H-2-associated specificity of virus-immune cytotoxic T cells fromH-2 mutant and wild-type mice: M523 (H-2K ka ) and M505(H-2K bd ) do,M504 (H-2D da ) and M506(H-2K fa ) do not crossreact with wild-typeH-2K orH-2D

B10.A(3R) (H-2K ^b ) mice infected with lymphocytic choriomeningitis virus (LCMV) or vaccinia virus generate cytotoxic T cells capable of specifically lysing virus-infected macrophage target cells fromH-2K ^b mutant mice M505 (H-2K ^bd ), and vice versa. Similarly, virus-immune B10.A(4R) (H-2K ^k ) T cells specifically lyse infected targets from M523 (H-2K ^ka ), and vice versa. In contrast, virus-specific cytotoxic T cells from neither M504 (H-2D ^da ) and B10.A(5R) (H-2D ^d ) nor M506 (H-2K ^fa ) and B10.M(11R) (H-2K ^f ) mutually crossreact at the cytotoxic effector-cell level. As far as tested, the crossreactivity patterns between wild-type and mutantK orD specificities are identical for LCMV- and vaccinia virus-immune spleen cells. Although this finding is no proof for either the altered self nor the dual recognition concept of T-cell recognition, it may be compatible with the latter model.     

Adoptive transfer of delayed-type hypersensitivity to sendai virus

TheH-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 usingH-2 mutant mice. Td and Tc lymphocytes exhibit the same fine specificity for self-recognition, for example, B6.C-H- 2^bm1 effector T cells were unable to recognize viral antigens in association with wild-type K^b and vice versa, B6.-H- 2^bm6 effector cells did not mediate a reaction against virus plus wild-type K^b but, on the other hand, T cells of wild-type K^b recognized virus plus K^bm6.BALB/c-H- 2^dm2 T cells lacked reactivity against virus in association with wild-type D^d, but again wild-type D^d effector cells recognized virus plus D^dm2.Abbreviations used in this paper DTH delayed-type hypersensitivity - EID₅₀ mean egg infective dose - FCS fetal calf serum - HAU hemagglutinating units - LPS lipopolysaccharide - Ly^(–) low amount of Ly antigens - MHC major histocompatibility complex - 2-ME 2-mercaptoethanol - PBS phosphate-buffered saline - Tc cytolytic T cell - Td T cell which mediates a delayed-type hypersensitivity reaction     

Host-determined T cell fine specificity for self-H-2 in radiation bone-marrow chimeras of standard C57BL/6 (H-2b) mutantHz1 (H-2ba), and F1 mice

Reciprocal radiation bone-marrow chimeras were produced between the standard C57BL/6 (=B6) and the mutant B6.C-H-2 ^ba (=Hz1) strain. When infected with vaccinia virus, these chimeras, as well as an (Hz1 × B6)= Hz1 chimera, produced cytotoxic cells that killed vaccinia-infected H-2K^kH-2D^b target cells but failed to kill virus-infected H-2K^bH-2D^d cells. Virus-infected (Hz1 × B6)F₁ B6 chimeras, however, killed both types of target. These experiments demonstrate strict T-cell specificity capable of differentiating between two molecules that apparently differ by a single amino acid substitution.     

H-2 class I loci determine sensitivity to MCMV in macrophages and fibroblasts

Peritoneal (PM) and bone marrow-derived (BMM) macrophages and lung fibroblasts (LF) from inbred, intra-H-2 recombinant, H-2 mutant, and hybrid mice were infected with murine cytomegalovirus (MCMV) under centrifugal enhancement. At the concentration of virus employed, peritoneal macrophages from strains carrying Kd, Kb, D^d, KS and/or D^s, K4 and/or D4 alleles could be infected to a level of 80%–100%, as assessed by viral antigen expression or loss of Fc receptors. Cells lacking these haplotypes and carrying K^k, K^f, D^k, Df, or Db were resistant, yielding levels of infection below 20% . The background (non-H-2) and class II genotype and the S allele did not influence the proportions of cells infected. Furthermore, sensitivity was dominant in the F, progeny of H-2 b x H-2 k and H-2d x H-2 k crosses, and was not compromised by thebm1, bm3, bm10, or bm14 mutations in the al or2 regions of Kb orD ^b. The proportions of cells able to release infectious virus were low, but paralleled the frequencies of viral antigen expression. The class I genotype also determined susceptibility to MCMV infection in BMM and LF, although up to 35% of H-2 k BMM and 46% of H-2 k LF could be infected. The findings are consistent with an association between K and D antigens and a cellular receptor for MCMV on all three cell types.     

