Class I MHC molecules rather than other mouse genes dictate influenza epitope recognition by cytotoxic T cells

Influenza nucleoprotein (NP) is an important target antigen for influenza A virus cross-reactive cytotoxic T cells (Tc). Here we examine the NP epitope recognized by cloned and polyclonal BALB/c Tc and the genetics of this recognition pattern. We can define NP residues 147–161 as the epitope seen in conjunction with K ^d , the only H-2^d class I responder allele for NP restriction. H-2 ^d /H-2 ^b F₁ mice (C57BL × DBA/2) primed by influenza infection lyse only H-2^d target cells treated with peptide 147–161 while H-2^b targets are recognized only after treatment with NP residues 365–379 (previously found to be recognized by D^b restricted Tc cells). Tc cell recognition of NP peptide 147–161 is entirely dictated by expression of K ^d and not by other B10 or OH background genes of congenic mice. Restriction of a unique NP sequence by each responder class I major histocompatibility complex (MHC) allele suggests that antigen and class I MHC interact for Tc recognition.     

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

Individual mice were tested for their proliferative T-cell response to H-Y- and H-3-incompatible stimulator cells in secondary mixed lymphocyte culture. Responders expressing the H-2 ^bhaplotype were restricted in their response to stimulators presenting H-Y and H-3 in the context of H-2 ^b. Lymphocytes from individual B10 females proliferated in response to H-Y presented with I-A ^band D ^b. The ratio of I-A ^b/D ^b-restricted responses varied between individual responders, indicating significant qualitative variation between genetically identical responders. The majority of the proliferative response in all tested mice was restricted to the entire H-2 ^bhaplotype suggesting complementation of I-A ^b- and D ^b-region genes in presenting the H-Y antigen. Similar observations were made in the response of individual B10.LP mice to the H-3 antigen. H-3-specific, proliferating T cells were restricted to H-3 antigen presented with K ^bA^band D ^bwith significant variation between individuals in their preference for H-3 plus K ^bA^band D ^b. In contrast to the response to H-Y, the proliferative response to H-3 plus H-2 ^bcould be accounted for by the summation of the proliferative responses to H-3. plus K ^bA^band D ^b. These observations demonstrate that the proliferative response to non-H-2 H antigens in the context of I-region determinants is not a sine qua non for the T-cell response to these antigens. Further, the individual qualitative and quantitative variation observed with individual genetically identical mice has strong implications for our knowledge of intrastrain variation in immune responsiveness and the characterization of inbred strains for immune responsiveness.     

Genetic control of sensitivity to Moloney leukemia virus in mice V. Role of H-2-linked genes in the control of anti-FMR cytolytic T lymphocytes

Three H-2-linked genes, Rmv1, Rmv2, and Rmv3 control the resistance of mice against Moloney virus (MLV)-induced leukemias. It has been shown previously that they function as immune response (Ir) genes regulating the level of antivirus antibodies. In the present experiments, the cell-mediated anti-tumor response has been studied in a series of inbred strains selected for their resistance or sensitivity to the MLV-induced disease. We failed to detect any relationship between resistance and sensitivity and the ability to produce cytolytic T lymphocytes (CTL) directed against the virus-induced FMR cell surface antigen. Furthermore, the role of each Rmv gene has been studied separately using congenic pairs of mice differing at only one of these loci: we failed to evidence any influence of these genes in the cell-mediated antitumor reactions as measured by the ability to lyse syngeneic FMR(+) target cells. Nevertheless a gene mapping in the D region of the MHC but probably different from Rmv3 controls the response of a subset of anti-FMR CTL restricted by the H-2K^k antigens, with higher response in H-2D^d than in H-2D^b animals. This observation confirms the existence of H-2D region associated Ir genes regulating the CTL-mediated antitumor immune responses by choosing the subset of responder CTL, and suggests that a fourth H-2-linked gene plays some role in the genetic control of the anti-FMR immune response.     

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.     

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.     

