Detection of an altered I-A beta polypeptide in the murine Ir mutant, B6.C-h-2bm12.
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Animals↗
Biomedical subjects
T H Hansen
Publications and source records attributed to T H Hansen.
I-A mutation resulted in a selective loss of an antigen-specific Ir gene function.
The immune responses to several antigens were compared in the I-A mutant mouse strain B6.C-H-2bm12 and the wild-type strain C57BL/6. With a lymph node cell proliferation assay, the response to two of these antigens, beef insulin and (TG)A-L, was demonstrated to be controlled by a gene in the I-Ab region. B6.C-H-2bm12 mice failed to respond to beef insulin, while their responses to (TG)A-L, DNP-OVA and PPD were comparable with those of the wild-type strain C57BL/6. Taken together with previous studies, these data suggest that the product of a single pleiotropic I-A gene, an Ia molecule, functions as a histocompatibility, Ia, and MLR antigen, as well as a necessary component for Ir gene function. Furthermore, the data reported here demonstrate that Ia molecules have multiple functional "Ir determinants," one of which has been altered in the B6.C-H-2bm12 mutant. The B6.C-H-2bm12 mice, therefore, represent a powerful analytical tool for the understanding of the cellular and molecular basis for Ir gene control of the immune response.
Alleles↗
Selective loss of antigen-specific Ir gene function in IA mutant B6.C-H-2bm12 is an antigen presenting cell defect.
Immune responses to several soluble antigens were compared between B6.C-H-2bm12 mutant and wild-type B6 mice by using a lymph node T-cell proliferation assay. B6.C-H-2bm12 mice failed to respond to beef insulin whereas other IA gene-controlled responses, such as response to poly(L-Tyr, L-Glu)--poly(DL-Ala, L-Lys) and collagen, were indistinguishable between mutant and wild-type mice. The responses to multideterminant antigens such as ovalbumin and purified protein derivative of tuberculin were also found to be comparable in B6.C-H-2bm12 and B6 mice, thus indicating that this mutation resulted in a selective loss of the ability to respond to a certain antigen(s)--e.g., beef insulin. Populations depleted of adherent cells have been used to examine the mechanism by which Ia molecules mediate Ir gene control of antigen recognition. We show that the nonresponsiveness to beef insulin in the mutant mouse is the result of defective antigen presentation. In addition, we find that F1 hybrids between two nonresponders--B6.C-H-2bm12 and B10.A or B10.AKM (IAk) mice--become responders to beef insulin, thus demonstrating gene complementation. These findings taken together with other serologic and biochemical studies in the B6.C-H-2bm12 present convincing genetic evidence for the direct association of the A beta polypeptide chain of the Iab molecules with the expression of immune responsiveness to beef insulin. Study of the B6.C-H-2bm12 mouse should provide new insight into the cellular and molecular mechanisms by which Ir genes determine the nature of the immune response.
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Studies on monoclonal antibodies to mouse MHC products.
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Animals↗
Ia specificities on parental and hybrid cells of an I-A mutant mouse strain.
Splenic B cells and B cell blasts from the I-A mutant mouse strain B6.C-H-2bm12 were tested by serology with a series of new monoclonal anti-Iab antibodies. Four out of 5 of those monoclonal antibody-defined specificities that are determined by wild-type I-Ab antigens were undetectable on B6.C-H-2bm12 cells. Specificities both present and absent on mutant cells appear to be determinants on the same wild-type molecule, as indicated by sequential precipitation experiments with soluble H-2b antigens. The lack of expression of certain Ia specificities on mutant cells was found not to be the result of disparate control by the Xid gene, which was previously shown to control the expression of Ia.W39, another specificity absent in B6.C-H-2bm12 mice. Serologic testing of Ia specificities on cells and blasts from F1-hybrid mice suggested that the Iabm12 antigens are codominantly expressed, indicating a failure to detect trans regulation or complementation of the mutant phenotype. Another monoclonal antibody-defined Ia specificity dependent on the expression of the E beta polypeptide was normally expressed in B6.C-H-2bm12 mice. These data thus suggest that the lesion of these mutant mice occurred in the A alpha and/or A beta structural gene, resulting in the loss of several Ia specificities.
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Flow microfluorometric analysis of H-2L expression.
The cell surface expression of H-2L, a major transplantation antigen, was compared by flow microfluorometry to the expression of products of H-2K and H-2D loci, using monoclonal antibodies. By this methodology, the ontogeny and tissue distribution of Ld antigens were found to be indistinguishable from those of the K and D antigens. In a reciprocal blocking assay, using fluorescein-labeled test reagents, it was shown that monoclonals anti-H-2.65 and anti-H-2.64 did not inhibit the binding of each other. These results suggest that the alloantigenic determinants H-2.64 and H-2.65 are located at distinct sites on Ld molecules. Quantitative comparisons using the fluorescein-labeled monoclonal reagents indicated that Ld molecules are expressed at 2- to 3-fold lower levels on the cell surface compared with K and D molecules. These findings give new credence to a "3-locus" model for the major histocompatibility complex of man and mouse, where H-2L and HLA-C share several homologies that are unique and distinguish them from the other histocompatibility loci.
