Natural resistance of irradiated 129-strain mice to bone marrow allografts: Genetic control by theH-2K region

A major genetic determinant of natural resistance to bone marrow allografts, designated asHh-3, was mapped to theH-2K region. This gene may code for or regulate the expression of cell surface structures selectively expressed on donor hemopoietic cells and recognized by naturally occurring cytotoxic effectors. Resistance was observed as failure of donor cell growth in the spleen of irradiated 129-strain (H-2 ^bc ) recipients of H-2^k bone marrow cells. The mapping was accomplished by substituting donor cells bearingk alleles throughout theH-2 complex with cells of recombinant mouse lines bearingk alleles at definedH-2 regions. The host antigraft reaction underlying resistance was abrogated by pretreating 129-strain mice with either rabbit antimouse lymphocyte serum or the antimacrophage agent silica. Grafting of H-2K^k cells into mice ancestrally unrelated to 129 but sharing theH-2 ^bc or the similarH-2 ^b haplotype, and intoH-2 ^b/k ,H-2 ^k/bc , andH-2 ^k/d F₁ hybrids revealed that resistance was unique to 129 mice, since mice of the other strains, including F₁ hybrids, were susceptible to the grafts. Thus,Hh-3 incompatibility was a necessary but insufficient condition for the manifestation of allogeneic resistance; other genetic factors not associated withH-2 conferred responder status to 129-strain mice and nonresponder status to D1.LP, B10.129(6M), B10, B6, and possibly to F₁ hybrid mice. The possible relationships between allogeneic resistance to H-2^k marrow grafts, hybrid resistance to H-2^k lymphomas, and F₁ hybrid antiparental H-2^k cytotoxicity induced in vitro are discussed.     

Immunogenetic analysis ofH-2 mutations

Spleen cells carrying theH-2K ^b allele and sensitized against TNP-modified stimulator cells in vitro displayed a cytotoxic effect against TNP-modified target cells carrying a mutation in theH-2K ^b allele (haplotypesH-2 ^ba ,H-2 ^bd , andH-2 ^bf ). Similar crossreactivity in TNP-CML was observed in the reciprocal direction. Spleen cells carrying theH-2K ^k allele and sensitized against TNP-modified stimulators displayed a cytotoxic effect against TNP-modified target cells carrying a mutation in theH-2K ^k allele (haplotypeH-2 ^ka ) and vice versa. The effector cells in these assays were sensitive to anti-T cell serum in the presence of complement, and supernatants from immune cultures did not induce nonimmune cells to display a cytotoxic effect. Titration of effector cells from mutant and wild-type strains of theH-2 ^b haplotype indicated no detectable quantitative differences in their activities. These data demonstrate that crossreactivity in TNP-CML occurs in closely related allogeneic strains that have recently undergone mutation in theH-2 complex.     

Antigen-presenting cells that induce anti-H-2K T-cell responses: Differences in stimulator-cell requirements for induction of proliferation and cell-mediated lympholysis

Splenic T or B cells, which have been depleted of adherent cells by passage through Sephadex G10 columns, fail to stimulate allogeneic lymph-node cells (LN) in primary mixed lymphocyte reactions (MLR) both when the stimulating antigens are H-2 plus Ia and H-2K only. This failure cannot be ascribed to lack of viability of G10-passed cells, since by dye exclusion they are 95 percent viable and can be induced to proliferate in vitro by exposure to LPS or allogeneic cells. Stimulation of MLR activity could be restored by addition of small numbers of plastic-adherent spleen cells (SAC) which had to be syngeneic with the G10-passed stimulator cells. Further, SAC alone without G10-passed cells induced MLR activity which was, on a cell-for-cell basis, 40 times more effective than that induced by unfractionated spleen cells. If the SAC were first depleted of Ia⁺ cells, no stimulation was obtained. This result was observed both in cases where responder and stimulator strains differed across the entireH-2-gene complex and in a mutant-wild type combination (CBA and H-2^km1) in which the difference between the two strains has been mapped to theK region only. These results indicate that Ia⁺ SAC contain a subset(s) of cells which are responsible for stimulation in MLR, regardless of whether the alloantigenic differences involve either Ia or H-2K. In contrast to the inability of G10-passed splenic cells to stimulate MLR activity, these cells were able to stimulate CTL from cytotoxic T lymphocyte precursors (CTL.P) in combinations where the antigenic differences between responder and stimulator were at the entireH-2 complex or atH-2K only. However, SAC were more potent stimulators of cell-mediated lympholysis (CML) activity on a cell-for-cell basis. Thus, either CTL.P can be stimulated by nonadherent spleen cells or they are specifically sensitive to a small subpopulation of contaminating cells that cannot readily be removed by G10 passage.     

