Alloimmune reactions among recently wild syrian hamsters and classical inbred strains include alloantibody production

Partially inbred lines of Syrian hamsters, the descendants of animals caught wild in Syria within the past decade, have been studied to determine their immunogenetic relationships to the long established domestic inbred strains. These new sources of hamster genes display strong alloimmune reactions when confronted with tissues from the domestic inbred strains and vice versa: acute skin graft rejection, strong mixed lymphocyte reactions, and intense graft-versus-host reactions. While these forms of reactivity have also characterized the disparities among the domestic inbred strains, no evidence of alloantibodies specific for putative transplantation antigens has ever been found in hamsters. We report, for the first time, that immunization of recently wild hamsters with tissues from domestic inbred strain donors elicits high titer cytotoxic antibodies directed at alloantigens. Similarly, animals of the domestic inbred strains produce comparably strong alloantibody responses when immunized with tissues from the recently wild hamsters. Availability of these alloantisera will make the possibility of defining the major histocompatibility complex equivalent in this species much more likely.     

Analysis of the major histocompatibility complex in Syrian hamsters. III. Cellular and humoral immunity to alloantigens.

Among the genetic loci incorporated into the major histocompatibility complex in every species studied to date have been prominent genes encoding for strong histocompatibility determinants that elicit detectable alloantibody responses and which are the chief antigenic targets of cell-mediated cytotoxicity reactions. The K and D regions of the H-2 complex in the mouse and the A, B, and C regions of the HLA complex in man are representative examples. Syrian hamsters, as described in this report, do not make alloantibodies to antigens of this type and only very poorly do they carry out in vitro cell-mediated cytotoxicity to target cells putatively bearing these antigens. Since hamsters are quite capable of discriminating analogous antigenic differences in xenogeneic species, and xenogeneic sources cannot distinguish immunologically between the antigens encoded by the two hamster major histocompatibility alleles. Hm-1a and Hm-1b, we conclude that the hamster strains we work with are serologically indistinguishable by the methods used here. However, they obviously differ for determinants which elicit T cell-mediated responses, as evidenced by their ability to express acute skin graft rejection, mixed lymphocyte reactivity, graft-vs-host reactions, and cell-mediated cytotoxicity reactions. Such alloreactivity may reflect a mutation at an SD locus, affecting antigenic sites recognized only by T cells, or that the available hamster strains are SD identical, but differ at loci similar to the I region loci in mice. Alternatively, we cannot exclude the possibility that Syrian hamsters somehow fail to express properly the genes coding for SD determinants.     

Immune response to acute virus infection in the Syrian hamster

Syrian hamsters show evidence of classical T-cell-mediated immune reactivity to acute virus infection as judged by primary foot pad swelling, kinetics of in vitro cytotoxic activity, and virus specificity of cytotoxic effector cells. In spite of this, no evidence of genetic restriction is observed among the variety of allodisparate inbred strains tested. This virus-induced, cell-mediated killing extends across strain barriers despite strong cellular and serologic alloreactivity among some of the strains utilized. To account for the apparent lack of genetic restriction, we currently favor the hypothesis that all hamsters examined thus far share at least one class I MHC antigen. Since these animals differ at hamster loci which elicit MLR, GVHR, acute SGR, CML, and alloantibody, we presume class II MHC polymorphism exists in this species. The presence of putative class II MHC polymorphism without detectable class I MHC polymorphism is unusual among mammals examined to date, and of unknown biologic significance.     

Evidence for two populations of B-L (Ia-Like) molecules encoded by the chicken MHC

Two specific alloantisera detecting B-L (Ia-like) antigens on chicken lymphocytes of the B ⁶ and B ¹⁵ haplotypes were found to cross-react strongly. Anti-B-L6 and anti-B-L15 alloantisera both reacted with B-L molecules on B6 and B15 lymphocytes as demonstrated by immunofluorescence and SDS-PAGE analysis of ¹²⁵I-labeled B-L antigens isolated by incubation with anti-B-L alloantisera. Absorption studies showed that the anti-B-L alloantisera reacted with at least two kinds of antigenic determinant, one set shared by B-L6 and B-L15 molecules and another set specific for each haplotype. In spite of the absence of genetic evidence for more than one B-L locus in the chicken B complex, it was shown by sequential antibody incubations that these two different B-L antigenic determinants are associated with at least two separate species of B-L molecules, indicating the presence of at least two B-L loci within the MHC of the chicken.     

