Analysis of hybrid H-2D and L antigens with reciprocally mismatched aminoterminal domains: functional T cell recognition requires preservation of fine structural determinants

Studies of immune recognition of hybrid class I antigens expressed on transfected cells have revealed an apparent general requirement that the N(alpha 1) and C1(alpha 2) domains be derived from the same gene in order to preserve recognition by virus-specific H-2-restricted and allospecific T cells. One exception has been the hybrid DL antigen in which the N domain of H-2Ld has been replaced by that of H-2Dd. Cells bearing this molecule serve as targets for some virus and allospecific CTL. Because cells expressing the reciprocal hybrid LD (N domain of H-2Dd replaced by that of H-2Ld) antigen have not been available, it has not been possible to evaluate whether this exception stemmed from the relatedness of H-2Ld and H-2Dd or whether the DL antigen fortuitously preserved some function of the parent molecule as a rare exception. To assess this question, and to evaluate the contribution of the N and C1 domains of H-2Ld and H-2Dd to serologic and T cell recognition, we have constructed the reciprocal chimeric gene pLD (the N exon of H-2Ld substituted for that of H-2Dd), introduced this into mouse L cells by DNA-mediated gene transfer, and analyzed the expressed product biochemically, serologically, and functionally. Transformant L cells expressing either LD or DL antigens were both reactive with a number of anti-H-2Ld or anti-H-2Dd N/C1-specific monoclonal antibodies, indicating the preservation in the hybrid molecules of determinants controlled by discrete domains. Mab binding was generally greater with cells expressing hybrid DL antigen than with those transformants expressing LD molecules. Moreover, the amount of beta 2M associated with DL antigens was more than that associated with LD. Cells expressing hybrid DL antigens were recognized as targets by bulk and cloned allospecific anti-H-2Dd and anti-H-2Ld CTL, whereas cells expressing LD molecules were not recognized by any of the T cells tested. VSV-specific H-2Ld-restricted CTL failed to lyse VSV-infected targets expressing either DL or LD. These results indicate that T cell reactivity of cells expressing the DL hybrid antigen is an exception to the observed general requirement for class I antigens to possess matched N and C1 domains for functional T cell recognition by T cells restricted to parental antigens.(ABSTRACT TRUNCATED AT 400 WORDS)     

Recognition of the alpha-1 and alpha-2 domains of H-2 molecules by allospecific cloned T cells

Previously we had shown that allospecific bulk cultures of cytolytic T lymphocytes lysed the products of cloned class I major histocompatibility genes expressed after DNA-mediated gene transfer. In these experiments, performed by using cloned allospecific T cell effectors, a T cell hybridoma, and recombinant DNA technology, we have been able to map determinants recognized by these T cell clones to the alpha-1 domain of H-2Dd and the alpha-2 domain of H-2Ld (four of eight clones). Target cells used were L cells (H-2k), expressing wild type or hybrid H-2 antigens of H-2d origin. Thus, for the first time determinants recognized by cloned T cells are found in the recombined alpha-1 and alpha-2 domains.     

Adoptive transfer of delayed-type hypersensitivity to Sendai virus. III. Effect of H-2 mutations on recognition by K, D-region restricted T effector lymphocytes

The H-2 restriction pattern of cytolytic T lymphocytes (Tc) and T lymphocytes which mediate a delayed-type hypersensitivity response (Td) directed against infectious Sendai virus was investigated using H-2 mutant mice. Td and Tc lymphocytes exhibit the same fine specificity for self-recognition, for example, B6.C-H-2bm1 effector T cells were unable to recognize viral antigens in association with wild-type Kb and vice versa, B6.H-2bm6 effector cells did not mediate a reaction against virus plus wild-type Kb but, on the other hand, T cells of wild-type Kb recognized virus plus Kbm6 BALB/c-H-2dm2 T cells lacked reactivity against virus in association with wild-type Dd, but again wild-type Dd effector cells recognized virus plus Ddm2.     

Construction of novel class I histocompatibility antigens by interspecies exon shuffling

Human and mouse class I histocompatibility antigens share considerable structural homology at both the protein and DNA sequence level. This homology has allowed the production of hybrid class I molecules by the reciprocal exchange of DNA sequences corresponding to equivalent domains of HLA-B7 and either H-2Ld or H-2Dd. It is shown that these genes give rise to protein products that are stably expressed on the surface of murine L cells after DNA-mediated gene transfer. These proteins express only those monoclonal antibody-defined H-2 determinants that are expected based on their genetic construction. The molecules have allowed the localization of a number of polymorphic and monomorphic HLA-specific epitopes. In all but one case, expression of an epitope on a domain does not appear to be influenced by the replacement of adjacent human domains with their murine equivalents, suggesting a considerable degree of structural independence of the domains. Cells expressing the hybrid molecules have also been tested as targets for a panel of HLA-B7-specific cytotoxic T cell clones. The results show that the polymorphic determinants recognized by these clones map to the alpha 1 and alpha 2 domains of the HLA-B7 molecule. No evidence for an influence of species-related amino acid sequence differences in the third extracellular domain on T cell recognition was seen. The results are discussed in light of the proposed domain structure of the class I proteins and the potential use of such molecules for further functional studies.     

