Beta-chain broadens range of CD8 recognition for MHC class I molecule.

It is known that the alpha-chain of CD8 binds to a negatively charged loop composed of residues 223 to 229 on MHC class I Ag and that binding of CD8 alpha enhances Ag recognition of T cells. We have recently shown that the mouse CD8 alpha homodimer does not bind to either the HLA class I alpha 3 domain or a mutant of H-2Kb Ag containing a substitution of glutamine for methionine at residue 224, which brings this residue toward the human consensus. Here we report a complementary study of the CD8 beta-chain. The functional role of the CD8 beta-chain was analyzed by using four T cell hybridoma lines expressing mouse CD8 alpha and transfected with the mouse CD8 beta gene. As compared with the lines expressing only CD8 alpha, allorecognition of the chimeric H-2Kb Ag that contains the HLA class I alpha 3 domain was enhanced in lines expressing both CD8 alpha and -beta. This enhancement was blocked by either anti-CD8 mAb or anti-HLA class I alpha 3 domain mAb. In addition, we show that CD8 alpha beta binds the H-2Kb mutant Ag at residue 224. These results suggest that the beta-chain allows the CD8 alpha beta heterodimer to recognize the chimeric H-2Kb Ag. A model for the role of the beta-chain is presented.     

Structural relatedness of distinct determinants recognized by monoclonal antibody TP25.99 on beta 2-microglobulin-associated and beta 2-microglobulin-free HLA class I heavy chains

The association of HLA class I heavy chains with beta2-microglobulin (beta2m) changes their antigenic profile. As a result, Abs react with either beta2m-free or beta2m-associated HLA class I heavy chains. An exception to this rule is the mAb TP25.99, which reacts with both beta2m-associated and beta2m-free HLA class I heavy chains. The reactivity with beta2m-associated HLA class I heavy chains is mediated by a conformational determinant expressed on all HLA-A, -B, and -C Ags. This determinant has been mapped to amino acid residues 194-198 in the alpha3 domain. The reactivity with beta2m-free HLA class I heavy chains is mediated by a linear determinant expressed on all HLA-B Ags except the HLA-B73 allospecificity and on <50% of HLA-A allospecificities. The latter determinant has been mapped to amino acid residues 239-242, 245, and 246 in the alpha3 domain. The conformational and the linear determinants share several structural features, but have no homology in their amino acid sequence. mAb TP25.99 represents the first example of a mAb recognizing two distinct and spatially distant determinants on a protein. The structural homology of a linear and a conformational determinant on an antigenic entity provides a molecular mechanism for the sharing of specificity by B and TCRs.     

Species specificity in the interaction of CD8 with the alpha 3 domain of MHC class I molecules.

The alpha 1 and alpha 2 domains of the class I MHC molecule constitute the putative binding site for processed peptides and the TCR, although the alpha 3 domain has been implicated as a binding site for the CD8 molecule. Species specificity in the binding of CD8 to the alpha 3 domain has been suggested as an explanation for the low xenogeneic T cell response to class I molecules, but results on this point have been conflicting and controversial. We have addressed this issue using CTL lines from HLA-A2.1 transgenic mice that specifically recognize and lyse A2.1-expressing cells infected with influenza A/PR/8 or pulsed with influenza matrix peptide M1(57-68). Species specificity was examined using transfectants that expressed hybrid molecules containing the alpha 1 and alpha 2 domains from HLA-A2.1 and the alpha 3 domain from a murine class I molecule. Lower levels of M1(57-68) peptide were required to sensitize L cell transfectants expressing a chimera that contained an H-2Dd alpha 3 domain than targets expressing the intact A2.1 molecule. However, at high doses of peptide, lysis of these two targets was similar. However, no reproducible difference in sensitization was observed using EL4 or Jurkat transfectants expressing A2.1 or A2.1 chimeric molecules that contained an H-2Kb alpha 3 domain. In all cases, however, lysis of peptide-pulsed A2.1 expressing targets was more sensitive to inhibition with anti-CD8 mAb than lysis of cells expressing these chimeric molecules. Thus, under suboptimal conditions such as low Ag density or in the presence of anti-CD8 mAb, these CTL preferentially recognize class I molecules with a murine alpha 3 domain. This suggests that there is some species specificity in the interaction of CD8 with the alpha 3 domain of the class I molecule. However, CTL recognition was inhibited by point mutations in the alpha 3 domain of HLA-A2.1 that have been shown to inhibit binding of human CD8 and recognition by human CTL, suggesting that murine CD8 interacts to some degree with human alpha 3 domains, and that similar alpha 3 domain residues may be important for murine and human CD8 binding. The relevance of these results to an understanding of low xenogeneic responses is discussed.     

