Histocompatibility antigens controlled by theI region of the murineH-2 complex

Spleen cells from A.TH mice, presensitized in vivo by skin grafting, were restimulated in vitro by A.TL lymphocytes, and A.TH anti-A.TL effector cells were generated. The effector cells lysed, in the CML assay, A.TL blasts. This reaction, which was againstI-region antigens, could be inhibited by the addition to the reaction mixture of anti-La sera directed against A.TL antigens. The inhibition was specific, since normal mouse serum, reciprocal antiserum (A.TL anti-A.TH), and anti-H-2 sera did not have a significant effect on the reaction. The Ia antibodies also specifically inhibited the reaction of A.TH anti-A.TL effector cells against CBA targets. Con A blasts were significantly poorer targets inI-region CML than LPS blasts. As CML targets, macrophages and cells of a mammary adenocarcinoma were as good as, if not better than, the LPS blasts. The experiments support the notion that Ia antigens are the targets in theI-region CML.     

Induction of T-cell producers of the macrophage migration inhibitory factor by the products of the K-, I- and D-regions of the H-2 complex in mice

It has been demonstrated with the help of intravenous in-vivo immunization with irradiated splenocytes of A.AL anti-A.TL (difference at the K-region of H-2), A. TH anti-A. TL and A. TL anti-A. TH (difference at the I-region of H-2), C57BL/10Sn anti-B10.D2(R107) and B10.D2 (R107) anti-C57BL/10Sn (difference at the D-region of H-2) mice that T cells--MIF producers, induced at different times after induction of the alloimmune response, react to the products of the K- and I- but not to those of the D-region of the H-2 complex.     

Inhibition of secondary mouse mixed lymphocyte reaction by anti-Ia sera

Secondary mixed lymphocyte reaction (MLR-II) was studied in A.TH anti A.TL and A.TL anti-A.TH combinations in which stimulation was mainly due toH-2I-region differences. In both cases the MLR-II was specifically inhibited by the responder anti-stimulator Ia serum. The level of inhibition was dependent on the ratio of the amount of immune serum to the number of stimulating cells. The inhibitory activity and Ia antibodies were specifically absorbed and eluted together. The results confirm that the lymphocyte-activating determinants of the MLR-II (1) are carried by the Ia molecules and (2) are identical to the serologically defined Ia determinants. —Anti-Ia sera directed against private and public specificities of the stimulating cell induced a higher level of inhibition than anti-Ia sera directed only against public specificities, indicating that both private and public Ia specificities are involved in restimulation during MLR-II. — These results, in connection with others, suggest that the receptor of the proliferating T cell recognizes the same Ia determinant as the combining site of the Ia-recognizing antibody.Abbreviations used in this paper Lad lymphocyte-activating determinant - MLR mixed lymphocyte reaction - MLR-I, MLR-II primary, secondary MLR - PRC primed responder cells - LCT dye exclusion lymphocytotoxicity microtechnique - RR relative response     

Two Distinct Populations of Primary Cytotoxic Cells Infiltrating into Allografted Tumor Rejection Sites: Infiltration of Macrophages Cytotoxic against Allografted Tumor Precedes That of Multiple Sets of Cytotoxic T Lymphocytes with Distinct Specificity to Alloantigens

It has been reported that the rejection of tumor allografts is mainly mediated by cytotoxic T lymphocytes (CTLs). Here, we characterized the cytotoxic effector cells of C57BL/6 (B6; H-2^b) mice infiltrating into the rejection site of the i.p. allografted Meth A fibrosarcoma (or P815 mastocytoma) cells of H-2^d origin. Two types of cytotoxic cells (i.e., CD8⁺ CTLs and macrophages (Mφs)) were identified by flow cytometric fractionation of the infiltrates or by specific in vitro elimination of cells either with antibody (Ab)-coated beads or with an Ab-plus complement. Of particular interest, these effector cells showed distinct and unique target specificities. First, the CTLs were inactive against transplanted tumor (e.g., Meth A) cells, whereas they were cytotoxic against donor-related concanavalin A (Con A) blasts as well as CTLL-2 (H-2^b) cells transfected with a class I gene of H-2^d origin. A cold target competition assay suggested that the CTLs were composed of multiple sets of T cells, each of which specifically recognized different allo-antigens. Second, the Mφs lysed the allografted tumor cells but were inert toward the Con A blasts and the CTLL-2 transfectants. Unexpectedly, the infiltration of Mφs preceded the infiltration of CTLs by several days during the course of rejection. These results indicate that two distinct populations of unique cytotoxic cells (i.e., CTLs and Mφs) are induced in the allografted tumor rejection site, and that the infiltration of cytotoxic Mφs responsible for rejection precedes that of the CTLs cytotoxic against cells expressing donor-related allo-antigens.     

