Chain Allotype Research Articles

Two genetic loci control the murine immune response to A-gliadin, a wheat protein that activates coeliac sprue.

Coeliac sprue is a disease in humans that is characterized by small intestinal mucosal injury and malabsorption. Ingestion of dietary gluten by susceptible individuals results in damage to the small intestine. The gliadin fractions of wheat gluten and similar proteins in rye and barley are known to activate the disease process1. The major histocompatibility complex (MHC) alleles HLA-B8, Dw3 and DRw3 occur at a high frequency in coeliac sprue2–5, suggesting that immunoregulatory mechanisms may be important in the pathogenesis of the disease. We recently reported that murine antibody responses to A-gliadin, a well characterized protein known to activate coeliac sprue1,6–9, are regulated by genes that map to H–2, the murine MHC10. However, it appeared that an additional genetic locus also might be important in regulating the immune response to gliadin proteins. We show here that two separate genetic regions determine the murine antibody response to A-gliadin: H–2 on chromosome 17 and the immunoglobulin heavy chain (CH) allotype locus (Igh) on chromosome 12. Possible ways in which these two loci could determine the response against A-gliadin are discussed.

Multiple sequences related to a constant-region kappa light chain gene in the rabbit genome

Four allelic genes control kappa light chain allotypes in the rabbit. Amino acid sequence studies have revealed an extensive divergence (22%–33%) in the alternative forms of the kappa constant region (b4, b5, b6 and bg). Furthermore, independent studies have shown that a rabbit could express a wrong allotype. To assess the hypothesis that b allotypes are encoded by duplicated genes with a polymorphic control mechanism, we have analyzed the DNAs of four different homozygous rabbits using the Southern blot hybridization technique, with a cloned b4 C κ probe. DNAs of individual b4, b5, b6 and b9 rabbits were cleaved with Eco RI, Kpn I and Pst I restriction endonucleases. Comparative analysis of restriction patterns shows that all four rabbit DNAs contain multiple DNA fragments hybridizing with the probe under low- and high-stringency washing conditions. In addition, each restriction pattern is distinct, suggesting that the C κ genes are organized differently in animals expressing different allotypes.

A subpopulation of small pre-B cells in rabbit bone marrow expresses kappa light chains and exhibits allelic exclusion of b locus allotypes.

The expression of immunoglobulin heavy and light chains by pre-B cells and B lymphocytes was examined by two-color immunoflourescence in heterozygous b5b9 rabbits. Allelic exclusion of b5 and b9 kappa light chain allotypes was observed for both surface immunoglobulin-negative pre-B cells and surface immunoglobulin-positive B lymphocytes. In newborn bone marrow, pre-B cells and immature B lymphocytes expressing b9 were as numerous as those expressing b5. In contrast, circulating B cells and bone marrow plasma cells expressing the b5 marker outnumber b9+ cells by 2 to 1 in adult b5b9 animals. Whereas most B lymphocytes expressed kappa light chain b allotypes, approximately 80% of the micro heavy chain-positive pre-B cells did not. The pre-B cells that expressed detectable light chains were relatively small lymphocytes. A model is presented which includes a "transitional" pre-B cell that expresses both micro chains and kappa chains.

Production of IgG-specific auto-anti-allotypes in b4.2-suppressed rabbits.

Rabbits were completely suppressed for kappa chain allotype b4.2, and autoantibodies against b4.1 or b4.2 could be raised in 2 out of 3 animals. The animal immunized with b4.1 produced anti-b4 and anti-Ms3, two activities which have never as yet been found together in one antiserum. Both autoantisera lacked the capacity to bind b4.2-IgM, whereas they precipitated 4.2-IgG very well. In one animal, anti-b4 IgM activity appeared after the sixth immunization, at the age of 14 months. Allotype suppression was maintained in both rabbits till the end of their lives whereas all control animals recovered within 6 months. The autoantisera which were specific for b4 IgG could not induce suppression in vivo. However, specific inhibition of b4 IgG secretion was observed in vitro.

Construction of a partial rabbit spleen cDNA library and identification of immunoglobulin clones.

