Roger W. Melvold

Eight new histocompatibility mutations associated with theH-2 complex

Eight newH-2 mutations are reported, five derived fromH-2b and three fromH-2d. TheH-2bmutants (H-2bg3,H-2bi,H-2bj,H-2bk, andH-2bn) were all of the gain+loss type, and all exceptH-2bn were at the same locus asH-2ba, associated with theK end. In addition,H-2bg3 was histocompatible with previously reported mutationsH-2bg1 andH-2bg2. The MST for mutant grafts on normal hosts was 2 to 5 weeks. TheH-2d mutations consisted of two losses (H-2db,H-2bc and one gain + loss (H-2dd). They involved at least two, possibly three, loci and have MSTs of less than two weeks. All mutations were recovered in (C57BL/6Kh x BALB/cKh)F1 hybrids, exceptH-2db, which was isolated on a pure BALB/c background.

Balb/c-h-2db. A new h-2 mutant in balb/ckh that identifies a locus associated with the d region.

A newH-2 mutant, BALB/c-H-2db, is described. This mutant originated in BALB/c, is inbred, and is coisogenic with the parental BALB/cKh strain. The mutation is of the loss type since BALB/c-H-db rejects BALB/c, but not vice versa. Complementation studies have localized the mutation to theD region of theH-2 complex. A cross between BALB/c-H-2db and B10.D2-H-2da failed to complement for either BALB/c or B10.D2 skin grafts, indicating that these are two separate mutations at the same locus (Z2). Direct serological analysis and absorption studies revealed that, with one exception, theH-2 andIa specificities of BALB/c and BALB/c-H-2db are identical. In particular,H-2.4, the H-2Dd private specificity, is quantitatively and qualitatively identical in the two strains. The exception is that of the specificities detected by antiserum D28b: (k×r)F1 anti-h, which contains anti-H-2.27, 28, and 29. These specificities appear to be absent from theH-2db mutant since they are not detected directly or by absorption. Other public specificities are present in normal amounts,e.g., the reaction with antisera to H-2.3, 8, 13, 35, and 36. The reaction with antiserum D28 (f×k)F1 anti-s, which contains antibodies to H-2.28, 36, and 42, is the same in both strains. Antiserum made between the two strains (H-2db anti-H-2d) reacts like an anti-H-2 serum, in that it reacts with both T and B cells by cytotoxicity, but is not a hemagglutinating antibody. The serum reacts as does the D28b serum in both strain distribution and in cross-absorption studies. We conclude that theH-2db mutation occurred at a locus in theD region, resulting in the loss of the H-2.28 public serological specificity and of a histocompatibility antigen. Whether these are one and the same antigen is not yet known. The data, in view of other evidence, imply that the public and private specificities are coded for by separate genes.

Dermal histocompatibility and in vitro lymphocyte reactions of three newH-2 mutants

Histocompatibility of skin grafts and in vitro lymphocyte reactions of three new H-2 mutants (B6-H-2bg1, B6-H-2bg2, and B6-H-2bh) are described. Complementation tests indicated thatH-2bg1,H-2bg2, andH-2bh were mutations at the same locus (z-1) and allelic withH-2ba. Two of the lines, B6-H-2bg1 and B6-H-2bg2, appeared identical, although they were of spontaneous and independent origin. The third line, B6-H-2bh, was unique. Such graft rejection was paralleled by reactivity in the MLR and by the generation of cytotoxic lymphocytes in vitro. The pattern of crossreactivity among antigens specified by these mutant genes supports the notion that thez-1 locus controls multiple specificities. The results indicate that a single point mutation can simultaneously affect histocompatibility, MLR, and CML reactions.

Evidence suggesting the existence of two h-y antigens in the mouse.

An exceptional (C57BL/6 × BALB/c)F1 male mouse, from an X-irradiated father, and lacking the H-Y (“male”) antigen, on the basis of skin-graft testing, was found. Conventional serological tests for H-Y antigen, however, were positive. His karyotype contained only 39 chromosomes, the Y chromosome apparently being absent. Although the testes were small, lacked germ cells, and were essentially Leydig cell tumors, the mutant was normal with respect to external male phenotype, accessory glands, and sexual behavior. The data suggest that the histogenic and serological tests for H-Y antigen may detect two different antigens, paralleling findings with mutants of theH-2 complex, where histoincompatibility can be generated without accompanying serological changes.

