H-2 antigen variants in a cultured heterozygous mouse leukemia cell line

In previous studies on the emergence of H-2 antigen loss variants from an H-2^b/H-2^d heterozygous mouse leukemia cell line, it has been shown that stable variants could be obtained after a single-step selection procedure with antibody and complement. Selection against the K-end alleles revealed an asymmetry in the types of variant obtained. This paper deals with the results of selection against theD-end alleles. Once again, an asymmetry is noticed. Selection against the H-2D^d gene product results in the isolation of stable variants that have lost H-2D^d, either alone or in combination with the linked H-2K^d antigens. Selection against H-2D^b, on the other hand, has not, so far, resulted in the isolation of any stable variants. These experiments have not demonstrated unequivocally the mechanism by which these H-2 antigen loss variants are generated. However, certain preliminary considerations suggest the possibility that these variants may not, in fact, be true genetic mutants. The alternative -that these variants arose through some nongenetic, but stable mechanism — must be considered seriously.     

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.     

Genetic control of contact sensitivity in mice: Effect ofH-2 and nonH-2 loci

The genetic control of delayed-type hypersensitivity in mice was investigated by contact sensitization with picryl chloride. Distribution patterns of contact sensitivity in 11 inbred strains of mice showed significant differences among strains. Comparison of levels of response between congenic-resistant lines and their inbred partners, at 9 to 11 weeks of age, revealed a clear association betweenH-2 haplotype and the magnitude of response. Testing ofH-2 recombinants further suggested the influence of two genes mapping at either end of theH-2 complex. While theH-2K ^d andH-2D ^k alleles were associated with a high response, theH-2K ^k ,H-2K ^b ,H-2D ^d , andH-2D ^b alleles were associated with a low response. Analysis of the ontogeny of response suggested that theH-2 haplotype manifests its effect through the maturation of contact sensitivity. On both the C57BL/6By and C57BL/10Sn backgrounds, theH-2 ^d haplotype was associated with early maturation of response, while theH-2 ^b haplotype was associated with late maturation. Analysis of the response of congenic lines with different genetic backgrounds and of CXB recombinant-inbred lines further revealed the marked effects of yet other genes on this trait.     

Fine specificity of cytotoxic T lymphocytes directed againstH-2L d

The fine specificity of cytotoxic T lymphocytes (CTL) directed againstH-2L ^d was analyzed by studying the lytic activity of BALB/cH- 2^dm2 (H-2L ^d loss mutant) anti-BALB/c-H-2 ^d CTL, generated in secondary mixed lymphocyte culture (MLC) against a panel of target cells of differentH-2 haplotypes. Target cells of allH-2 haplotypes tested, except that of the MLC responder, were lysed by anti-L^d CTL, although to a widely varying extent. The genes coding for antigens detected by anti-L ^d CTL were mapped to distinct regions in theH-2 ^d ,H- 2^dm1,H-2 ^q ,H-2 ^k , andH-2 ^b haplotypes. The sequence of lysis intensity against the variousH-2 haplotypes and theH-2 regions involved were as follows:L ^d ,D ^q L ^q ,D ^dm1 L^dm1,K ^k ,D ^b L ^b ,r, p, f, s, C3H.OH (K ^d D ^k L ^k ), strong lysis occurring againstL ^d and weak lysis againstH-2 ^s and C3H.OH.By monolayer adsorption and cold target inhibition experiments, it was shown that anti-L ^d CTL contained a CTL subset directed against a private L^d specificity, hitherto undetected by anti-L ^d antibodies. This subset of CTL was separate from the CTL subsets reacting againstH-2 ^q and against the mutant haplotypeH- 2^dm1. The reactions against the latter two haplotypes were also mediated by separate CTL subsets. It is concluded that the L^d molecule, to a varying extent, shares target antigens for CTL with K- and/or D-end H-2 molecules of all haplotypes tested. These antigens are detected by multiple subsets of anti-L ^d CTL. One CTL subset is directed against a target structure unique forL ^d (L^d private specificity).     

