Mapping of the structural gene for the second component of complement with respect to the human major histocompatibility complex.

Families have been HLA typed, and allotypes of the second component of complement and properdin factor B determined. The lod score for the C2 structural gene and HLA-B from the study of 11 families and 55 informative meioses was 14.39 at maximum likelihood estimate of the recombination fraction of .02. This is related to other estimates of the distance between these two genes. The relative kinetic activities of the C2 allotypes were studied and no differences were demonstrated. No crossovers between Bf and C2 were observed.     

The role of lymphoid cells in antibody-induced suppression of the fourth component of guinea pig complement

Many laboratories have demonstrated that immunoglobulin production by B cells is controlled by networks of interacting lymphocytes and their products. Our laboratory has demonstrated that complement components produced by macrophages are also regulated by networks of interacting cells and humoral factors. Treatment of mice in vivo or guinea pig cells in vitro with anticomponent antibody specifically inhibits synthesis and secretion of the component by macrophages. We have further characterized the cellular basis for in vitro suppression of the fourth component of guinea pig complement. C4 suppression has been accomplished with dispersed spleen cells as well as intact splenic fragments. This facilitated examination of the cells responsible for long-term C4 suppression. The data suggested that C4 suppression required either cell contact or sufficient concentrations of soluble factors. Long-term suppression of C4 depends upon a lymphoid cell contained in the spleen and in lymph nodes but absent or in insufficient concentration in the peritoneum. The lymphocyte that actively maintains suppression was negative for the guinea pig T-cell marker detected by the monoclonal antibody mc8BE6. Therefore, the critical cell is either another T-cell subset or non-T lymphocyte. These data demonstrate that a network of interacting cells analogous to that proposed to regulate antibody synthesis is also involved in regulating some nonlymphoid cell products.     

Linkage of the preimplantation-embryo-development (Ped) gene to the mouse major histocompatibility complex (MHC)

The preimplantation-embryo-development (Ped) gene influences the rate of cleavage division of preimplantation mouse embryos. The location of the Ped gene in the mouse major histocompatibility complex (MHC), the H-2 complex, has been inferred from the analysis of cleavage rates of embryos from mouse strains congenic at the H-2 complex. In this paper, formal genetic linkage studies were undertaken to evaluate linkage of the Ped gene to the H-2 complex. Co-segregation of Ped gene phenotype and H-2 haplotype was found in back-cross embryos. These data support the hypothesis that the Ped gene is linked to the H-2 complex.     

Serological studies on the major histocompatibility complex of new inbred strains of the guinea pig.

New inbred strains of guinea pigs, JY 1, JY 2, JY 3, JY 6, JY 9 and JY 10 have been established in this Institute. Serologic studies of guinea pig leukocyte antigens (GPLA antigens) were carried out in order to examine their major histocompatibility complex (GPLA complex). Antisera specific for Ia antigens were raised by cross-immunization of NIH strain 2 (NIH 2) and NIH strain 13 (NIH 13) guinea pigs, well known inbred guinea pigs. The sera identified four distinct Ia specificities, which were designated as Ia.2a, Ia2b, Ia.13a and Ia.13b. Six antigenic specificities different from the above Ia specificities were identified by sera obtained by appropriate immunization of the inbred guinea pigs and were designated as P.1, P.2, P.3, P.4, P.5 and P.6. Antigenic specificities of GPLA antigens recognized in inbred guinea pigs were : NIH 2 (Ia.2a, Ia.2b, P.1, P.2, P.O, P.4), NIH 13 (Ia.13a, Ia.13b, P.1, P.2, P.3, P.4), JY 1 (Ia.13a, Ia.13b, P.5), JY 2 (Ia.2b, Ia.13b, P.3, P.4, P.6), JY 3 (Ia.13a, Ia.13b, P.2, P.4, P.5), JY 6 (Ia.2b, Ia.13b, P.3, P.6), JY 9 (Ia.13a, Ia.13b, P.4, P.5), JY 10 (Ia.13a, Ia.13b, P.2, P.3, P.4, P.6), JY 9 (Ia.13a, Ia.13b, P.4, P.5), JY 10 (Ia.13a, Ia.13b, P.2, P.3, P.4, P.6). The correspondence of these specificities to those already reported was discussed and the P.2 or P.4 was considered to be an additional specificity of GPLA antigens that have not been reported yet. As the new inbred strains of guinea pigs were thus found to possess characteristic GPLA complex, which differ from each other and from those of NIH 2 and NIH 13 strain, they should be useful for studies of roles of the major histocompatibility complex in the immune system.     

