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.     

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.     

Studies of two H-2Db mutants: B6. C-H-2bm13 and B6.C-H-2bm14

Two new C57BL/6 H-2 mutants, B6.C-H-2bm13 and B6.C-H-2bm14 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 the H-2Db gene. However, 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 X bm14)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-2Db 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.     

Mutation at I-A beta chain prevents experimental autoimmune myasthenia gravis

Immune response (Ir) gene(s) at the I-A subregion of the mouse H-2 complex influence susceptibility to experimental autoimmune myasthenia gravis (EAMG). To determine the importance of the Ir gene product, the Ia antigens, in EAMG pathogenesis, we studied the degree of EAMG susceptibility of an I-A mutant strain, the B6.C-H-2 ^bm12 (bm12), and its parent B6/Kh. According to the cellular, humoral, biochemical, and clinical manifestations of EAMG, the I-A mutation converted an EAMG susceptible strain (B6/Kh) into a relatively resistant strain (bm12). The relative resistance to EAMG induction in bm12 may be due to the lack of Ia.8 and/or la.39 determinants and/or quantitative expression of la antigens.Abbreviations used in this paper MG myasthenia gravis - AChR acetylcholine receptors - EAMG experimental autoimmune myasthenia gravis - Ir immune response - B6 C57BL/6J - bm12 B6.C-H-2 ^bm12 - CFA complete Freund's adjuvant - LNC lymph node cells - PPD purified protein derivative     

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.     

The definition of a new IA antigenic specificity using the B6.C-H-2 bm12 I-region mutant strain

A new Ia specificity has been defined using theIA-subregion mutant B6.C-H-2 ^bm12. The immunization to produce the antiserum wasbm12 anti-A.BY, as all other immunizations, such asbm12 anti-C57BL/6, failed to produce antibody. By selecting strains of C57BL origin for testing, it was shown that, (a) the serum was only weakly cytotoxic but gave substantial reactions using a rosetting assay; (b) the antibody reacted with B cells and not T cells; (c) strains of theb, d, p andq H–2 haplotypes were positive, whereasf, k, r ands were negative; (d) absorption studies demonstrated only a single specificity to be present and by testing recombinant strains, the reaction mapped to theIA subregion; (e) SDS-PAGE demonstrated that the antiserum reacted with a molecule of MW 33 000. Preliminary studies indicate this new specificity, present on C57BL/6 and lost frombm12, is present on the same molecule as other I-A specificities.This work was supported by funds obtained from the N. H. and M. R. C. (Australia) and the National Institutes of Health, Grant No. CA-21224.     

Reactivity of the B6.C-H-2bm−1 mutant to the wild-type tumor EL4: Characterization of the serological response

Hyperimmunization of B6.C-H-2^bm?1 (H-2^bm?1), a congenic mutant of C57Bl/6J (B6), with the C57Bl lymphoma EL4 resulted in the induction of antibodies with apparent EL4 specificity. EL4 reactivity was demonstrable in H-2^bm?1 anti-EL4 sera by complement-mediated cytotoxicity, absorption, and enzyme-linked immunosorbent assay. By these same serological tests, H-2^bm?1 anti-EL4 serum was found to be nonreactive with B6 normal lymphoid cells, embryonic fibroblasts, and two fibrosarcomas previously induced in B6 mice by methylcholanthrene. These data suggest that the serological response of H-2^bm?1 to EL4 is directed against tumor-associated antigens on EL4. These findings indicate that congenic mutants which differ from the wild-type strain at MHC Class I subloci, but which do not evoke serological responses to MHC components, may provide convenient sources for preparing serological reagents directed against tumor-specific antigens.     

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.     

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.     

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.     

Use ofH-2 mutations to quantitate alloreactivity: Alloreactivity is strongest against H-2 antigens which are closest to self

Lymph-node cells fromH-2 allogeneic, intra-H-2 recombinant andH-2 mutant congenic strains were sensitized in limiting dilution cultures to quantitate the cytotoxic T-lymphocyte precursor frequencies (CTL.Pf) against antigens encoded by different regions of theH-2 complex. When fourH-2K ^b mutants of C57BL/6 (B6) were tested, we observed anti-B6 CTL.Pf that were as high or higher than those of recombinant strains which differ from B6 at theK end of theH-2 complex. Relative to strains completelyH–2 allogeneic to B6, the CTL.Pf inH-2 ^bm1,H-2 ^bm3 andH-2 ^bm5 averaged 40–50 percent, andH-2 ^bm8 averaged 140 percent. Recombinant strains B10.A (4R) and B10.D2 (R103), which differ from B6 at theK end of theH-2 complex, averaged 60 percent of the completelyH-2 allogeneic value. Since the mutant and wild-type gene products have no serological and minimal structural differences relative to other alleles atH-2K, these results indicate that the CTL.Pf does not increase with increasing H-2 antigenic disparity between any two strains. Rather, the data suggests that the T-cell receptor repertoire recognizes those H-2 molecules or determinants closest to self.     