Relationship between trinitrophenyl and H-2 antigens on trinitrophenyl-modified spleen cells. I. H-2 antigens on cells treated with trinitrobenzene sulfonic acid are derivatized.

Spleen cells were treated with TNBS in order to determine if cell surface H-2 antigens are derivatized with TNP. By labeling the cell membrane of the TNP-modified cells with 125I, followed by detergent lysis and immune precipitation with anti-TNP, it was determined that no H-2 antigenic activity remained in the supernatant. Further, by the use of an antibody-induced antigen redistribution assay it was found that previous exposure to TNP-modified cells to anti-TNP in the absence of complement rendered these cells resistant to lysis by anti-H-2 in the presence of complement. Together these data indicate that at the concentration of TNBS used for modification, H-2 antigens are derivatized with TNP. However, in addition to H-2, other proteins including immunoglobulin were also derivatized with TNP. Anti-TNP cytotoxic effector cells were blocked from their cytotoxic activity by anti-TNP antiserum. These data indicate that TNP directly couples to H-2 antigens on the cell surface of TNP-modified cells and that TNP is associated with the antigenic determinant that the cytotoxic T cell recognizes.     

Immunogenetic analysis ofH-2 mutations

Spleen cells carrying theH-2K ^b allele and sensitized against TNP-modified stimulator cells in vitro displayed a cytotoxic effect against TNP-modified target cells carrying a mutation in theH-2K ^b allele (haplotypesH-2 ^ba ,H-2 ^bd , andH-2 ^bf ). Similar crossreactivity in TNP-CML was observed in the reciprocal direction. Spleen cells carrying theH-2K ^k allele and sensitized against TNP-modified stimulators displayed a cytotoxic effect against TNP-modified target cells carrying a mutation in theH-2K ^k allele (haplotypeH-2 ^ka ) and vice versa. The effector cells in these assays were sensitive to anti-T cell serum in the presence of complement, and supernatants from immune cultures did not induce nonimmune cells to display a cytotoxic effect. Titration of effector cells from mutant and wild-type strains of theH-2 ^b haplotype indicated no detectable quantitative differences in their activities. These data demonstrate that crossreactivity in TNP-CML occurs in closely related allogeneic strains that have recently undergone mutation in theH-2 complex.     

Immunogenetic analysis ofH-2 mutations

A.BY, B10.LPa, and B10.129(5M) mice were presensitized in vivo against B10.A(5R) cells and then restimulated in vitro by the same cells in the standard CML assay. The effector cells thus generated lysed not only B10.A(5R), but also C57BL/6 targets, indicating that, in addition to anti-H-2D_d response [measured on the B10.A(5R) targets], response to minor histocompatibility (H) antigens (measured on the C57BL/6 targets) also occurred. The latter response was directed against multiple minor H antigens in the case of the A.BY effectors, and against H-1 and H-3 antigens in the case of B10.129(5M) and B10.LPa effectors, respectively. The sensitization against minor H antigens occurred in the context of H-2K^b H-2D^d antigens, but by testing the response on C57BL/6 targets, only cells reacting with minor H antigens in the context of H-2K^b were assayed. The same effector cells were then tested against H-2^b mutant strains, in which theH-2K ^b allele was replaced by a mutant one. All three effector types [A.BY, B10.LPa, and B10.129(5M)] behaved in a similar way: they all reacted with theH-2 ^bg1 mutant to the same degree as withH-2 ^b, they did not react at all or reacted only weakly with theH-2 ^bd andH-2 ^bh mutants, and they reacted moderately or strongly with theH-2 ^ba mutant. The degree of crossreactivity with the mutants reflects, with one exception, the degree of relatedness of these mutants toH-2 ^b, as established by other methods. The one exception is theH-2 ^ba mutant, which is the most unrelated toH-2 ^b, and yet it crossreacted strongly. Further testing, however, suggested that in this instance the crossreactivity was probably directed against H-2 antigens: the anti-H-2D^d effectors apparently crossreacted with the H-2K^ba antigens. This finding is an example of cell-mediated crossreactivity between the products of two differentH-2 genes (H-2K andH-2D). It is also an example of anH-2 mutation generating an antigenic determinant known to be present in another strain.     