Analysis by NMR spectroscopy of the structural homology between the linear and the cyclic peptide recognized by anti-human leukocyte antigen class I monoclonal antibody TP25.99*

The anti-human leukocyte antigen (HLA) class I monoclonal antibody (mAb) TP25.99 has a unique specificity since it recognizes both a conformational and a linear determinant expressed on the beta(2)-mu-associated and beta(2)-mu-free HLA class I heavy chains, respectively. Previously, we reported the identification of a cyclic and a linear peptide that inhibits mAb TP25.99 binding to the beta(2)-mu-associated and beta(2)-mu-free HLA class I heavy chains (S. A. Desai, X. Wang, E. J. Noronha, Q. Zhou, V. Rebmann, H. Grosse-Wilde, F. J. Moy, R. Powers, and S. Ferrone, submitted for publication). The linear X(19) and cyclic LX-8 peptides contain sequence homologous to residues 239-242, 245, and 246 and to residues 194-198, respectively, of HLA class I heavy chain alpha(3) domain. Analysis by two-dimensional transfer nuclear Overhauser effect spectroscopy of the induced solution structures of the linear X(19) and cyclic LX-8 peptides in the presence of mAb TP25.99 showed that the two peptides adopt a similar structural motif despite the lack of sequence homology. The backbone fold is suggestive of a short helical segment followed by a tight turn, reminiscent of the determinant loop region (residues 194-198) on beta(2)-mu-associated HLA class I heavy chains. The structural similarity between the linear X(19) and cyclic LX-8 peptides and the lack of sequence homology suggests that mAb TP25.99 predominantly recognizes a structural motif instead of a consensus sequence.     

The alpha 1/alpha 2 domains of class I HLA molecules confer resistance to natural killing

The expression of transfected HLA class I Ag has previously been shown to protect human target cells from NK-mediated conjugation and cytolysis. In this same system, transfected H-2 class I Ag fail to impart resistance to NK. In this study, we have mapped the portion of the HLA class I molecule involved in this protective effect by exploiting this HLA/H-2 dichotomy. Hybrid class I genes were produced by exon-shuffling between the HLA-B7 and H-2Dp genes, and transfected into the class I Ag-deficient B-lymphoblastoid cell line (B-LCL) C1R. Only those transfectants expressing class I Ag containing the alpha 1 and alpha 2 domains of the HLA molecule are protected from NK, suggesting the "protective epitope" is located within these domains. Since a glycosylation difference exists between HLA and H-2 class I Ag within these domains (i.e., at amino acid residue 176), the role of carbohydrate in the class I protective effect was examined. HLA-B7 mutant genes encoding proteins which either lack the normal carbohydrate addition site at amino acid residue 86 (B7M86-) or possess an additional site at residue 176 (B7M176+) were transfected into C1R. Transfectants expressing either mutant HLA-B7 Ag were protected from NK. Thus, carbohydrate is probably not integral to a class I "protective epitope." The potential for allelic variation in the ability of HLA class I Ag to protect C1R target cells from NK was examined in HLA-A2, A3, B7, and Bw58 transfectants. Although no significant variation exists among the HLA-A3, B7, and Bw58 alleles, HLA-A2 appears unable to protect. Comparison of amino acid sequences suggests a restricted number of residues which may be relevant to the protective effect.     