TheD17Tu5 locus in thet complex: implications for the origin oft haplotypes and inbred strains

The mouse × Chinese hamster cell line R4 4-1 contains only one mouse chromosome, the bulk of which corresponds toMus musculus chromosomes 17 and 18 (MMU17 and MMU18, respectively). A genomic library was prepared from the R4 4-1 DNA, and a mouse clone was isolated from the library, which—with the help of somatic cell hybrids-could be mapped to the MMU17. A locus defined by a 2.7-kb longBam HI probe from this clone was designatedD17Tu5 (Tu for Tübingen). The locus proved to be polymorphic among inbred strains and wild mice. By testing of recombinant inbred strains and partialt haplotypes, theD17Tu5 locus could be mapped to a position between theD17Leh66E andD17Rp17 loci within thet complex. Two alleles were found at this locus,D17Tu5 ^a andD17Tu5 ^b , defined byTaq I restriction fragment length polymorphism. Both alleles are present among inbred strains and wild mice of the speciesM. domesticus. All completet haplotypes tested carry theD17Tu5 ^a allele and all tested wild mice of the speciesM. musculus, with the exception of those bearingt haplotypes, carry theD17Tu5 ^b allele. Additional alleles are found in some populations of wild mice and in other species of the genusMus. The distribution of the two alleles among the inbred strains correlates well with their known or postulated genealogy. Their distribution between the two species ofMus and among the mice withT haplotypes suggests a relatively recent origin of thet haplotypes.     

Syrian hamsters express diverse MHC class I gene products

MHC class I glycoproteins are highly diverse in most species. The Syrian hamster has long been thought to express monomorphic MHC class I molecules and thus be an exception to this rule. Here we show that Syrian hamsters express diverse MHC class I gene products. The nucleotide sequences of the alpha 1 and alpha-2 domains of classical Syrian hamster MHC class I molecules are highly variable and show evidence of having been under selective pressures at their Ag recognition sites. Interestingly, none of the Syrian hamster class I genes was closely related to their counterparts in the mouse. These observations suggest that Syrian hamsters in the wild may express diverse MHC class I molecules.     

Polymorphism of the major histocompatibility locus in the wild Norway rat

Specific alloantisera against the eight Ag-B groups found in inbred strains of rats were capable of reacting with all wild Norway rats (Rattus norvegicus) tested. Absorption studies, antisera production, and progeny testing involving wild rats showed that the antigenic specificities detected in the wild rat population were similar, if not identical, to the Ag-B antigens present in inbred strains. Xenoantisera prepared in rabbits against rat erythrocyte antigens (Ag-C1 and/or C2) reacted with erythrocytes from each wild rat tested. Progeny testing involving these erythrocyte antigens was identical to that observed in inbred strains. The restricted genetic polymorphism of theAg-B alleles in the wild rat population suggests that the functional and evolutionary significance of the major histocompatibility complex in the rat may not depend upon a high degree of genetic variability.     

The expression of Mls c determinants on Mls a,Mls b,and Mls x prototypic strains

In the mouse sytem, specific determinants other than major histocompatibility complex (MHC) gene products are capable of inducing strong primary proliferative responses in naive T cells. These determinants are encoded by at least two gene loci designated as minor lymphocyte stimulatory (Mls) loci. In order to elucidate the biological role of the Mls system, an effort has been initiated to clarify the fundamental immunogenetic characteristics of the Mls system. In this report, we describe the unexpected finding that Mls ^c determinants are expressed on splenocytes of strains including those which have been used as prototypic examples of three other Mls types: Mls ^a (DBA/2, DBA/1), Mls ^b , (BALB/c), and Mls ^x (PL/J). The expression of Mls ^c by these strains was demonstrated both by the response patterns of unprimed T cells from MHC-identical inbred or F₁ hybrid strains and by the responses of a panel of Mls-specific T-cell clones. The experimental results reported here also suggest that the expression of Mls determinants may be influenced by multiple other genes, including MHC-linked genes.Abbreviations used in this paper MHC major histocompatibility complex - MLR mixed lymphocyte reaction - Mls minor lymphocyte stimulating locus antigen - MMC mitomycin C - NNT nylon wool nonadherent T cells     