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 antigen variants in a cultured mouse leukemia cell line

We have investigated the effect of immune selection against a single gene product on a cultured mouse Friend leukemia cell line. The clonal cell line used is heterozygous at theH-2 complex and expresses theH-2 ^d andH-2 ^b haplotypes. The genes selected against were theH-2K locus alleles. Variants were obtained after a single-step selection using either antiH-2K^b or anti-H-2K^d serum. The phenotypes of the variants obtained showed an interesting asymmetry between the two haplotypes. Selection against theH 2K ^b allele resulted in the isolation of the two expected types of variant-those that had lost only H-2K^b and those that had lost both H-2K^b and the linked H-2D^b. Selection against H-2K^d yielded, exclusively, variants that had lost both the selected antigen and the linked H-2D^d. None of the variants showed an alteration in expression of antigens intrans configuration. Karyotypic analyses of the variants revealed that all the cells had retained both copies of chromosome 17 present in the wild-type cells. The results suggest that the variants did not emerge through chromosome loss.     

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.     

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     

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.     

Cytotoxic T-cell responses to H-Y:Ir genes and associative antigens map inH-2

The secondary cytotoxic responses to the male-specific antigen (H-Y) in mice showH-2 restriction so that the cytotoxic female cell must share the K- and/or D-end antigen with the male target cells. The association with the K and/or D end varies with differentH-2 haplotypes,e.g., H-2 ^b cytotoxic cells require the H-2D^b antigen(s) on the target cells, while cytotoxic cells fromH-2 ^b/H-2 ^d F₁ mice sensitized toH-2 ^d male cells kill only male targets having H-2K^d antigen(s). This association of H-Y with appropriate K/D antigens seems to be needed also in the induction of the cytotoxic response. Of the independent haplotypes, onlyH-2 ^b strains are capable of making secondary anti-H-Y responses and this trait seems to be dominant,i.e., the F₁ strains with oneH-2 ^b parent are able to produce anti-H-Y cytotoxic cells against both theH-2 ^b parent and the nonresponder parent. The mating of the two nonresponder strains may produce F₁ mice which are responders, thus suggestingIr gene complementation. Mapping data indicates that at least one of these complementary genes is located in theI-C region fork/s complementation.     

Time course study of H-2 and other antigen expression by hybrids of a myeloma cell line with inflammatory macrophages

The hybrids (the CANS lines) between inflammatory macrophages from C57BL/6N (B6) mice (H-2^b) and BALB/c mouse (H-2^d)-derived myeloma cell line NS1 in the early period after cell fusion showed no macrophage functions. However, most of the hybrids expressed these functions after prolonged cultivation accompanied with chromosome loss. In contrast, the hybrids initially displaying myeloma functions ( light chain production) lost this function when they exhibited macrophage functions. We studied the expression of cell-surface antigens in these hybrids and found that hybrids in the early period after cell fusion codominantly expressed both parental cell H-2 antigens (H-2K^b, H-2K^d, and H-2D^d) but not the H-2D^b antigen. On the other hand, aged hybrids strongly expressed the H-2 d antigen but lacked the H-2K^b antigen. Alternatively, these aged hybrids with macrophage functions expressed antigen(s) as detected with antiaged CANS-196 cell sera and asialo GM1 antigen, both of which were thought to be found exclusively on macrophages. Thus, the expression of cell-surface antigens in these hybrids was greatly altered after cell fusion.     

Effect of anH-2 mutation on cytotoxic T cell recognition. Specific antigen can determine the pattern ofH-2 restriction