The allorecognition of H-2Kb-specific CD4-CD8- T cell hybridomas is influenced by the substitution at residue 256 of MHC class I molecules

Our previous studies demonstrated that allorecognition of HTB176.10 and HTB177.2, H-2Kb-reactive CD4-CD8- T cell hybridomas is markedly influenced by the exchange of the alpha 3 domain between H-2Kb and H-2Dp. The recombinant genes of the exon 4 between H-2Kb and H-2Dp were constructed to determine the residues of the alpha 3 domain that influence the allorecognition of these T cell hybridomas. Seven recombinant genes of the exon 4 were generated by in vivo recombination in Escherichia coli. Chimeric genes containing these recombinants were transfected into L cells and the transfectants expressing equivalent amounts of chimeric molecules were selected by flow cytometry. Studies on responses of these T cell hybridomas to the chimeric molecules confirmed our previous observation that the primary structure of the alpha 3 domain influences the allorecognition by the hybridomas. Moreover, it was indicated that residue 256 on the alpha 3 domain markedly affects the allorecognition by the T cell hybridomas, although substitutions at residues 184, 193, 195, 197, 262, and 264 exerted some effects on the T cell recognition. Further studies with the use of a single amino acid mutant of H-2Kb at residue 256 confirmed the effect of substitution at residue 256 on allorecognition of the T cell hybridomas. Taken together, results of this study demonstrated that polymorphism of the alpha 3 domain is indeed involved in the formation of allodeterminants recognized by TCR.     

Identification of monoclonal antibody defined epitopes on human leukocyte antigens utilizing phage display peptide libraries

Summary Utilizing phage display peptide libraries, we have identified and mapped the antigenic determinants recognized by mouse monoclonal antibodies (mAb) on two sets of immunologically important molecules, HLA class I and class II antigens. Anti-HLA class I mAb TP25.99 recognizes a conformational and a linear determinant on distinct regions of the HLA class I α3 domain. Anti-HLA class I mAb HO-4 recognizes a conformational determinant on the α2 domain of HLA-A2 and A28 allospecificities. Anti-HLA-DR1,-DR4,-DR6,-DR8,-DR9 mAb SM/549 recognizes a conformational determinant on the β chain of HLA class II antigens. These results indicate the versatility of phage display peptide libraries to characterize antigenic determinants recognized by anti-HLA mAb.     

Phenotypical and functional characterization of the CD8+ T cell repertoire of HLA-A2.1 transgenic, H-2KbnullDbnull double knockout mice.

Homozygous HLA-A2.1 transgenic H-2KbnullDbnull double knockout (KO) mice were created. Their potential to develop HLA-A2. 1-restricted cytolytic responses was compared with that of their classical transgenic counterparts, which still express H-2Kb, Db molecules. On cell surfaces, both strains express similar amounts of chimeric (alpha 1 alpha 2 domains of human, alpha 3 cytoplasmic domains of mouse) HLA-A2.1 molecules in noncovalent association with mouse beta 2-microglobulin. Compared with mice that are totally deprived of histocompatibility class Ia molecules (H-2KbnullDbnull double KO), the expression of HLA-A2.1 in transgenic/double KO mice resulted in sizeable increase in the periphery of CD8+ T cells with a normally diversified TCR repertoire. A biased education in favor of HLA-A2.1, ascribable to the absence of H-2 class Ia molecules, was evidenced in these transgenic/double KO mice by their improved capacity to mount HLA-restricted cytolytic responses, regardless of whether they were virally infected or injected with synthetic epitopic peptide. HLA class I transgenic, H-2 class Ia KO mice should represent useful animal models for the preclinical evaluation of vaccine formulations aiming at the induction of HLA class I-restricted CTL responses.     

