Interactions between the amino-terminal domains of MHC class II molecules have a profound effect on serologic and T cell recognition: an analysis using recombinant HLA-DR/H-2E molecules.

A series of transfected L cell lines were generated expressing the products of wild-type or recombinant HLA-DR1/H-2Ek beta-chain-encoding genes paired to DR alpha or E alpha. The recombinant genes were created by reciprocal exchange of the gene segments encoding the amino (NH2)-terminal and carboxy (COOH)-terminal halves of the beta 1 domain and the beta 2 domain. The majority of the serologic determinants, predicted from the genetic composition of the class II dimers, were expressed indicating that no gross conformational changes were induced by the creation of the interspecies recombinant molecules. Subtle conformational variation was detected by the anti-H-2Eb,k,s mAb Y17. Epitope expression was dependent on the presence of the E alpha-chain and NH2-terminal sequence from the beta 1 domain of H-2Ek. Substitution of DR1 sequence in either region led to loss of recognition by Y17. This pattern of reactivity maps the Y17 epitope either to the E alpha-chain or to an exposed sequence on the fourth strand of the beta sheet of the beta 1 domain. If the Y17 epitope is located on the E alpha-chain this raises the interesting possibility that the conformation of this chain, which is invariant by sequence, may vary according to the beta-chain with which it is coexpressed. The ability of the recombinant class II dimers to present Ag to the pigeon cytochrome c-specific, H-2Ek-restricted T cell hybridoma 2B4 was assessed. Transfected L cells expressing E beta k paired to E alpha or DR alpha presented Ag with equal efficiency, and the beta 2 domain of H-2Ek could be substituted with the equivalent region from DR1 without any loss of response. Wild-type DR1 failed to function as a restriction element, however, substitution of the COOH-terminal portion of the beta 1 domain with the equivalent sequence from H-2Ek was sufficient to produce a partial recovery of Ag recognition. Cells expressing a recombinant beta 1 domain comprising the COOH-terminal sequence from H-2Ek and the NH2-terminal sequence from DR1 presented Ag when paired to DR alpha but failed to do so when paired to E alpha. This indicates that a subtle conformational disturbance caused by mismatching of the NH2-terminal region of the beta-chain and the alpha-chain can have pronounced effects on T cell recognition.(ABSTRACT TRUNCATED AT 400 WORDS)     

The molecular basis of alloreactivity in antigen-specific, major histocompatibility complex-restricted T cell clones

We have studied the relationship between major histocompatibility complex (MHC)-restricted antigen recognition and alloreactivity by examining T cell receptor (TCR) alpha and beta gene expression in cytochrome c-specific, Ek alpha:Ek beta (Ek)-restricted helper T cell clones derived from B10.A mice. The clones could be segregated on the basis of four distinct alloreactivity patterns. Clones cross-reactive for three different allogeneic la molecules (As alpha:As beta [As], Ab alpha:Ab beta [Ab], Ek alpha: Eb beta [Eb]) expressed the same V alpha and V beta gene segments, generating the distinct alloreactive specificities via unique V alpha-J alpha and V beta-D beta-J beta joining events. Ek alpha:Es beta (Es)-alloreactive B10.A clones expressed the same V alpha, J alpha, and V beta segments as an Es-restricted, Ek-alloreactive, cytochrome c-specific, H-2-congenic B10.S(9R) clone. This homology between TCRs mediating allorecognition of la molecules and recognition of the same la molecules as restriction elements associated with nominal antigen suggests that MHC-restricted recognition and allorecognition represent differences in the affinity of the TCR-MHC molecule interaction.     