I-A Mutation resulted in a selective loss of an antigen-specific Ir gene function

The immune responses to several antigens were compared in the I-A mutant mouse strain B6.C-H-2^bm12 and the wild-type strain C57BL/6. With a lymph node cell proliferation assay, the response to two of these antigens, beef insulin and (TG)A-L, was demonstrated to be controlled by a gene in the I-A^b region. B6.C-H-2^bm12 mice failed to respond to beef insulin, while their responses to (TG)A-L, DNP-OVA and PPD were comparable with those of the wild-type strain C57BL/6. Taken together with previous studies, these data suggest that the product of a single pleiotropic I-A gene, an la molecule, functions as a histocompatibility, la, and MLR antigen, as well as a necessary component for Ir gene function. Furthermore, the data reported here demonstrate that la molecules have multiple functional “Ir determinants,” one of which has been altered in the B6.C-H-2^bm12 mutant. The B6.C-H-2^bm12 mice, therefore, represent a powerful analytical tool for the understanding of the cellular and molecular basis for Ir gene control of the immune response.     

Recognition of H-2Kb mutant target cells by Moloney virus-specific cytotoxic T lymphocytes from bm13 (H-2Db mutant) mice. I. Full recognition of Kbm11 by Kb-restricted CTL

In C57BL/6 (B6, H-2b) mice, the secondary in vitro CTL response against Moloney leukemia virus is restricted and regulated by the H-2Db locus. B6.C-H- 2bm13 ( bm13 ) mice, however, carrying a mutation at the Db locus, show an increased H-2Kb-restricted CTL response without a demonstrable CTL component restricted by the mutant Dbm13 molecule (D----K shift). These purely Kb-restricted bm13 virus-specific CTL were incubated with a series of Kb mutant virus-infected target cells to study the effect of the mutations at the target cell level. Of six Kb-mutant virus-infected target cells tested, bm1 cells were not recognized and bm8 cells were recognized only marginally by bm13 virus-specific CTL, whereas bm3 , bm5 , bm6 , and bm11 cells were fully recognized. Thus, the bm3 , bm5 , bm6 , and bm11 Kb mutants fully share the relevant H-2K restriction specificities with H-2Kb, whereas the bm1 mutant totally and the bm8 mutant almost completely lack these specificities. This result differs markedly from the restriction site relationships among B6 and these Kb mutants in other antigenic systems. The most striking example concerns the bm11 mutant, which is fully recognized by Moloney-specific CTL, but not at all by Sendai, minor H (H-3.1, H-4.2), and sulfhydryl hapten-specific CTL. Monoclonal anti-H-2Kb antibody B8-3-24 inhibited virus-specific lysis by bm13 CTL of all Kb virus-infected mutant target cells to which this antibody binds. Lysis of bm5 and bm11 but not of bm3 target cells was inhibited, in line with the fact that B8-3-24 antibody does not bind bm3 . On the other hand, not only bm5 and bm11 but also bm3 virus-infected target cells blocked virus-specific lysis to the same extent as syngeneic bm13 target cells. Therefore, bm13 virus-specific CTL populations do not recognize the discrete cluster alteration in the Kbm3 molecule, as identified by antibody B8-3-24. The bm1 and the bm8 mutations, which have structural alterations in completely different sites of the Kb molecule, show complete or almost complete loss, respectively, of Kb-Moloney restriction sites. This finding supports the notion that these virus-specific CTL recognize conformational determinants rather than linear amino acid sequences.     

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     

Effects of fourH-2K mutations on virus-induced antigens recognized by cytotoxic T cells

Lysis of ectromelia- or LCM virus-infected macrophage target cells by virus-specific cytotoxic T cells from mice immunized with the homologous virus occurred only where donors of T cells and target cells shared eitherH-2K orH-2D genes. With both viruses, use of T cell or target cell donors bearing mutations (B6.C-H-2^ba, B6-H-2^bh, B6-H-2^bg1, and B6-H-2^bg2), all of which apparently occurred in the same single genetic element in theH-2K^b region, abolished (H-2^ba) or impaired (H-2^bh,H-2^bg1 andH-2^bg2) lysis in T cell-target cell combinations that shared (apart from the mutations) all other genes in theK, I-A, orI-B regions of theH-2 complex. The data suggest that virus-induced antigenic patterns on infected B6.C-H- 2^ba (mutant) cells are more different antigenically from those on C57BL/6 (wild type) cells than are those on infected cells from the other mutants -B6-H-2^bh, B6-H-2^bg1, and B6-H-2^bg2. (B6.C-H-2^ba× B6 -H-2^bh)F₁ mice behaved like B6-H-2^bh, indicating no complementation, and confirming that theH-2K gene(s) involved in recognition of virus-infected cells by virus-specific T cells behave as a single element. These findings are discussed in relation to the nature of virus-induced antigenic patterns that are recognized by virus-specific cytotoxic T cells.     

