Gillian M. Morgan

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.

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.

Detection of mouse alloantibodies by rosetting with protein a-coated sheep red blood cells.

SUMMARY Staphylococcal protein A has an affinity for the Fc portion of the IgG molecule of different species and can therefore be used to detect cell-bound immunoglobulin. Using this property, protein A coupled to sheep red blood cells via chromic chloride can detect alloantibodies to mouse H-2, Thy-1, Ly-1, 2, 4, 5, 6, and 7, and la antigenic specificities bound to the surface of lymphocytes by the formation of rosettes. In comparison with other rosetting and cytotoxicity assays, the protein A assay shows a greater sensitivity than does cytotoxicity using spleen cells as the target, as does the sheep anti-mouse Ig rosetting assay, whereas cytotoxicity shows greater sensitivity with some antisera on thymocytes. The major advantages of the protein A assay are that constant low reproducible backgrounds are obtained, there is no need to remove surface Ig by capping prior to antiserum treatment, and that viable cells can be recovered. The ability of Staphylococcal protein A to bind to the Fc portion of the IgG molecule of several species has proven to be useful in a number of different immunological studies. For example, protein A has been used in the purification of IgG by affinity chromatography (5), for the coprecipitation of soluble cell surface antigens ( 1), and for the detection of cell surface Ig using radioiodinated protein A (2). In addition, cell surface IgG has been detected using a rosetting method, and heterologous anti-IgG wherein protein A was coupled to glutaraldehyde-fixed cells (3). We now report on the detection of cell surface alloantigens in the mouse using a rosetting method involving protein A coupled to sheep red blood cells (SRBC) with chromic chloride. This method is rapid and reproducible, and compares favourably with both cytotoxicity and the recently reported rosetting method using antiglobulin coupled to SRBC (10).

Mutation in the ia subregion of the murine h-2 complex.

The I region of the major histocompatibility complex (MHC) of the mouse contains genes coding for a number of functions: Ir genes, Ia specificities, histocompatibility genes, and other products (Shreffler and David 1975). The interrelationships of these genes, particularly of Ir genes and cell-membrane Ia antigens, has been studied mainly by the use of H-2 recombinant strains and, by this means, a large number of complexities in the expression of lr-gene functions has been revealed (Shreffler and David 1975, Klein 1975). A second approach to the fine dissection of these genetic relationships would involve studies of / region mutations such as those already used in studies of the H-2K, H-2D, and H-2L alloantigens. Of the 22 H-2 mutations previously described, all have involved H-2K, H-2D and H-2L loci IKohn et al. 1978) except one. We report here the first H-2 mutation involving the I -A subregion. The B6.C-H-2 bmaz congenic mutant strain carries a spontaneous mutation (H-2 bma2, formerly H-2 bin) which originally occurred in the H-2 b haplotype of a (C57BL/6Kh x BALB/cKh)F1 female mouse. The mutation is of the gain and loss type, as parental and mutant strains reciprocally reject skin grafts within 14 davs a n d gene complementation studies mapped the bm12 mutation to the K I A region of the H-2 complex, although the ability of the H 2 K b and bml2 mutants to complement each other for C57BL/6 grafts indicated that the mutation had not occurred in the H 2 K b gene (McKenzie et al. 1979) Serological studies indicated that the H-2K/D specificities of bml 2 were the same as those of C57BL/6 including H-2.33, the private H-2K b specificity, although the initial studies suggested that H-2.33 was defective in bml2 as the antisera used contained both anti-H-2.33 and anti-Ia antibodies. However, subsequent studies indicated that the bml2 mutant lacked Ia specificities (McKenzie et al. 1979). The C57BL/6 (H-2 b] strain carries the Ia specificities 3, 8, 9, 15, 20, all coded for by the I -A subregion Ino I-E/C products

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

Two new C57BL/6H-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 theH-2Db 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-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.

Implication of the H-2L locus in hybrid histocompatibility (Hh-1).

The Hh-1 (hybrid histocompatibility) effect in which F1 hybrids with heterozygosity at the “D end” of the H-2 complex can reject parental grafts of the H-2b haplotype was examined in four H-2 mutants wherein the mutation had affected the H-2Db, H-2Dd, or H-2Ld genes. In bone marrow grafting experiments it was shown that two separate mutations affecting the H-2Db locus did not affect the Hh-1 gene, suggesting that H-2Db and Hh-1 are probably two different genes. By contrast, experiments with two mutants, one affecting the H-2Dd and H-2Ld loci (B10.D2-H-2dml) and the other a loss mutation at the H-2Ld locus (BALB/c-H-2dm2), demonstrate an alteration in the hybrid histocompatibility phenomenon and the presumption is made that the Hh-1 and H-2Ld locus are identical. This presumption was supported by studies of Hh-1 using the EL4 tumor grafting model where marked differences in growth were noted in the (B10 BALB/ c)F1 and (B10 BALB/c-H-2dm2)F1 hybrids. By contrast, the functionally related natural killer (NK) phenomenon appeared to be the same in the BALB/c parent and dm2 mutant. Hybrid histocompatibility is a complex phenomenon but at this time we conclude that the H-2L locus is related to, if not identical to the Hh-1 gene but the H-2L locus is distinct from genes affecting the NK phenomenon.