Intra-H-2 recombination in the mouse

Serological characterization of threeK-S interval recombinant strains, TBR2 (H-2 ^at2 ), TBR3 (H-2 ^at3 ) and AIR1 (H-2 ^a2 ) was performed using anti-H-2, Ia, Ss and Slp antisera. The data presented here reveal that the crossover events in both TBR2 and TBR3 occurred between theI-A andI-E subregions. In both cases, theH-2K andI-A subregions were derived from theH-2 ^t1 chromosome, while theI-E, S andH-2D regions were derived from theH-2 ^b chromosome (K ^s A ^k E ^b S ^b D ^b ). TheH-2 ^a2 chromosome resulted from a crossover event between theH-2 ^a1 andH-2 ⁱ⁹ chromosomes. Ia and Ss typing of AIR1 suggested that theK toI-E regions originated fromH-2 ^a1 and theS andD regions originated fromH-2 ⁱ⁹ (K ^k A ^k E ^k S ^b D ^d ).     

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     

Immunogenetic analysis ofH-2 mutations

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

Serology and polymorphism ofH-2L- locus encoded antigens

Thirty B10.W congenic lines were analysed serologically, both by direct cytotoxicity and by absorption, for the presence of H-2L antigens. Three new H-2L antigens, 73, 74, and 75, were discovered. The B10.W lines and the inbred strains can be classified into at least six H-2L phenogroups on the basis of their reactivity withH- 2^dm2 anti-H- 2^d serum: BALB/c, B10.BUA1, B10.GAA37, B10.BUA16. B10.KPB128, and the negative group. Twenty-oneD-end recom-binants were analysed for the possible separation ofH-2D andH-2L loci. The failure to find such a separation indicates that theH-2D andH-2L loci are tightly linked. Serological analysis also indicated that theH- 2^dm1 has lost most of its H-2L antigens but retained at least one specificity which can be detected byH- 2^dm2 anti-H- 2^d serum.     

Teratocarcinoma transplantation rejection loci: Genetic localization of the Gt-1 locus on chromosome 17 and the expression of alternate alleles

A series of congenic mice on the BALB/c genetic background have been employed to localize a teratocarcinoma transplantation rejection locus, Gt-1, to the K side of the H-2 locus on chromosome 17. Previous studies have placed the Gt-1 ^sv allele about 8 centimorgans away from the H-2 ^b or H-2 ^bv1 locus. Teratocarcinomas derived from 129/sv mice, Gt-1 ^sv (H-2K ^bv1/H-2D ^bv1), are rejected by BALB/c (H-2K ^d/H-2D^d) and BALB-G mice (H-2K ^d/H2D-^b, but form tumors in BALB-B (H-2K ^b/H2D ^b) and BALB/5R5 mice (H-2K ^b/H2D ^d). In the reciprocal tumor-rejection test, a BALB/c teratocarcinoma was rejected by immunized BALB·B mice, but formed tumors in the immunized isogenic BALB/c mouse. These studies demonstrate the reciprocal expression of two Gt-1 alleles, one Gt-1 ^c, in BALB/c mice, and the other, Gt-1 ^sv, in the congenic BALB·B mice. Shedlovsky and co-workers have placed the Gt-1 locus in a similar location on the K side of the H-2 locus on chromosome 17.     

Biochemical analysis of an MHC-linked hematopoietic cell surface antigen,Qa-2

The region of the murine 17th chromosome telomeric to H-2D encodes a group of serologically defined cell surface antigens termed Qa-1-5. These antigens are of interest because their expression is restricted to hematopoietic cells. In addition, the molecular weight and subunit structure (ie, association with β-2 microglobulin) of Qa-2 molecules are similar to H-2 and TL antigens. In the present studies, we have prepared isotopically labeled Qa-2 and H-2 molecules from mitogen-stimulated C57BL/6 spleen cells. Comparative peptide mapping of tryptic peptides from Qa-2 and H-2 molecules (K^b, D^bK^k, D^d) reveal that Qa-2 has a unique primary structure. However, considerable homology is indicated since 30–40% of the Qa-2 peptides cochromatograph with peptides derived from H-2K^b, H-2D^b, H-2K^k, and H-2D^d. Studies by other investigators have demonstrated that similar levels of structural homology are observed when H-2K, H-2D, and H-2L tryptic peptides are analyzed. We conclude from these studies that the Qa-2 alloantigen is structurally related to a class of cell surface molecules (ie, H-2) that play critical roles in immune recognition processes. These data further suggest that the genes encoding Qa-2 and H-2 molecules have arisen from a common primordial gene.     