Immune response to the H-X antigen on P815-X2

In a preceding report, the detection of an H-2-linked immune response to the H-X ^d antigen on the P815-X2 mastocytoma was demonstrated by the significantly increased survival of (C57BL/6 × DBA/2)F₁ (B6D2F1) male hybrids (H-X ^b ) compared with female siblings (H-X ^{b/H-X d} ) after injection with the histocompatible tumor (H-X ^d ). This interpretation was supported by the absence of this sex effect in reciprocal D2B6F1 hybrids (H-X ^d and H-X ^{d/H-X b} ). Additional findings presented in this paper support the conclusion that this sex effect is due to a true immunological response to H-X ^d : (a) Reciprocal (DBA/2 × C57BL/6 H-2 mutant)F₁ hybrids, as well as D2B6F1, failed to exhibit the sex effect: (b) the demonstration of the sex effect in (BALB/c × DBA/2)F₁ and (BALB/c-H-2 ^dm2 × DBA/2)F₁ hybrids and in (C57BL/10 × DBA/2)F₁ hybrids was consistent with the known H-X incompatibilities between the strains BALB/c and DBA/2 and C57BL/10 and DBA/2, respectively, previously demonstrated by skin grafting; and (c) the sex effect was not abrogated by castration of male B6D2F1 hybrids. Variability in the presence or absence of the sex effect was observed in various [recombinant inbred (RI) × DBA/2]F₁ hybrids and may be attributed to the influence of a regulatory non-H-2 gene which is closely linked to the gene coding for mouse kidney-androgen-regulated protein (KAP) but androgen-independent, or to variability in inheritance of the H-X ^b allele among the RI lines. It is proposed that the P815-X2 model may be utilized to type RI lines derived from a cross between C57BL/6 and DBA/2 for their H-X genotypes.Abbreviations B C57BL/6 origin allele - B6 C57BL/6 - B10 C57BL/10 - B6D2F1 (C57BL/6 × DBA/2)F₁ - B6 ^m D2F1 (C57BL/6 H-2 mutant × DBA/2)F₁ - bm10 B6.C-H-2 ^bm10 - C BALB/c - D DBA/2 origin allele - D2 DBA/2 - dm2 BALB/c-H-2 ^dm2 - H-X X chromosome-determined histocompatibility antigen of the mouse - Ir gene, immune response gene - KAP kidney androgenregulated protein - MST median survival time - RI recombinant inbred - SDP strain distribution pattern     

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.     

Localization of anionic constituents in mast cell granules of brachymorphic (bm/bm) mice by using avidin-conjugated colloidal gold

We used the egg avidin gold complex as a polycationic probe for the localization of negatively charged sites in the secretory granules of mouse mast cells. We compared the binding of this reagent to mast cell granules in wild-type mice and in congenic brachymorphic mice in which mast cell secretory granules contained undersulfated proteoglycans. We localized anionic sites by post-embedding labeling of thin sections of mouse skin and tongue tissues fixed in Karnovsky’s fixative and OsO₄ and embedded in Araldite. Transmission electron microscopy revealed that the mast cell granules of bm/bm mice had a lower optical density than those of wild-type mice (P<0.001) and a lower avidin gold binding density (by approximately 50%, P<0.001). The latter result provided additional evidence that the contents of mast cell granules in bm/bm mice were less highly sulfated than in those of wild-type mice. In both wild-type and bm/bm mast cells, the distribution of granule equivalent volumes was multimodal, but the unit granule volume was approximately 19% lower in bm/bm cells than in wild-type cells (P<0.05). Thus, bm/bm mast cells develop secretory granules that differ from those of wild-type mice in exhibiting a lower optical density and slightly smaller unit granules, however the processes that contribute to granule maturation and granule-granule fusion in mast cells are operative in bm/bm cells.     

Study ofH-2 mutations in mice

The serologically defined H-2.5 specificity was tested on spleen cells and red blood cells (RBC) of theH-2 ^b haplotype and a number of its mutants. Thebm8 (bh) mutant was barely distinguishable fromb in a variety of tests made. On spleen cells ofbm1 (ba) the H-2.5 specificity seemed to be unchanged, while it was virtually absent from RBC of this mutant. Mutantsbm4 (bf),bm5 (bg1), andbm6 (bg2) were similar tobm1, with slight differences between them. The mutantbm3 (bd) retained an unchanged quantity of H-2.5 on its spleen cells, while the specificity was substantially increased on its RBC. The H-2.5 ofbm3 is not identical to that ofH-2 ^a . Possible mechanisms causing differential serology of theH-2 ^b mutants are discussed.