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.

High titres of antibody to murine leukaemia virus in lymphocyte (ly) alloantisera.

The radioimmune precipitation (RIP) assay was used to examine the antibody titres against endogenous AKR murine leukaemia virus (MuLV) in a number of antisera to lymphocyte (Ly) alloantigens. The sera from normal donor and unimmunized recipient mice used in raising the alloantisera were also examined for anti-MuLV activity. It was found that all the antisera had high anti-MuLV titres and that in all but one case alloantigen immunization augmented the anti-viral titres. The degree of augmentation did not appear to be related to the anti-MuLV titre in the donor strain sera. Three I-region antisera were also examined for anti-MuLV antibodies and were found to have lower anti-viral titres than the Ly antisera even though immunization to I-region products greatly augmented the anti-viral titre. These results caution against the use of Ly antisera in characterizing the phenotype of lymphoid tumour cells without prior virus absorption.

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-2bm12. 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.

Studies of H-2Kb mutant mice. II. Definition of new H-2 specificities (H-2.68, 69, 71, 72) using the H-2Kb mutant, B6.C-H-2bm10.

Using theH-2Kb mutant strain, B6.C-H-2bm10, antibodies were produced which define four new H-2 specificities. This is in contrast to otherH-2Kb mutant strains in which attempts to produce antibodies against the mutation have failed with the exception ofH- 2bm3 in which a new specificity, H-2.62, was described by absorption analysis of an H-2.33 antiserum. With the mutant strain as immunization donor in the combination (C57BL/10 × 129)F1 anti B6.C-H-2bm10, two new H-2 specificities, H-2.68 and H-2.69, were produced. H-2.68 is found only on B6.C-H-2bm10, while H-2.69 is present on B6.C-H-2bm10 and on all cells of theH-2k haplotype. By testing the appropriateH-2 recombinant strains, the reaction with theH-2k haplotype could be mapped to theH-2D locus, whereas in B6.C-H-2bm10 it must have occurred in theH-2Kb gene. The reciprocal immunization of congenic strains failed, but with B6.C-H-2bm10 anti-A.BY, the specificities H-2.71 and H-2.72 were produced: H-2.71 was present on cells of theb, d, andk haplotypes, while H-2.72 was found only on theb haplotype. Genetic mapping usingH-2 recombinant strains showed the H-2.72 reaction to be associated with theH-2Kb,H-2Dd andH-2Kk genes. The specificities described were shown to be of theH-2 type by their reactivity withH-2 congenic and recombinant strains, their segregation in a backcross and their ability to precipitate a molecule of 45000 daltons molecular weight on SDS-PAGE. Therefore, the mutation carried by B6.C-H-2bm10, which resulted in a marked alteration of the H-2Kb specificities H-2.33 and H-2.5, is such that specific antibodies were able to be produced against both the gain and loss alterations produced by the mutation.Using the H-2Kb mutant strain, B6.C-H-2bm10, antibodies were produced which define four new H-2 specificities. This is in contrast to other H-2Kb mutant strains in which attempts to produce antibodies against the mutation have failed with the exception of H-2bm3 in which a new specificity, H-2.62, was described by absorption analysis of an H-2.33 antiserum. With the mutant strain as immunization donor in the combination (C57BL/10 x 129)F1 anti B6.C-H-2bm10, two new H-2 specificities, H-2.68 and H-2.69, were produced. H-2.68 is found only on B6.C-H-2bm10, while H-2.69 is present on B6.C-H-2bm10 and on all cells of the H-2k haplotype. By testing the appropriate H-2 recombinant strains, the reaction with the H-2k haplotype could be mapped to the H-2D locus, whereas in B6.C-H-2bm10 it must have occurred in the H-2Kb gene. The reciprocal immunization of congenic strains failed, but with B6.C-H-2bm10 anti-A.BY, the specificities H-2.71 and H-2.72 were produced: H-2.71 was present on cells of the b,d, and k haplotypes, while H-2.72 was found only on the b haplotype. Genetic mapping using H-2 recombinant strains showed the H-2.72 reaction to be associated with the H-2Kb, H-2Dd and H-2Kk genes. The specificities described were shown to be of the H-2 type by their reactivity with H-2 congenic and recombinant strains, their segregation in a backcross and their ability to precipitate a molecule of 45,000 daltons molecular weight on SDS-PAGE. Therefore, the mutation carried by B6.C-H-2bm10, which resulted in a marked alteration of the H-2Kb specificities H-2.33 and H-2.5, is such that specific antibodies were able to be produced against both the gain and loss alterations produced by the mutation.