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.     

Genetic analysis of theT-H-2 region in non-t Chromosomes

A general breeding protocol useful in the construction of congenic lines of mice disparate in the 15 cMT-H-2 region of chromosome 17 in non-t chromosomes is described. Two such congenic lines, B6.TC2/Rn and B6.TC3/Rn, were derived from the C57BL/6J and B6.C-H-2 ^d/By strains using this protocol. Both B6.TC2 and B6.TC3 dissociate the quantitative activity locus for glyoxalase I (Qglo-1) from theH-2 complex, and hence possess BALB/cBy DNA centromeric toH-2. However, neither new strain is able to map anyH-2-associated restriction fragment length polymorphism with anH-2 cDNA probe even though both strains are recombinant in the 2 cMQglo-1-H-2K interval.     

Mutations in H-2K b influence the specificity of alloreactive effector cells included in the repertoire of H-2D b -restricted cytotoxic T lymphocytes against Moloney leukemia virus

Moloney leukemia virus-specific cytotoxic T lymphocytes (CTL), generated by secondary in vitro stimulation of spleen cells with syngeneic virus-infected cells, frequently lysed not only syngeneic virus-infected cells, but also noninfected allogeneic target cells. This phenomenon was studied with B6(H-2 ^b ) responder cells and a series of H-2K ^b -mutant responder cells. Thus, B6 Moloney-specific CTL lysed noninfected K ^b -mutant cells, but not B6 cells, whereas K ^b -mutant Moloney-specific CTL lysed noninfected B6 cells and not noninfected cells of the same mutant. Cold-target-inhibition studies showed that the CTL reactions against different allogeneic cells were mediated by different subpopulations of virus-specific CTL: lysis of allogeneic target cells was fully inhibited only by the same allogeneic and by syngeneic virus-infected cells, but not by another allogeneic cell, also lysed by the same effector-cell population. Lysis of syngeneic virus-infected cells could not be inhibited by allogeneic target cells. These data imply that a minority of virus-specific CTL shows cross-reactivity with a given allogeneic target cell. It is concluded that limited amino acid substitutions in the K^b molecule alter the repertoire of Moloney virus-specific CTL, as reflected in alloreactive CTL populations, even though the virus-specific CTL response. of B6 and all K ^b mutants is mainly D^b-restricted. Thus, the development of tolerance to self class-I major histocompatibility complex (MHC) molecules affects the repertoire of self-restricted cytotoxic T cells.     

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

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

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.     

Serological and complementation studies in four C57BL/6H-2 mutants

C57BL/6 (H-2 ^b ) mice, and four mutants (B6.C-H-2 ^ba , B6-H-2 ^bg1 , B6-H-2 ^bg2 , B6-H-2 ^bh ) derived from this strain after separate mutations had occurred at the same locus within theH-2 complex, were analyzed to determine whether the mutations had led to anyH-2 (or Ia) difference which could be detected serologically. The strains were typed directly with antisera specific for H-2K and H-2D public and private specificities and for the Ia specificities; quantitative absorption studies were also performed for the relevant H-2K^b, H-2D^d and Ia^b specificities. In no case was any quantitative or qualitative difference detected serologically between any of the strains. In addition, by using a variety of techniques to produce and assay for antibody, we failed to produce any antisera between the parental strains and the four mutants. TheH-2 mutations therefore appear to give rise to a type of antigenic specificity which is recognized byT cells and which generateT, but notB cell responses; nor are they recognized by H-2 or Ia alloantisera. The location of the mutating locus within theH-2 complex was shown by the complementation method to be within theK orIA region and not in theIB region, since crosses of the mutant strains with B10.A(4R) or D2.GD failed to complement for a subsequent C57BL/6 skin graft.     

