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.     

Secondary structure of the murine histocompatibility alloantigen H-2Kb: relationship between heavy chain, beta 2-microglobulin, and antigenic reactivity

The far-ultraviolet circular dichroism (CD) spectra of the extracellular portion (papain-cleaved fragment) of the histocompatibility antigen H-2Kb and its noncovalently associated components, heavy chain and beta 2-microglobulin (beta 2m), indicate that the antigen is highly structured, containing about 30% alpha-helix, 41% beta-sheet, and 29% random coil. Separation of beta 2m from the heavy chain produced a decrease in heavy chain alpha-helix and beta-sheet structure which correlated with a loss of alloantigenic reactivity. Reconstitution of the heavy chain-beta 2m complex resulted in an increase in secondary structure which was greater than the sum of the free chains and the recovery of considerable alloantigenic reactivity. This suggests that some of the secondary structure and much of the alloantigenic reactivity may depend on conformation associated with the binding of beta 2m to heavy chain. A prediction of heavy chain secondary structure based on Chou-Fasman analysis of the primary amino acid sequence agreed with results from CD measurements and suggested that the segments of alpha-helix and beta-sheet structure are distributed throughout the molecule.     

Regulation of Qa-1 expression and determinant modification by an H-2D-linked gene,Qdm

We examined the regulation of cell surface expression of the Qa-1 alloantigens using a panel of monoclonal anti-Qa-1 cytotoxic T-lymphocyte (mCTL) lines. In contrast to previous reports of tissue-specific expression, we found that Qa-1 was widely expressed, resembling the prototypical class I H-2K/D molecules. We further found that an H-2D-linked gene, which we termed Qdm for Qa-1 determinant modifier, controlled expression of certain CTL-defined Qa-1 antigenic determinants. H-2D ^khomozygous haplotypes expressed a recessive allele of the modifier, Qdm ^k, whereas all other H-2 haplotypes tested expressed a dominant allele, Qdm ⁺. The Qdm ⁺ allele regulated in trans Qa-1 epitope expression from a Qdm ^kchromosome, modifying expression of particular CTL-defined Qa-1 antigenic determinants rather than affecting levels of cell surface expression. Mechanisms of Qdm function may include either a novel protein modification system or an unprecedented case of antigen recognition restricted by a nonclassical major histocompatibility complex molecule.     

Multiple CTL subsets generated by H-2.L locus products stimulation: evidence for an antigenic determinant private to H-2.Ld

Analysis of the fine specificity of CTL subpopulations raised by an H-2.L locus products stimulation (H-2dm2 anti-H-2d) was performed by absorption experiments by using monolayers of macrophages of H-2m, H-2q, H-2b, and H-2k haplotypes. The results show the existence of four CTL subsets. The pattern of reactivity of three of them could be correlated with that of antibodies present in H-2dm2 anti-H-2d antisera (anti-H-2.64, anti-H-2.65, and anti-H-2.Kk). The fourth CTL subset reacted with a specificity unique to H-2.Ld molecules (a private specificity?), absent on cells from H-2m, H-2q, H-2b, and H-2k haplotypes, and undescribed as yet by serologic methods. These data support the hypothesis that the H-2.L locus products are comparable in their antigenic properties to those of the H-2.K and H-2.D loci.     

Immunoselection of structural H-2Kb variants: Use of cloned cytolytic T cells to select for loss of a CTL-defined allodeterminant

Functional studies concerning the unique interaction between class I H-2 allodeterminants and cytolytic T lymphocyte (CTL) antigen receptors have benefitted from the development of H-2K^b mutant mouse strains and somatic H-2 variants selected with monoclonal antibody. Here, we describe the development of a novel approach to immunoselection of somatic H-2K^b variants employing a K^b-specific CTL clone as the negative selective agent. The rationale for this method is that the use of an alloreactive CTL clone as the selective agent should enable us to detect the emergence of structural K^b variants based on their loss of the relevant CTL-defined allodeterminant. Thus, these structural variants are well suited to an in-depth analysis of the functional relationship between H-2 antigens and receptor recognition by CTL. Using this approach, we successfully isolated two types of structural K^b variants, as well as numerous Kb-loss variants. The functional studies described in this paper indicate that these structural variants exhibit alterations in expression of both CTL-defined and serologically defined H-2K^b allodeterminants. The structural characterization of such variants should enable us to identify the precise amino acid residues responsible for the creation of the relevant CTL-defined K^b allodeterminants.     