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

C57BL/6 (H-2b) mice, and four mutants (B6.C-H-2ba, B6-H-2bg1, B6-H-2bg2, B6-H-2bh) 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-2Kb, H-2Dd and Iab 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.

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

In an earlier paper, we presented evidence that two independent mutants of the bg series, B6-H-2bm5 (bm5) and B6-H-2bm16 (bm16) carry identical mutations such that tyrosine at residue number 116 of the H-2Kb 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-2bm6 (bm6), B6.C-H-2bm7 (bm7), and B6.C-H-2bm9 (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 Kb molecule.

Biochemical studies on the H-2K antigens of the MHC mutantsbm3 andbm11

Biochemical analyses of the H-2K-gene products of the C57BL/6 mutant strains, bm3 and bm11, have been carried out in order to characterize the structural relationships among these antigens. From comparative tryptic peptide mapping of cyanogen bromide fragments from the mutant and parent Kb glycoproteins and from preliminary amino-acid sequence analyses, a number of discrete differences have been discerned. Two sites of difference at amino residues 77 and 89 in the Kbm3 glycoprotein are noted relative to Kb. An alteration at residue 77 similar or identical to that seen in Kbm3 is present in the K antigen of thebm11 mutant. Because our techniques sample only 75 to 80 percent of the extracellular portion of H-2Kb, other undetected changes are possible. However, our present findings are most consistent with the conclusion that only very limited differences exist between mutant and parent molecules. Further, taken together with CML (cell-mediated-lymphocytotoxicity) reaction patterns (Melief et al. 1980), the biochemical data support the hypothesis that the proposed structural alterations in theKb mutant glycoproteins are directly related to their observed immunological specificity.

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-2Kb mutants analyzed.

The relationship between theH-2 loss mutations ofH-2(da) andH-2 (db) in the mouse.

The mouse strain B10.D2-H-2da carries the mutantH-2da allele, derived after chemical induction, and this has been shown to be a gain and loss mutation involving theH-2Dd locus.BALB/c- H-2db, derived spontaneously, is a loss mutation only, and appears not to involve theH-2Dd, but rather theH-2Ld locus. The two mutations effectboth graft rejection and serologically detected H-2 specificities (Type II mutation). In the experiments described in this study, theloss mutations in theH-2da andH-2db mutants have been compared by skin grafting, and by direct and absorption serological techniques: (1) By skin grafting, using the well established complementation method, it has been shown thatH-2da andH-2db do not complement each other, i.e., the mutation in both occurred at the same ‘locus.’ However, by appropriate selection of donor and recipient, it has become clear thatH-2da had a greater loss than didH-2db, althoughH-2da includes the loss found inH-2db. (2) Serological studies have demonstrated that H-2D.4 was altered inH-2da, but not inH-2db; ‘H-2.28’ (detected by D-28b and D-29) was decreased or lost in both mutants;H-2db anti-BALB/c failed to react withH-2da; both mutants reacted similarly with D-28 sera. In addition, sera made usingH-2da as donor did not contain an anti-H 2.28 antibody. The loss mutation involvingH-2da therefore appears to have led also to the loss of H-2.28 as found inH-2db. We conclude that theH-2da strain arose after a complex mutation or recombination event which involvedboth theH-2Dd locus and the closely linkedH-2Ld locus, whereasH-2db affects only theH-2L locus.

H-2 and non-H-2 interaction in expression of mutant histocompatibility geneH(KH-11)

H(KH-11) is a mouse mutant histocompatibility gene, the expression of which, as detected by skin graft rejection, requires the presence of a second gene in the graft donor which is associated with theH-2b haplotype, but not withH-2d. The mutant gene is not linked to theH-2 complex and may be carried and transmitted with or without expression, as predicted by classical Mendelian genetics.