Biochemical studies of H-2K antigens from a group of related mutants. II. Identification of a shared mutation in B6-H-2 bm6 , B6.C-H-2 bm7 , and B6.C-H-2 bm9

In an earlier paper, we presented evidence that two independent mutants of the bg series, B6-H-2 ^bm5 (bm5) and B6-H-2 ^bm16 (bm16) carry identical mutations such that tyrosine at residue number 116 of the H-2K^b molecule from the parent strain C57BL/6Kh is replaced by a phenylalanine in each of the two mutant molecules. In this paper, we demonstrate, using similar techniques, that the independent bg series mutants B6-H-2 ^bm6 (bm6), B6.C-H-2 ^bm7 (bm7), and B6.C-H-2 ^bm9 (bm9), which share biological properties with bm5 and bm16, can be grouped together because they share two identical mutations, one of which is common to bm5 and bm16, a Tyr to Phe interchange at residue number 116. In addition, a second mutation is at residue number 121, where a Cys in the H-2K molecule from 136 is substituted with an Arg in the mutant. Since all of the bg series mutants arose independently and share biological and biochemical characteristics, it is anticipated that study of these mutants could lead to some understanding of the high mutation rate in the K^b molecule.     

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.     

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.     

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-2 ^b haplotype and the same differential minor histocompatibility alleles as the immunizing strain. That is, B10.C(47N) (H-2 ^b ,H-7 ^b ) anti-C57BL/10 (H-2 ^b ,H-7 ^a ) cytotoxic cells lysed C57BL/10 targets but not B10.BR (H-2 ^k ,H-7 ^a ) targets, nor BALB.B (H-2 ^b ,H-7 ^b ) 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-8 ^b ) 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.     

Multigenic control of immune responses of inbred mice against the terpolymers poly(Glu57Lys38Ala5) and poly(Glu54Lys36Ala10) and linkage withH-2 haplotype

Results of immunizations of recombinant inbred and congenic strains of mice with the random polymers poly(glu⁵⁷ lys³⁸ala⁵) or GLA⁵ and poly(glu⁵⁴lys³⁶ala¹⁰) or GLA¹⁰ indicate that there is an association of the responsiveness with theH-2 haplotype. Although the C57BL/6J mice (H-2 ^b haplotype) are “non responders”, the C57BL/6By originally derived from mice of the same haplotype are responders. The immune response pattern of recombinant strains carrying haplotypes derived by crossing over within theH-2 complex indicate that the responsiveness is under control of anIr gene which maps to the left of theIB subregion. Studies with the backcross mice indicated multigenic control of the responsiveness, with one locus beingH-2 linked and another locus segregating independently ofH-2.     

Studies of twoH-2D b mutants: B6.C-H-2 bm13 and B6.C-H-2 bm14

Two new C57BL/6H-2 mutants,B6.C-H- 2^bm13 and B6.C-H- 2^bm14 are described. They arose independently in C57BL/6 as spontaneous mutations of the gain and loss type. Complementation studies map the mutations in both bm13 and bm14 to theH-2D ^b gene. How ever, these two mutant strains are not identical, but occurred as independent mutations at the same locus, as shown by reciprocal graft rejection and by the inability of the (bm13 × bm114)F₁ hybrid to accept C57BL/6 grafts. Serological studies by direct testing (cytotoxicity and hemagglutination) and by quantitative absorption demonstrated a decrease in the H-2D^b private specificity H-2.2 in both bm13 and bm14 when compared to C57BL/6. This was confirmed by SDS-PAGE analysis using antisera detecting the H-2.2 specificity. Attempts to produce antibodies to either the gained or lost specificities of the two mutant strains failed.     

