Minor Histocompatibility Loci Research Articles

Genetics of histocompatibility in mice. II. Survey for interactions between minor (non-H-2) antigens by skin grafting.

Twenty-five congenic mouse strains differing at distinguishable minor (non-H-2) histocompatibility loci were paired in 71 different combinations. F1 offspring were used as skin-graft donors for more than 4000 recipients to test whether immune responses to parental strain antigens were statistically independent. Thirty-four (48 percent) of the 71 combinations were predicted adequately by an independent response hypothesis. A simple additive model was consistent with 39 (55 percent) of the observed responses, although 18 of these were among those in agreement with the independent hypothesis. A synergistic response faster than that predicted by either the independent or additive response model was seen in 12 (17 percent) of the combinations. The remaining 5 percent were not well described by any of these models. No strain was represented with unusual frequency among those involved in synergistic interactions.

Definition of a new T lymphocyte cell surface antigen, Ly 11.2.

The present communication defines a new cell surface antigen, Ly 11.2, which appears from its strain and tissue distribution to be distinct from all other previously defined normal or virally coded antigens or traits of the mouse. Lymphocytes of the T cell lineage, but not B cells or nonlymphoid cells, bear this marker. Although the locus coding for this antigen appears to be closely linked to the minor histocompatibility locus H-30 and to the locus coding for Ly 6.2, a chromosome assignment has not yet been made for my of these loci.

Reversal of experimental acute hepatic failure in the rat

A suitable animal model of acute fulminant hepatic failure was developed in the rat by the administration of the selective hepatotoxin, galactosamine hydrochloride. A dosage of 2.6 g/kg given intraperitoneally to Lewis rats resulted in reproducible, potentially reversible hepatic necrosis and high mortality. The ability of dispersed single-cell suspensions of syngeneic hepatocytes, allogeneic hepatocytes, and bone marrow to provide “support” so that liver recovery could occur was explored in this model. Syngeneic hepatocytes (4 × 10 7 cells) given intraperitoneally 48 and 60 hr after toxin administration increased survival to 70 and 66%, respectively, compared to a control of 0%. Hepatocytes prepared from Fischer strain animals which differ from Lewis strain at minor histocompatibility loci similarly improved survival (62.5%). Syngeneic bone marrow cells (4 × 10 7 cells) given intraperitoneally 24 hr after toxin administration improved survival to 62.5% compared to a control group of 0%. This effect is not mediated by a mature macrophage or lymphoid cell population, but is specific to the cells derived from bone marrow, since single-cell suspensions of splenocytes, thymocytes, and peritoneal exudate cells had no effect on increasing survival. The mechanism of action of hepatocytes and bone marrow cells and whether they act by the same or different mechanisms remain to be defined. Cellular transplantation may offer a new simple measure in the treatment of acute hepatic failure.

Systemic immune unresponsiveness induced in adult mice by anterior chamber presentation of minor histocompatibility antigens.

The ability to introduce carefully controlled numbers of viable cells into the anterior chamber of mouse eyes made it possible to examine the interrelationship between presentation of antigens into the anteior chamber and into conventional body sites and their synergistic/antagonistic effects on the immune system. P815 mastocytoma (DBA/2; H-2d) cells are syngeneic with BALB/c hosts at the major histocompatibility locus, but differ at multiple minor histocompatibility loci. When P815 cells were injected subcutaneously, they were rejected by BALB/c recipients who became specifically immune. By contrast, when P815 cells were injected intracamerally, they grew progressively into massive intraocular tumors; moreover, these BALB/c hosts proved subsequently unable to reject subcutaneously injected P815 cells, and, more impressively, failed to reject DBA/2 skin allografts placed orthotopically. Minor histocompatibility antigens, presented first through the anterior chamber of mouse eyes, elicit a suppressive rather than an aggressive host immune response that protects cells that bear these antigens from a destructive alloimmune reaction at both intracameral and systemic sites.

