, but only one of these five (that was HLA-DR2 restricted and cross-reactive with M. tuberculosis) recognized the recombinant C-terminal half (amino acids 278-621) of the M. leprae hsp 70 molecule and also recognized the recombinant M. tuberculosis hsp 70. We therefore have used the 5' part of the M. leprae hsp 70 gene that we have cloned recently. This fragment (that encodes amino acids 6-279) was indeed recognized by the other four M. leprae-specific T cells that were all HLA-DR3 restricted and did not cross-react with the highly homologous (95%) M. tuberculosis hsp 70. These results suggest that this novel fragment is a relevant T cell-stimulating Ag for leprosy patients. A panel of other recombinant Ag, including hsp 18 was tested. The majority of T cell clones appeared to recognize antigenic fractions distinct from hsp. In conclusion, T cells of leprosy patients see a large variety of different Ag including non-hsp, and one newly recognized moiety is the N-terminal M. leprae hsp 70 fragment.(ABSTRACT TRUNCATED AT 400 WORDS)