Biochemical studies on the H-2K antigens of the MHC mutantbm1

Biochemical analysis of the H-2K-gene product from the MHC mutant strainbml and from the C57BL/6 parent strain has been carried out in order to characterize the structural differences between parent and mutant K-gene products. Based on comparative tryptic peptide mapping of the cyanogen bromide fragments from these glycoproteins, two peptide differences were localized to the CN-Ia fragment. Partial amino-acid sequence analysis revealed two alterations in the primary structure of K^bml involving substitutions of tyrosine for arginine at position 155, and tyrosine for leucine at position 156. Both of these amino-acid replacements require a minimum of two nucleotide base changes at the nucleic acid level. These changes were the only alterations noted differentiating the K^bml and K^b glycoproteins. However, because our techniques allow us to analyze only 75 to 80 percent of the extra cellular portion of H-2K^b, it is possible there are other undetected changes. Nonetheless, the biochemical data are consistent with the hypothesis that the structural alterations noted in the K^bml mutant glycoprotein are directly related to the observed immunological specificity relative to the parent K^b molecule. Peptide comparisons of the K^b molecules of two C57BL/6 sublines and of the H-2^b lymphoblastoid cell line, EL-4, disclosed no difference.     

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     

Studies ofH-2K b mutant mice

Three newH-2 ^b mutant strains, B6.C-H-2 ^bm9 , B6.C-H-2 ^bm10 and B6.C-H–2 ^bm11 , are described. The three mutant strains are of the gain and loss type as they reject skin grafts reciprocally with the parental C57BL/6Kh. The mutations, which arose independently, are all allelic at the same locus as 11 other mutant strains already described. By complementation and other studies the mutated gene has been shown to beH-2K ^b . The strains were typed directly and by absorption with antisera specific for H-2K^b and H-2D^b private and public specificities and for Ia^b specificities. Each strain typed differently with these sera. The strain B6.C-H-2 ^bm9 was found to be serologically identical with C57BL/6. The strains B6.C-H-2 ^bm10 and B6.C-H-2 ^bm11 were found to have alterations in the private H-2K^b specificity, H-2.33, and in the public specificity, H-2.5, but to a different extent. B6.C-H- 2^bm10 had a marked decrease in the amount of H-2.33 expressed on the splenic cell surface as compared to C57BL/6 and also has a marked decrease in the expression of H-2.5 on both spleen and red blood cells. In comparison, B6.C-H-2 ^bm11 has a decrease in the expression of H-2.33 but an increase in the expression of H-2.5 on both splenic and red blood cells. The other H-2^b specificities appeared to be unaltered as compared with C57BL/6.     

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     

Genetic analysis of anH-2 mutant,B6.C-H-2 ba,using cell-mediated lympholysis: T- and B-cell dictionaries for histocompatibility determinants are different

B6.C-H-2 ^ba [H (z1)] is a mutant derived from C57BL/6. The two strains mutually reject their skingrafts and are incompatible in the mixed leucocyte reaction (MLR) and in cell-mediated lympholysis (CML) assays. They are serologically indistinguishable. This report shows that H(z1) carries a new, privateK end CML specificity clearly distinguishable from that of B6 by a third party strain, HTG. Antisera directed against the private H-2K specificity of B6 present on H(z1) cells) can block CML between the two strains in either direction. The new CML specificities of H(z1) cross-react with (public) CML specificities controlled by bothK andD regions of other unrelated haplotypes. The results suggest that H(z1) carries a mutation in theH-2K locus itself or in a closely linked gene, the product of which is also physically associated with the H-2K molecule corresponding to the cis-configuration of the alleles in both loci. These findings indicate that T- and B-cell dictionaries for histocompatibility determinants are different.     

Selective destruction by formaldehyde fixation of an H-2Kb serological determinant involving lysine 89 without loss of T-cell reactivity