A partial cDNA library was constructed from total poly A(+)-RNA isolated from the spleen of a rabbit (kappa allotype b5; heavy chain allotypes a3d12e15) that had been hyperimmunized with Streptococcus pneumoniae (type III). In spite of the absence of either specific DNA probes for rabbit immunoglobulin (Ig) sequences or cross-hybridizing mouse Ig DNA probes, recombinant clones containing cDNA sequences of rabbit gamma heavy chain and kappa light chains were identified by a combination of screening techniques: (a) colony hybridization using labeled mRNA; (b) mRNA hybridization selection and translation and (c) hybridization to electrophoretically fractionated poly A(+)-RNA ("Northern" analysis). Sequencing of three kappa light chain recombinant DNA sequences, including part of the 3' untranslated (UT) region, has confirmed the fact that recombinant DNA for kappa light chain mRNA has been identified. An unexpectedly high degree of homology between the 3' UT region sequence of this DNA from a rabbit of b5 allotype and the published 3' UT sequence from a b4 rabbit was found. It appears that 3' UT sequences from b4 and b5 alleles have diverged less than the coding sequences for the constant regions. The functional significance of this conservation of 3' UT sequences remains to be elucidated.

Rat Kappa Chain Allotypes. IV. Monoclonal Antibodies to Distinct RI-1b Specificities

Four hybridomas are described which produce antibodies to the RI-1b rat kappa chain allotype. Three are rat alloantibodies of KG2b isotype, one is a mouse heteroantibody of KG2a isotype. These antibodies were labeled by incorporation of 75Se-selenomethionine and their specificity determined by a plate-binding assay. Inhibition studies using partially cross-reactive sera from Asian and Australian Rattus show that these four hybridomas detect four distinct RI-1b specificities. Differences in the slopes of the inhibition curves indicate that each of the specificities may exist in more than one form. In addition, in the form of 125I-labeled antibodies, two of these hybridomas show a high degree of specificity in binding to Ig-bearing lymphocytes from spleen and lymph nodes. These hybridomas will therefore be useful not only in studies on the phylogenetic distribution of RI-1 specificities but also in replacing conventional antisera for immunochemical and cellular immunological studies.

Cytotoxic T cells specific for antigens expressed on surface immunoglobulin-positive cells.

C.B-20 mice were immunized with splenocytes or B leukemia cells (BCL1) from Ig H chain allotype congenic strains. Spleen cells from these immunized mice were rechallenged in vitro to generate H-2-restricted cytotoxic T cells that were specific for target antigens controlled by genes linked to the Ig H chain locus. The anti-Ig H cytotoxic T cells detected an antigen(s) expressed only on surface Ig+ cells. Thus, T cell lymphoblasts, eight BALB/c myeloma cell lines, and a T cell lymphoma were not lysed by the effector cells. In contrast, B cell lymphoblasts and the surface Ig+ BCL1 cells were sensitive to lysis. A surface Ig- hybridoma (which secretes the IgM from the BCL1 cells) generated by fusing BCL1 cells to X63 myeloma cells was not killed by the effector cells. These data indicate that cytotoxic T cells specific for antigenic determinants on either surface IgM+ or IgD+ or on a molecule that is coordinately expressed on IgM+ or IgD+ cells can be generated and that such cells might play a role in regulating the growth of normal B cells or surface Ig+ tumor cells in vivo.

Idiotypes of anti-Ia antibodies. I. Expression of the 14-4-4S idiotype in humoral immune responses.

The idiotype of a mouse monoclonal anti-I-E antibody, 14-4-4S, has been studied using a heterologous anti-idiotypic reagent. This antibody recognizes Ia. 7, an antigenic specificity present in all strains expressing a product of the I-E subregion. Expression of the 14-4-4S idiotype in humoral immune responses was analyzed by an idiotype-specific enzyme-linked immunosorbent assay system. The idiotype was readily detectable in C3H.SW anti-C3H alloantisera, the same immunization combination from which the hybridoma was derived. Absorption analysis demonstrated the anti-I-E specificity of the idiotype-positive molecules in these alloantisera. Penetrance of idiotype expression was high among individual C3H.SW immune mice (9 of 10 tested). To examine genetic requirements for idiotype expression, an immunization was performed using as responders CWB mice, congenic with C3H.SW but differing at the heavy chain allotype loci. Immune sera of individual CWB mice contained very little or no idiotype, demonstrating that levels of idiotype expression are influenced by allotype-linked genes, although the influence of other genes has not been ruled. The 14-4-4S idiotype therefore represents a shared idiotype of anti-Ia antibodies and provides opportunities for analysis of the idiotypes of cellular receptors for the corresponding Ia antigen.

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 Ighc haplotype, and the second, identified by the alloantiserum A anti-IgA of I/St strain hybridoma ID150, correlates with Ighc and Ighb 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 alpha 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 alpha allotypic determinants have been mapped to the Igh region on chromosome 12.