Genetic control of immune responses of inbred mice: Responses against terpolymers poly(glu57lys38ala5) and poly(glu54lys36ala10)

The immune response patterns of inbred and congenic strains of mice against terpolymers poly(glu⁵⁷lys³⁸ala⁵) and poly(glu⁵⁴lys³⁶ala¹⁰) have been studied. Initial recognition of the polymers is ascribed to ‘GA’ receptors (Ir-GA gene product) on T cells of mice ofH-2 haplotypes,a,b,f,k ands, and ‘GL’ receptors (Ir-GL gene product) of mice ofH-2 ^p,H-2 ^q andH-2 ^j haplotypes, and to GA and/or GL receptors of mice ofH- 2^d andH- 2^r haplotypes. The specificity of the antibody is directed predominantly against GL. The inability to elicit antibody with GA specificity has been ascribed to the lack of significant concentrations of GA sequences in the polymers to interact with appropriate receptors on B cells. The weakest responders were mice of H-2^b haplotype. F¹ hybrids (responders×nonresponders) were all responders demonstrating the dominant character of responsiveness. Wide variations in antibody levels produced among strains of mice of theH-2 ^k andH-2 ^b haplotypes are ascribed to genes not linked toH-2.     

Do histocompatibility antigens recognize themselves?

In the Simonsen spleen weight assay, theH-2K ^ba mutant does not respond against theH-2K ^bd mutant orH-2K ^bd /H-2K ^b hybrid, while the parentalH-2K ^b haplotype does respond. TheH-2K ^ba /H-2K ^b hybrid reacts strongly to bothH-2K ^bd andH-2K ^bd /H-2K ^b , indicating that the donor genotype could influence the reactivity against the same antigenic difference. The response of theH-2 ^ba mutant against a number of unrelated H-2 antigens does not differ from that of the parental haplotype. TheH-2K ^bd mutant reacts againstH-2K ^b andH-2K ^ba , and theH-2K ^b parent reacts against both theH-2K ^ba andH-2K ^bd mutants. The specific defect of reactivity in theH-2K ^ba mutant is effectively complemented by crossing with a number of unrelatedH-2 haplotypes. TheH-2 ^ka andH-2 ^fa mutants complement poorly compared to corresponding parental strains CBA and A.CA, while the B10.M (H-2 ^f ) strain does not complement at all (which is probably attributable to an undetectedH-2 mutation in the last strain). The data strongly suggest that the product of theH-2K locus-which is known to function as a transplantation antigen, lymphocyte activating determinant, and serologically defined antigen-also influences the immune response capacity against a mutant histocompatibility determinant.     

Serological characterization of previously unknown H-2 molecules identified in the products of theK d andD k region

The molecular relationship between H-2 private and some public specificities was studied in C3H.OH (H-2 ⁰² ) mice using surface-antigen re-distribution methods. Besides the K^d- and D^k-region antigens, which can be capped by antisera against the private and public specificities characteristic for a given allele, a previously unknown type of molecule was found in the products of both theK ^d andD ^k regions. These can be capped by the respective anti-private serum but not by antisera against some public specificities. The two K^d-region molecules are provisionally named H-2K1^d and H-2K2^d. We detected them onH-2 ⁰² (K ^d ,I ^d ,S ^d ,D ^k ) and also onH-2 ^dx (K ^d ,I ^f ,S ^f ,D ^dx ) T lymphocytes. Similarly, the two types of molecules detected on the products of theD ^k region are provisionally named H-2D1^k and H-2D2^k. The serological characteristics of these molecules are described. When compared with the products of theD ^d region, in which we previously described three different molecules (H-2D^d, H-2M^d, and H-2L^d), the mutual relationship between H-2K1^d and H-2K2^d as well as between H-2D1^k and H-2D2^k appears to be similar to that between H-2D^d and H-2M^d. In the absence of relevant recombinants or informative biochemical data, it is, however, difficult to establish homology between molecules produced by differentK- andD-region alleles.     