In preparation for functional analyses, a study of the binding of H-2K^b-specific monoclonal antibodies (mAb) to formaldehyde (FOR)-fixed H-2^b spleen or tumor cells revealed that three of nine mAb tested had lost reactivity with the FOR-fixed cells, whereas the reactivity of the other mAb generally did not diminish. Comparison of the reactivity of these mAb on untreated H-2K ^bm mutant cells and on FOR-treated H-2K^b cells suggests that for three mAb the total loss of reactivity on the latter could be a consequence of the alteration by FOR of lysine 89, which is substituted by alanine in mutant bm3. H-2KP^b-specific alloreactive polyclonal or monoclonal CTL, all of which had retained reactivity with bm3 target cells, had also retained reactivity with FOR-fixed H-2^b cells as indicated by cold target inhibition studies. The H-2K^b-specific CTL were probably reactive with conformational determinants of H-2K^b, which are dependent on the integrity of both the 1 and the 2 domains of the H-2K^b molecule. Results are compatible with FOR treatment selectively affecting a serological determinant in the 1 domain without affecting conformational-type CTL determinants.Abbreviations used in this paper CTL cytotoxic T lymphocyte - FOR formaldehyde - PBS phosphatebuffered saline - FCS fetal calf serum - mAb monoclonal antibody - TNBS trinitrobenzene sulfonate     

Cross-reactivity patterns of vaccinia-specific cytotoxic T lymphocytes from H-2K b mutants

Limit-dilution cultures were used to select vaccinia-immune T-cell populations from bml and bm3 mutant mice that were not lytic for virus-infected targets expressing the K^b and D^b MHC glycoprotein. Approximately 30% of virus-immune CTL were restricted in each case to K^bm1 and K^bm3, rather than to D^b. Evidence of extensive cross-reactivity was found for these virus-immune CTL. Bm3 and bmll mice sharing one amino acid mutation from wild-type but differing by a second mutation seen only in bm3 are the most cross-reactive pair in their presentation of vaccinia. The bm1 and bm10 pair with dissimilar mutations from wild-type affecting the same CNBr fragment are also largely cross-reactive. However, 30% cross-reactivity is also found for bm1 and bm3, which differ in separate CNBr fragments. That mutants expressing amino acid substitutions in the same region of the peptide tend to show more evidence of cross-reactivity does not necessarily mean the T cells see linear arrays of amino acids on the MHC glycoprotein. For instance, K^bm1 and K^bm10 differ for three amino acids, but bm1 T cells are highly lytic for bm10 virus-infected targets. However, there is no cross-reactivity for K^bm1 and K^b, which differ at only two amino acids. The key to further understanding may rest with defining the nature of the conformational differences among the K^bm1, K^bm10, and K^b glycoproteins.     

Susceptibility to db gene and streptozotocin-induced diabetes in C57BL mice: control by gender-associated,MHC-unlinked traits

H-2 haplotype differences distinguish the related C57BL/KsJ (BKs) and C57BL/6J (B6) inbred strains. BKs mice are more susceptible to diabetes induction by a recessive obesity gene, diabetes (db), or by multi-dose streptozotocin (MSZ) administration. The purpose of this study was to evaluate whether the H-2 differences were the important genetic background modifiers determining inbred strain susceptibility or resistance to these diabetogenic stresses. Diabetes susceptibility of BKs.B6-H-2 ^b congenic mice was compared with that of the parental BKs and B6 stocks. In addition, diabetes severity was studied in (B6 × BKs)F₁ and F₂ db/db mice and an H-2 segregation analysis was performed. BKs susceptibility genes expressed in a dominant fashion in the F₁ generation, and were transmitted to F₂ db/db males without apparent segregation. No association between H-2 ^b haplotype and B6-type diabetes resistance was found in response to either the db mutation or to MSZ. Insulitis, associated with development of hyperglycemia in BKs males, also occurred in the H-2 ^b congenic stock. However, an apparent interaction between H-2 ^b haplotype, the db mutation (on chromosome 4), and male gender (Y chromosome?) was indicated by a segregation ratio distortion in recovery of this genotype. A more moderate diabetes in some F₂ db/db females suggested that non-MHC-linked genes controlling sex steroid metabolism were the important determinants of diabetogenic sensitivities in the C57BL stocks. In support of the latter, strain differences were demonstrated in activity levels of steroid sulfatase, which is regulated by a sex-linked gene likely expressed on both the X and Y chromosome, and which may control tissue levels of active androgens and estrogens. We show that the diabetes-susceptible F₁ hybrids exhibit the higher activity associated with the BKs strain.     