Hz1 (H-2 ^bm1 ) mice, an H-2 mutant strain derived from C57BL/6(H-2 ^b ), were either injected with vaccinia virus or had their spleen cells sensitized in vitro with syngeneic TNP-modified cells. The cytotoxic cells generated were tested for their activity against target cells that were either infected with vaccinia virus, TNP-modified, or both vaccinia infected and TNP-modified.Hz1 anti-TNP cytotoxic cells specifically lysed syngeneic target cells that were trinitrophenylated but not infected with vaccinia virus, while anti-vaccinia cells specifically lysed vaccinia infected target cells but not TNP-cells. Hz1 (H-2K ^bm1 D ^b ) anti-TNP effector cells killed B10.A(5R)-TNP (H-2K ^b D ^d ) targets, indicating that there is cross-reactivity between TNP-H-2K^b and TNP-H-2K^bm1. On the other hand, there is no cross-reactivity between vaccinia-H-2K^b and H-2K^bm1, since Hz1 anti-vaccinia effector cells did not kill vaccinia infected B10.A(5R) targets.Since Hz1 anti-TNP effector cells lysed B10.A(5R) target cells that were first infected with vaccinia virus and then derivatized with TNP, virus does not mask cross-reactive determinants shared by TNP-H-2K^b and H-2K^bm1. Additional experiments showed that Hz1 anti-TNP effector cells lysed TNP-modified and vaccinia infected B10.A(5R) target cells irrespective of the virus concentration used for infection or the time of addition of virus. Further, there are no detectable quantitative differences between C57BL/6 and Hz1 anti-TNP effector cells in their ability to kill TNP-5R targets.The cytotoxic effect of Hz1 anti-TNP effector cells on B10.A(5R)-TNP targets could not be blocked with TNP derivatized inhibitor cells that carry theH-2D ^d region allele. Thus, the ability of anti-TNP H-2K^b effector cells to cross-react with H-2K^bm1 cannot be explained by a cross-reaction between H-2K^bm1 and H-2D^d.Abbreviations used in this paper TNP trinitrophenol - PFU plaque forming unit - Con A Concanavalin A - BSS balanced-salt-solution - FCS fetal calf serum - TNBS trinitrobenzene sulfonic acid - PBS phosphate-buffered-saline     

H-2 class I antigen expression on mouse teratocarcinoma cell lines

Immunity against PCC3 teratocarcinoma cells (129, H-2 ^b) was induced in allogeneic (C3H, H-2 ^k) mice by preimmunization with L cells (C3H, H-2 ^k) expressing cosmid-introduced K ^b or D ^b genes, but not with nontransfected L cells. In addition, the growth of PCC3 cells in sublethally irradiated (C3H × B6-H-2 ^bm1)F₁ and (C3H × B6-H-2 ^bm13 )F₁ mice bearing the K ^bm1 and D ^bm13 mutations, respectively, was either prevented, stopped, or delayed in comparison with the (C3H × B6)F₁ (k × b) mice, which failed to reject the PCC3 cells. The teratocarcinoma line OC15S was exceptional because it reacted specifically with K^b- and D^b-specific (but not I^b-specific) alloantisera, and because K^b- and D^b-specific antibodies could be absorbed by OC15S cells. The subpopulation of OC15S cells bearing the ECMA-7 antigen characteristic for embryonic carcinoma (EC) cells was isolated by the fluorescence-activated cell sorter and was shown to react specifically with K^b- and D^b-specific antisera. These experiments show that teratocarcinoma cells express antigens similar or identical to the K-and D-region products of differentiated cells. The lack of expression of class I antigens is thus neither a condition nor a consequence of the pluripotentiality of the EC cells. The exact nature of the major histocompatibility complex antigens on EC cells has yet to be established using the methods of molecular biology and biochemistry.     

NK gene complex and chromosome 19 loci enhance MHC resistance to murine cytomegalovirus infection

