Studies on recombination within the mouse H-2 complex: IV. Characterization of new recombinant haplotypes H-2t7, H-2t8, H-2as2, and H-2as3.

Four new intra-H-2 recombinants were characterized serologically and functionally. In two of these recombinants, B10.ASR1 and B10.ASR7, crossing over occurred between the A alpha and E alpha subregions, very probably in E beta since most intra-I region recombinants thus far investigated at the DNA level appear to involve recombination within the E beta gene. In the other two recombinants, B10.ASR2 and B10.ARS8, crossing over occurred between the S and D subregions. B10.ASR2 and B10.ASR8, crossing over occurred between the S and D subregions. B10.ASR7, which is serologically indistinguishable from B10.BASR1 and B10.S(8R), slightly stimulates and strongly responds to both of these strains in MLR. The probable location of the B10.S(8R) stimulatory product is E beta. The H-2 composition of B10.ASR1 is closest to that of B10.S(9R) and B10.HTT; therefore, precise definition of the cross-over point at the DNA level will be of particular interest.

Immunogenetic analysis of H-2 mutations. V. Serological analysis of mutations H-2da, H-2ra, and H-2ka1.

The H-2 and Ia antigenic composition of strain pairs B10.D2 (H-2d) and M504 (H-2da). A.CA (H-2f) and M506 (H-2fa), and CBA (H-2k) and M523 (H-2ka) was compared by testing their cells against a battery of oligospecific antisera, by performing absorption analysis, and by cross-immunization. The two strains of each pair are congenric and differ in taht the second strain of the pair carries a mutation that occurred in the H-2 haplotype of the first strain. The Ia composition of each mutant haplotype was found to be the same as that of the haplotype from which the mutant was derived. Several differences in the serologically detectable H-2 antigens were found. The H-2d and H-2da haplotypes were found to differ in that the latter lost at least one and gained another antigen. The affected antigens were demonstrated to be classic H-2 antigens controlled by the H-2D locus. The H-2t and H-2fa haplotypes were found to differ in that antigens 26, 37, and 39, controlled by the latter, bound their respective antibodies less firmly than those controlled by the former haplotype. Since all three antigens are coded for by the H-2K locus, since no change was found in the D-region controlled antigens, and since the H-2fa mutation maps in the K end, we conclude that most likely the mutation occurred in the K region. The H-2k and H-2ka haplotypes were found to differ in that the latter lost one antigen encoded by the H-2Kk allele. This mutation, therefore, must have occurred in the H-2K locus. The data tip the scale of evidence in favor of the interpretation that each of the H-2 mutations occurred in a single region, either K or D. No evidence for a second mutation within any of the other H-2 regions was found.

A method for detection of Ia antigens in the absence of appropriate H-2 recombinants.

A method is described which obviates the use of H-2 recombinant strains for production and detection of Ia antibodies. The antibodies are produced by immunization with spleen cells in a strain combination in which the donor differs from the recipient either in the K end of the H-2 complex or in the whole complex. If the right schedule is used, this immunization produces antibodies against both H-2 and Ia antigens. The H-2 antibodies are absorbed out with erythrocytes or T lymphocytes rendering the antiserum specific for Ia antigens. The presence of Ia Ia antibodies is asserted by determining the tissue distribution of the antigens detected with the absorbed antiserum. Another antiserum is then prepared in the same strain combination by immunization with tissue that does not contain Ia but does contain H-2 antigens (e.g., a sarcoma of the proper genotype). The second antiserum contains only H-2 antibodies and the presence of these antibodies is again asserted by determining the tissue distribution of the corresponding antigens. The molecular distinctiveness of the putative Ia and H-2 antigens is then demonstrated by the newly developed technique of antibody-mediated induction of resistance to cytotoxicity (lysis). If the antigens detected with the two antisera move independently in the cell membrane, and if the antigens detected with the first antiserum do indeed have Ia-like properties, it is concluded that the antiserum detects Ia antigens. This method should prove to be useful for detection of Ia antigens in H-2 haplotypes for which no intra-H-2 recombinants are known, and for detection of Ia-like antigens in other mammalian species, particularly in man.

Induction of resistance to antibody-mediated cytotoxicity. H-2, Ia, and Ig antigens are independent entities in the membrane of mouse lymphocytes.

Mouse spleen or thymus lymphocytes incubated with monospecific H-2 or Ia alloantisera and then coated with a xenogeneic antimouse Ig serum become specifically resistant to the alloantiserum (and complement) they have been incubated with. This so called "lysostrip method" was used to investigate the molecular interrelationships of antigens in the mouse lymphocyte membrane. The results of this investigation confirm that H-2K and H-2D antigens are carried by two distinct populations of molecules. They provide evidence that the Ia antigens move in the membrane independently of both H2-K and H-2D antigens; and finally they demonstrate absence of any physical linkage between Ig receptors in B cells, on the one hand, and Ia, H-2K, and H-2D molecules on the other hand.

Serological distinction of mutants B6.CH(zl) and B6.M505 from strain C57BL-6.

A quantitative serological difference was found between strains Hz1 and M505 carrying mutant H-2 haplotypes ba and bd, respectively, and the original strain B6(H-2(b)). The finding suggests that the mutations occurred in the H-2K(b) gene, and together with data on MLR and CML challenges the current concept of H-2 regions' involvement in immune reactions.