In vitro alloreactivity of mouse bone marrow lymphocytes

Before characterizing alloreactive cells of the bone marrow, it was necessary to reevaluate the alloantigen response in this tissue. The results of previous studies using the parental-F 1 system in the mixed-lymphocyte reaction (MLR) are open to question because of the recently documented proliferation of F 1 stimulator cells (W. H. Adler, T.Takiguchi, B. Marsh, and R. T. Smith, J. Immunol. 105, 984, 1970; P. F. Piguet, H. K. Dewey, and P. Vassalli, J. Exp. Med. 146, 735, 1977). The culture system was optimized for measuring the MLR of bone marrow lymphocytes enriched on sucrose density gradients. The proliferative response of the enriched fraction (BML) to 2000-R irradiated allogeneic spleen cells was three times as high as the response of unfractionated bone marrow. For maximal responses, antigen concentration had to be twice as high for the BML as for the lymph node, and in a time course study the highest [ 3H]TdR uptake occurred on Day 3 in BML cultures and on Day 5 in the LN. In lymph node semiallogeneic cultures stimulator cell proliferation can be disregarded, while semiallogeneic BML MLR err significantly on the high side. When BML were matched with allogeneic stimulator cells at the H-2 locus, they gave good MLR responses, provided there was a minor Mls histocompatibility locus difference, while in the lymph node the response was greatly diminished in similar mixtures. The differences in the BML and lymph node alloantigen responses with respect to antigen concentration, kinetics and susceptibility to F 1 and Mls stimulation, suggest that the bone marrow alloantigen response is mediated by a cell population that is different than alloresponsive cells in the lymph node.

H GENE THEORY OF INHERITED AUTOIMMUNE DISEASE

Autoimmune disease in inbred mice is probably determined by co-dominant genes associated with both the major and minor histo-compatibility loci. It is postulated that the genes involved are histocompatibility-antigen (H) genes themselves, which delete complementary clones in fetal life, in accord with Burnet's theory of clonal selection. Such deletions cause perturbations in the paratope-idiotope network reactions envisaged by Jerne. As well as having negative effects on immune-response capacity, the perturbations have positive effects, because the deletion of clones with specificity for the idiotopes of other clones permits immune responses which would otherwise be absent. Such perturbations can influence the chance of emergence of a forbidden clone by somatic mutations occurring in the V genes of dividing immunocytes and so can provide a genetic predisposition to autoimmune disease.

Combined effects of antineoplastic agents and anti-lymphoma allograft reactions

Combined effects of chemotherapy and anti-lymphoma allograft responses were studied in mice inoculated intraperitoneally (i.p.) with leukemia cells incompatible for multiple minor histocompatibility loci ( MMHL) and treated with graded doses of 1,3-bis( 2-chloroethyl)- 1-nitrosourea (BCNU), vincristine (VCR), cyclophosphamide ( Cy), 5-(3,3-dimethly- 1-triazeno)-imidazole- 4-carboxamide (DTIC) or hexamethyl-melamine (HMM). The results showed that: (a) no appreciable difference in survival times was found, as a rule, in leukemic compatible or allogeneic mice not subjected to drug treatment; (b) both BCNU and Cy markedly prolonged the survival times of compatible leukemic mice, but only treatment with BCNU resulted in chemo-immune collaborative activity when the drug was administered to allogeneic hosts; (c) DTIC was moderately active in compatible mice and no additive or synergistic effects were found in allogeneic hosts; (d) VCR, little or no effect in prolonging the survival times of leukemic compatible mice, proved to be highly efficient when given to allogeneic recipients; (e) HMM was totally ineffective in prolonging the survival time of either compatible or allogeneic mice. These studies evidenced that no obvious relationship could be found between the anti-leukemic activity of different drugs and their antineoplastic effectiveness when combined with antitumor immune responses.

Combined effects of BCG or Candida albicans (CA) with antitumor agents against a virus-induced lymphoma in mice

Moloney-virus-induced lymphoma LSTRA of BALB c origin was inoculated i.p. into histocompatible CD2F 1 mice or into allogenic BD2F 1 hosts incompatible for multiple minor histocompatibility loci (MMHL). All untreated mice died with comparable median survival times (MST). However, when recipients were subjected to i.p. treatment with BCG or inactivated Candida albicans (CA), significant antitumor effects were detected if the non-specific immunoadjuvants (IA) were given before (on day − 14) and after (on day + 1) tumor challenge. The efficiency of IA was found to be higher in MMHL incompatible mice than in CD2F 1 hosts. Chemotherapy experiments were conducted in histocompatible mice using IA given according to various treatment schedules, in combinations with 3 nitrosoureas of clinical interest (i.e. BCNU, CCNU and Me-CCNU). Synergistic antitumor effects were evidenced when the antineoplastic agents were associated with IA administered on the “ − 14 + 1” regimen. These results pointed out that the antilymphoma effects of chemotherapy could be amplified by IA only when the treatment schedule included adjuvants administration prior to tumor challenge.

