Histocompatibility-2 system in wild mice

Ninety-six wild mice trapped at 13 localities in the state of Texas were tested in the dye-exclusion cytotoxic test with a battery of 49 oligospecific H-2 antisera. The antisera detected 36 class I (K and D) and 10 class II (Ia) antigens. The phenotypic frequencies of private class I antigens ranged from 1 to 20%, the majority of them being in the range between 1 and 5%. At least some of the higher frequencies resulted from the presence of more than one antibody in the typing reagents, and from other factors complicating the typing. We estimate that the frequencies of most of the class I alleles among Texas wild mice are 1% or less. This estimate leads to the prediction that at least 200 alleles exist in Texas mice at theH-2K locus, and another 200 alleles exist at theH-2D locus. Frequencies of most of the class I public antigens were in excess of 20%. In the sample of 96 mice, 46 different phenotypic combinations of private class I antigens were found, and the frequency of blanks (mice unreactive with any of the antibodies to private class I antigens) was 27%. The frequencies of private class II antigens ranged from 5 to 15%. Some of the public class II antigens, in particular those controlled by theE region, occurred with frequencies of 80% or higher. The class II antigens were found in 26 phenotypic combinations. No striking linkage disequilibrium was found either between K and D antigens, or between class I and class II antigens. The polymorphism of theK, A, andD region appears to be higher than that of the corresponding regions of the human or rat major histocompatibility complex. The polymorphism of theE region is significantly lower than that of theA, K, andD regions. The polymorphism of theA region is extensive.     

Sixteen newH-2 haplotypes derived from wild mice

Wild mice captured in Texas, Scotland, Federal Republic of Germany, Denmark, Spain, Greece, Israel, Egypt, and Chile were mated to inbred strains and through successive backcross matings and H-2 typing lines homozygous for wild-derived H-2, haplotypes were established. The lines, which are neither congenic nor inbred, were then typed with antibodies defining known H-2 alleles at class I and class II loci. In addition, antisera were produced by the immunization of inbred strains with tissues of the new lines. Sixteen of the lines were characterized in this manner. The characterization resulted in the identification of 16 new H-2 haplotypes, 11 new K alleles, 10 new D alleles, and 21 new class I antigenic determinants, most of them of the private type. Most of the haplotypes represent natural recombinants sharing segments of the H-2 complex with previously identified haplotypes. A number of haplotypes are recombinants between the K and the A loci, which in genetic studies have proved difficult to separate. The lines, however, also provide evidence for preservation of blocks of genes in the H-2 complex, particularly in the class II region. Some of class I alleles previously found in wild mice from Michigan have now been found again in these mice. Several class II alleles of these lines appear to be the same as those found in inbred strains. Identical or nearly identical class I and class II alleles thus commonly occur in different populations. These findings strengthen the argument that in populations, H-2 alleles are relatively stable.     

H-2 polymorphisms in outbred strains of mice

We studied H-2 polymorphisms of five outbred strains, CF1, ddY, ddN, ICR and KUNM, using 15 alloantisera to H-2 class I private antigens. All individuals had at least one private antigen at H-2K and/or H-2D. The number of H-2 class I private antigens in each strain were three in ddY, four in ICR, five in CF1, and seven in ddN and KUNM. The number of H-2 phenotypes were five in ddY, six in CF1 and ICR, twelve in ddN and seventeen in KUNM. Each strain had strain specific antigen(s) which discriminated each strain from the other strains. The CF1 strain had the H-2.23 and H-2.32 antigens controlled by the H-2k haplotype at a frequency of about 70%. The ddN strain had H-2.4 (H-2Dd) at a frequency of 37.8%. The H-2.9 antigen (57%) governed by H-2Df was distributed only in the KUNM strain. Both the ddY and ICR strains possessed H-2.17 and H-2.30 antigens (85-97%) which were controlled by the H-2q haplotype. The H-2 locus will be useful to characterize each strain and distinguish between strains, and for monitoring of outbred strains by H-2 gene frequency.     