Role and strain distribution of genes controlling light chains needed for the expression of an intrastrain cross-reactive idiotype.

Several strains of mice were tested for their capacity to provide immunoglobulin L chains required for the expression of the major cross-reactive idiotype (CRIA) associated with p-azophenylarsonate-specific antibodies of strain A mice. To facilitate testing, mice were bred that were homozygous for Igh-Ce and Lyt-2a, 3a, i.e., they possessed genes controlling H chains but not L chains required for expression of the CRIA. Such male mice were mated to females of various strains and their offspring were tested; expression of CRIA indicated the presence in the female parent of genes controlling the appropriate L chains. All females bearing the Lyt-2a, 3b or Lyt-2b, 3b genotype yielded offspring, most of which were CRIA+, whereas all the offspring of females that were Lyt-2a, 3a were CRIA-. The female parents included mice of several strains that are congenic for Lyt-2a, 3a, Lyt-2b, 3b or Lyt-2a, 3b, thus demonstrating very close linkage between the Lyt loci and the expression of CRIA. In addition, doubly congenic strains of mice with the heavy chain allotype of the CRIA+ AL/N strain and the Lyt-2a, 3a genotype on a BALB/c background failed to express CRIA. The data provide further evidence for the similarity of repertoires of L chains in Lyt-3b mice of various strains. When genes were present controlling A/J H chains and L chains of C57BL/6 or BALB/c origin, the quantitative expression of CRIA was only slightly lower than that observed in A/J mice. Mice possessing genes controlling the H or L chains required for CRIA expression, but not both, did not express CRIA but synthesized Ar-specific antibodies which contained low but significant concentrations of the idiotype-associated chain.

Allelic polymorphism of murine IgE controlled by the seventh immunoglobulin heavy chain allotype locus.

Two alloantisera against hybridoma-derived IgE detected allotypic determinants expressed on the murine epsilon chain. An antiserum raised in BALB/c mice against monoclonal IgE of C57BL/6 origin reacted exclusively with IgE of strains having Igh-1b (IgG2 a) allotype. The second antiserum, C57BL/6 anti-BALB/c monoclonal IgE, reacted with IgE of strains having Igh-1a, Igh-1d, Igh-1e and Igh-1j allotypes. The genetic studies of (BALB/c x C57BL/6)F1 and backcross F2 animals indicated that the locus controlling the IgE allotype is linked to the Igh-1 locus. This was further confirmed by the possession of respective IgE allotypes by Igh-C congenic mice, BALB/c and BAB-14, C3H.SW/Hz and CWB/Hz. Thus, the allotype detected on the epsilon chain is controlled by the seventh murine immunoglobulin allotype locus, and should be designated as the Igh-7 allotype.

Genetics of the anti-dextran B512 and the autoanti-idiotypic response: Codominant expression in F1 hybrids and dichotomy of response and allotype-linked idiotype

The IgM plaque-forming response to the alpha 1-6 epitope of dextran B512 is linked to the Ig-1 heavy chain allotypes j and b characteristic of CBA and C57BL strains, respectively, and the response typically induces the formation of autoanti-idiotypic antibodies that can distinguish between anti-dextran antibodies of CBA and C57BL origin. Nevertheless, some substrains of Balb/c mice (allotype a) and some Bailey recombinant stains give a PFC response although they do not possess allotypes j or b. The anti-dextran antibodies in these strains lack the idiotypes characteristic of either CBA and C57BL antibodies to dextran, but they possess their own particular idiotype. F1 hybrids between two responder strains possessing different idiotypes on their antibodies against dextran, produce both idiotypes and two different autoanti-idiotypic antibodies. CBA(Ig-1b) mice were high responders to dextran and possessed the idiotype of C57BL, whereas C57BL/6(Ig-1a) mice were low responders. The V(H) recombinant strains BAB.14 and CB-8KN that possess the Ig-1b allotype of C57BL, but have some of the V(H) genes from Balb/c and the rest from C57BL/6 were high responders to dextran, but did not possess the C57BL idiotype, suggesting that the genes determining the response against dextran and the idiotype may have different locations in the heavy chain locus.