Ek beta mutant antigen-presenting cell lines expressing altered Ak alpha molecules

Antigen-presenting cells (APC) expressing mutant Ek beta and Ak alpha proteins were isolated after chemical mutagenesis of TA3 cells and negative immunoselection for altered Ek beta molecules. Mutant clones were analyzed for biosynthesis, assembly, and cell surface expression of altered Ia molecules, and were assayed for antigen-presenting function by using a variety of T cell clones. Three types of mutants were detected: type 1, which had lost expression of the Ek beta chain and produced altered Ak alpha chains; type 2, which also expressed altered Ak alpha chains, and which expressed Ek beta proteins that had lost reactivity to the 17.3.3 and 74D monoclonal antibodies (mAb), but retained reactivity to other anti-Ek beta mAb; and type 3, which had lost expression of both Ek beta and Ak beta: Ak alpha surface molecules. Thus, all of the mutant clones that produced modified Ak alpha proteins also displayed either total loss or serologic modification of the Ek beta molecule. Ek beta:E alpha-reactive T cell clones were not stimulated when type 1 or type 3 cells were used as APC, but all such T cells were fully reactive with type 2 mutant APC. Most Ak beta:Ak alpha-reactive T cell clones could respond to type 1 and 2 APC, and none were responsive to type 3 APC. However, two autoreactive Ak beta:Ak alpha-specific T cell hybridomas were stimulated only very weakly by type 1 and type 2 cells expressing modified Ak alpha proteins. These results demonstrate that Ia mutations can have highly selective effects on antigen presentation to T cells as well as on mAb binding, and thus suggest that individual Ia molecules may be composed of many different functional subsites.     

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.     

Dissection of H-2Db-restricted cytotoxic T-lymphocyte epitopes on simian virus 40 T antigen by the use of synthetic peptides and H-2Dbm mutants.

Five distinct cytotoxic T-lymphocyte (CTL) recognition sites were identified in the simian virus 40 (SV40) T antigen by using H-2b cells that express the truncated T antigen or antigens carrying internal deletions of various sizes. Four of the CTL recognition determinants, designated sites I, II, III, and V, are H-2Db restricted, while site IV is H-2Kb restricted. The boundaries of CTL recognition sites I, II, and III, clustered in the amino-terminal half of the T antigen, were further defined by use of overlapping synthetic peptides containing amino acid sequences previously determined to be required for recognition by T-antigen site-specific CTL clones by using SV40 deletion mutants. CTL clone Y-1, which recognizes epitope I and whose reactivity is affected by deletion of residues 193 to 211 of the T antigen, responded positively to B6/PY cells preincubated with a synthetic peptide corresponding to T-antigen amino acids 205 to 219. CTL clones Y-2 and Y-3 lysed B6/PY cells preincubated with large-T peptide LT220-233. To distinguish further between epitopes II and III, Y-2 and Y-3 CTL clones were reacted with SV40-transformed cells bearing mutations in the major histocompatibility complex class I antigen. Y-2 CTL clones lysed SV40-transformed H-2Dbm13 cells (bm13SV) which carry several amino acid substitutions in the putative antigen-binding site in the alpha 2 domain of the H-2Db antigen but not bm14SV cells, which contain a single amino acid substitution in the alpha 1 domain. Y-3 CTL clones lysed both mutant transformants. Y-1 and Y-5 CTL clones failed to lyse bm13SV and bm14SV cells; however, these cells could present synthetic peptide LT205-219 to CTL clone Y-1 and peptide SV26(489-503) to CTL clone Y-5, suggesting that the endogenously processed T antigen yields fragments of sizes or sequences different from those of synthetic peptides LT205-219 and SV26(489-503).     