Restriction in adoptive transfer of resistance to Listeria monocytogenes. II. Use of congenic and mutant mice show transfer to be H-2K restricted

Adoptive transfer of cell-mediated immunity to the facultative intracellular bacterium Listeria monocytogenes is restricted by the H-2 complex of mice. Using C57BL/10 and C57BL/6 congenic strains of mice it was shown that compatibility of the H-2K locus, not the I region, was essential and sufficient for adoptive transfer and that H-2D compatibility was not relevant. Mutation at the H-2K locus prevented adoptive transfer, while mutation at the Ia-1 locus, as in the B6.C-H-2bm12 mutant of C57BL/6, did not affect adoptive transfer. The contrast between these findings and the previously accepted I region restriction of adoptive transfer of Listeria immunity is discussed.     

Effects of H-2 on neural tube defects in congenic mice.

Pregnant mice congenic with C57BL/10 (B10.A, B10.BR, B10.D2, B10.A[2R], B10.A[5R], B10.A[15Rd, B10.A[1R], B10.A[18R], and B10.0L) were fed Purina Mouse Chow or the same diet plus 200 IU of vitamin A daily. The pregnant dams were sacrificed on the eighteenth day of gestation, and the fetuses were sexed and examined for defects in neural tube development. The frequency of neural tube defects was low (mean frequency of all strains, 0.36%) and was not affected by the addition of vitamin A (200 IU/day) to the diet. Twenty-seven of the 29 defects observed occurred in the anterior tube (exencephaly); fourteen were identified in female fetuses, but the sex could not be determined in the other 15 cases because of fetal death and early autolysis. Variations in frequency among the strains suggest that a locus between E beta and H-2D has a moderate influence on the occurrence of neural tube defects. Strains that had H-2d alleles in this segment of the H-2 complex had relatively high frequencies, and those with H-2b or H-2k alleles had significantly lower frequencies.     

Transplantation of cultured thymic fragments. VI. H-2 recombinant donors

Athymic (nude) mice were transplanted with cultured thymic fragments from syngeneic, allogeneic, and partially allogeneic (recombinant) mice. Lymphocyte proliferation and cytotoxicity in vitro were measured to assess immunologic reconstitution. Transplanted nude mice were immunocompetent whether donor and recipient were disparate for class I, class II, or both H-2 gene types. Furthermore, allotolerance for thymic H-2 class I antigens was achieved independently of class II antigen allotolerance. Class I antigen tolerance was not broken during lymphocyte responses to unrelated alloantigens, ruling out insufficient help as the tolerance mechanism. Splenocytes, isolated from nude mice transplanted with fully allogeneic or syngeneic thymic fragments and stimulated in vitro with trinitrophenyl-modified cells, displayed H-2-restricted, hapten-specific cytotoxicity. Cytotoxic cells from allotolerant mice were restricted to either host or thymic H-2 antigens, depending on the stimulating cell haplotype. Response levels for thymic and host trinitrophenyl-modified cells were comparable. We have shown that allogeneic thymic epithelium transplanted into adult nude mice can induce allotolerance to class I and II H-2 antigens equally, and permits T lymphocyte interaction with cells bearing thymic donor or host H-2 antigens. Our results are consistent with a model wherein T lymphocyte self-receptors retain their genomic repertoire but can be selectively mutated or expanded by appropriate H-2 antigen presentation by the thymus.     

Leishmania donovani infection in heterozygous and recombinant H-2 haplotype mice

On a B10 (Lshs) genetic background the development of acquired T-cell-mediated immunity to Leishmania donovani infection in mice is under H-2-linked genetic control. Three phenotypic patterns of recovery were previously observed: "early cure" (H-2s, H-2r), "cure" (H-2b) and "noncure" (H-2d, H-2q, H-2f), with cure behaving as a recessive trait in H-2b/H-2d mice. In this study the long-term response to L. donovani is followed over 130 days of infection in eight recombinant haplotype strains and in six further heterozygous haplotype combinations. Noncure in B10.HTG mice, which carry d alleles for loci at the K end and b alleles for loci at the D end of H-2, confirms that H-2-linked genetic control of the acquired response to L. donovani infection is located in the K end. The complex pattern of dominance relationships observed in the additional heterozygous haplotypes studied, the variable phenotypic response of H-2k mice and of recombinant haplotype strains carrying IEk in common, and the differential early curing activity observed in heterozygotes involving the s but not the r early cure haplotype and in recombinant haplotype mice carrying s alleles to the left of IE suggest, however, that more than one subregion (IE and presumably IA) are involved. Results are interpreted in the light of immunoregulatory T-cell populations previously demonstrated in noncure, cure, and early cure strains.     