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.     

Evidence for a third,Ir-associated histocompatibility region in theH-2 complex of the mouse

Skin grafts transplanted from B10.HTT donors onto (A.TL × B10)F₁ recipients are rapidly rejected despite the fact that the B10.HTT and A.TL strains should be carrying the sameH-2 chromosomes and that both the donor and the recipient contain the B10 genome. The rejection is accompanied by a production of cytotoxic antibodies against antigens controlled by theIr region of theH-2 complex. These unexpected findings are interpreted as evidence for a third histocompatibility locus in theH-2 complex,H-2I, located in theIr region close toH-2K. The B10.HTT and A.TL strains are postulated to differ at this hypothetical locus, and the difference between the two strains is explained as resulting from a crossing over between theH-2 ^t1 andH-2 ^s chromosomes in the early history of the B10.HTT strain. TheH-2 genotypes of the B10.HTT and A.TL strains are assumed to beH-2K ^s Ir ^s / ^k Ss ^k H-2D ^d andH-2K ^s Ir ^k Ss ^k H-2D ^d , respectively. Thus, theH-2 chromosomes of the two strains differ only in a portion of theIr region, including theH-2I locus. The B10.HTT(H-2 ^tt) and B10.S(7R)(H-2 ^th) strains differ in a relatively minor histocompatibility locus, possibly residing in theTla region outside of theH-2 complex.     

Intra-H-2 recombination inH-2b/H-2t1 heterozygotes in the mouse

Four cases of intra-H-2 recombination were detected during serological screening of 1066 backcross animals produced fromH-2^b/H-2^t1 heterozygous mice. Three of the intra-H-2 recombinants received theK region fromH-2^t1 and theD region from theH-2^b parental chromosome. The remaining recombinant received theK region from theH-2^b parental chromosome and theD region fromH-2^t1. Three of the four recombinants have been developed into inbred lines TBR2, TBR3, and TBR4 and were assigned the haplotype designations at², at³, and at⁴. Ss typing revealed that TBR2 and TBR3 originated fromK- S interval crossover events, while the remaining two recombinants resulted from crossing over in theS- D interval.     

Aberrancy in immunogenicity and cell-surface expression of H-2 antigens on erythrocytes

Immunogenicity for T cell-independent B-cell response assessed by splenic plaque-forming cell (PFC) response and cell-surface expression measured by laser flow cytometry of various class I H-2 antigens on mouse red blood cells (RBC) were compared. It was found that the order of magnitude of both immunogenicity and cell-surface expression on RBC is H-2D^d H-2D^b > H-2K^d, H-2K^b. Furthermore, H-2^d public antigens and H-2L^d antigens were neither immunogenic nor easily demonstrable on RBC. These findings contrasted with poor immunogenicity for PFC response (Nakashima et al. 1982, 1983) and proportionally strong expression of H-2 antigens on lymphoid cells. Immunogenicity and cell-surface expression of H-2D^d antigen on RBC were not shown to be controlled by the action of genes outside H-2D. It was therefore suggested that a number of H-2 antigens, including H-2K^d private, H-2K^b private, and H-2^d public specificities are at least functionally defective on RBC. This is possibly due to the structural characteristics of the antigens. Since immunogenicity and cell-surface expression were in parallel, the expression of H-2 antigens on RBC must be dictated by a subset of B cells whose activity was assessed by PFC response. This finding supports the view that the H-2 molecules display a new category of activity which is different from their ability to activate T cells and depends on their expression on RBC.     

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.     

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.