Separation of spleen colony-forming units and prothymocytes by use of a monoclonal antibody detecting an H-2K determinant

The density of H-2K antigens was determined on both the mouse hemopoietic stem cell, using an assay for spleen colony-forming units (CFU-S), and the prothymocyte, using a thymus repopulation assay. This was done by light-activated cell sorting of bone marrow cells labeled first with a biotinylated antibody against H-2Kk and then with avidin-fluorescein isothiocyanate. Almost all CFU-S were found to be present among the 4% bone marrow cells with high forward light scatter (FLS), low perpendicular light scatter (PLS), and bright immunofluorescence. Thymus regeneration by this brightly fluorescent fraction was delayed 3 days compared to thymus regeneration by unsorted cells, although the same number of CFU-S was present in each cell suspension. This delay indicates that differentiation from CFU-S to prothymocytes takes 3 days. The fraction of cells in the FLS/PLS window with dull anti-H-2Kk fluorescence contained few CFU-S and gave rise to a transient thymus regeneration. These findings indicate that the prothymocyte carries fewer H-2K antigens than does the CFU-S. The H-2K antigen is a marker with which CFU-S and prothymocytes can be separated. Therefore, during early T-cell differentiation, the number of H-2K molecules on the cell surface decreases (CFU-S----prothymocyte----cortical thymocyte). During maturation of T cells, a reexpression of H-2K molecules occurs, since lymph node cells and spleen cells were shown to be brightly positive for H-2K antigen.     

Identification of the Cloned Gene for the Murine Transplantation Antigen H-2Kb by Hybridization with Synthetic Oligonucleotides

The H-2K^b gene is a member of the large major histocompatibility complex class I gene family. Since many members of this family cross-hybridize with class I cDNA probes, the cloned H-2K^b gene was identified by hybridization with specific oligonucleotide probes. This clone was definitively shown to encode the H-2K^b polypeptide by partial DNA sequencing and by serological and tryptic peptide analyses of the expressed product.     

Increased sensitivity to MHC-nonrestricted lysis of BL6 melanoma cells by transfection with class I H-2Kb gene

An H-2Kb- negative clone of BL6 melanoma (BL6-8) was transfected with neor, H-2Kb, or H-2IAk genes. In an 18-h cytotoxicity assay clones with high levels of H-2Kb Ag expression were found more sensitive to lysis by spleen cells of syngenic and allogeneic mice than H-2Kb low clones. NK cells were involved in the lysis of H-2Kb+ BL6 melanoma clones, with spleen cell cytotoxicity of mice increased after poly I:C stimulation or decreased after pretreatment with anti-asialo GM1 serum or NK1.1 mAb. Anti-TNF Ab were also able to reduce the cytotoxicity of normal spleen cells and completely abolished the cytotoxicity of the NK-depleted spleen cells suggesting involvement of NC cells in lysis of H-2Kb+ BL6 melanoma clones. Increase in sensitivity of H-2Kb+ BL6 cells to natural cell-mediated cytotoxicity was associated with the appearance of NK recognizable determinants as assessed by the cold target inhibition assay. All BL6 clones, irrespective of sensitivity to natural cell-mediated cytotoxicity, showed high sensitivity to lysis by LGL-derived granules. In contrast, all H-2Kb low BL6 clones were resistant and all H-2Kb highly positive clones were sensitive to lysis by TNF-alpha. When an H-2Kb highly positive clone was selected in vitro for resistance to TNF, it concomitantly showed increased resistance to cytotoxicity by spleen cells, confirming the importance of TNF in spleen cell cytotoxicity against H-2Kb+ melanoma cells. Taken together, the data indicate that class I H-2Kb but not class II H-2IAk gene product could increase the sensitivity of BL6 cells to lysis by NK and natural cytotoxic cells as well as TNF. We hypothesize that these effects could be due to pleiotropic effects of H-2Kb gene products on various biologic properties of BL6 melanoma cells some of which may be more directly involved in regulation of tumor cell sensitivity to lysis by NK and/or natural cytotoxic cells.     