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.     

H-2-restricted graft-versus-host reaction: Foreign determinants and restriction elements

Nylon-wool-purified T cells from radiation chimeras cause a lethal graft-versus-host reaction (GVHR) in irradiated, bone-marrow-protected recipients only if the recipient shares a restriction element with the T-cell donor and also expresses antigens foreign to the donor. Class I molecules (H-2K and H-2D) can act as restriction elements, but restriction to class II molecules could not be demonstrated. However, class II molecules as well as H-2K and some non-H-2 determinants could serve as foreign antigens.     

Regulatory mechanisms in cytotoxic T lymphocyte development. III. Induction, specificity, and genetic restriction of an in vitro suppressor T cell

We addressed questions pertaining to the immunogenetics of an in vitro alloinduced suppressor T cell (Ts) previously shown to inhibit cytotoxic T-lymphocyte (CTL) development by suppressing CTL precursor proliferation. Using intra-MHC recombinant strains of B10 congenic mice, the requirements for H-2 differences to induce Ts activity, the antigen specificity of the Ts, and the genetic restriction of Ts function were studied. It was found that differences at the K, D, or I regions alone can induce strong suppressor activity. Suppression of CTL development does not appear to be genetically restricted since the Ts inhibit CTL from responder cells disparate at K, K and D, I, or K and I. The alloinduced Ts is specific for the antigen stimulating its induction, but also inhibits CTL responses against immunologically unrelated determinants, even between class I and class II antigens, provided those determinants are carried on cells expressing the original inducing antigen. Ts can be triggered by antigens present on the responder cells but absent on the stimulator cells, indicating that the suppressive signal may be exerted directly on the responder population without specific interaction with stimulator cells.     

Polymorphism of minor histocompatibility genes in wild mice

H-2^b-restricted cytolytic T lymphocytes (CTL) were generated against H-1, H-3, and H-4 antigens and tested against target cells of F₁ hybrids between wild mice and inbred H-2 ^b mice. The congenic strain combinations for the CTL production were such that they tested one allele each at the H-1 and H-4 loci and four alleles at the H-3 locus. Most of the wild mice tested came from Southern Germany, but a few mice came from other European countries and Egypt and Israel. Virtually all wild mice typed as positive with CTL directed against H-3^b and H-4^b antigens; 32% of the F₁ hybrids tested reacted with anti-H-1^cCTL and 9% reacted with anti-H-3^d CTL. The positive results were not caused by cross-reaction with allogeneic H-2 antigens controlled by the major histocompatibility complex (Mhc) genes of the wild mice. At least some of the H-3 and H-4 antigens detected by the CTL in the F₁ hybrid were not identical with antigens of the immunizing strains. These results suggest a relatively low degree of polymorphism of the tested minor H loci in wild mice and further support the notion that minor H loci are unrelated to the Mhc.     

Restricted recognition of H-2 subregion coded alloantigens in delayed-type hypersensitivity

Subcutaneous (sc) immunization of mice with H-2K, I, or D incompatible spleen cells induces a state of host-versus-graft (HvG) delayed-type hypersensitivity (DTH). The DTH reaction is elicited by challenging the immunized mice in a hind foot with similar allogeneic spleen cells and is measured as the subsequent foot swelling. DTH effector T cells specific for H-2I-coded alloantigens, but not for H-2K/D-coded alloantigens, can be induced in a graft-versus-host (GvH) model as well. In this paper we report that under HvG as well as under GvH conditions the recognition of class II antigens by DTH effector T cells is restricted by class I molecules. Furthermore, DTH effector T cells induced by sc immunization with class I antigens appear to be restricted by class II molecules.     