Clonal analysis of B and T cell responses to Ia antigens. IV. Proliferative T cell clones recognizing E beta and/or E alpha allodeterminants

The allospecific T cell recognition of the I-Ek molecule was assessed by using eight A. TH anti-A. TL proliferative T cell clones, all of which expressed the Thy-1-2+, Lyt-1+, Lyt-2-, Ia-, and p94,180+ cell surface phenotype. The use of panels of stimulating cells from homozygous of F1 hybrid strains indicated each T cell clone exhibited specificity for distinct alloactivating determinants including: i) a private E beta k-controlled determinant expressed in cis- or trans-complementing E beta kE alpha strains; ii) an apparently nonpolymorphic E alpha determinant resembling the serologic specificity Ia.7, i.e., present in all strains carrying E alpha and E beta expressor alleles; and iii) a series of conformational I-E determinants, the expression of which required a precisely defined combinatorial association of E beta plus E alpha chains. Two clones were found to be reactivated by cis- but not trans-complementing E beta k E alpha k strains, and another recognized an allodeterminant shared by the I-Ab molecule. Various I-Ek-reactive monoclonal antibodies (mAb) directed to epitopes presumably expressed on either E alpha (epitope clusters I and II) or E beta (epitope cluster III) chains inhibited the proliferative responses of seven clones recognizing private E beta k or unique E beta E alpha conformational activating determinants. By contrast, the restimulation of the clone directed to a nonpolymorphic E alpha determinant was selectively blocked by anti-Ia.7 mAb defining epitopes on the E alpha chains but not by those directed to the E beta chain. On the basis of these data, it was concluded that the recognition sites of most anti-I-Ek proliferative T cells were expressed on the E beta chain or the E beta plus E alpha interaction products, and that a minority of such alloreactive T cells could be activated through recognition of the E alpha chain per se.     

T cell recognition of Mlsc. I. Influence of MHC gene products in Mlsc-specific T cell recognition

Monospecific T cell clones have been proven to be powerful tools for the characterization of T cell recognition in many Ag-specific as well as allo-specific T cell responses. In this report, in order to elucidate the mechanism of T cell recognition of minor stimulating locus Ag (Mlsc) determinants, Mlsc-specific cloned T cells were employed together with primary T cell responses to clarify the role of MHC-gene products in Mlsc-specific T cell recognition. The results indicated that T cells recognize Mlsc determinants in conjunction with I-region MHC gene products. Moreover, certain MHC haplotypes (e.g., H-2a and H-2k) appear to function efficiently in the "presentation" of Mlsc, whereas other haplotypes (e.g., H-2b and H-2q) function poorly if at all in presenting Mlsc. Experiments with the use of stimulators derived from F1 hybrids between the low stimulatory H-2b, Mlsc strain, C3H.SW, and a panel of Mlsb, H-2-different or intra-H-2 recombinant strains strongly suggested that expression of E alpha E beta molecules on stimulators plays a critical role for Mlsc stimulation. The functional importance of the E alpha E beta product in Mlsc recognition was further demonstrated by the ability of anti-E alpha monoclonal antibody to inhibit the response of cloned Mlsc-specific T cells. Inhibition of the same Mlsc-specific response by anti-A beta k antibody suggests that the A beta product may also play a role in T cell responses to Mlsc.     

Arsonate-specific murine T cell clones. IV. Properties of I-E- and I-A-restricted clones

The T cell antigen L-tyrosine-p-azobenzenearsonate is unique in being a simple determinant that can be presented in the context of both I-A and I-E. I-E-restricted T cell clones derived from B10.A(5R) mice were found to fall into three groups: Type I clones recognized antigen only in the context of syngeneic apcs, Type II clones recognized antigen with the same highly specific major histocompatibility complex restriction but in addition proliferated in response to allogeneic stimuli; Type III clones were "degenerate" in their major histocompatibility complex-restricted recognition of antigen and proliferated when antigen-presenting cells bearing Eb beta Ek alpha (syngeneic), Ek beta Ek alpha, or Ed beta Ed alpha were used. These observations allow some conclusions to be drawn about sites on the I-E molecule that may be functionally significant in the presentation of this antigen. By using the B cell hybridoma LK35.2 as target cells, some of these T cell clones act as cytotoxic cells in the Class II-restricted manner predicted from the results of proliferative assays. Class II-restricted cytotoxicity can therefore be controlled by both I-A and I-E mouse Ir gene loci.     