Cross-reactive recognition of mouse cells expressing the bm3 and bm11 mutations within H-2Kb by H-2Kb-restricted herpes simplex virus-specific cytotoxic T lymphocytes

Cytotoxic T lymphocytes, generated in C57BL/6 mice in response to herpes simplex virus type 1 (HSV) and known to be restricted in their recognition of HSV-encoded antigen(s) in association with the class I H-2Kb gene product, were consistently found to contain a subpopulation that recognized and lysed uninfected, SV40-transformed cells that expressed the H-2Kbm3 and H-2Kbm11 mutant class I gene products on their cell surface. The mutant cell lines, designated Lgbm3SV and Kbm11SV, share a common amino acid substitution at position 77, with the bm3 mutation having an additional amino acid substitution at position 89. Cross-reactive lysis was observed only after in vivo priming with HSV, suggesting an important role for an antigen-dependent driving step in the expansion of these cross-reactive CTL. The phenotype of the cross-reactive effector population was further confirmed as a T lymphocyte by negative-selection techniques. Limiting dilution analysis of the frequency of cross-reactive CTL precursors suggested that cross-reactivity was mediated by a subpopulation of HSV-specific CTL, and this was confirmed by clonal analysis of the reactivity patterns of short-term, HSV-specific CTL clones. However, analysis of the specificity of the cross-reactive CTL population by cold-target inhibition of bulk culture-derived CTL, or by Spearman ranking analysis of limiting dilution-derived CTL, indicated that the specificity of the cross-reactive population for HSV-infected H-2b target cells and for uninfected bm3 or bm11 target cells was quite distinct. These findings suggested that the cross-reactive CTL population played little, if any, role in the HSV-specific CTL response as measured in vitro. The findings also suggested that the HSV-specific CTL clones able to mediate cross-reactive recognition of the bm3 and bm11 targets had a higher intrinsic avidity for the foreign target than for the inducing antigen.     

In vivo selection of a lymphocytic choriomeningitis virus variant that affects recognition of the GP33-43 epitope by H-2Db but not H-2Kb

CD8 T cells drive the protective immune response to lymphocytic choriomeningitis virus (LCMV) infection and are thus a determining force in the selection of viral variants. To examine how escape mutations affect the presentation and recognition of overlapping T-cell epitopes, we isolated an LCMV variant that is not recognized by T-cell receptor (TCR)-transgenic H-2Db-restricted LCMV GP33-41-specific cytotoxic T lymphocytes (CTL). The variant virus carried a single-amino-acid substitution (valine to alanine) at position 35 of the viral glycoprotein. This region of the LCMV glycoprotein encodes both the Db-restricted GP33-43 epitope and a second epitope (GP34-42) presented by the Kb molecule. We determined that the V-to-A CTL escape mutant failed to induce a Db GP33-43-specific CTL response and that Db-restricted GP33-43-specific CTL induced by the wild-type LCMV strain were unable to kill target cells infected with the variant LCMV strain. In contrast, the Kb-restricted response was much less affected. We found that the V-to-A substitution severely impaired peptide binding to Db but not to Kb molecules. Strikingly, the V-to-A mutation did not change any of the anchor residues, and the dramatic effect on binding was therefore unexpected. The strong decrease in Db binding explains why the variant virus escapes the Db GP33-43-specific response but still elicits the Kb-restricted response. These findings also illustrate that mutations within regions encoding overlapping T-cell epitopes can differentially affect the presentation and recognition of individual epitopes.     

Induction of high-grade anti-tumor immunity by use of a recombinant H-2Kb/avian erythroblastosis virus erbB gene transfectant

Summary The recombinant H-2K^b-erbB gene, encoding for a part of the H-2 class I antigen and the kinase domain of the V-erbB peptide, was successfully introduced into murine mastocytoma P815 variant P1.HTR cells, which resulted in low but significant cell-surface expression of the hybrid gene product. When the chimeric gene transfectant was inoculated into the CDF₁ mice, it soon grew but regressed thereafter. The tumorigenicity of this transfectant was lower than the H-2K^b gene transfectant that expressed the H-2K^b antigen at a comparable level. These CDF₁ mice that had received the chimeric gene transfectant obtained a high-grade anti-tumor immunity against the challenge of a high dose of parental tumor. Corresponding to these observations, anti-tumor cytotoxic T lymphocytes, which lyse parental P1.HTR cells but not syngeneic L1210 or NS-1 tumor cells, were developed in the peritoneal cavity of mice that had been inoculated with the transfectant and parental tumor. Definite antibody activity binding to parental P1.HTR tumor cells was also demonstrated in the sera of these mice, precipitating 40-kDa, 74-kDa and 98-kDa molecules from the surface of the radiolabeled P1-HTR tumor cells. The results suggested that the chimeric H-2-erbB gene transfectant efficiently triggers both cellular and humoral anti-tumor immune responses.     