Species-specific structural differences in the alpha 1 + alpha 2 domains determine the frequency of murine cytotoxic T cell precursors stimulated by human and murine class I molecules

The frequency of murine CTL precursors (CTLp) that recognize the human histocompatibility Ag HLA-A2 and HLA-B7 was measured and found to be approximately two orders of magnitude lower than the frequency of CTLp that recognize murine H-2 alloantigens. The possible contribution of other cell surface molecules to this difference in response was addressed by expression of the H-2Ld molecule on a human cell and the HLA-B7 molecule on a murine cell. It was found that both human and murine H-2Ld expressing cells elicited comparable levels of H-2Ld specific CTL. Although murine HLA-B7 positive cells stimulated a higher frequency of HLA-B7-specific CTLp than did human cells, this appeared to be largely due to stimulation of CTLp that recognized HLA-B7 in the context of H-2 molecules; consequently, it was concluded that the difference in the frequency of murine CTLp elicited by human and murine class I Ag is due to species specific structural differences in these molecules. The regions of the class I molecule that were responsible for this difference were mapped using chimeric class I molecules constructed to replace domains of the human molecule with their murine counterparts. It was found that the frequency of CTLp is controlled by structures within the alpha 1 and alpha 2 domains of the molecule. These results are discussed in the light of models for T cell recognition of class I Ag and the diversification of the T cell receptor repertoire.     

Analysis of the HLA-Cw3-specific cytotoxic T lymphocyte response of HLA-B7 X human beta 2m double transgenic mice

The cytolytic responses of either normal (non transgenic), HLA-B7 (single transgenic) or HLA-B7 x human beta 2 microglobulin (double transgenic) DBA/2 mice induced by transfected HLA-Cw3 P815 (H-2d) mouse mastocytoma cells were compared, to evaluate whether the expression of an HLA class I molecule in responder mice would favor the emergence of HLA-specific, H-2-unrestricted CTL. Only 8 of 300 HLA-Cw3-specific CTL clones tested could selectively lyse HLA-Cw3-transfected cells in an H-2-unrestricted manner, all having been isolated after hyperimmunization of double transgenic mice. These clones also lysed HLA-Cw3+ human cells. Unexpectedly, the lysis of the human but not that of the murine HLA-Cw3 cells was inhibited by Ly-2,3-specific mAb. Despite significant expression of HLA-B7 class I molecules on transgenic lymphoid cells, including thymic cells, limiting dilution analysis and comparative study of TCR-alpha and -beta gene rearrangements of the eight isolated clones (which suggested that they all derived from the same CTL precursor) indicated that the frequency of HLA-Cw3-specific H-2 unrestricted cytotoxic T lymphocytes remained low (even in HLA-B7 x human beta 2-microglobulin double transgenic mice). This suggests that coexpression of HLA class I H and L chain in transgenic mice is not the only requirement for significant positive selection of HLA class I-restricted cytotoxic mouse T lymphocytes.     

Expression of class I HLA genes by trophoblast cells. Analysis by in situ hybridization

Evaluation of trophoblast cells by immunohistology has shown that subpopulations of trophoblast cells express class I HLA differently from one another and differently from embryonic and adult cells. Placental syncytial trophoblast does not express detectable levels of class I HLA; chorion membrane cytotrophoblasts bind one mAb to monomorphic determinants of class I Ag, W6/32, but not a second, 61D2. In the present study, sections of normal term placentae and matching extraplacental membranes were evaluated by in situ hybridization procedures for cells containing class I HLA mRNA using pHLA1.1, which is complementary to HLA-B. Class I Ag expression was identified by immunohistology using two mAb to class I HLA (W6/32, 61D2) and the mAb 4E to identify HLA-B. Placental syncytial trophoblast contained low to undetectable levels of class I mRNA and failed to bind all three mAb. Chorion membrane cytotrophoblast cells contained moderate levels of class I HLA mRNA and were positive with the mAb W6/32 but were negative with 61D2 and 4E. In adjacent tissues, fetal mesenchymal cells and maternal decidual cells contained high levels of class I mRNA and were positive with all three mAb. The results suggest that syncytial trophoblast may not express class I HLA because of low steady-state levels of class I HLA mRNA. In contrast, chorionic cytotrophoblast cells may express truncated versions of class I HLA or nonclassical HLA-A,B,C-like Ag. Regulation of the expression of class I HLA gene products may be essential to the development of a satisfactory immunologic relationship between the mother and her semiallogeneic fetus during pregnancy.     