Recognition of EBV plasma membrane protein expressed on murine cells after gene transfer

The immune response to B lymphocytes infected with Epstein-Barr virus (EBV) prevents their overgrowth in normal humans. A murine model is now described for analyzing the T cell immune response to Epstein-Barr virus genes expressed in murine lymphoblasts by gene transfer. In mice, a 60,000 dalton virus-encoded protein characteristically found in the plasma membrane of latently infected human lymphocytes readily induces both proliferative and cytolytic T lymphocytes specific for both the EBV protein and murine major histocompatibility proteins. Longterm cultures of L3T4+ cells, some of which were cytolytic, were found to be restricted by H-2I-Ed and the latent membrane protein. Similarly, Lyt-2+ cells were cytolytic and were restricted by H-2Ld and the lymphocyte membrane protein gene product. The similarity in murine and human effector cell responses suggests that this is a useful experimental model, and the EBV latent infection membrane protein may be an important antigen in the immune restriction of growth transformed latently infected lymphocytes.     

The alpha-3 domain of class I MHC proteins influences cytotoxic T lymphocyte recognition of antigenic determinants located within the alpha-1 and alpha-2 domains

An interspecies class I MHC molecule, Kb1+2/A2 (in which the alpha-1 and alpha-2 domains of the H-2Kb molecule have been linked to the alpha-3, transmembrane and intracytoplasmic domains of the HLA-A2 molecule) has been expressed on both human and mouse target cells by gene transfer. Maintenance of serologic determinants has been demonstrated. However, decreased lysis by allospecific CTL populations of cell lines that expressed a hybrid interspecies class I molecule, Kb1+2/A2, as compared with lines that expressed the native Ag, H-2Kb, has been described. An analysis with a limited panel of H-2Kb allospecific clones demonstrated that not all H-2Kb-specific CTL can lyse cells that express Kb1+2/A2 Ag. This suggested that the reduction of lysis by CTL populations was due to the loss of specific alloreactive clones in the population. Each clone used in this study was then defined as having high or low affinity characteristics. No correlation between the affinity of the CTL and the ability to recognize the interspecies hybrid molecule could be shown. Rather, these data suggest that antigenic determinants that are located within the polymorphic domains, alpha-1 and alpha-2, may be conformationally influenced by the alpha-3 domain.     

Selection of the T-cell repertoire in transgenic mice expressing a transplantation antigen in distinct thymus subsets

Transgenic mice that expressed a transplantation antigen, H-2Kb, in an unusual tissue distribution have been developed. Gene-regulatory elements from the immunoglobulin heavy-chain locus (Emu enhancer and heavy chain promoter) were linked to the class I Kb gene and the construct microinjected into fertilized mouse eggs of a different haplotype. It was expected that such gene-regulatory elements would direct expression of the foreign class I molecules only to B and T lymphocytes. However, expression was also detected in a subset of thymus medullary epithelium. The Kb molecules expressed on this thymic subset were unable to positively select T cells for passage to the periphery. The mice were, however, tolerant of the cell types expressing the foreign Kb molecules and were also tolerant of Kb presented as skin grafts. These results suggest that not all components of thymic epithelium are involved in positive selection of T cells and that transplantation antigens expressed on non-dendritic cells can induce tolerance.     

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.     

Antibody-induced redistribution of normal and tumor associated surface antigens

Redistribution (capping) of normal and tumor-associated surface antigens was studied on murine and human cells by the indirect membrane immunofluorescence (MIF) technique. The capping of H-2 isoantigens was compared on normal mouse T-lymphocytes and on YAC cells, a Moloney leukemia virus (MLV) induced lymphoma. H-2 and Moloney virus induced cell surface antigen (MCSA) capping was compared on three YAC lines with different MCSA concentrations. H-2 and tumor-associated surface antigen capping was compared on two polyoma induced sarcoma lines and five methylcholanthrene induced sarcoma lines. In the human system, IgM-capping was compared on normal lymphocytes and on the Burkitt lymphoma derived Daudi line. Capping of HL-A and the Epstein-Barr virus (EBV) determined membrane antigen (MA) was compared on the Burkitt lymphoma derived line Maku and on EBV-superinfected Daudi cells. H-2 antigens on normal murine cells capped more promptly and on a larger fraction of the cell population on the various tumor cells. Surface associated IgM showed a better capping on normal lymphocytes than on Daudi cells. All tumor associated antigens except MCSA, showed good capping. MCSA was almost completely refractory to capping. Increasing concentrations of MCSA appeared to inhibit the capping of H-2 on the YAC sublines with different concentrations of MCSA. The polyoma induced ascites sarcoma (SEWA) did not cap either with regard to H-2 or the polyoma determined surface antigen.     