Influence of xenogeneic beta2-microglobulin on functional recognition of H-2Kb by the NK cell inhibitory receptor Ly49C

NK cells maintain self-tolerance through expression of inhibitory receptors that bind MHC class I (MHC-I) molecules. MHC-I can exist on the cell surface in several different forms, including "peptide-receptive" or PR-MHC-I that can bind exogenous peptide. PR-MHC-I molecules are short lived and, for H-2K(b), comprise approximately 10% of total MHC-I. In the present study, we confirm that signaling through the mouse NK inhibitory receptor Ly49C requires the presence of PR-K(b) and that this signaling is prevented when PR-K(b) is ablated by pulsing with a peptide that can bind to it with high affinity. Although crystallographic data indicate that Ly49C can engage H-2K(b) loaded with high-affinity peptide, our data suggest that this interaction does not generate an inhibitory signal. We also show that no signaling occurs when the PR-K(b) complex has mouse beta(2)-microglobulin (beta(2)m) replaced with human beta(2)m, although replacement with bovine beta(2)m has no effect. Furthermore, we show that beta(2)m exchange occurs preferentially in the PR-K(b) component of total H-2K(b). These conclusions were reached in studies modulating the sensitivity to lysis of both NK-resistant syngeneic lymphoblasts and NK-sensitive RMA-S tumor cells. We also show, using an in vivo model of lymphocyte recirculation, that engrafted lymphocytes are unable to survive NK attack when otherwise syngeneic lymphocytes express human beta(2)m. These findings suggest a qualitative extension of the "missing self" hypothesis to include NK inhibitory receptors that are restricted to the recognition of unstable forms of MHC-I, thus enabling NK cells to respond more quickly to events that decrease MHC-I synthesis.     

Mapping the orientation of an antigenic peptide bound in the antigen binding groove of H-2Kb using a monoclonal antibody.

The major histocompatibility complex class I molecules are receptors for intracellular peptides, both of self and non-self origin. When non-self peptides (eg., pathogen derived) are bound to the class I molecules, they form ligands for T cell receptors resulting in antigen specific lysis of the infected cells by cytotoxic T lymphocytes. Therefore, an understanding of the process of antigen recognition requires the precise definition of the structural features of the bimolecular complex formed by a single well defined antigenic peptide bound to the class I molecule. A strategy using antibodies was developed to probe the structural features of the H-2Kb containing a defined peptide in the antigen cleft. We report that the binding surface area of a Kb specific monoclonal antibody (28-13-3s) includes residues in the alpha 1 (Gly56 and Glu58) and alpha 2 (Trp167) helices of Kb thus, binding across the antigen binding groove. When cells treated with the antigenic peptide of vesicular stomatitis virus, N52-59, and its alanine substituted analogs were tested for 28-13-3s binding, it was found that position 1 of the peptide also forms a part of the antibody binding site. This finding strongly supports the positioning of the N-terminus of N52-59 proximal to pocket A, thus, assuming an orientation parallel to the alpha 1 helix.     

Selective activation of immunoregulatory T-cell circuits by an Ia+ antigen-presenting cell line

ORA I-a, a cloned Ia+ monocyte tumor line, interacts with distinct immunoregulatory T-cell subsets. ORA cells present soluble and alloantigen to primed lymph node T cells and alloantigen to antigen-activated T-cell clones. However, they induce dose-dependent suppression during primary mixed lymphocyte cultures. Activation of a mixed lymphocyte response (MLR) suppressor pathway is mediated by Ly 1+ T cells. This T-cell subset proliferates in response to ORA when Ly 2+ cells are depleted. Furthermore, once activated, Ly 1+ T cells induce effectors of suppression within fresh T-cell populations. These studies indicate that antigen presentation to distinct T-cell subsets during different stages of an immune response may be mediated by unique antigen-presenting cell subpopulations. Immune homeostasis may thus be controlled not only by regulatory T cells, but also by unique antigen-presenting cells which are responsible for their selective activation.     