The complementarity-determining region-like loops of CD8 alpha interact differently with beta 2-microglobulin of the class I molecules H-2Kb and thymic leukemia antigen, while similarly with their alpha 3 domains

The murine CD8 glycoprotein interacts with both classical MHC class I molecules and some nonclassical molecules, including the thymic leukemia Ag (TL). TL binds preferentially to CD8alphaalpha homodimers with a 10-fold higher affinity than H-2K(b) class I molecules. To understand the molecular basis for this difference, we created a panel of CD8alpha mutants and tested the ability of the CD8alphaalpha homodimers to bind to H-2K(b) tetramers and TL tetramers. Mutations in three CD8 residues located on the complementarity-determining region-like loops contacting the negatively charged loop in the alpha3 domain of MHC class I greatly reduced binding to both tetramers. Because TL and H-2K(b) class I sequences are highly conserved in the alpha3 domain of MHC class I, this suggests that CD8 contacts the alpha3 domain of TL and H-2K(b) in a similar manner. In contrast, mutations in residues on the A and B beta strands of CD8 that are involved in contact with beta(2)-microglobulin affected interaction with the H-2K(b) tetramer, but not the TL tetramer. Therefore, the orientation of interaction of TL with CD8 appears to be different from that of H-2K(b). The unique high affinity binding of TL with CD8alphaalpha is most likely a result of amino acid differences in the alpha3 domain between TL and H-2K(b), particularly at positions 198 (K to D) and 228 (M to T), which are contact residues in the CD8alphaalpha-H-2K(b) cocrystal.     

Enhancing effect of anti-HLA class I monoclonal antibodies on T cell proliferation induced via CD2 molecule

The mAb 131 to a determinant preferentially expressed on the gene products of the HLA-A locus, the mAb Q6/64 and 4E to determinants preferentially expressed on the gene products of the HLA-B locus, the anti-HLA-A2,A28 mAb CR11-351, HO-2, HO-3, HO-4, and KS1, and the anti-HLA-B7 cross-reacting group mAb KS4 enhanced proliferation of T cells in most, if not all, the PBMC preparations stimulated with the anti-CD2 mAb 9-1 + 9.6. The mAb CR10-215, W6/32, and 6/31 to distinct monomorphic determinants of HLA class I antigens enhanced CD2-induced T cell proliferation in at most 30% of the PBMC preparations tested. The anti human beta 2-microglobulin (beta 2-mu) mAb NAMB-1 displayed no detectable effect on the proliferation of T cells stimulated with the mAb 9-1 + 9.6. The enhancing effect of anti-HLA class I mAb is specific, is dose dependent, is not abrogated by the addition of exogenous IL-1 and IL-2 to the cultures, and reflects the interaction of anti-HLA class I mAb with T cells. Enhancement of CD2 mediated proliferation of T cells is not a unique property of anti-HLA class I mAb, since the anti-HLA class II mAb Q5/6 and Q5/13 also had a similar effect. Analysis of the kinetics of the enhancing effect of anti-HLA class I mAb suggests that they modulate an early event of T cell activation and may affect the interaction of T cells with mAb 9-1. Phenotyping of T lymphocytes activated by mAb 9-1 + 9.6 in the presence of anti-HLA class I mAb suggests that the enhancing effect of anti-HLA class I mAb may reflect the recruitment of a higher percentage of T cells. The present study has shown for the first time that certain, but not all, the determinants of the HLA class I molecular complex are involved in the proliferation of T cells stimulated with the anti-CD2 mAb 9-1 + 9.6. Furthermore, the inhibitory effect of mAb CR11-351, KS1, Q6/64, and W6/32 on the proliferation of T cells stimulated with mAb OKT3 or with mAb BMA 031 indicates that the same determinants of HLA class I antigens play a differential regulatory role in T cell proliferation induced via the CD2 and CD3 pathway.     