Qa-1-Associated Antigens

Abstract The Qa-1b allele is assigned to strains whose peripheral lymphocytes are not lysed by a standard anti-Qa-1 serum. In order to investigate whether Qa-1b represents a single allele or several alleles that have been grouped together as “not a,” we studied the activity of cytotoxic T lymphocytes (CTL) specific for Qa-1b-associated antigens. Cold target inhibition experiments were performed to test the ability of various Qa-1b targets to inhibit the lysis of 51Cr-labeled B10 blasts by B10.BR anti-C3H and C57BR anti-AKR CTL. Qa-1a targets did not inhibit lysis relative to controls syngeneic with the CTL. In contrast, Qa-1b cold targets strongly inhibited the lysis of labeled B10 targets. We found no differences in inhibition among 16 Qa-1b strains representing six H-2 haplotypes and nine different backgrounds. These data indicate that all of these Qa-1b strains express the Qa-1 antigens shared by B10 and C3H or AKR, and thus suggest that Qa-1 is markedly less polymorphic than H-2. Experiments were also performed to test the possibility that Qa-1-associated antigens are involved in the recognition of minor histocompatibility determinants. CTL were generated in secondary cultures between strains identical at Qa-1 and at H-2, but differing at minor histocompatibility loci. These CTL were tested for their ability to lyse targets bearing the minor histocompatibility antigens of the stimulators, and sharing either Qa region or H-2 antigens with the stimulators. In contrast with the well established role for H-2K and H-2D antigens in the recognition of conventional determinants, we found that in both Qa-1a and Qa-1b strain combinations, Qa region homology was neither necessary nor sufficient for minor locus-specific lysis.

Nonspecific suppressor elements in murine allogeneic radiation chimeras

Spleen cells from long-term mouse allogeneic radiation chimeras were tested for their ability to modulate the graft-versus-host (GVH) or plaque-forming cell (PFC) response of normal lymphocytes transplanted in lethally X-irradiated recipients. In vivo GVH proliferation of normal lymphocytes (syngeneic to donor cells of the chimera) against antigens of host-type in which the chimeric state had been established was reduced by chimera cells. Inhibition varied, some chimeras suppressing GVH more than others and a few not suppressing at all. The suppressive effect was abrogated if the chimera cells were treated with anti-θ; treatment with anti-IgM did not eliminate this activity. When mixtures of normal donor lymphocytes and chimera cells were given to irradiated recipients genetically different from host or donor, reduction of donor cell GVH also occurred. Further, chimera cells reduced the GVH activity of normal host cells in irradiated recipients differing from the host at one H-2 locus and from the donor at minor histocompatibility loci. The modulating effect of spleen cells from chimeras on the PFC response by normal lymphocytes also varied. Six chimeras induced a 25 to 90% suppression, two enhanced the response, and one showed no effect. Where suppression occurred, treatment of chimera cells with anti-θ most often, but not always, restored PFC production. Our results show that the suppressive action of splenic lymphoid cells by chimeras is highly nonspecific and variable in expression. We suggest that tolerance in chimeras may be mediated by nonspecific suppressor elements leading to unresponsiveness to a variety of antigens including SRBC.

In Vitro Analysis of Allogeneic Lymphocyte Interaction

Abstract A helper allogeneic effect factor (AEF) was produced across an incompatibility at the minor histocompatibility loci. This AEF is genetically restricted in its activity since it helps B cells only of the stimulator haplotype and of haplotypes that share both an Mls and I-region identity with the stimulator haplotype. The I-region genes involved here map to the I-A and/or I-C subregions. An anti-LyM immunoadsorbent column but neither an anti-H-2 nor an anti-Ia column adsorbed AEF helper activity. It is suggested that the activation of T helper cells by a positive allogeneic effect across a minor H locus difference and their genetically restricted interaction with allogeneic B cells may in part be due to the acquisition by alloactivated T cells of stimulator cell-derived LyM and/or Mls determinants. The data presented indicate that helper T cells recognize Mls locus alloantigens in the milieu of self Ia antigens.