Evidence for differentiation of NK1+ cells into cytotoxic T cells during acute rejection of allogeneic bone marrow grafts

The ability of lethally irradiated C57BL/6 mice to acutely reject H-2^d bone marrow is due to a lymphocyte population that is NK1⁺, ASGM1⁺, CD4^–, CD8^–, CD3⁺. Transfer of spleen cells from C57BL/6 mice expressing these antigens into nonresponder 129 mice adoptively transfers the ability to reject H-2^d marrow grafts. The specificity of this rejection maps to the H-2D major histocompatibility complex (MHC) region. Transplantation of high doses of H-2^d marrow into C57BL/6 overrides the acute rejection mechanism leading to graft survival. During growth of the graft, a cytolytic activity develops that is due to ASGM1⁺, CD8⁺ cytolytic T lymphocytes (CTLs) with H-2L^d specificity. The possibility that the ASGM1⁺, CD8⁺ CTLs are descendents of the CD3⁺, NK1⁺, ASGM1⁺, CD8^– cells responsible for acute rejection is investigated by adoptive cell transfer experiments. We show that beige mice that lack NK1⁺ cells as well as the ability to acutely reject H-2^d marrow fail to generate specific CTLs after transplantation with a high dose of H-2^d marrow. Transfer of highly purified NK1⁺ cells from B6.PL-Ly-2 ^a /Ly-3 ^a (Lyt-2.1) into beige mice together with H-2^d marrow leads to generation of Lyt-2.1 CTLs from donor NK1⁺ cells. These results show that specific CTLs are generated from NK1⁺ cells during acute marrow graft rejection. Offprint requests to: G. Dennert.     

Activation of natural killer (NK) cells in vivo with H-2 and non-H-2 alloantigens

Intraperitoneal inoculation of allogeneic lymphoid cells rapidly activates cytotoxic cells in the peritoneum which are nonadherent and express the NK-1, asialo-GM₁, and Thy-1 antigens. Allogeneic spleen cells were very efficient at activating these natural killer (NK) cells, while allogeneic thymocytes were much less effective. Heat-killed allogeneic cells or sonicates also could augment NK activity. — Incompatibility atH-2K, H-2I-A, orH- 2D readily evoked NK cell activity, whileH-2S- andH-2I-E/C-associated disparities did not. Non-H- 2 differences also stimulated NK activity and augmentation was particularly evident inMls-disparate combinations. Thus, the same alloantigens which efficiently activate T cells also activate NK cells.     

Genetic resistance to murine leukemia induced by different radiation leukemia virus variants; a comparative study on the role of theH-2 complex

Association of resistance to leukemogenesis with theH-2 complex was investigated. Three variants of the radiation leukemia virus: D-RadLV, RadLV and RS-RadLV were tested. Using congenic and recombinant mouse strains, the region includingH-2K andI-A/B was found to be involved with resistance to all three viruses. In addition,H-2D-region linked resistance was found in leukemogenesis in two of the three passages. The phenotypic expression of resistance associated withH-2D seemed to depend on the simultaneous presence ofH-2K-H-21-linked resistance alleles. TheH-2 haplotypes associated with resistance or sensitivity to the different RadLV variants were different for each passage, suggesting that there is a large degree of heterogeneity both inH-2-linked resistance and between the radiation-induced leukemia viruses.     

Blocking of MLC stimulation by anti-Ia sera: studies with the virus plaque assay.

The development of congenic mouse strains identical at the H-2K and H-2D loci but differing by I-region associated (Ia) determinants has permitted an association to be established between Ia determinants and stimulation in mixed lymphocyte culture reactions (MLR). The present experiments were undertaken to establish whether the Ir-coded control of MLR operated at the level of recognition or of stimulation. Reciprocal MLR were established between A.TH and A.TL mouse spleen cells in the presence or absence of anti-Ia sera directed either at determinants of the stimulating or responding cells. The number of T cells responding was assessed by the virus plaque assay. Anti-Ia sera directed against the responding cells were no more inhibitory of the MLR than normal mouse serum. In contrast, anti-Ia sera directed against determinants of the mitomycin-treated stimulating cells markedly inhibited activation of T cells in the MLR.     