An H-2^k MHC locus is critical for murine cytomegalovirus (MCMV) resistance in MA/My mice and virus control is abolished if H-2^k is replaced with H-2^b MHC genes from MCMV-susceptible C57L mice. Yet, H-2^k resistance varies with genetic background; thus, modifiers of virus resistance must exist. To identify non-MHC resistance loci, spleen and liver MCMV levels and genome-wide genotypes were assessed in (C57L × MA/My) and (MA/My × C57L) F₂ offspring (representing 550 meioses). Significantly, a non-Mendelian frequency of MHC genotypes was observed for offspring of the latter cross. Quantitative trait loci (QTL) and their interaction potential in MCMV resistance were assessed in R/qtl; QTL on chromosomes 17, 6, and 19 affected MCMV levels in infected animals. A chromosome 6 QTL was linked with the NK gene complex and acted in an additive fashion with an H-2^k MHC QTL to mitigate spleen MCMV levels. We provide biological confirmation that this chromosome 6 QTL provided MCMV control independent of H-2^k via NK cells. Importantly, both chromosome 6 and 19 QTLs contribute to virus control independent of H-2^k. Altogether, MHC and non-MHC MCMV-resistance QTL contribute in early resistance to MCMV infection in this genetic system.     

H-2-linked control of resistance to Ectromelia virus infection in 1310 congenic mice

Several B 10 strains of mice, recombinant at theH-2 locus, have been shown to differ in their resistance to infection with ectromelia virus, a natural mouse pathogen. Of 10 strains, 1310, B 10.A(2R), B10.A(4R) and B10.D2 were the most resistant, while B10.G and B 10.A(5R) were the most susceptible. Other strains were intermediate between these extremes. Several genes conferring resistance have been mapped toD ^b in B10.A(2R),K ^k I-A ^k I-B ^k in B10.A,I-J ^b in B10.A(2R) and toD ^d in B 10.T(6R). In general, death among susceptible strains was not a consequence of acute liver necrosis as in other non-B10 strains, and occurred randomly from 8–14 days after infection. The exact cause of death is unknown but is characterized by persisting high titers of virus in the spleen and sometimes the liver, despite an ongoing immune response indicated by strong cytotoxic T-cell activity detectable in the spleens of all mice. The most resistant B10 and B10.A(2R) strains cleared virus from the spleen and liver by 8 days after infection. Analysis of infection in chimeric mice indicates thatH-2 genes, which determine susceptibility to virus persistence in the spleen, operate via radiosensitive cells of the lymphomyeloid system. This evidence, together with several examples ofH-2-linked differences in cytotoxic T-cell responsiveness between resistant and susceptible strains, is consistent with the hypothesis that the mechanism by whichH-2 genes control resistance to ectromelia virus in B10 strain mice is by their influence on the effectiveness of a cell-mediated immune response.     

Effect of mouse thymic medullary epithelial cell line on thymocyte development and functional maturationin vivo

Thymic medullary type epithelial cell line (MTEC1), which expressed H-2D^d and Ia^d, was derived from BALB/c mouse. MTEC1 cells were introduced by intrathymic injection into irradiated H-2^b mice reconstituted with H-2^bxd F1bone marrow cells. Two months later, the injected MTEC1 cells were found to be still present in the recipient thymus. Splenocytes from chimeric mice, inin vitro functional assays, were analyzed to investigate whether the MTEC1 cellsin vivo could induce the production of H-2^d restricted antigen-specific T cells. The H-2^d restricted VSV-antigen specific proliferating and IL-2 producing T cells as well as H-2^d restricted influenza virus specific cytotoxic T cells were found in chimeric mice injected with MTEC1 cells, and these cells were shown to be tolerant to H-2^d selfantigen. On the contrary, H-2^d restricted antigen-specific and H-2^d self-antigen tolerant T cells were not shown in control mice injected with saline. These results suggest that intrathymically injected MTEC1 cells could induce T lineage cell development and functional maturation in the intact thymus. A hypothesis of “second thymic selection” in thymic medulla has been postulated and its implication discussed.     

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.     

An improved preparation of murine histocompatibility antigens (H-2b) and a novel membrane binding form of H-2Kb

A modified preparation of H-2^b is described which results in a greater overall yield, higher proportion of H-2D^b and better preservation of the heavy chain - β₂ microglobulin complex than were obtained previously (9). In the absence of inhibitors of proteolysis, a novel low molecular weight H-2K^b heavy chain is obtained. Results indicate that a portion of the H-2K^b has lost a 3000 dalton peptide from the carboxyl terminus not including the hydrophobic membrane binding peptide.