Comparative genetics of hamster amylases

Syrian (Mesocricetus auratus) and Chinese (Cricetulus griseus) hamsters were phenotyped by electrophoresis for salivary and pancreatic amylases. Syrian hamsters possess two salivary amylase electromorphs, the more anodal (fast) being invariant in 250 outbred and 17 representatives of 5 highly inbred lines. The slow electromorph had activity equal to that of the fast amylase (heavy), or had distinctly less activity (light), or was absent (null). The slow electromorph is inherited as an autosomal semidominant trait with two alleles, Amy ^s and Amy ^o . Amy ^s homozygotes produce heavy, Amy ^o homozygotes null, and heterozygotes light phenotypes, respectively. Five inbred strains of hamsters were homozygous Amy ^o . Pancreatic amylase was monomorphic. Eight outbred Chinese hamsters showed no salivary amylase activity with electrophoresis, but slight activity with long incubation on starch-agar plates. However, pancreatic amylase activity in the Chinese hamster exceeded that in Syrian hamsters. Site duplication and apparent null alleles for amylase genes occur in muroid rodents. The evolutionary implications are discussed.George H. Bunch Chair Professor of Biology     

New mouse immunoglobulin a heavy chain allotype specificities detected using the hybridoma-derived IgA of I/St Mice

Immunizations of C57BL/6 and A mice with IgA derived from the I/St mouse strain yield alloantisera which detect two allotypic determinants of immunoglobulin A. The two determinants display discrete strain distributions. The first, identified by the alloantiserum C57BL/6 anti-IgA of I/St strain hybridoma ID150, follows the Igh ^c haplotype, and the second, identified by the alloantiserum A anti-IgA of I/St strain hybridoma ID150, correlates with Igh ^c and Igh ^c haplotypes. Absorption with monoclonal IgM, which has the same idiotype as the ID150 IgA clone, removed idiotype-specific antibodies from both alloantisera. The remaining antibodies are directed against determinants associated with the chain constant region, as shown by absorption with monoclonal IgA. By use of recombinant inbred strains of mice and mice congenic at the Igh locus, the loci controlling both C allotypic determinants have been mapped to the Igh region on chromosome 12.Abbreviations used in this paper Ig immunoglobulin - NMS normal mouse serum (sera) The genetic nomenclature of Green (1979) for mouse immunoglobulin loci was used in this report.     

Molecular and immunogenetic analysis of major histocompatibility haplotypes in northern bobwhite enable direct identification of corresponding haplotypes in an endangered subspecies,the masked bobwhite

The major histocompatibility complex (MHC) is a group of genetic loci coding for haplotypes that have been associated with fitness traits in mammals and birds. Such associations suggest that MHC diversity may be an indicator of overall genetic fitness of endangered or threatened species. The MHC haplotypes of a captive population of 12 families of northern bobwhites (Colinus virginianus) were identified using a combination of immunogenetic and molecular techniques. Alloantisera were produced within families of northern bobwhites and were then tested for differential agglutination of erythrocytes of all members of each family. The pattern of reactions determined from testing these alloantisera identified a single genetic system of alloantigens in the northern bobwhites, resulting in the assignment of a tentative genotype to each individual within the quail families. Restriction fragment patterns of the DNA of each bird were determined using the chicken MHC B‐G cDNA probe bg11. The concordance between the restriction fragment patterns and the alloantisera reactions showed that the alloantisera had identified the MHC of the northern bobwhite and supported the tentative genotype assignments, identifying at least 12 northern bobwhite MHC haplotypes. Eighteen northern bobwhite alloantisera were then used to detect a minimum of 17 masked bobwhite MHC haplotypes. Subsequent restriction fragment pattern analyses using cDNA probes for chicken MHC genes were in agreement with agglutination patterns displayed by the antisera, showing that the immunogenetically identified alloantigen system constituted the MHC of the masked bobwhite. These data demonstrate that a non‐endangered species may be used to provide antisera for differentiating MHC haplotypes in a closely related endangered species, thus providing a practical basis for long‐range monitoring of MHC haplotypes of birds surviving in their native habitats. Zoo Biol 18:279–294, 1999. © 1999 Wiley‐Liss, Inc.     