Role of accessory cells in the induction of a secondary cytotoxic response to Moloney murine sarcoma virus-induced tumors

The role of Ia-positive accessory cells in the generation of a secondary cytotoxic response to tumor-associated antigens induced by Moloney murine sarcoma virus (M-MSV) was evaluated. Spleen cells from M-MSV-immune A.TL mice, depleted of accessory cells by anti-Iak serum plus C treatment and stimulated in secondary mixed leukocyte tumor cell culture (MLTC) with syngeneic Ia-negative A6ATL Moloney leukemic cells, failed to generate virus-specific cytotoxic T lymphocytes (CTL). CTL generation in Ia-depleted MLTC may be reconstituted by the addition of nonimmune Ia-positive spleen or peritoneal cells obtained not only from syngeneic A.TL but also from I-incompatible A.TH mice. This lack of restriction observed in accessory cell function is explained in terms of a nonspecific mechanism of CTL triggering mediated by soluble factors. In fact, IL 2 as well as supernatants obtained from I region-incompatible cultures consisting of M-MSV-immune, Ia-depleted A.TL spleen cells and A.TH Ia-positive cells, reconstituted secondary virus-specific CTL generation.     

Rapid murine mixed lymphocyte cultures assessed by emergence of T-cell insulin receptors

Murine T lymphocytes incubated in a mixed lymphocyte culture with allogeneic lymphocytes incompatible for theH-2 region or subregions thereof rapidly develop insulin receptors. In contrast, T cells cultured with syngeneic orH-2- andMlslocus compatible cells do not develop insulin-binding sites. The emergence of insulin receptors is probably an early premitotic event.     

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.     

A new locus (H-2T) at theD end of theH-2 complex

A.TH (H-2 ^t2) anti-A.TL (H-2 ^t1) effectors, obtained after in vitro restimulation of in vivo sensitized cells, react in the CML assay not only withH-2 ^t1, but also with a number of other targets carrying unrelatedH-2 haplotypes. The broad cross-reactivity can be explained by postulating the presence among the effectors of at least two populations of cells, one reacting with antigens controlled by theI region, and the other directed against antigens controlled by a locus at theD end, outside theH-2 complex. The existence of the two cell populations is also supported by cold-target inhibition data. The locus coding for the D-end CML antigens maps betweenQa-2 andTla. The locus is assigned the symbolH-2T. TheH-2T-locus CML is seen only after in vivo presensitization, but the killing is not K/D-restricted.     

Spleen cells from animals neonatally tolerant to H-2Kk antigens recognize trinitrophenyl-modified H-2Kk spleen cells

A.TL mice injected with (A.AL × A.TL)F₁ cells within 24 hours after birth were rendered tolerant to H-2K^k antigens, as evidenced by acceptance of A.TL skin grafts. When spleen cells from these tolerant animals were cocultured with A.AL stimulator cells, no cytotoxic effector cells were generated in a cell-mediated lympholysis assay. However, when the A.AL stimulator cells were derivatized with trinitrophenol, effector cells that displayed a cytotoxic effect against trinitrophenyl-modified H-2K^k target cells were generated. These data indicate that animals tolerant to H-2 determinants but chimeric to only a minor extent possess cytotoxic precursor cells in sufficient frequency to mount a primary in vitro response against trinitrophenol in the context of an allogeneicH-2K region.     

Inhibition of secondary mouse mixed lymphocyte reaction by anti-Ia sera

Secondary mixed lymphocyte reaction (MLR-II) was studied in A.TH anti A.TL and A.TL anti-A.TH combinations in which stimulation was mainly due to H-21-region differences. In both cases of MLR-II was specifically inhibited by the responder anti-stimulator Ia serum. The level of inhibition was dependent on the ratio of the amount of immune serum to the number of stimulating cells. The inhibitory activity and Ia antibodies were specifically absorbed and eluted together. The results confirm that the lymphocyte-activating determinants of the MLR-II (1) are carried by the Ia molecules and (2) are identical to the serologically defined Ia determinants. - Anti-Ia sera directed against private and public specificities of the stimulating cell induced a higher level of inhibition than anti-Ia sera directed only against public specificities, indicating that both private and public Ia specificities are involved in re-stimulation during MLR-II. - These results, in connection with others, suggest that the receptor of the proliferating T cell recognizes the same Ia determinant as the combining site of the Ia-recognizing antibody.     

Immune response deficiency of BSVS mice

The genetic control of the low response of BSVS mice to streptococcal Group A carbohydrate (GAC) was studied in crosses with responder A/J mice. F₁ mice were responders. In the backcross (BSVS × A/J)F₁ × BSVS mice, there were equal numbers of anti-GAC responder and nonresponder mice, indicating genetic control by a small number of major loci. The anti-GAC responses of the backcross mice showed no obligate linkage between responder status and A/JH-2 orIgC _H alleles. However, it was observed that the average anti-GAC titers were higher in backcross mice heterozygous at these loci. The above data, a lack of low-responder F₂ animals, and the segregation of a non-H-2-, non-IgC _H -linked locus in the first and second backcross mice, indicate that the defect in the BSVS anti-GAC responsiveness involves three loci: one linked toH-2, another linked toIgC _H , and a third locus —tentatively namedIr-GAC- not linked toH-2, IgC _H , orHbb.     