The major histocompatibility complex of the guinea pig. I. Serologic and genetic studies.

Serologic and genetic studies of the antigens which comprise the guinea pig MHC have demonstrated three distinct but linked genetic regions. Antisera to the B region were raised by cross-immunization of random-bred animals; this region controls antigens B.1, B.2, B.3, and B.4 which behave as alleles at a single locus and which resemble the products of the murine D or K region genes in their tissue distribution and molecular characteristics. Cross-immunization of inbred strain 2 and strain 13 animals, both of which bear the B.1 antigen, leads to sera which identify antigens which resemble the products of the I region of the murine MHC. Specific absorption experiments have demonstrated four distinct I region antigens. In addition to the B and I regions, inbred strain 2, strain 13, and some outbred animals bear an antigen (S.1) which is the product of a third genetic region and which also resembles the murine D or K region gene products in molecular size. The results of these studies should facilitate the use of the guinea pig as an experimental model for studies of genetic control of the immune response and the function of the histocompatibility-linked Ir genes.     

Proteolysis of the heavy chain of major histocompatibility complex class I antigens by complement component C1s

The major histocompatibility complex (MHC) class I antigens contain a light chain β₂-microglobulin, non-covalently associated to the transmembrane heavy α-chain carrying the allotypic determinants. Since the C1q complement component is known to associate with β₂-microglobulin, and we recently found that activated C1s complement was capable of cleaving β₂-microglobulin, we decided to investigate the proteolytic activity of C1 complement towards the heavy chain of class I antigens. Our results demonstrate that human C1s complement cleaves the heavy chain of human class I antigens into at least two fragments, with apparent molecular weights of 22 000 and 24 000 g/ mol on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), under both reducing and non-reducing conditions. The cleavage of the heavy chain is inhibited by the presence of C1 esterase inhibitor. The molecular weights of the fragments are in agreement with the cleavage located in the area between the disulphide loops of the α₂-andα₃-domains of the heavy chain. In addition human C1s complement is able to cleave H-2 antigens from mouse in a similar fashion but not rat MHC class I antigen or mouse MHC class II antigen (I-A^d). Mouse MHC class I antigen-specific determinants could also be detected in supernatant from mouse spleen cells incubated with C1r and C1s. These results indicate the presence in the body fluids of a non-membrane-bound soluble form of the α₁andα₂-domains which represent the binding site for atnigenic peptide.     

Restriction by the major histocompatibility complex of antigen-induced T lymphocyte proliferation in new inbred guinea pig strains.