MHC recognition by clones of Mls specific T-lymphocytes

To test whether M1s determinants, like other non-MHC or nominal antigens, are recognized by T-cells in association with H-2 determinants, the in vitro proliferative responses of T-cell lines and clones were studied. Lines and clones were prepared by soft agar cloning (B10.BR x BALB/c)F₁ (H-2^k/H-2^d, M1s^b/M1s^b) T-cells responding in a primary MLR to AKD2F1 (H-2^k/H-2^d, M1s^a/M1s^a) stimulator cells. All the T-cell clones obtained could respond equally well in a proliferative assay to the Mls^a determinant in association with the H-2 haplotype of either parent, i. e., DBA/2 (H-2^d, M1s^a), and AKR (H-2^k, M1s^a) both stimulated equally well. When the T-cell lines and clones were screened against stimulators from recombinant inbred (RI) strains, it became apparent that strains exhibiting the H-2^b, M1s^a genotype stimulated poorly or not at all. This shows that the T-cell response to M1s^a involves MHC recognition, and raises the possibility that the response to M1s^a can involve recognition of H-2 specificities shared between the H-2 ^k and H-2 ^d haplotypes.Abbreviations used in this paper MHC major histocompatibility complex - MLC mixed lymphocyte culture - IL-2 interleukin 2 - Con A concanavalin A - RI recombinant inbred Howard Hughes Medical Institute     

Evolutionary relationships between the t and H-2 haplotypes in the house mouse

Thirty-three mouse strains carrying t haplotypes were typed with a large battery of monoclonal and polyclonal antibodies specific for class I and class II antigens controlled by the H-2 complex. Among these t haplotypes were representatives of the six complementation groups defined previously and of eight new groups defined by us recently. The typing resulted in the identification of the H-2 haplotypes of these strains and of their alleles at K, D, A, and E loci. Nineteen of the 33 strains proved to carry a mutation that prevents the expression of the E molecule on the cell surface. All H-2 haplotypes of the t strains are related in terms of sharing certain antigenic determinants, most of which have not, as yet, been found in inbred strains or in wild mice that do not carry t haplotypes. According to the degree of serological relatedness, the haplotypes can be arranged into a pedigree presumably reflecting the evolutionary history of the t chromosomes. The ancestral t chromosome from which the 33 chromosomes derive was presumably present in the mouse population before the divergence of the Mus musculus and Mus domesticus species. The E° mutation, too, is apparently ancient because it occurs in different branches of the evolutionary tree.     

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.     

Analysis of the B-G antigens of the chicken MHC by two-dimensional gel electrophoresis

The B-G antigens of the chicken major histocompatibility complex (MHC) have been analyzed by high resolution two-dimensional (2-D) gel electrophoresis. Monoclonal antibodies recognizing a widely shared B-G determinant were used for immunoprecipitating the B-G antigens from radioiodinated, detergent-solubilized erythrocyte membrane preparations. The B-G antigens produce a variety of patterns on 2-D gels. The number of polypeptides within a B-G pattern varies among haplotypes from single polypeptide arrays showing slight microheterogeneity to complex patterns which contain as many as four or five polypeptide arrays differing in relative mobility and isoelectric point. Many of the patterns, but not all, include a polypeptide of Mr =48 kd focusing near pH 6.9. At present it is not understood whether the multiple polypeptides within some B-G patterns represent the expression of multiple B-G genes or whether they are the result of modifications of single gene products during biosynthetic processing. 2-D gel analyses were also used to confirm the assignment of the same B-G haplotype in several different inbred flocks and the fate of the B-G antigens in two B system recombinant haplotypes. The 2-D gel patterns of these highly polymorphic antigens provide evidence for a complexity of the B-G locus not previously demonstrated. This technique may serve to define more objectively the diverse chicken MHC haplotypes which are now recognized and characterized only by serological techniques using alloantisera and monoclonal antibodies with varying cross-reactivities.     