Rabbit T cells with and without Fc receptors

Earlier, indirect evidence for rabbit subpopulations differing in Fc receptors and in response to mitogen has been directly tested. T cells were purified from spleen suspension by removal of adherent cells, followed by removal of Ig-bearing cells on petri dishes coated with antibody, directed against the light chain allotype of Ig receptors. The purified cells were further fractionated by formation of EA rosettes and separation on Ficoll-Hypaque. T cells which lacked Fc receptors had a larger response when stimulated with Con A or PHA than did T cells which possessed Fc receptors. Both subpopulations responded more when irradiated nonadherent B cells were added to the mixture, but the extent of help was the same for both cell populations. T cells which contained both Fc receptor-bearing cells and cells which lacked the receptor had a response which was intermediate between that of the two separated subpopulations.

Polyclonal activation of Ts cells with antiserum directed against an IGH‐1 linked candidate for a T‐cell receptor constant region marker

An anti-T cell serum raised in allotype congenic mice recognizes the product of a new locus coding for a heavy chain-linked polypeptide found on a subpopulation of T cells. Anti-Tsd raised in BALB/cAnN mice against selected C.AL-20 T cells reacts with a cell surface antigen in virgin animals that is found on 25% of mature thymocytes and Lyt-bearing T cells, but not on prothymocytes, Lyt1 T cells or B cells. The antigen is restricted to strains bearing the Ig-1d and Ig-1e heavy chain allotype haplotypes, and is expressed in the F1 animal. The antigen is unlinked in expression to the Lyt2, H-2, or kappa light chain loci. The antigen is not detected in the hematopoietic cells in the bone marrow and appears to mark only the mature peripheral pool of T cells. As previously reported, the antiserum blocks the binding of suppressor T cells to the cross-reactive idiotype for arsonate, while reagents specific for Fab, Fc and Ig were ineffective. It seems probable that the marker may represent a T cell constant region marker analogous to the Igh products on immunoglobulin. Antiserum against this marker induces in vivo triggering of Ts cells for a wide variety of T-dependent antigens. All subclasses of anti-hapten antibodies are suppressed; no affinity restrictions or clonotype specificity is observed in suppressed adult mice. Results suggest that precursor T cells regulating major serum idiotypes regulate individual idiotypes.

Multigenic regulation of the primary immune response to ovalbumin in mice

At least four genes regulate the primary immune response to ovalbumin in mice. The ability to be sensitized to transfer delayed type hypersensitivity to ovalbumin is controlled by two genes. One gene,OVA-β, is linked to theH-2 complex and maps to the left ofI-E. The linkage of the other gene,OVA-Bg1, has not been determined, but it segregates independently of theLy M locus, of the heavy chain allotype genes and of certain genes controlling coat color. At least two genes regulate the ability to respond with a primary antiovalbumin antibody response. One gene,OVA-α, is linked to theH-2 complex and maps to the right ofI-E. Discordance of the minimum dose of antigen needed to elicit delayed type hypersensitivity response and antibody suggests that non-H-2 gene(s) regulating the primary antibody response are different fromOVA-Bg1.

A mature T lymphocyte subpopulation marker closely linked to the Ig‐1 allotype CH locus

An antiserum raised in BALB/c AnN mice against selected CAL.20 T cells reacts with a cell surface antigen in virgin animals that is found on 25% of mature thymocytes and Lyt-2-bearing T cells, but not on prothymocytes, Lyt-1 T cells or B cells. The antigen is restricted to strains carrying the Ig-1d or Ig-1e heavy chain allotype haplotypes. It is expressed in F1 mice. The antiserum blocks the binding of suppressor T cells to the cross-reacting idiotype for arsonate while reagents specific for Fab, Fc or Ig were ineffective. We suggest that the antigen described represents a determinant on the product of a new locus coding for a heavy chain-linked polypeptide found on a subpopulation of T cells.

A cross-over chromosome combiningIg heavy chain genes of two mouse strains

Two congenic strains of mice were bred in Konstanz that bear theIg heavy chain allotype gene of the C57BL/6 (Ig-1 b) in a BALB/c background genome. One line (CB-8K) underwent eight backcross generations to BALB/c before sister-brother mating was initiated. For the other line (CB-16KN) backcrossing was continued for eight further generations, then a homozygousIg-1 b/Ig-1 b strain was produced by sister-brother mating. Both lines were tested for four VH markers of the BALB/c and one of the C57BL parent. The CB-16KN strain expressed the C57BL marker VHNPb together with the C57BL allotype marker, and failed to express the three BALB/c markers, VHDEX, VHS117, VHphOx and VHNPa. It thus resembled the CB-20 strain.Strain CB-8K expressed the VHNPb marker of C57BL but also all the four BALB/c markers that were tested. The strain appeared more heterogeneous than the CB-16KN strain, and a subline was bred from two exceptional mice that did not express the VHNPb marker. This subline (CB-8KN) expressed the BALB/c marker VHNPa regularly, and was negative for the VHNPb marker. It thus resembles the BAB-14 line.The crossing over event thus must have happened in one of the two meioses, which led to the CB-8K line. As BAB-14 is derived in an analogous manner to a branch of the backcross of Potter and Lieberman, which ended up in CB-20, the unexpected finding is discussed that two independent crossing over events (in CB-8KN and BAB 14) within theIg heavy chain gene region have taken place at approximately the same stage of two breeding programs.