Recognition of H-2Kb mutant target cells by Moloney virus-specific cytotoxic T lymphocytes from bm13 (H-2Db mutant) mice. I. Full recognition of Kbm11 by Kb-restricted CTL

In C57BL/6 (B6, H-2b) mice, the secondary in vitro CTL response against Moloney leukemia virus is restricted and regulated by the H-2Db locus. B6.C-H- 2bm13 ( bm13 ) mice, however, carrying a mutation at the Db locus, show an increased H-2Kb-restricted CTL response without a demonstrable CTL component restricted by the mutant Dbm13 molecule (D----K shift). These purely Kb-restricted bm13 virus-specific CTL were incubated with a series of Kb mutant virus-infected target cells to study the effect of the mutations at the target cell level. Of six Kb-mutant virus-infected target cells tested, bm1 cells were not recognized and bm8 cells were recognized only marginally by bm13 virus-specific CTL, whereas bm3 , bm5 , bm6 , and bm11 cells were fully recognized. Thus, the bm3 , bm5 , bm6 , and bm11 Kb mutants fully share the relevant H-2K restriction specificities with H-2Kb, whereas the bm1 mutant totally and the bm8 mutant almost completely lack these specificities. This result differs markedly from the restriction site relationships among B6 and these Kb mutants in other antigenic systems. The most striking example concerns the bm11 mutant, which is fully recognized by Moloney-specific CTL, but not at all by Sendai, minor H (H-3.1, H-4.2), and sulfhydryl hapten-specific CTL. Monoclonal anti-H-2Kb antibody B8-3-24 inhibited virus-specific lysis by bm13 CTL of all Kb virus-infected mutant target cells to which this antibody binds. Lysis of bm5 and bm11 but not of bm3 target cells was inhibited, in line with the fact that B8-3-24 antibody does not bind bm3 . On the other hand, not only bm5 and bm11 but also bm3 virus-infected target cells blocked virus-specific lysis to the same extent as syngeneic bm13 target cells. Therefore, bm13 virus-specific CTL populations do not recognize the discrete cluster alteration in the Kbm3 molecule, as identified by antibody B8-3-24. The bm1 and the bm8 mutations, which have structural alterations in completely different sites of the Kb molecule, show complete or almost complete loss, respectively, of Kb-Moloney restriction sites. This finding supports the notion that these virus-specific CTL recognize conformational determinants rather than linear amino acid sequences.     

Role of the H-2I region in the generation of an antiviral cytotoxic T-cell response in vitro

The participation of H-2I gene products in generating virus-specific proliferative and/or cytotoxic T-lymphocyte (CTL) responses was investigated. Spleen cells from mice infected with vaccinia virus were restimulated secondarily in vitro with syngeneic virus-infected peritoneal exudate cells (PEC) and then restimulated in tertiary cultures with virus-infected PEC from syngeneic and partially histoincompatible strains of mice. Based on the finding that comparable proliferative responses resulted when stimulating the responding cells were histocompatible at the H-2K, I, or D region of the major histocompatibility complex (MHC), the additively enhanced, but not potentiated, proliferation caused by identity at two or three H-2 regions was analyzed. Enhancement of proliferation followed when the H-2K/D components plus virus and the H-2I components plus virus were either on the same, or alternatively on two, stimulating cells. This suggests that H-2K, D, and I plus virus trigger distinct T-cell subsets. A virus-specific CTL response was generated in vitro when spleen cells from virus-primed mice and even unprimed mice were stimulated with cells sharing only H-2K and/or H-2D of the MHC. Identity at the H-2I region did not stimulate a CTL response, nor did it influence the magnitude of the $\text{K}\text{D}$ restricted response. Nevertheless, the presence of anti-Ia antiserum in cultures of syngeneic stimulators and responders inhibited the cytotoxic response to a great extent. Therefore, H-2I region products seem to participate in the generation of virus-specific CTL in vitro.