H-2-restricted cytolysis of L cells doubly transformed with a cloned H-2Kb gene and cloned retroviral DNA

We report the construction of target cells by the double transformation of mouse L cells with a cloned H-2Kb gene and molecular clones of Akv or Gross murine leukemia virus (MuLV) or the cloned gag gene of Akv MuLV. Cytolytic T lymphocytes (CTL) generated in BALB.B mice specific for Gross MuLV (closely related to Akv MuLV) kill these doubly transformed cells. This is a direct indication that a CTL subpopulation recognizes H-2Kb antigen in association with a viral antigen encoded by the gag gene of Gross MuLV.     

Wheat germ agglutinin-resistant variant of EL4 containing altered oligosaccharides as a target cell for cytotoxic T cells

A lectin-resistant variant of the murine EL4 lymphocytic leukemia cell line was selected in the presence of wheat germ agglutinin for low levels of cell-surface sialic acid. H-2Kb was the major internally radiolabeled H-2b molecule on the cell-surface of WD1, and it was not sialylated, as determined by two-dimensional gel analysis. Endo-beta-N-acetylglucosaminidase H treatment of the WD1 membrane fractions suggested that the oligosaccharides on the cell-surface H-2Kb molecule were complex, but nonsialylated. Monoclonal antibody inhibition of the allogeneically primed cell-mediated cytotoxicity (CMC) reaction indicated that the T cells (BALB/c anti-EL4; H-2d anti-H-2b) were specific only for the H-2Kb target cell antigen. These WD1 variant cells were used as targets in the CMC assay using anti-H-2Kb T cells and compared with the parent EL4 in vitro line. The change in the cell-surface oligosaccharide did not affect the susceptibility to lysis by the cytotoxic T lymphocytes even though there were 2.5-fold more H-2Kb antigens on the WD1 variant cell (1.5 X 10(5) sites/cell) than on the parent EL4 in vitro cell (5.9 X 10(4) sites/cell). It was possible to isolate highly purified preparations of H-2Kb from either the EL4 or the WD1 line using a monoclonal antibody affinity column. Interestingly, the variant WD1 cell would no longer grow in the peritoneal cavity of the syngeneic C57BL/6 mouse.     

Resistance to cell-mediated cytotoxicity is correlated with reduction of H-2K gene products in AKR leukemia

AKR leukemia cell lines differing in the amount of H-2K and H-2D antigens expressed on the cell surface were used to assess cell-mediated immune responses in syngeneic mice against Gross/AKR murine leukemia virus (MuLV)-induced tumors. Leukemic cells with reduced expression of H-2K^k antigens were inactive as inducers of Gross-MuLV/H-2^k-specific cytotoxic T lymphocytes (CTL) and resistant to lysis by CTL raised against H-2K^k positive AKR leukemia cells. H-2K^k positive leukemias induced cytotoxic effectors, which upon restimulation in vitro, lysed the stimulating and other H-2K^k positive leukemia cells. In antibody inhibition experiments, T-cell-mediated cytotoxicity to these leukemias could only be inhibited by antisera and monoclonal antibodies specific for the H-2K^k antigens. Due to this specific role of H-2K^k antigens in T-cell cytotoxicity to Gross/AKR MuLV-induced tumors, reduced expression of H-2K^k antigens on spontaneous AKR leukemic cells could have important implications for surveillance of these neoplastic cells.Abbreviations used in this paper CTL cytotoxic T lymphocytes - MuLV murine leukemia virus     

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.     

Differential lack of class I H-2d antigen expression by sublines of the BALB/c S49 T cell lymphoma

Five different sublines of the BALB/c murine S49.1 T cell lymphoma were found to exhibit distinct patterns of absence of detectable H-2d class I major histocompatibility antigen expression. The results were demonstrated and verified by a) the generation of H-2Kd-, H-2Dd,Ld-, and H-2Ld-specific cytotoxic T lymphocytes that were assayed on S49.1 target cell lines, b) antibody-mediated cytotoxicity with the use of anti-H-2d monoclonal reagents, and c) flow microfluorometry. The five lines investigated were S49.1, T-25, T-25ADH, Thy-1-, and 100/0. None of these lines expressed detectable levels of Ld. S49.1 expressed both Kd and Dd, T-25 and T-25ADH expressed Dd but not Kd or Ld, Thy-1- expressed Kd but not Dd or Ld, and 100.0 did not express any detectable amounts of Kd, Dd, or Ld. These results indicate that K and D (and L) antigens can be expressed independently of each other and suggest that expression of class I antigens is controlled in a locus-specific manner.     