Expression of b 4 and b 5 chi light chain allotypes by B and pre-B cells in allotype-suppressed and neutralized b4b5 rabbits.

AbstractIn previous studies, we described a primitive lymphoid cell found in fetal liver and in the bone marrow of older rabbits which contained cytoplasmic IgM but lacked surface IgM detectable by immunofluorescence. In heterozygous b4b5 rabbits, the pre‐B cells in which we could detect these kappa chain allotypes appeared to exhibit allelic exclusion. In the present study, we investigated the effects of allotype suppression and its neutralization on the expression of the b4 and b5 allotypes by B and pre‐B cells from the spleens and bone marrow of b4b5 rabbits. We found that in young allotype suppressed rabbits, pre‐B cells of the suppressed allotype persist in bone marrow when B cells of the suppressed allotype are absent or severely depleted. The persistence of pre‐B cells of the suppressed type supports the view that pre‐B cells differ in their responsiveness to external influences such as anti‐Ig compared to B lymphocytes. Injection of serum with b5 immunoglobulin into b4b5 animals suppressed 14‐23 days previously for b5 was followed by the appearance of increased proportions of b 5 B cells in spleen within 24 h. Surviving pre‐B cells are a likely source of these rapidly appearing B cells as well as of the B cells bearing surface immunoglobulin of the suppressed allotype which appear during the recovery phase of allotype suppression.

Antigen- and receptor-driven regulatory mechanisms. II. Induction of suppressor T cells with idiotype-coupled syngeneic spleen cells.

Anti-p-azobenzenearsonate (ABA) antibodies, coupled covalently to normal syngeneic spleen cells and then given intravenously to normal animals, were found to be potent tolerogens for delayed-type hypersensitivity (DTH) to ABA. The ability of the antibody-coupled cells to induce tolerance was determined to be a result of the cross-reactive idiotype (CRI+) fraction of the antibodies, because anti-ABA antibodies lacking the CRI+ components when coupled to spleen cells were unable to cause any significant inhibition. Furthermore, genetic analysis revealed that the ability of CRI-coupled cells to inhibit ABA-specific DTH is linked to Igh-1 heavy chain allotype, in as much animals which possess heavy chain allotypes similar to that of A/J were sensitive to this inhibition. Adoptive transfer experiments provided evidence that CRI-coupled cells induce suppressor cells, and spleen cells or thymocytes from animals received CRI-coupled cells were able to transfer suppression to naive recipients. In addition, treatment with anti-Thy1.2 serum plus complement completely abrogated their ability to transfer suppression. Thus, this active suppression is a T-cell-dependent phenomenon. In investigating the specificity of these suppressor T cells, it was found that they functioned in an antigen-specific manner and were unable to suppress the development of DTH to an unrelated hapten 2,4-dinitro-1-fluorobenzene.

Gm, Am and Km immunoglobulin allotypes of two populations in Tunisia.

Gm, Am and Km allotypes were investigated in two Tunisian populations (236 samples from Mahdia and 142 samples from Sfax). These populations descend from immigrants and, therefore, the results were compared with those obtained in other populations living in the Near East and in North Africa. The subclass heavy chain allotypes G1m, G2m, G3m and A2m are inherited in fixed combinations. There were five main and four minor Gm-Am haplotypes that could be deduced from the phenotypes. This led to the conclusion that the populations studied are Caucasoids with some African admixture (about 10%) and a very low oriental contribution. Furthermore, there were 11 samples which showed 8 uncommon Gm-Am phenotypes. These could be explained by the assumption of five different uncommon Gm-Am haplotypes. Four of these may have arisen by equal crossing over of prevalent haplotypes. The fifth may be the result of unequal crossing over of prevalent haplotypes. The fifth may be the result of unequal crossing over, since it was proven, by family study, that more markers are transmitted together than are present in the prevalent haplotypes.