Molecular studies of a rare DR2/LD-5a/DQw3 HLA class II haplotype. Multiple genetic mechanisms in the generation of polymorphic HLA class II genes

A cDNA library was constructed from a homozygous B lymphoblastoid cell line (REM) obtained from an individual of a long isolated American Indian tribe, the Warao. The REM cell line expresses serologically defined determinants, DR2 and DQw3, and the T lymphocyte-defined (Dw/LD) specificity, LD-5a. T cells can recognize differences between FJO (a DR2/DQw1 cell line that expresses the Dw specificity MN2) and REM for both DR and DQ molecules. cNDA clones encoding the polymorphic DR beta 1-, DR beta 2-, DQ beta-, and DQ alpha-chains were sequenced and compared with other DR and DQ gene sequences. The DR beta 1-sequence of REM is identical to the DR beta 1-sequence of FJO; the DR beta 2-sequence is also identical to that of FJO except for one amino acid difference at position 67 in the polymorphic first domain (Leu in REM, Phe in FJO) due to a single point mutation. The DQ beta-sequence is identical to that of DR4/DQw3 haplotype; the DQ alpha is different from the DQ alpha of DR4/DQw3 haplotype and identical to the DQ alpha of both the DR3/DQw2 haplotype of a Raji cell line and the DR5/DQw3 haplotype in deduced amino acid sequence. Taken together, these findings suggest that: 1) a single amino acid difference (position 67) in the third hypervariable region of the first domain of the DR beta 2-chain in the DR2 haplotype is apparently sufficient for stimulating T cell responses; 2) the DQw3 serologic specificity may be defined mainly by the DQ beta-rather than DQ alpha-chain; and 3) multiple genetic events have probably occurred to generate the rarely found REM (DR2/LD-5a/DQw3) haplotype.     

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.     

Cell surface expression of hybrid murine/human MHC class II beta alpha dimers. Key influence of residues in the amino-terminal portion of the beta 1 domain

To aid in the identification of key residues responsible for the control of class II MHC beta-alpha dimer assembly and expression, a series of cotransfections of human plus mouse beta- and alpha-genes was performed. The resulting expression data were correlated with the sequences of the relevant proteins to identify residues that played critical roles in these processes. For the I-E/DR homologues good expression was seen for both E beta DR alpha and DR beta E alpha combinations involving several allelically variable beta-chains of each species. These results are consistent with the sequence conservation seen for I-E and DR gene products, and indicate that the species-specific differences that do exist play little role in controlling dimer formation or transport. For A beta chains, a more complex picture was seen. A beta d, but not A beta k or A beta b, was found to coexpress with human alpha-chains. Not only did A beta d show expression with the homologous DQ alpha-chain, but it also was expressed with DR alpha and DP alpha. These data indicate that species-specific residues do not control dimer expression under these conditions and confirm that allelically polymorphic residues have a crucial role in this process. Mapping studies using recombinant A beta genes established the importance of the residues in the amino-terminal half of the beta 1 domain in the differences observed among the A beta alleles. Sequence comparison of DR beta, DP beta, DQ beta, E beta, and A beta chains in this region revealed a single residue (position 12) conserved in most chains and differing in a nonconservative fashion between A beta d vs A beta b or k. A beta d has the conserved lysine at this position, whereas A beta b has methionine and A beta k has glutamine. To test whether this residue actually was important physiologically, a lysine codon was created in a recombinant A beta gene possessing the amino-terminal sequence of the kappa haplotype, and the ability of this mutant chain to be expressed with various mouse A alpha-chains was examined. This mutant chain was shown to gain the ability to be efficiently expressed with A alpha d without losing its ability to be expressed with A alpha k. These data reemphasize the special role played by allelically polymorphic residues in Ia expression and identify one such polymorphic site as position 12.(ABSTRACT TRUNCATED AT 400 WORDS)     