Studies ofH-2K b mutant mice

Three newH-2 ^b mutant strains, B6.C-H-2 ^bm9 , B6.C-H-2 ^bm10 and B6.C-H–2 ^bm11 , are described. The three mutant strains are of the gain and loss type as they reject skin grafts reciprocally with the parental C57BL/6Kh. The mutations, which arose independently, are all allelic at the same locus as 11 other mutant strains already described. By complementation and other studies the mutated gene has been shown to beH-2K ^b . The strains were typed directly and by absorption with antisera specific for H-2K^b and H-2D^b private and public specificities and for Ia^b specificities. Each strain typed differently with these sera. The strain B6.C-H-2 ^bm9 was found to be serologically identical with C57BL/6. The strains B6.C-H-2 ^bm10 and B6.C-H-2 ^bm11 were found to have alterations in the private H-2K^b specificity, H-2.33, and in the public specificity, H-2.5, but to a different extent. B6.C-H- 2^bm10 had a marked decrease in the amount of H-2.33 expressed on the splenic cell surface as compared to C57BL/6 and also has a marked decrease in the expression of H-2.5 on both spleen and red blood cells. In comparison, B6.C-H-2 ^bm11 has a decrease in the expression of H-2.33 but an increase in the expression of H-2.5 on both splenic and red blood cells. The other H-2^b specificities appeared to be unaltered as compared with C57BL/6.     

Detection of H-2Db antigen on osmotic shock-treated friend leukemia virus particles

Antisera specific for either H-2K^b, H-2D^b, H-2K^k or H-2D^k antigenic determinants were examined for their capacity to neutralize Friend virus (FV) collected from the serum of infectedH-2 ^b /H-2 ^k heterozygous mice. Neutralizing activity was detected (1) only withanti-H-2D ^b antisera, (2) only when the surface of virus particles had been mildly deranged by osmotic shock treatment and (3) only in the assay for the defective spleen focus-forming virus component of FV.     

H-2 effects on cell-cell interactions in the response to single non-H-2 alloantigens

Immune response (Ir) genes mapping in theI region of the mouseH-2 complex appear to regulate specifically the presentation of a number of antigens by macrophages to proliferating T cells. We have investigated the possibility that similarIr genes mapping in theH-2K andH-2D regions specifically regulate the presentation of target antigens to cytotoxic effector T cells. We report that the susceptibility of targets expressing specific non-H-2 H alloantigens to lysis by H-2-compatible, H-antigen-specific cytotoxic effector T cells is controlled by polymorphicH-2K/D genes. This control of susceptibility to lysis is accomplished through what we have defined operationally as antigen-specific regulation of non-H-2 H antigen immunogenicity. High immunogenicity of the H-4.2 alloantigen is determined by a gene mapping in theH-2K region ofH-2 ^b . However, high immunogenicity of H-7.1 is determined by a gene mapping in theH-2D region ofH-2 ^b . High immunogenicity of the H-3.1 alloantigen is determined by genes mapping in both theH-2K andH-2D regions ofH-2 ^b . Therefore, genes mapping in theH-2K andH-2D regions serve a function in presenting antigen to cytotoxic effector T cells. This function is analogous to that played byI-regionIr genes expressed in macrophages which present antigen to proliferating T cells. We present arguments for classification of theseH-2K/D genes as a second system ofIr genes and discuss the implications of twoH-2-linkedIr-gene systems, their possible functions, and their evolution.     