Identification of monoclonal antibody defined epitopes on human leukocyte antigens utilizing phage display peptide libraries

Utilizing phage display peptide libraries, we have identified and mapped the antigenic determinants recognized by mouse monoclonal antibodies (mAb) on two sets of immunologically important molecules, HLA class I and class II antigens. Anti-HLA class I mAb TP25.99 recognizes a conformational and a linear determinant on distinct regions of the HLA class I 3 domain. Anti-HLA class I mAb HO-4 recognizes a conformational determinant on the 2 domain of HLA-A2 and A28 allospecificities. Anti-HLA-DR1, -DR4, -DR6, -DR8, -DR9 mAb SM/549 recognizes a conformational determinant on the chain of HLA class II antigens. These results indicate the versatility of phage display peptide libraries to characterize antigenic determinants recognized by anti-HLA mAb.     

The Q7 alpha 3 domain alters T cell recognition of class I antigens.

In this study we have analyzed the role of the alpha 3 domain of class I molecules in T cell recognition. Using the laboratory engineered molecules LLQQ (alpha 1/alpha 2 from Ld, alpha 3, and phosphatidyl inositol (PI) linked C terminus from Q7) and LLQL (alpha 1/alpha 2 from Ld, alpha 3 from Q7, transmembrane (TM) and cytoplasmic domains from Ld) we show that these molecules are not recognized by primary Ld-specific CTL. The cell membrane expression of both Ld and LLQL are upregulated by co-culture with an exogenously supplied murine cytomegalovirus-derived peptide indicating that the Q7 alpha 3 domain does not interfere with binding of Ag to alpha 1/alpha 2. However, only peptide pulsed Ld but not LLQL target cells are recognized by Ld-restricted-peptide specific CTL. In contrast to the above results, LLQL and LLQQ molecules can be recognized by bulk alloreactive anti-Ld CTL and 2/3 of CTL clones derived from in vivo primed mice. The fact that these secondary CTL recognize LLQQ indicates that a PI linkage is permissive for presentation of class I epitopes to alloreactive CTL. These secondary CTL are resistant to blocking at the effector stage by mAb against CD8 and express relatively low levels of membrane CD8 molecules compared to CTL from unprimed mice. Further, culture of unprimed CTL precursors in the presence of CD8 mAb also allows for the generation of CD8-independent CTL that recognize LLQL. Taken together, these data indicate that the alpha 3 domain of Q7 (Qa-2) prevents CD8-dependent CTL from recognizing Ld, regardless of whether the class I molecule is attached to the cell surface by a PI moiety or as a membrane spanning protein domain. We hypothesize that this defect in recognition is most likely due to an inability of CD8 to interact efficiently with the Q7 alpha 3 domain and could account for why Q7 molecules do not serve as restricting elements for virus and minor H-Ag-specific CTL.     

Cell surface expression and alloantigenic function of a human class I MHC heavy chain gene (HLA-B7) in transgenic mice

We have introduced the gene encoding the heavy chain of the human MHC class I Ag HLA-B7 into transgenic mice. The gene was shown to be expressed at both the RNA and protein level. Cell surface HLA-B7 was detected on whole spleen cells by immunoprecipitation and on purified T cells by flow cytometry (FACS). Normal mice immunized with H-2-syngeneic B7-transgenic spleen cells generated CTL capable of killing transgenic cells and B7-expressing human JY cells. Anti-HLA mAb blocked the killing of JY cells. These results indicate that the human class I Ag HLA-B7 can be expressed at the surface of transgenic spleen cells in the absence of human beta 2-microglobulin, and that a significant fraction exists in a form recognizable by nontransgenic CTL as a major histocompatibility Ag unrestricted by H-2.     