Responses against antigens encoded by theH-3 histocompatibility locus: Antigens stimulating class I MHC- and class II MHC-restricted T cells are encoded by separate genes

The purpose of this work was to study the genetic basis of histocompatibility antigens encoded by the mouse minor histocompatibility (H) locusH-3. Both class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL) and class II MHC-restricted helper T cells (TH) specific for antigens encoded by genes within theH-3 locus were isolated and analyzed. Typing a number of mouse strains for expression of antigens recognized by these TH and CTL suggested that there was a different strain distribution pattern of expression of the antigens recognized by TH compared with those recognized by CTL. Separation of the genes whose products stimulate TH from those whose products stimulate CTL was suggested by: (1) analysis of the strain B10.FS(92NX)/Grf that has undergone recombination within theH-3 region; (2) genetic segregation studies of (B10.UW-H-3 ^b/Sn×C57BL/10Sn)F₂ mice; and (3) F₁ complementation studies in which CTL specific for products of the TH-defined gene(s) could not be detected, even in the absence of immune responses to products of the CTL-defined genes. Taken together, these data suggest that in addition to two genes (B2m andCd-1) within theH-3 region whose products typically stimulate class I MHC-restricted CTL, there is at least one additional gene whose product selectively stimulates class II MHC-restricted TH. This new gene is located telomeric from the CTL-defined genes and between the lociwe andun on chromosome 2. These data demonstrate a novel degree of complexity of theH-3 “locus” and suggest selective presentation of minor H gene products in the context of class I or class II MHC proteins.     

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.     

Polygenic control of the immune response to F antigen

The ability to produce an autoimmune response to F antigen in mice is underH-2-linked and non-H- 2-linkedIr-gene control. There is an absolute requirement for ak allele atH-2K orI-A in order to produce anti-F antibodies. Low and high responsiveness is controlled by a non-H-2-linkedIr gene which behaves in a similar fashion toIr-3, in that as the dose of F-antigen is lowered, low responders behave as high responders and vice versa. This conversion from low to high responsiveness also occurs within a month after ATX.— Most F₁ hybrids derived from (responder x nonresponder) parents bearing identical F-types behave as dominant nonresponders. As a result of ATX, such F₁ mice convert to high responders. This conversion occurs if the animals are not immunized before day 90. If they receive F antigen prior to that time, they remain nonresponders for 7–9 months. One F₁ combination — AKD2 — behaves as a dominant high responder. Genetic analysis showed that in the presence of ak allele atH-2K orI-A, a non-H-2-linkedIr gene inherited from the AKR mice determined dominant responsivenss. No manipulation of the immune response or combination of genes converted nonresponders lacking ak allele into responders. Such complex genetic control suggests regulation by a number of independently segregating loci whose function it is to limit the autoimmune response to F antigen.     

Role of H-2 antigens in the host response to methylcholanthrene-induced tumors

H-2 loss variant sublines of a sarcoma (M-AS), induced by methylcholanthrene in an (A × A.SW)F₁ mouse, were used to study the role of the MHC products in the recognition of MC-TSTA. The two reciprocal variant sublines (M-A and M-S) were found to express the TSTA of the original tumor as shown by cross-reactions in graft rejection experiments performed in (A × A.SW)F₁ mice. In the A/Sn and A.SW mice the presence of the reciprocal parental H-2 antigens on the immunizing cells decreased the response against the tumor antigens. An admixture of lymphocytes derived from hyperimmune mice inhibited the outgrowth of the tumor cells. The growth inhibition was mediated by T cells and was H-2 restricted. Cells derived from hyperimmune syngeneic mice inhibited the outgrowth of the variant subline used for immunization but had no effect on the reciprocal variant subline.     

Effector cell expression of NK1.1, a murine natural killer cell-specific molecule, and ability of mice to reject bone marrow allografts

The rejection of Hh-1 incompatible bone marrow cells in irradiated mice is mediated by NK cells and is genetically regulated. We tested the role of the NK-specific gene, NK1.1, in regulating the rejection of allogeneic bone marrow cell grafts. NK1.1+ mice, that are known to display strong resistance against Hh-1 incompatible grafts, were crossed to H-2/Hh-1 identical NK1.1-, poor responder mice, and the progeny were backcrossed to the poor responder parent. The segregating mice were individually typed for their expression of NK1.1 and the ability to resist Hh-1 incompatible bone marrow cells (BMC). A strong correlation was noted between expression of NK1.1 and rejection of H-2d/Hh-1d BMC. Our results support the idea that NK1.1 is one of the genes responsible for strong resistance to Hh-1d (determinant 2) but not for Hh-1j (determinant 3) BMC grafts. We suggest that the NK1.1 molecule functions as an accessory molecule in the cellular interactions involving the recognition of Hh-1 determinants.