Cross-reactivity patterns of vaccinia-specific cytotoxic T lymphocytes from H-2K b mutants

Limit-dilution cultures were used to select vaccinia-immune T-cell populations from bml and bm3 mutant mice that were not lytic for virus-infected targets expressing the K^b and D^b MHC glycoprotein. Approximately 30% of virus-immune CTL were restricted in each case to K^bm1 and K^bm3, rather than to D^b. Evidence of extensive cross-reactivity was found for these virus-immune CTL. Bm3 and bmll mice sharing one amino acid mutation from wild-type but differing by a second mutation seen only in bm3 are the most cross-reactive pair in their presentation of vaccinia. The bm1 and bm10 pair with dissimilar mutations from wild-type affecting the same CNBr fragment are also largely cross-reactive. However, 30% cross-reactivity is also found for bm1 and bm3, which differ in separate CNBr fragments. That mutants expressing amino acid substitutions in the same region of the peptide tend to show more evidence of cross-reactivity does not necessarily mean the T cells see linear arrays of amino acids on the MHC glycoprotein. For instance, K^bm1 and K^bm10 differ for three amino acids, but bm1 T cells are highly lytic for bm10 virus-infected targets. However, there is no cross-reactivity for K^bm1 and K^b, which differ at only two amino acids. The key to further understanding may rest with defining the nature of the conformational differences among the K^bm1, K^bm10, and K^b glycoproteins.     

Recognition of Db and Kb gene products by influenza-specific cytotoxic T cells

A series of 16 H-2^b-restricted, A influenza virus-specific cytotoxic T-cell clones are described and characterized. One is K^b restricted, the others D^b restricted. The factors governing K^b or D^b restriction patterns seen in the mixed populations from which clones are derived are investigated. The K^b-restricted clone does not recognize K^b mutant bm1 and influenza and all 15 D^b-restricted clones do not recognize D^b mutant bm14 and A influenza virus; these results are discussed in the light of findings in a variety of other viral systems. Representative K^b- and D^b-restricted clones were used to assess the functional properties of cloned cosmids containing either K^b or D^b genes expressed in transformed L-cells (κ haplotype). The expression products of both cosmids functioned efficiently as mutually exclusive restriction elements for A influenza virus recognition.     

Neonatal tolerance induction acrossH-2 mutational disparity: Induction and specificity of tolerance

Mutational disparities derived from alleles of theH-2K andH-2D loci vary widely in their ability to induce neonatal tolerance. The more subtle mutations, such asK ^bm5 andK ^bm8, proved to be excellent tolerogens, but theK ^bm3 mutant (M505) turned out to be the poorest tolerogen yet studied of all H-2 alloantigens. By challenging tolerant animals with skin grafts from related mutants, it was found that expression of tolerance was highly specific. Although a minority of tolerant animals failed to discriminate between the K^b, K^bm5 and K^bm8 antigens, they never failed to discern K^b, K^bml and K^bm3 as distinctly different alloantigens.     

Studies of twoH-2D b mutants: B6.C-H-2 bm13 and B6.C-H-2 bm14

Two new C57BL/6H-2 mutants,B6.C-H- 2^bm13 and B6.C-H- 2^bm14 are described. They arose independently in C57BL/6 as spontaneous mutations of the gain and loss type. Complementation studies map the mutations in both bm13 and bm14 to theH-2D ^b gene. How ever, these two mutant strains are not identical, but occurred as independent mutations at the same locus, as shown by reciprocal graft rejection and by the inability of the (bm13 × bm114)F₁ hybrid to accept C57BL/6 grafts. Serological studies by direct testing (cytotoxicity and hemagglutination) and by quantitative absorption demonstrated a decrease in the H-2D^b private specificity H-2.2 in both bm13 and bm14 when compared to C57BL/6. This was confirmed by SDS-PAGE analysis using antisera detecting the H-2.2 specificity. Attempts to produce antibodies to either the gained or lost specificities of the two mutant strains failed.     