H-2 class I loci determine sensitivity to MCMV in macrophages and fibroblasts

Peritoneal (PM) and bone marrow-derived (BMM) macrophages and lung fibroblasts (LF) from inbred, intra-H-2 recombinant, H-2 mutant, and hybrid mice were infected with murine cytomegalovirus (MCMV) under centrifugal enhancement. At the concentration of virus employed, peritoneal macrophages from strains carrying Kd, Kb, D^d, KS and/or D^s, K4 and/or D4 alleles could be infected to a level of 80%–100%, as assessed by viral antigen expression or loss of Fc receptors. Cells lacking these haplotypes and carrying K^k, K^f, D^k, Df, or Db were resistant, yielding levels of infection below 20% . The background (non-H-2) and class II genotype and the S allele did not influence the proportions of cells infected. Furthermore, sensitivity was dominant in the F, progeny of H-2 b x H-2 k and H-2d x H-2 k crosses, and was not compromised by thebm1, bm3, bm10, or bm14 mutations in the al or2 regions of Kb orD ^b. The proportions of cells able to release infectious virus were low, but paralleled the frequencies of viral antigen expression. The class I genotype also determined susceptibility to MCMV infection in BMM and LF, although up to 35% of H-2 k BMM and 46% of H-2 k LF could be infected. The findings are consistent with an association between K and D antigens and a cellular receptor for MCMV on all three cell types.     

Biochemical studies on the H-2K mutant B6.C-H-2 bm10

The H-2K glycoprotein from the MHC mutant bm10 was analyzed biochemically to determine where primary structural differences distinguished it from the parental standard molecule, K^b. Comparative peptide maps showed differences in two peptides known to be part of the parental CNBr fragment spanning amino acids 139 to 228. Partial sequence analyses of CNBr fragments and tryptic peptides identified two tightly clustered amino acid substitutions at amino acids 165 (Val to Met) and 173 (Lys to unknown). The substitutions in bm10 represent the most carboxy-terminal substitutions characterized in the K^b molecules of the spontaneous, histogenically active H-2 mutants.     

Possible role of Ab b gene in mouse resistance to EL4 metastases

S (survivor) mutants were produced in mice for genetic analysis of host resistance to metastatic cancer. S-mutants S-27 and S-31 resist transplantation of lymphoma EL4 of parental C57BL/6J (B6) mice while they accept parental skin grafts. Mutant S-27 also resists formation of spontaneous metastases from intradermally growing EL4 tumor into lymp nodes; mutant S-31 is highly susceptible to EL4 metastases. Another mutant. H-2 ^bm26 (bm26), resists EL4 and rejects B6 skin grafts. Major histocompatibility complex (MHC) class I and class II gene expression was compared in these mutants and normal B6 mice. All three mutants tested, S-27, S-31, and bm26, expressed a low amount of K ^b mRNA in organspecific fashion. Mutant bm26 and S-31 expressed a low amount of Ab ^b mRNA and of A^b antigen on their spleen cells. Some oligonucleotide probes designed to hybridize to the second exon of the clss II MHC gene Ab ^b did not hybridize with DNA from all three mutants. These findings suggest extensive sequence alternations in the Ab ^b gene in mutants S-27, S-31, and bm26; they also suggest a major role of MHC in the control of host resistance to spontaneous metastases of the EL4 tumor. Address correspondence and offprint request to: I. K.. Egorov.     

Recognition of Db and Kb gene products by influenza-specific cytotoxic T cells

A series of 16 H-2^b-restricted, A influenza virus-specific cytotoxic T-cell clones are described and characterized. One is K^b restricted, the others D^b restricted. The factors governing K^b or D^b restriction patterns seen in the mixed populations from which clones are derived are investigated. The K^b-restricted clone does not recognize K^b mutant bm1 and influenza and all 15 D^b-restricted clones do not recognize D^b mutant bm14 and A influenza virus; these results are discussed in the light of findings in a variety of other viral systems. Representative K^b- and D^b-restricted clones were used to assess the functional properties of cloned cosmids containing either K^b or D^b genes expressed in transformed L-cells (κ haplotype). The expression products of both cosmids functioned efficiently as mutually exclusive restriction elements for A influenza virus recognition.