Changes of the immunogenic properties of K36 lymphoma treated in vivo with 5(3,3-dimethyl-1-triazeno) imidazole-4-carboxamide (DTIC)

Mice of AKR strain bearing syngeneic K 36 lymphoma were treated with DTIC for a number of transplant generations. The lymphoma line (K36/DTIC) became resistant to DTIC treatment and weakly immunogenic for AKR or (AKR × DBA/2)F 1 hosts. Previous findings, however, showed that DTIC-treated H-2 b or H-2 b lymphomas became DTIC-resistant as well, but acquired strong immunogenicity for histocompatible hosts. Transplantation resistance of allogeneic mice against K 36 or K36/DTIC lines injected i.p. or i.v. was also investigated. Both lines inoculated i.p. were rejected by either H- 2-incompatible recipients, or H- 2-compatible mice incompatible for minor histocompatibility loci (MIH). When the tumors were injected intravenously, H- 2-compatible MIH-incompatible mice were more susceptible than H- 2-MIH-incompatible recipients to lymphoma challenge. Moreover K36/DTIC line elicited stronger transplantation resistance than K 36 tumor. Unexpectedly H- 2-MIH-incompatible mice homozygous for the H-2 d haplotype were partially susceptible to the i.v. challenge with K 36 lymphoma cells. However, strong transplantation resistance was found in the same hosts against K36/DTIC line. In conclusion the limited increase of tumor cell immunogenicity obtained by treatment of K 36 lymphoma with DTIC was detectable in syngeneic, hybrid and allogeneic mice.

Quantitative pathology of oral mucosal allografts in the rabbit

Quantitative analysis of four morphological components of the rejection reaction revealed differences between skin and mucosal allografts and between allografts and autografts of these tissues. Orthotopic skin or mucosal allografts were exchanged between pairs of rabbits matched or mismatched at the RLA locus of the major histocompatibility complex. Similar autografts were placed as controls. Quantitative microscopic examination of paraffin sections of all grafts, in which a standardized microscopic field was analysed, gave mean values for: (1) percentage of vessels with damaged walls; (2) fraction of vessel lumen occupied by blood cells or thrombus; (3) extravascular lymphocyte counts; (4) extravascular granulocytes counts. Significant differences were found between mismatched mucosal and mismatched skin allografts for all variables except lymphocyte counts. Mucosal and skin autografts differed significantly only in lymphocyte and granulocyte counts. Comparisons between mismatched allografts and autografts showed significant differences in all four variables in both mucosa and skin, whereas matched allografts and autografts of both tissues differed significantly for all variables except for the granulocyte count. Differences between autografts and allografts, whether RLA matched or mismatched, are clearly attributable to histocompatibility barriers in allografts. If the RLA locus had a significant effect on the 4 pathological components analysed, differences between matched and mismatched allografts would be evident. There being no such differences it is concluded that the minor histocompatibility loci play an important role in this type of immunological reaction. Differences in the reaction in oral mucosa and skin may be due to differences in the expression of histocompatibility antigens or tissue-specific antigens in these tissues and to factors of the local environment.

Genetic variation at a locus (TAM-1) for submaxillary gland protease in the mouse and its location on chromosome No. 7.

Electrophoretic and activity variants for a testosterone-induced esteroprotease have been discovered in submaxillary glands from inbred strains of mice. The enzyme is tentatively designated tamase [TAM-1] and the variant genetic locus is Tam-1. The alleles Tam-1a and Tam-1b determine electrophoretically distinct zones of tamase activity, while Tam-lc produces no detectable enzyme activity. Data from recombinant inbred strains and B6AF1 X B6 and B6D2F1 X B6 backcrosses established linkage of Tam-1 to glucose phosphate isomerase (Gpi-1), pink-eyed dilution [p] and beta-hemoglobin (Hbb) on chromosome 7. The gene order is Gpi-1--Tam-1--p--HBB. Analysis of congenic resistant strains indicates that Tam-1 is closely linked to the minor histocompatibility locus, H-4. TAM-1 was not cross-reactive with antisera to mouse nerve growth factor, submaxillary renin, or tamases A and D.