Study ofH-2 mutations in mice

The serologically defined H-2.5 specificity was tested on spleen cells and red blood cells (RBC) of theH-2 ^b haplotype and a number of its mutants. Thebm8 (bh) mutant was barely distinguishable fromb in a variety of tests made. On spleen cells ofbm1 (ba) the H-2.5 specificity seemed to be unchanged, while it was virtually absent from RBC of this mutant. Mutantsbm4 (bf),bm5 (bg1), andbm6 (bg2) were similar tobm1, with slight differences between them. The mutantbm3 (bd) retained an unchanged quantity of H-2.5 on its spleen cells, while the specificity was substantially increased on its RBC. The H-2.5 ofbm3 is not identical to that ofH-2 ^a . Possible mechanisms causing differential serology of theH-2 ^b mutants are discussed.     

Dissociation ofH-2 recognition by antibody and cytotoxic T cells of a cloned murine leukemia cell line

The differential expression of H-2 specificities recognized by antibody and by cytotoxic T lymphocytes (CTL) has been studied using a clone (FY7) of the C57BL/6 leukemia cell line FBL-3 (H-2 ^b /H-2 ^b ). Unlike C57BL/10 spleen cells, EL-4 lymphoma cells and Y57-2C leukemia cells (allH-2 ^b /H-2 ^b ), FY7 failed to induce the primary in vitro generation of anti-H-2^b CTL by (B10.A x A)F₁ (H-2 ^a /H-2 ^a or (B10.D2 x BALB/c)F₁ (H-2 ^d /H-2 ^d ) responder spleen cells. In addition, FY7 was not lysed by, and did not competitively inhibit anti-H-2^b CTL. Quantitative absorption tests with H-2K^b and H-2D^b antisera revealed that FY7 expressed these antigens in quantitatively similar amounts to EL-4. The H-2K^b product of FY7 appeared to be identical with that of C57BL/10 spleen cells both in apparent molecular weight and isoelectric point. Yet FY7 failed to inhibit anti-H-2K^b CTL competitively in a cold target inhibition assay. Possible mechanisms are discussed for the lack of T-lymphocyte recognition of the H-2K^b-gene product expressed by FY7.Abbreviations used in this paper CTL cytotoxic T lymphocytes - MHC major histocompatibility complex - MLC mixed lymphocyte culture - PAGE polyacrylamide gel electrophoresis     

Multiple cytolytic T-cell clones with distinct cross-reactivity patterns coexist in anti-self + hapten cell lines

C57BL/10 T cells sensitized with TNBS-treated syngeneic cells and maintained in culture by repeated stimulations exhibit high cytolytic activity toward syngeneic TNBS-treated target cells with marked cross-reactivity on TNBS-treated target cells from mice of independent H-2 haplotypes (ten tested). The analysis of the reactivities of 48 T-cell clones derived by limiting dilution from such T-cell populations revealed three types of cytotoxic T-cell clones: (1) clones restricted by H-2K^b + TNP without cross-reaction on TNBS-treated or untreated target cells of other tested mouse haplotypes; (2) clones that lysed H-2^b + TNP and also TNBS-treated target cells from not more than one, two or three different H-2 haplotypes; (3) clones that lysed untreated H-2^k target cells. No T-cell clone was found to exhibit the wide pattern of cross-reactivity on any TNBS-treated mouse cell, characteristic of the original T-cell populations, indicating that these were composed of individual T-cell clones specific for TNP + private or for TNP + distinct public H-2 determinants. Correlation with described serological public H-2 specificities was possible for some cytotoxic T-cell clone reactivity, but not for others. The general pattern of T-cell reactivity as revealed by clonal analysis in this study, as well as in published work, includes cross-reactions between self H-2^a + X and allogeneic H-2ⁿ + X, between self H-2^a + X and unmodified allogeneic H-2ⁿ, or between allogeneic H-2ⁿ and allogeneic H-2^m + X, and is consistent with the hypothesis that MHC-class restriction is the main rule in T-cell recognition.Abbreviations used in this paper CTL cytotoxic T lymphocyte - TNBS trinitrobenzene sulfonate - TNP trinitrophenyl - TNCB trinitrochlorobenzene - H-2 mouse major histocompatibility complex - IL-2 interleukin 2 - Con A concanavalin A - FCS fetal calf serum - SCA IL-2 containing supernatant - LPS E. coli lipopolysaccharide - X a non-H-2 conventional antigen     

Cross-reactivity patterns of murine cytotoxic T lymphocytes.