The E3/19K protein of adenovirus type 2 binds to the domains of histocompatibility antigens required for CTL recognition

The E3/19K protein of human adenovirus type 2 binds to HLA class I antigens and blocks their terminal glycosylation and cell surface expression. The nature of this interaction is non-covalent and involves neither disulfide bridges between the two molecules nor their carbohydrates. The murine H-2 Kd antigen associates with the E3/19K protein in a similar fashion to human HLA antigens whereas the allelic product H-2 Kk does not. Hybrid genes between the Kd and Kk alleles were constructed and their products were expressed in embryonic kidney cells together with the E3/19K protein. This allowed us to identify the alpha 1 and alpha 2 domains as the essential structures of the histocompatibility antigens for binding the viral protein. Interestingly, these domains are also crucial for T cell recognition. The implications for the evolution of adenoviruses and their ability to cause persistent infections are discussed.     

Cross-reactivity between human and murine lymphocyte antigens: III. Reactivity of H-2 allo- and xenoantisera with human lymphoid cells

H-2 alloantisera and antimouse lymphocyte xenoantisera react with 14%–100% of human lymphocytes from a panel of at least 80 unrelated people. Population and family studies did not reveal HL-A specificity of such lymphocytotoxic antibodies but indicated that the antibodies are directed against polymorphic antigenic determinants inherited in association with HL-A antigens. H-2 allo- and xenoantisera absorbed with human lymphoid cells and a panel of platelets bearing all the known HL-A specificities were still cytolytic when tested against murine lymphocytes, suggesting that only a small proportion of the heterogeneous population of H-2 antibodies react with human lymphocytes. On the other hand, HL-A alloantisera could be absorbed by lymphocytes from certain murine strains. These results suggest that the crossreactivity between human and murine lymphocytes is caused by antigens common to several HL-A (or H-2) types or by antigens linked to HL-A but not identical with them.     

Immunodominance in the T cell response to multiple non-H-2 histocompatibility antigens. III. Single histocompatibility antigens dominate the male antigen

Immunization of mice with multiple non-H-2 histocompatibility antigens results in the generation of cytolytic T lymphocytes that are specific for a limited number of immunodominant antigens. The experiments presented in this communication were designed to reveal immunodominance in pairwise combinations of autosomal and sex-linked non-H-2 histocompatibility (H) antigens. Priming and boosting responders with the male antigen, H-Y, paired with the H-4.2, H-7.1, or H-3.1 antigens, resulted in the generation of cytolytic T cells specific for the autosomal H antigens but not the H-Y antigen. Furthermore, co-immunization and boosting of C57BL/6 female responder spleen cells with BALB.B male cells resulted in the generation of cytolytic T cells specific for the BALB.B immunodominant antigens but not H-Y. No dominance was observed in H-4-plus H-7-incompatible combinations. Co-immunization of three different H-3 congenic strains with H-3.1 plus H-Y demonstrated that an efficient anti-H-3.1 T cell response is required for observing H-3.1 immunodominance over H-Y. Co-expression of H-3.1 and H-Y on the same priming and boosting cells was required for immunodominance. In fact, immunization with H-3.1 and H-Y presented on different cells resulted in normal generation of H-Y-specific cytolytic T cells, but no generation of H-3.1-specific cytolytic T cells resulted unless H-Y-specific cells were stimulated in the mixed lymphocyte cultures. These observations suggest that in vitro T cell responses to paired, non-H-2 H antigens may be independent, competitive, or synergistic, depending on the identity of the antigens and the priming and boosting conditions.     

T cells from naive mice suppress the in vitro cytotoxic response against endogenous Gross virus-induced tumor cells

Syngeneic normal lymphoid cells added in co-culture of immune lymphocytes and tumor cells reveal a suppressive activity inhibiting the generation of cytolytic T lymphocytes. The suppression was specific for the response directed against endogenous virus-induced or x-ray-induced tumor cells expressing endogenous C type virus antigens. Thymocytes, spleen cells, or lymph node cells from naive mice were able to express this suppressive activity. The same cells displayed no suppressive activity on killer cells directed against exogenous C type virus-induced tumor cells. The suppressor cells were Thy-1+, Lyt-1- 2+. Our results strongly suggested that the spontaneous suppressor cells exert their activity by interacting with an early step on the CTL response, probably at the level of the helper T cell function. The suppressive activity was mediated by soluble factor(s) that were antigen specific and possibly H-2 restricted. The possible implications of these spontaneous suppressor T lymphocytes in the development of endogenous virus-induced tumors and their possible implications in tolerance to self antigens are discussed.