Analysis ofH-2 mutants: Evidence for multiple CML target specificities controlled by theH-2K b gene

C57BL/6 (H-2 ^b ) mice and two mutants derived from this strain, B6.C-H-2 ^ba (Hz1) andE6-H-2 ^bd (M505), were studied in a number of functional tests, in vitro and in vivo, that assay for differences at theH-2 complex. All three strains give rise to reciprocal mixed lymphocyte reactivity (MLR) and cell-mediated lympholysis (CML) in vitro as well as graft-host reactivity (GVHR) and skin graft rejection in vivo. Analysis for cross-reactivity between these strains in CML revealed that the gained antigens in each mutant do not cross-react, and that Hz1 has lost an antigen shared by C57BL/6 and M505 strains. In addition, spleen cells from B10.A(4R) mice, which differ from theH-2 ^b haplotype only at theK end of theH-2 complex, recognize a common antigen shared by all three strains tested. Provided that the mutations occurred in theH-2K ^b gene, these data indicate that a) there are at least three antigenic specificities coded for by theH-2K ^b gene(s) that serve as targets for receptors on thymus-derived (T) cells in CML; b) since C57BL/6 strain mice and the mutants are serologically indistinguishable on a qualitative basis, the antigens recognized by the receptors on T cells and by humoral H-2 antibody are nonidentical; and c) mutation in theH-2K ^b locus itself can give rise to allogeneic recognition phenomena such as MLR and GVHR.     

Biochemical studies of H-2K antigens from a group of related mutants. I. Identification of a shared mutation in B6-H-2 bm5 and B6-H-2 bm16

Structural studies of the H-2 gene products from a group of five closely related but independent C57BL/6 H-2 mutant mice were undertaken. Each of the mutants exhibits reciprocal graft rejection with the parent. The group is remarkable, however, because each member of this group can accept skin grafts from any other member. The results of biochemical analysis of the H-2 glycoproteins from two of these related mutants, bm5 and bm16, are presented in this report. Evidence is given that the H-2K molecules from these two mutants are identical to each other based on comparative tryptic peptide mapping profiles with the parent. From partial amino acid sequence analysis, K products of both mutants have at least one common difference from the parental type located at residue number 116. Definitive studies established that in both bm5 and bm16 a tryosine found in the parent molecule is substituted with a phenylalanine in the mutant. These results show that a biochemical difference between the K products of the two mutants and of the parent can be detected, that the mutants appear to be identical with one another even though they arose independently, and that they differ from the other H-2K ^b mutants analyzed.Abbreviations used in this paper B6 C57BL/6Kh - bm5 B6-H-2^bm5 - bm6 B6-H-2 ^bm6 - bm7 B6.C-H-2 ^bm7 - bm9 B6.C-H-2 ^bm9 - bm16 B6-H-2 ^bm16 - D H-2D - K H-2K - MHC major histocompatibility complex     

The role ofH-2 regions in overcoming tissue incompatibility

Neonatal transplantation tolerance to the products of theH-2 ^b complex was induced in B10.A (H-2 ^a ) mice. On the basis of the survival of skin allografts it was found that antigens determined by theD region of theH-2 ^b complex (of the B10.A(2R) strain) were most easily overcome and that tolerance to the products of theD end of theH-2 complex (of the B10.A(4R) strain) was also easy to induce. The antigens produced by theK end ofH-2 (of the B10.A(5R) and B10.A(3R) strains) represented a stronger incompatibility barrier and a difference in the entireH-2 ^b complex caused strongest resistance to tolerance induction. When tolerance to the products of the entireH-2 ^b complex was induced in newborn B10.A mice, and the neonatally treated animals were grafted simultaneously with five different grafts, those disparate at theK end ofH-2 and in the entireH-2 region were rejected in some animals, while the grafts disparate at theD end of H-2 remained intact in the same mice. No dependence on theI-J subregion was observed in this system. Furthermore, tolerance was more easily inducible in male than in female B10.A mice.     

Study of anH-2K d mutant strain: C.B6-H-2 dm4

A new-H-2 mutant involving theH-2 ^d haplotype is described — C.B6-H- 2^dm4 (dm4). This mutant strain carries a gain and loss mutation which maps to theK ^d gene of theH-2 complex. Serological testing comparing the mutant and the parental BALB/cKh strain failed to detect any difference between the two strains and no antibodies could be produced, although a reciprocal mixed lymphocyte reaction was observed between mutant and parent.     