Cross-reactivity of TNP-immune, virus-immune, and alloreactive murine cytotoxic thymus-derived (T_c) cells was investigated at the level of target cell lysis. Alloreactive T_c cells cross-reacted on TNP-modified and unmodified third-party targets and on syngeneic TNP-modified targets but did not cross-react on syngeneic virus-infected targets. TNP-immune T_c cells showed marked cross-reactivity on certain allogeneic targets modified by TNP (loss of H-2 restriction) and also on certain unmodified allogeneic targets but did not cross-lyse virus-infected syngeneic targets. Targets treated with TNP-Sendai virus were not lysed by TNP-immune T_c cells, but T_c cells stimulated by cells treated with TNP-Sendai virus lysed such targets readily. These results are consistent with the view that T_c-cell recognition of foreign H-2 antigens and TNP-modified self-H-2 antigens are mechanistically similar (possibly via one receptor), whereas recognition of viral plus H-2 antigens is different (possibly via two receptors).Virus-immune T_c cells ubiquitously exhibited strong cross-reactivity on syngeneic TNP-modified targets using pox-, arena-, alpha-, myxo-, and paramyxoviruses for T_c-cell induction. The lysis of virus-infected targets by virus-immune T_c cells could be inhibited by cold TNP-modified competitors, thus establishing that some individual virus-immune T_c cells could recognize both types of target cells. This genuine cross-reactivity at the effector level was not observed at the level of induction of secondary responses, since the cross-reactive subset of virus-immune memory T_c cells could not be activated by TNP-modified stimulator cells but could be activated by virus-infected stimulators. These results implied that requirements for stimulation of precursor T_c cells are sometimes different from antigenic requirements for recognition and lysis of effector T_c cells.     

Negative selection experiments support the idea that T-T help is required for the H-Y-specific cytotoxic T cell response

Antigen-primed female thoracic duct lymphocyte (TDL) populations (B6, H-2K^bI-A^bD^b) were depleted of T cells specific for the male H-Y antigen presented in the context of I-A^b, by negative selection through irradiated male H-2K^bI-A^bD^b recipients. This ocedure substantially removed the capacity of such TDL to respond to H-Y-H-2D^b. The response can be reconstituted by adding either smaller numbers of unfiltered, H-Y-primed, B6 female TDL or anti-Ly-2-treated memory T cells. The results thus support the idea that concurrent presence of helper T cells specific for H-Y-H-2-I-A^b is required for the generation of secondary cytotoxic T lymphocyte responses to H-Y-H-2D^b. This is discussed in the context of similar, though not identical, experiments with the vaccinia virus model.     

Selective destruction by formaldehyde fixation of an H-2Kb serological determinant involving lysine 89 without loss of T-cell reactivity

In preparation for functional analyses, a study of the binding of H-2K^b-specific monoclonal antibodies (mAb) to formaldehyde (FOR)-fixed H-2^b spleen or tumor cells revealed that three of nine mAb tested had lost reactivity with the FOR-fixed cells, whereas the reactivity of the other mAb generally did not diminish. Comparison of the reactivity of these mAb on untreated H-2K ^bm mutant cells and on FOR-treated H-2K^b cells suggests that for three mAb the total loss of reactivity on the latter could be a consequence of the alteration by FOR of lysine 89, which is substituted by alanine in mutant bm3. H-2KP^b-specific alloreactive polyclonal or monoclonal CTL, all of which had retained reactivity with bm3 target cells, had also retained reactivity with FOR-fixed H-2^b cells as indicated by cold target inhibition studies. The H-2K^b-specific CTL were probably reactive with conformational determinants of H-2K^b, which are dependent on the integrity of both the 1 and the 2 domains of the H-2K^b molecule. Results are compatible with FOR treatment selectively affecting a serological determinant in the 1 domain without affecting conformational-type CTL determinants.Abbreviations used in this paper CTL cytotoxic T lymphocyte - FOR formaldehyde - PBS phosphatebuffered saline - FCS fetal calf serum - mAb monoclonal antibody - TNBS trinitrobenzene sulfonate