Detection of two allotype-(Ig-1)-linked minor histocompatibility loci by the use ofH-2-restricted cytotoxic lymphocytes in congenic mice

Cytotoxic lymphocytes (CTL) were generated betweenIg-1-congenic strains BALB/c(H-2(d),Ig-1(a)) andC.B-17(H-2(d),Ig-1(b)) by cross-immunization in both directions and rechallenge in vitro. The effector cell populations specifically lysed target cells sharing both theH-2 haplotype and theIg-1 allele of the sensitizing strain. B- and T-cell blasts were equally good targets, suggesting thatH-2-restricted cytotoxic lymphocytes are not directed against serologically defined conventional allotypic determinants, but probably against minor histocompatibility antigens controlled by genes linked to theIg-1 complex. Competition experiments using cold target cells from a series ofIg-1(b)-congenic strains of the BALB/c background (BAB-14, C.B-17, C.B-26) revealed two not yet described minor histocompatibility loci linked to theIg-1 complex: We could demonstrate that BALB/c anti-C.B-17 effector cells recognize at least two distinct antigenic determinants on C.B-17 target cells, but only one on target cells from BAB-14, which carries a recombinantIg-1 complex. From these results we conclude that one of the minor histocompatibility antigens, designated as H(C(H)), is encoded by a gene linked to the heavy-chain constant-region (C(H)) genes, whereas the second minor histocompatibility antigen, designated as H(V(H)), is coded for by a gene linked to the heavy-chain variable-region (V(H)) genes. These two new genetic markers may be useful for further analysis of the mouseIg-1 complex because the analysis of the H(C(H)) and H(V(H)) genes may facilitate the search for recombinants in that chromosomal region.

Effect of 6-Mercaptopurine on Response to Major and Minor Histocompatibility Antigens

Pregraft treatment of chicks with 6 mercaptopurine-palladium complex (6-MP-Pd) resulted in a 16% survival of skin allografts incompatible at the major histocompatibility locus B. These incompatible grafts survived beyond 16 weeks postgrafting and showed profuse feather growth. No form of postgraft treatment was necessary. All nontreated incompatible grafts were rejected prior to 4 weeks postgrafting with no evidence of feather development.The same treatment, however, did not significantly suppress rejection of B-system compatible grafts which differed at minor histocompatibility loci. The rate of feather growth in successful B-system compatible grafts was significantly enhanced by the 6-MP-Pd treatment through 2 weeks postgrafting. Though the drug appears to affect the cellular immune system, the humoral immune response was unaffected as evidenced by a high antibody titer following turkey erythrocyte challenge.

Genetics of graft-versus-host reactions

Intracardiac injection of lymph node cells into newborn allogeneic recipients incompatible with congenic donors at multiple loci within theH-2 complex (B10→B10.A) led to GVH splenomegaly, retarded growth, and mortality. In contrast,H-2I region differences [B10.A(4R)→B10.A(2R)] required fortyfold more cells to produce a comparable splenomegaly, and did not produce runting or GVH mortality despite a strong proliferative response in the spleen. However, when 4R donors were preimmunized with host lymphoid cells, 51 percent of 2R recipients died of allogeneic disease. GVHR was also observed in the reciprocal combination (2R→4R), in contrast to previous reports. The phenotypic determinants governing GVHR in the 4R→2R combination are probably coded for byI-C subregion genes. Disparities at theI-B orI-J subregions did not evoke detectable GVHR. The data indicate thatI-C subregion loci have the impact of so-called minor or moderate histocompatibility (MoH) loci. Possible implications for allogeneic human bone marrow transplantation are discussed.