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 ).     

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.     

Immune response to the Slp alloantigen in the mouse:H-2-linked qualitative and quantitative control

Immunization of inbred mouse strains lacking the Slp allotype results in the production of Slp antibodies in some strains but elicits no detectable response in other strains. Analysis of standard inbred and congenic resistant strains reveals that both the qualitative and quantitative ability to respond to the Slp allotype is associated with theH-2 haplotype of the recipient. Three different response phenotypes can be identified utilizing complement fixation and quantitative immunodiffusion tests. Strains which carry theH-2 ^q haplotype are high responders,H-2 ^k strains are intermediate in response, whileH-2 ^b andH-2 ^v strains produce no detectable antibody. The characteristic response patterns of high and intermediate responders were manifest by day 35 of immunization and continued as discrete response types after a second booster. Quantitative data in the immune response of the intra-H-2 recombinant B10.A(4R) suggest that the recombination event which established theH-2 ^h4 chromosome disturbs the proper function of the genetic determinant controlling response to Slp.     

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.     

TheH-2 class II genes and the susceptibility to the development of pulmonary adenoma in mice

To determine the locus in theH-2 complex that affects susceptibility to the development of pulmonary adenomas in mice,H-2 congenic and recombinant strains of mice with A/Wy, BALB/c, C3H, and B10 backgrounds were subjected to treatment with urethane. The average number and the incidence of adenoma foci were recorded five months after the treatment. InH-2 congenic strains on the A/Wy background, the average number of adenoma foci per mouse was significantly higher in mice of the A/Wy, A/J, and A-Tla ^b (H-2 ^a ) strains than in A.BY (H-2 ^b ) mice. In BALB/c and C3H congenic strains, the strains carrying theH-2 ^k haplotype were more susceptible than those carrying theH-2 ^b haplotype. InH-2 congenic strains on the B 10 background, the average number and incidence of foci was also higher in haplotypesa, h2, k, andj than in haplotypesb, s, f, d, r, h4, i3, i5, and4. The average numbers of adenoma foci in (A/J × A.BY)F₁ (H-2 ^a /H-2 ^b ) and (B10 × B10.A)F₁ (H-2 ^b /H-2 ^a ) were intermediate between the numbers in the parental strains. In [B10.A (4R) × B10.A (3R)]F₁ (H-2 ^h4 /H-2 ⁱ³ ) and [B10.A (4R) × B10.A (5R)]F₁ (H-2 ^h4 /H-2 ⁱ⁵ ), the numbers of adenoma foci were higher than in resistant parental recombinants. These patterns of response to urethane matched the patterns of the immune response to lactate dehydrogenase-B (LDH-B) and immunoglobulin gamma 2a (IgG2a) proteins. These differences between mice in their susceptibility to the development of pulmonary adenomas is probably due to the polymorphism of the class II genes in theH-2 complex.     

Variable effect of anH-21 barrier on response to skin allografts in the mouse

(AQR×B10)F₁ mice were grafted with skin from donors differing in theK, I, KI, andISD regions of theH-2 complex. A dichotomy was observed in the fate of theH-2I-disparate grafts: either they were rejected acutely within the second week or were accepted indefinitely. Acceptances were much more common among male than female hosts. Acceptor status was limited to the I group, was unpredictable in occurrence, was not well-correlated with positive serum anti-Ia titers, and did not confer protection of grafts that were alike atH-2I but different atH-2K orH-2D. Since theH-2I barrier studied here elicited such divergent responses in genetically identical hosts, it is unlikely that any histocompatibility typing test could predict graft fate.Abbreviations used in this paper are MST median survival time - MHC major histocompatibility complex - CTL cytotoxic T lymphocyte - B10 C57 BL/10 - 6R BIO.T(6R) - B10.A BIO. ASn - H-2-Ia serologically detected antigens coded in theI region ofH-2 This term is used in preference toIa, since it has recently been shown that Ia-like alloantigens may be coded outside the MHC (Dickleret al. 1975).     

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.