Clonal analysis of H-2Kb + TNP recognition by T cells with the use of H-2Kbm mutants and H-2Kb-specific monoclonal antibodies

Eleven long-term cytotoxic T lymphocyte (CTL) clones derived from C57BL/10 T cells sensitized in vivo and in vitro with trinitrobenzene sulfonate- (TNBS) treated syngeneic cells were all restricted to the K end of H-2b. The fine specificity of these CTL clones was analyzed by using H-2Kbm mutant target cells and H-2Kb-specific monoclonal antibodies (mAb). Seven distinct patterns of reactivity of the T cell clones could be observed with the use of six H-2Kbm mutant target cells. Further heterogeneity could be detected in terms of the ability of anti-Lyt-2 mAb to inhibit CTL activity. Cross-reactivity between H-2Kb + TNP and H-2Kbm + TNP was observed for all clones tested for bm5 and bm6, but less frequently for bm3 (8/11), bm8 (7/10), bm4 (4/11), and bm1 (3/11). It was further observed that amino acid substitutions located in the first domain only (one clone), or in the second domain only (six clones), or in either the first or the second domain (three clones) of the H-2Kb molecule could affect target cell recognition by a given T cell clone. the latter type of reactivity suggested that some clones recognized "conformational" determinants of the H-2 molecule, or that amino acid substitutions in one domain might influence the structure of the next domain. One H-2Kb + TNP-reactive clone exhibited a heteroclitic behavior with decreasing avidities for target cells expressing H-2Kbm8 + TNP, H-2Kb + TNP, and H-2Kbm8, which further extends the various patterns of T cell cross-reactions observed within a given class of MHC products. The use of H-2Kb-specific mAb in blocking studies as an attempt to define further the H-2Kb epitopes recognized by CTL clones indicated that: a) TNBS treatment may affect the antigenicity of the H-2Kb molecule as assessed by some mAb; and b) that the T cell clone-target cell interaction may or may not be inhibited by a given mAb, depending on structural variations of the H-2Kb molecule (use of H-2Kbm mutants) that do not affect the interaction itself. These results indicate that this type of analysis does not permit correlation of serologic- and T cell-defined epitopes.     

CD8+ T cells are necessary for recognition of allelic, but not locus-mismatched or xeno-, HLA class I transplantation antigens

Although HLA transgenic mice (HLA TgM) could provide a powerful approach to investigate human MHC-specific T cell responsiveness, the extent to which these molecules are recognized by the mouse immune system remains unclear. We established TgM expressing HLA class I alleles A2, B7, or B27 in their fully native form (HLAnat) or as hybrid molecules (HLAhyb) of the HLA alpha1/alpha2 domains linked to the H-2Kb alpha3, transmembrane, and cytoplasmic domains (i.e., to maintain possible species-specific interactions). Comparison of each as xeno- (i.e., by non-TgM) vs allo- (i.e., by TgM carrying an alternate HLA allele) transplantation Ags revealed the following: 1) Although HLAhyb molecules induced stronger xeno-CD8+ T cell responses in vitro, additional effector mechanisms must be active in vivo because HLAnat skin grafts were rejected faster by non-TgM; 2) gene knockout recipients showed that xenorejection of HLAnat and, unexpectedly, HLAhyb grafts doesn't depend on CD8+ or CD4+ T cells or B cells; 3) each HLAhyb strain developed tolerance to "self" but rejected allele- (-B27 vs -B7) and locus- (-B vs -A) mismatched grafts, the former requiring CD8+ T cells, the latter by CD8+ T cell-independent mechanisms. The finding that recognition of xeno-HLAhyb does not require CD8+ T cells while recognition of the identical molecule in a strictly allo context does, demonstrates an alpha1/alpha2 domain-dependent difference in effector mechanism(s). Furthermore, the CD8+ T cell-independence of locus-mismatched rejection suggests the degree of similarity between self and non-self alpha1/alpha2 determines the effector mechanism(s) activated. The HLA Tg model provides a unique approach to characterize these mechanisms and develop tolerance protocols in the context of human transplantation Ags.     