A novel association of DQ alpha and DQ beta genes in the DRw10 haplotype. Determination of a DQw1 specificity by the DQ beta-chain

The association of the class II genes of the DRw10 haplotype from a cell line, NASC, initiated from a member of a well characterized family, was analyzed by sequencing cDNA clones corresponding to DR beta I, DQ alpha, and DQ beta genes. An identical haplotype was also identified in the Raji cell line. In addition to typing as DRw10 and DQw1 with HLA typing sera both, the NASC and Raji cell lines were shown to react strongly with the monoclonal antibodies 109d6 (specific for DRw10 beta 1 and DRw53 beta 2 gene products) and Genox 3.5.3 (specific for DQw1) and exhibited the restriction fragment length polymorphism indicative of a DRw10, DQw1 haplotype. The DR beta 1 gene corresponding to the DRw10 specificity was found to have a first domain sequence different from all other DR beta I genes. Sequence analysis of the 3'-untranslated region of this DR beta-chain gene showed a significant divergence from the 3' untranslated region of the DRw53 family of haplotypes and a lesser divergence from that of the DRw52 and DR1/DR2 families. The sequence of the DQ beta genes corresponding to the DQw1 specificity in the DRw10 haplotype was found to be identical to the DQ beta gene from a DR1, DQw1 haplotype. Surprisingly, however, the DQ alpha gene did not resemble other DQw1-like DQ alpha genes, but was identical in sequence to the DQ alpha gene found in DR4 haplotypes. The novel association of DQ alpha and DQ beta genes in the DRw10 haplotype revealed in these studies may result from a double recombinational event. More consequentially, these studies strongly suggest that the DQw1 specificity recognized by Genox 3.5.3 is determined by the DQ beta chain and is not affected by the DQ alpha-chain.     

Experimental allergic encephalomyelitis mediated by cloned T cells specific for a synthetic peptide of myelin proteolipid protein. Fine specificity and T cell receptor V beta usage.

Proteolipid protein (PLP) is the major protein of central nervous system myelin. SJL (H-2s) mice immunized with a synthetic peptide corresponding to PLP residues 139-151 develop acute EAE. In this study, 6 IAs-restricted, CD4+, TCR alpha beta-bearing T cell clones were derived from SJL/J mice after immunization with this synthetic peptide. The clones responded in in vitro proliferative assays to the whole PLP molecule and to PLP peptide 139-151, but not to irrelevant Ag. They also responded to truncated and overlapping forms of the peptide but five distinct reactivity patterns were observed using these peptides. A panel of anti-TCR V beta mAb and TCR V beta-specific cDNA probes were used to determine the TCR V beta usage of the clones. Five clones were found to use four different V beta (V beta 2, V beta 6, V beta 10, or V beta 17a), whereas the V beta on the sixth clone could not be identified. Five of the clones induced EAE of varying severity upon adoptive transfer into naive syngeneic mice or mice pretreated with irradiation and pertussis and one clone was nonencephalitogenic. The Ag-specific proliferative response of all but the nonencephalitogenic clone could be blocked by an anti-CD4 mAb. Thus, the clones showed differences in their fine specifity, TCR V beta usage, sensitivity to antibody blocking, and encephalitogenic potency. These data demonstrate that the T cell response to the encephalitogenic PLP peptide 139-151 is heterogeneous.     

Thymic deletion of V beta 11+, V beta 5+ T cells in H-2E negative, HLA-DQ beta+ single transgenic mice