Effect of thymocyte-stimulating factor-containing supernatants on the surface antigens of murine thymocytes.

Incubation of murine thymocytes in thymocyte-stimulating factor (TSF)-containing supernatants causes a four- to fivefold increase in the expression of the H-2^k and H-2^d antigens and a similar decrease in the expression of the TL antigen (in TL⁺ strains) on the surface of these cells. Experiments with antisera directed toward the private H-2K and H-2D antigens showed that TSF-containing supernatants cause approximately the same increase in the expression of the H-2K and H-2D antigens of thymocytes of the d and b haplotypes. With thymocytes of the k haplotype, only an increase in the expression of the H-2D antigens takes place, while no significant increase was found for the H-2K antigens. TSF-containing supernatants cause no significant change in the expression of the following antigens on the surface of thymocytes: Thy-1.1, Thy-1.2, Ly-1.2, Ly-2.2, Ly-6.2, Th-B, Ia-1,2,3,7, and G_IX. A factor similar to murine TSF, produced by human peripheral blood leukocytes, does not affect appreciably the expression of the H-2 antigens on the surface of murine thymocytes. The factor(s) causing the increased expression of the H-2 antigens on the surface of murine thymocytes appears to be produced by T lymphocytes. The factor(s) is eluted from a Sephadex G-100 column in at least two broad peaks with molecular weights of 300,000 and 90,000-25,000. Most of the activity enhancing the expression of the H-2 antigens is lost at pH 2, while most of it is maintained at pH 11.5 and at 56 °C. On the basis of these properties, it is concluded that the factor under study is probably different from the factor enhancing the phytohemagglutinin responsiveness of thymocytes.     

The histocompatibility-2 system in wild mice

The I-region gene products of 29 wild-derivedH-2 haplotypes on a B10 background (B10.W congenic lines) were typed with alloantisera which detect 17 inbred I-region antigens. Five new I-region antigens were defined by expanding the inbred line panel ofH-2 haplotypes to includeH-2 ^u , H-2^v, andH-2 ^j . Based on serological analyses of the inbred and B10.W lines, the polymorphism of theIA gene (or genes) is estimated to be at a minimum of 15 alleles and theIE gene (or genes) at a minimum of 4 alleles. These results indicate that theIA subregion is more polymorphic than theIE subregion. By combining the I-region typing data with theH-2K andH-2D region typing data reported previously, a total of 11 new natural recombinants of inbredH-2 alleles were detected among the B10.W lines.     

Parasite-accessory cell interactions in murine leishmaniasis. II. Leishmania donovani suppresses macrophage expression of class I and class II major histocompatibility complex gene products

In the present study, we examined the modulation of MHC class II and class I gene products on BALB/c macrophages infected with the obligate intracellular protozoan Leishmania donovani. Our findings indicated that this organism suppressed macrophage expression of both classes of MHC antigens. These effects varied somewhat, depending on whether cells were in the basal state or were stimulated with interferon-gamma. Thus, class II density on interferon-gamma-treated infected macrophages was suppressed by as much as 90%, relative to lymphokine-stimulated control cells. Induction of H-2K and H-2D by lymphokine treatment of infected macrophages was also markedly reduced. In the basal (non-lymphokine-treated) state, infected cells also showed reduced expression of H-2K and H-2D, but not I-A or I-E. The latter result was related to minimal levels of class II molecules on normal, in vitro cultured macrophages. Suppression of MHC gene products correlated with both the duration and intensity of leishmania infection and could not be overcome by increasing doses of interferon-gamma. Culture of cells under conditions of cyclooxygenase inhibition completely abolished elevated synthesis of prostaglandin E2 by infected macrophages and augmented their responsiveness to lymphokine induction of class II antigens by 60 to 80%. These results indicate that L. donovani is capable of subverting a critical macrophage accessory function required for the induction of T lymphocyte immunity. This mechanism could account, at least in part, for defective parasite-specific cell-mediated immunity seen during infections with this protozoan.     