Deletion of antigen-specific immature thymocytes by dendritic cells requires LFA-1/ICAM interactions.

An in vitro assay was used for assessing the participation of various cell surface molecules and the efficacy of various cell types in the deletion of Ag-specific immature thymocytes. Thymocytes from mice expressing a transgenic TCR specific for the male Ag presented by the H-2Db class I MHC molecule were used as a target for deletion. In H-2d transgenic mice, cells bearing the transgenic TCR are not subjected to thymic selection as a consequence of the absence of the restricting H-2Db molecule but, nevertheless, express this TCR on the vast majority of immature CD4+8+ thymocytes. In this report we show that CD4+8+ thymocytes from H-2d TCR-transgenic mice are preferentially killed upon in vitro culture with male APC; DC were particularly effective in mediating in vitro deletion when compared with either B cells or T cells. Deletion of CD4+8+ thymocytes by DC was H-2b restricted and could be inhibited by mAb to either LFA-1 alpha or CD8. Partial inhibition was observed with mAb to ICAM-1, whereas mAb to CD4 and LFA-1 beta were without effect. These results are the first direct evidence of LFA-1 involvement in negative selection and provide further direct support for the participation of CD8/class I MHC interactions in this process. Like the requirements for deletion, activation of mature male-specific CD4-8+ T cells from female H-2b TCR-transgenic mice was also largely dependent on Ag presentation by DC and required both LFA-1/ICAM and CD8/class I MHC interactions; these results support the view that activation and deletion may represent maturation stage-dependent consequences of T cells encountering the same APC. Finally, our results also support the hypothesis that negative selection (deletion) does not require previous positive selection because deletion was observed under conditions where positive selection had not occurred.     

Human MHC class I transgenic mice deficient for H2 class I expression facilitate identification and characterization of new HLA class I-restricted viral T cell epitopes

Although mice transgenic (Tg) for human MHC (HLA) class I alleles could provide an important model for characterizing HLA-restricted viral and tumor Ag CTL epitopes, the extent to which Tg mouse T cells become HLA restricted in the presence of endogenous H2 class I and recognize the same peptides as in HLA allele-matched humans is not clear. We previously described Tg mice carrying the HLA-B27, HLA-B7, or HLA-A2 alleles expressed as fully native (HLA(nat)) (with human beta(2)-microglobulin) and as hybrid human/mouse (HLA(hyb)) molecules on the H2(b) background. To eliminate the influence of H2(b) class I, each HLA Tg strain was bred with a H2-K(b)/H2-D(b)-double knockout (DKO) strain to generate mice in which the only classical class I expression was the human molecule. Expression of each HLA(hyb) molecule and HLA-B27(nat)/human beta(2)-microglobulin led to peripheral CD8(+) T cell levels comparable with that for mice expressing a single H2-K(b) or H2-D(b) gene. Influenza A infection of Tg HLA-B27(hyb)/DKO generated a strong CD8(+) T cell response directed at the same peptide (flu nucleoprotein NP383-391) recognized by CTLs from flu-infected B27(+) humans. As HLA-B7/flu epitopes were not known from human studies, we used flu-infected Tg HLA-B7(hyb)/DKO mice to examine the CTL response to candidate peptides identified based on the B7 binding motif. We have identified flu NP418-426 as a major HLA-B7-restricted flu CTL epitope. In summary, the HLA class I Tg/H2-K/H2-D DKO mouse model described in this study provides a sensitive and specific approach for identifying and characterizing HLA-restricted CTL epitopes for a variety of human disease-associated Ags.