DQw6b transgenic mice have been generated by microinjecting a linearized cosmid clone containing 34-kb DQb genomic DNA, isolated from HLA-homozygous B cell line AKIBA (DR2, Dw12, DQw6), into embryos of (CBA x B10.M)F2 or (SWR x SJL)F2. Among 85 mice screened, eight mice were transgene-positive. The transgene in seven of eight founders was germline-transmitted. FACS analysis and immunohistochemical studies with DQ beta-specific mAb demonstrated that DQ beta molecules in association with mouse A alpha f molecules are expressed on peripheral mononuclear cells, spleen cells, and in thymic medulla. More interestingly, V beta 11-, V beta 5.1-, and V beta 5.2-bearing T cells, but not V beta 8.2-bearing T cells, were clonally deleted in the H-2E-negative but DQ beta+ progeny of two selected founders (260-23 and 258-10). The deletion was found to take place intrathymically during the transition stage from immature to mature thymocyte development. We postulate that although human DQ genes are more homologous to mouse H-2A genes, A alpha f/DQ beta hybrid molecules may possess the same self-peptide- (or superantigen)-presenting epitope as E alpha/E beta molecules critical for deletion of V beta 11-, V beta 5.1-, and V beta 5.2-bearing T cells in thymus. Our results also confirm the previous findings that accessory molecules on thymocytes such as CD4 may be involved in thymic selection, and further suggest that an interaction of mousE CD4 and mouse A alpha chain is required for the clonal deletion.     

Limited T cell receptor beta-chain usage in the sperm whale myoglobin 110-121/E alpha dA beta d response by H-2d congenic mouse strains.

The specificity and TCR gene usage of a panel of sperm whale myoglobin (SpWMb)-reactive T cell clones from DBA/2 mice have previously been characterized, to study structure-function relationships between components of the ternary complex consisting of Ag, TCR, and MHC class II molecules, whose interaction leads to Th cell activation. These DBA/2 clones were specific for epitopes within the residue 110 to 121 region of SpWMb, in the context of the mixed isotype molecule E alpha dA beta d, and expressed the TCR V beta 8.2 gene element. SpWMb-specific T cell hybridomas from the H-2d-congenic B10.D2 mouse strain, which differs from the DBA/2 strain only in the non-MHC background, were generated and compared with the T cell hybridomas from DBA/2 mice, in order to investigate the influence of non-MHC genes on the specificity of the T cell response to the 110-121 epitope. V beta usage by these hybridomas was very homogeneous; three of three DBA/2 and eight of nine B10.D2 hybridomas specific for the 110-121 epitope, in the context of the mixed isotype molecule E alpha dA beta d, expressed the V beta 8.2 gene product. Nucleotide and amino acid sequences of D beta, J beta, and N regions were also similar. One 110-121/E alpha dA beta d-specific B10.D2 hybridoma used V beta 7, a V beta that is clonally deleted in DBA/2 mice. These experiments suggest that a similar set of TCR beta genes are used to respond to a given epitope, regardless of non-MHC background, and they support the hypothesis that, despite great variability between individuals in their non-MHC background genes, human HLA-associated diseases might result from the formation of a particular ternary complex consisting of a shared MHC molecule, a common "disease-associated" epitope, and a shared TCR.     

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.     

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.     

Stimulation of normal inducer T cell clones with antigen presented by purified Ia molecules in planar lipid membranes: specific induction of a long-lived state of proliferative nonresponsiveness

Culture of normal inducer T cell clones with antigen and purified Ek beta:Ek alpha incorporated into planar lipid membranes resulted in specific T cell activation as determined by cell volume increase and IL 3 production. However, in contrast to results obtained with T cell hybridomas, antigen presentation by planar membranes did not induce measurable IL 2 production, and proliferative responses were not detected. Rather, recognition of only Ek beta:Ek alpha and antigen resulted in the specific induction of a long-lived state of proliferative nonresponsiveness to subsequent stimulation by conventional APC and antigen. Induction of nonresponsiveness required protein synthesis, and was not simply due to the absence of IL 2. The antigen-nonresponsive cells could respond to either PMA plus ionomycin or IL 2, and they expressed normal levels of surface antigen-receptor molecules. These results demonstrate that recognition by normal T cell clones of antigen and Ia molecules in the absence of other accessory cell molecules and signals results in a prolonged state of proliferative nonresponsiveness, possibly similar to a state of T cell tolerance in vivo.