Antigen expression on early mouse trophoblast

THE mammalian foetus inherits paternal as well as maternal genes and can be considered to constitute a naturally occurring and highly successful allograft1. It has been suggested that an important factor in the survival of the foetus in an immunocompetent histoincompatible mother might be the presence of an antigenically deficient barrier of foetal trophoblast cells2–4. Cell surface antigens determined by minor (non-H–2) histocompatibility loci have been demonstrated on cleavage stage mouse embryos5, and subsequently major (H–2) as well as non-H–2 determinants have been detected on the trophectoderm of pre-implantation mouse blastocysts6,7. The expression of these antigens, however, is considerably reduced, below detectable levels in the case of H–2, on blastocytes which have been activated for implantation from a state of experimentally induced delay6,7. In addition, immunofluorescence8, immunoperoxidase7 and mixed haemadsorption (MHA)9 studies have failed to demonstrate histocompatibility antigens on the trophoblast component of blastocysts cultured as outgrowths in vitro. In contrast to all these findings, however, a recent report has claimed to demonstrate both maternally and paternally inherited antigens on blastocyst outgrowths by means of an MHA technique10. We have now confirmed this report and reconciled the apparently contradictory results by investigation of the identity of the antigens expressed by the trophoblast at this early stage of its differentiation in four mouse inbred strains.

Cytotoxic T lymphocytes specific for I region determinants do not require interactions with H-2K or D gene products

Gene products coded for by the major hisocompatibility complex (MHC) can serve as target antigens for cytotoxic T lymphocytes (CTL) (1). A variety of test systems are available which have yielded information consistently reinforcing the importance of this complex of genes in the generation and effector phases of the cytotoxic immune response. Originally, it was shown that allogeneically-induced CTL had specificity primarily for the products of the K and D loci of the mouse H-2 complex (2). More recently this has also been found to be the case for xenogeneic immunizations (3,4). Additional examples of T cell-mediated lysis have been reported involving viral-infected or chemically- modified syngeneic stimulating and target cells in which homology at H-2K or H-2D was required between the responding and target cells for appreciable lysis to occur (5-7). Moreover, CTL specific for minor histocompatability antigens are able to lyse only target cells bearing these membrane antigens and sharing a common H-2K or H2-D gene product with the effector (8,9). Two hypotheses have been proposed to explain the requirement for H-2 identity between effector and targets in these systems. CTL may recognize new antigenic determinants created by the interaction of the modifier with syngeneic K and D gene products. Alternately, a dual recognition system my exist, requiring an antigen-specific receptor as well as a second receptor with specificity for homologous H-2K or H-2D determinants (5). Neither model can be excluded at this time. The I region also contains genes coding for histocompatibility loci since animals differing at the I-A or I-C regions of the H-2 complex reject skin grafts (10-12), though less rapidly than mice differing at the H-2K or H-2D regions, Also CTL can be generated to I region determinants but less efficiently than CTL specific for H-2K or H-2D gene products (12-14). The question can therefore be raised, whether the I region minor histocompatibility loci function independently from the H-2K or H-2D loci or whether I region-specific cytolysis requires the participation of H-2K or H-2D gene products of the target cell. This communication illustrates the generation of CTL showing specificity for I region determinants in primary mixed lymphocyte cultures. Further, we demonstrate by genetic analysis and byt eh use of speficit alloantisera that CTL directed to Ia determinants (a) do not see these antigens as modifications of H-2K or H-2D gene products but as independent gene products coded for by the I region, and (b) they do not require interaction with target cells bearing the same H-2K or H-2D gene product as the effect CTL.

H-2 restriction of cytolysis after immunization of minorH congenic pairs of mice

Mice of three congenic resistant lines differing from C57BL/10 at theH-3, H-13, H-7, andH-8 minor histocompatibility loci were used to immunize, and were immunized with, C57BL/10. Cytotoxic cells which were capable of causing rapid lysis of cells from the immunizing strain were generated at least one-way in all combinations tested. In order for a target to be susceptible to cytolysis, it had to carry both the sameH-2b haplotype and the same differential minor histocompatibility alleles as the immunizing strain. That is, B10.C(47N) (H-2b,H-7b) anti-C57BL/10 (H-2b,H-7a) cytotoxic cells lysed C57BL/10 targets but not B10.BR (H-2k,H-7a) targets, nor BALB.B (H-2b,H-7b) targets. Crossreactions in the cytotoxic assay suggest that theH-3, H-13 congenic pair —B10.LP and C57BL/10 —may differ in at least two more minor histocompatibility loci which have not yet been defined. Although 6 x 1066 C57BL/10 lymphoid cells primed B10.D2(57N) (H-8b) mice for a secondary in vitro cytotoxic response, a tenfold higher dose apparently made them tolerant. It is concluded that all minor histocompatibility differences can generate cytotoxic T cells which show specificity both for the minor and major histocompatibility alleles.