Specificity and frequency of primary anti-HLA cytotoxic T lymphocytes in normal and HLA-B27.2-, HLA-B27.5-, and HLA-Cw3-transgenic mice. A transgenic model for MHC xenoantigen recognition

Previous studies have shown that the lymphocytes of naive mice produce a strong primary CTL responses in vitro to human MHC class I Ag presented by HLA-transgenic mouse (TGM) cells. A limiting dilution (LD) assay was used to analyze this xenoreactive CTL repertoire in mice. Frequencies of HLA class I-specific CTL precursors (CTLp) were estimated in naive normal and HLA-B27.2-, -B27.5- and HLA-Cw3-double TGM (i.e., mice expressing HLA and human beta 2-microglobulin (hu beta 2m]. The xenoreactive CTLp frequencies were compared to frequencies of CTLp to H-2 alloantigens estimated in naive normal mice. The results showed that the frequencies of HLA class I-specific CTLp are comparable with those of alloreactive CTLp. This overlap in CTLp frequencies suggests that HLA class I xenoantigens are recognized by primary mouse CTL as allelic variants of H-2K and H-2D. This was confirmed in split well analysis by the observation that the xenoreactive response was not restricted by self-MHC of the responding mouse. Thus, primary HLA class I-specific mouse CTL clones recognized their target Ag regardless of whether they were expressed on H-2-mismatched mouse cells or on human cells. The frequencies of HLA class I-specific CTLp in HLA-TGM were comparable to those in normal mice. We propose that MHC allo- and xenoreactive CTL responses are not caused by the activation of CTLp specific for self-MHC plus peptide but to the activation of CTLp recognizing MHC allo- and xenoantigens directly or as peptides presented by their native MHC molecules.     

Analysis of neonatally induced tolerance of H-2 alloantigens

Neonatal inoculation of mice with semi-allogeneic lymphohematopoietic cells produces a state of highly specific allograft tolerance. Phenotypically, by both in vivo and in vitro criteria, antigen-reactive cells specific for the tolerated antigens appear to be clonally deleted from intact, tolerant mice. However, a series of adoptive transfer experiments using mice rendered tolerant of variousH-2 alloantigens revealed that tolerance of Ia (class II) antigens is maintained by a different mechanism than tolerance of K/D (class I) antigens. Long-term acceptance of Ia-disparate grafts by recipients of Ia-tolerant lymphoid cells suggested that an active process (rather than passive clonal deletion) mediates and maintains this type of tolerance. No comparable success was achieved when tolerance of isolated class I or entireH-2 haplotype disparity was examined, suggesting that clonal deletion might be operative in these combinations. Modest prolongation of skin-graft survival was observed in adoptive transfer recipients of lymphoid cells from donors tolerant ofI-JECSD disparity. These data are compatible with the hypothesis that the centralI region (JE) promotes tolerance induction to associated strong IA- and D-region alloantigens by activating a suppression mechanism.With the technical assistance of Phoebe Strome.     

Anti-major histocompatibility complex immunity detected prior to intentional alloimmunization. I. Naturally occurring H-2-specific antibodies in C57BL/KaLwRij (H-2b) mice

Naturally occurring, H-2-specific, lymphocytotoxic antibodies were detected in 3-10% of young adult and in 10-40% of aged C57BL/KaLwRij (H-2b) mice. The antibodies were of the IgM class and occurred in low titers, but occasionally a high titer was found. The antibodies detected public lymphocyte-membrane antigens controlled by genes identical with, or closely linked to class-I H-2K and H-2D genes. Antibodies against 7 different allogeneic H-2 haplotypes were detected but sera of individual mice exerted different reaction patterns and some specificities occurred more frequently than others. Although the occurrence of the antibodies was age dependent, thymus involution, gammapathies, autoimmunity, the presence of other natural lymphocyte-specific antibodies, and polyclonal or nonspecific stimulation could not be related to the occurrence of natural H-2-specific antibodies. Several possible explanations of natural H-2-specific antibodies exist. We propose that determinants of complex altered self-MHC (MHC + X) antigen(s) triggered the production of H-2-restricted antibodies that recognize H-2-public determinants on normal allogeneic cells.