Employing new inbred guinea pig strains, JY 1, JY 2 and JY 3, established in this Institute in addition to strains 2 and 13, the authors investigated histocompatibility restriction in macrophage-T lymphocyte interaction. These five strains are known to possess distinct major histocompatibility complex (MHC) gene profiles (1, 2). This fact was supported by our results concerning the mixed leukocyte reaction (MLR) and cytotoxicity test with alloantisera. Using various combinations of T lymphocytes and peritoneal exudated cells (PECs) from these strains, in vitro proliferative responses of T lymphocytes from BCC-immune animals to PPD-pulsed normal PEC were tested. Successful activation of T cell response was observed not only in syngeneic combinations but also in allogeneic combinations among strains JY 1, JY 3 and strain 13 which share common Ia antigens detected by strain 2 anti-strain 13 alloantiserum. Because JY 1 and JY 3 seem to share a common B antigen differing from strain 13, it was suggested that identification in the I region of MHC is sufficient for effective antigen-presentation by the macrophage. Although a part of Ia is shared, no T lymphocyte activation was observed in the combination between JY 2 and JY 1 or JY 3, whereas strong MLR occurred in these allogeneic combinations. At the present stage of the study, it can be said that disparity in the part(s) of Ia antigens which is responsible for strong MLR cannot lead to effective T cell-macrophage interaction. These results support the concept that functional activation of primed, proliferating T lymphocyte requires the participation of gene products of macrophages coded for by the I region in MHC. By employing JY 1, JY 2 and strain 2, which appear to possess distinct B and Ia antigens, it was shown that the T lymphocyte and macrophage interactions essential for mitogen-induced T lymphocyte proliferation are not restricted by histocompatibility.     

Proteolysis of the heavy chain of major histocompatibility complex class I antigens by complement component C1s.

The major histocompatibility complex (MHC) class I antigens contain a light chain, beta 2-microglobulin, non-covalently associated to the transmembrane heavy alpha-chain carrying the allotypic determinants. Since the C1q complement component is known to associate with beta 2-microglobulin, and we recently found that activated C1s complement was capable of cleaving beta 2-microglobulin, we decided to investigate the proteolytic activity of C1 complement towards the heavy chain of class I antigens. Our results demonstrate that human C1s complement cleaves the heavy chain of human class I antigens into at least two fragments, with apparent molecular weights of 22,000 and 24,000 g/mol on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), under both reducing and non-reducing conditions. The cleavage of the heavy chain is inhibited by the presence of C1 esterase inhibitor. The molecular weights of the fragments are in agreement with the cleavage located in the area between the disulphide loops of the alpha 2-and alpha 3-domains of the heavy chain. In addition human C1s complement is able to cleave H-2 antigens from mouse in a similar fashion but not rat MHC class I antigen or mouse MHC class II antigen (I-Ad). Mouse MHC class I antigen-specific determinants could also be detected in supernatant from mouse spleen cells incubated with C1r and C1s. These results indicate the presence in the body fluids of a non-membrane-bound soluble form of the alpha 1-and alpha 2-domains which represent the binding site for antigenic peptides.     

Restriction fragment length polymorphism of the major histocompatibility complex of the pig

Human HLA cDNA probes were used to analyze the restriction fragment length polymorphism (RFLP) of the SLA major histocompatibility complex in swine. Cellular genomic DNA from 19 SLA homozygous pigs representing 13 different haplotypes was digested with restriction endonucleases Eco RI, Hind III, or Bam H1, separated by electrophoresis, and transferred onto diazobenzyloxymethyl paper by the Southern blot technique. The blots were probed with ³²P-labeled class I or beta-DR class II cDNA. Depending on the haplotypes and the endonucleases used, seven to ten restriction fragments hybridized with the class I probe, and five to seven with the beta-DR probe. Their sizes ranged from 3.4 to 22 kilobase-pairs. Few bands were common to all 13 haplotypes. With all but one haplotype, identical autoradiogram patterns were obtained from unrelated, but phenotypically SLA-identical pigs, suggesting that most of the RFLP revealed were controlled by the SLA region. Further polymorphism was found in a group of seven unrelated pigs which typed serologically as SLA A15 CI B18 homozygotes but could be divided into two subgroups, with five animals in one subgroup and two in the other, when the genomic DNA was hybridized with the class I probe. When the class 11 beta-DR probe was tested on the same seven pigs, another subdivision was seen, and this correlated with MLR data. These results demonstrate that HLA class I and class II probes can be used to identify certain well-established SLA haplotypes and to identify subclasses within at least one SLA haplotype.Abbreviations MHC major histocompatibility complex - MLR mixed lymphocyte reaction - kbp kilobase pair(s) - RFLP restriction fragment length polymorphism