Phosphoglucomutase electrophoretic variants in the mouse

The phosphoglucomutase (PGM) electrophoretic phenotype of the mouse (Mus musculus) consists of several distinct components which can be grouped into two major zones designated PGM-1 and PGM-2. Evidence presented here indicates that each zone is controlled by a single genetic locus denoted Pgm-1 and Pgm-2, respectively. Two variant forms segregated at the Pgm-1 locus. They were codominantly expressed and inherited as alleles at an autosomal locus. The alleles were termed Pgm-1 ^a (fast) and Pgm-1 ^b (slow). These alleles were separately fixed in a number of inbred strains of mice. Preliminary evidence based on wild mouse phenotypes indicates that variant forms also exist for PGM-2 which are inherited as alleles at an autosomal locus. Genetic linkage relationships have not been determined for these loci. PGM-1 variants and PGM-2 were expressed in mouse fibroblasts in vitro.Supported by U.S. Public Health Service grants GM-09966 and GM-07249 from General Medical Sciences and 5 F2 HD-35,531 from Child Health and Human Development; and Atomic Energy Commission contract AT(30-1)-3671.Postdoctoral Fellow of the U.S. Public Health Service.     

A serum protein polymorphism determinant on chromosome 9 of Mus musculus

Summary Genetic polymorphism for a previously undescribed serum protein has been found among inbred strains of Mus musculus. The new serum protein locus, gene symbol Sep-1, has been located on Chromosome 9, gene order Lap-1-Sep-1-Mpi-1-d-Mod-1, by utilizing information obtained from 52 recombinant inbred strains together with standard genetic backcrosses. The strain distribution pattern for this locus, supernatant malic enzyme, and transferrin, also on Chromosome 9, are given for 67 inbred strains. Because the genotype of SEP-1 can be determined for individual mice without killing them, Sep-1 is a very useful gene in linkage studies and experimental biology.     

Ia antigens in inbred rats and their relationship to the MLR phenotype.

Three different alloantisera were raised by using Ag-B/MLR disparate rats, and the cytotoxic activity remaining after absorption with erythrocytes to remove anti-Ag-B antibodies was examined. The alloantisera detected surface antigens present only on B cells and segregation studies demonstrated that the genes that code for these antigenic specificities were linked to the major histocompatibility complex. The reactivity of the alloantisera with splenic lymphocytes from a panel of strains representative of the currently known Ag-B groups showed that multiple specificities were present in two of the three antisera and that these specificities were shared by many inbred strains. The appropriate absorption studies showed, however, that each antiserum detected an unique specificity that was found only in those inbred strains that shared the same mixed lymphocyte reactivity (MLR) phenotype as the donor strain. The alloantiserum produced against the KGH strain inhibited the MLR reactions involving this strain only when it was used as the stimulating cell population. The antigens detected by the three alloantisera described here have the characteristics of Ia antigens, and they have tentatively been designated Ia.1 (ACI anti-KGH), Ia.3 (B3 anti-BN) and Ia.4 (MNR anti-DA).     

Genetic and immunological characterization of naturally occurring recombinant B3 rats

The B-stock population of rats was bred for homozygosity at the loci controlling coat color. In this process, theAg-B1 andAg-B3 haplotypes became fixed in Hardy-Weinberg equilibrium. Extensive immunization and absorption studies showed that the specificities in the B-stock rats homozygous for theAg-B1 haplotype were the same as those found in the inbred F344 strain (Ag-B1), and that the specificities in the rats homozygous for theAg-B3 haplotype were the same as those found in the inbred BN (Ag-B3) strain. A homozygous line derived from the rats carrying theAg-B3 haplotype (B3) has the mixed lymphocyte reactivity and antibody responsiveness to poly (Glu⁵²Lys³³Tyr¹⁵) characteristic of the inbred strains in theAg-B4 group. Thus, it represents a naturally occurring recombination between the loci controlling MLR and immune responsiveness, on the one hand, and those controlling the Ag-B antigens on the other. Antibody responsiveness segregated with theAg-B3 haplotype in crosses between the B3 homozygotes and the low responder BUF and M520 strains; hence, this recombination is a stable one. There was no linkage of antibody formation or haplotype to coat color. The finding of a strain with a naturally occurring recombination in the major histocompatibility complex between the loci controlling mixed lymphocyte reactivity and the Ag-B histocompatibility antigens provides evidence for the separateness of these loci. Since the portion of the genetically determined mechanism controlling antibody responsiveness which is linked to the MHC was that characteristic of the MLR type, it too must lie outside the region defined by the serological specificities of theAg-B haplotype.