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Immunochemical evidence in two haplotypes for at least three D region-encoded molecules, D, L, and R.
Sequential immunoprecipitation analyses have defined a new transplantation antigen, designated R, which in addition to the 2 previously isolated D and L molecules is encoded in the D region. All 3 of these gene products are 45,000 m.w. glycoprotein, and each bears a unique combination of specificities as recognized by monoclonal and/or conventional anti-H-2 sera. Three D region products, D, L, and R, have now been isolated from soluble antigens of both the H-2d and H-2q haplotypes. The resulted reported here also indicate that the loss-mutant BALB/c-H-2dm2 fails to express both Ld and Rd antigens. Further chemical comparisons of the primary structure of R molecules with D and L molecules will now be necessary to determine whether R antigens are the products of a unique gene, as opposed to a glycosylation or conformational variant of D and/or L molecules. In either case, the discovery of 3 D region-encoded gene products in certain haplotypes raises new questions about the evolution and regulation of expression of H-2 loci.
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Evidence for mutation in an I-A gene.
Mutant mouse strains are important tools for immunogenetic studies of the regulation, structure and function of major histocompatibility (H-2) antigens1-3. Several inbred strains have been established which carry H-2-linked mutations that cause changes in cell surface antigens as recognized by cytotoxic T cells and sometimes antibodies. Using intra-H-2 recombinant haplotypes in skin graft complementation studies, lesions in several of these mutant strains have been genetically mapped to genes encoded in the K- or D-end of the H-2 complex3. More precise mapping has often not been possible as many of the available mutant strains possessed lesions in the K-end of the H-2b haplotype and a recombinant separating the Kb and I-Ab regions was not previously available. The genetic locations of the lesions in some of these mutant strains could therefore only be inferred when a serological and/or structural alteration was detected (see refs 1, 2). We describe here the use of a newly established recombinant strain, B10.MBR (ref. 4), for skin graft complementation studies to map K-end mutations. These studies provide the first genetic evidence that the B6.C-H-2bm12 mutation involves an I-Ab gene and also confirm that the lesions in two other mutants can be mapped to a gene in the Kb region.
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Functional studies of the products of H-2L locus.
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Animals↗
Primary structural studies of an H-2L molecule confirm that it is a unique gene product with homology to H-2K and H-2D antigens.
Radiochemical methodology has been used in the isolation and preliminary biochemical characterization of the murine H-2Ld major histocompatibility complex gene product. The radiolabeled molecule was isolated by immunoprecipitation from the glycoprotein fraction of detergent-solubilized H-2d tumor cells. Six major CNBr fragments were isolated from a papain fragment of this molecule; three of the fragments are connected by disulfide bonds. Due to the high degree of homology between major transplantation antigens, it was possible to align the fragments by comparison of their amino acid sequences to that of the H-2Kb gene product. Of the positions available for comparison between H-2Ld and H-2Kb, H-2Dd, and H-2Kd gene products, 61 out of 80 (78%), 45 out of 55 (82%), and 12 out of 15 (80%), respectively, are identical. Differences between the Ld and Kb and Dd molecules are distributed throughout the amino acid sequence. These data indicate that the H-2Ld gene product is a molecular species distinct from, but homologous to, the H-2K and H-2D gene products.
Amino Acid Sequence↗
Monoclonal antibodies to mouse MHC antigens. II. Antibodies to the H-2Ld antigen, the products of a third polymorphic locus of the mouse major histocompatibility complex.
In order to study the fine structure of H-2L, the third H-2 locus of the mouse major histocompatibility complex, we have produced and characterized the monoclonal anti-Ld antibodies secreted by 3 different hybridoma cell lines. Two antibodies were obtained by a specific immunization directed to Ld determinants. Another antibody obtained from an immunization including, but not specific for, an L locus difference was found to detect Ld, and not Kd or Dd molecules. Although the fusions involved an H-2d myeloma variant line, no apparent adverse "autoreaction" was observed in these anti-H-2d antibody-producing hybridoma cells. All 3 antibodies exhibited cross-reactions with the antigens of certain haplotypes, and the reactivity patterns observed corresponded to the previously described alloantigenic specificities H-2.64 and H-2.65. This concordance with known H-2 specificities contrasts with the broad, unique specificity patterns exhibited by most monoclonal antibodies to H-2 antigens and may reflect a difference in the complexity of the polymorphism of the L locus relative to that of the K and D loci. Immunoprecipitation analyses using 1 of the monoclonal antibodies showed that the molecules precipitable by this reagent were indistinguishable from those precipitable by anti-Ld alloantisera. In addition, cross-reactions of Ld, Dq, and Lq molecules were revealed by sequential immunoprecipitation analyses, supporting a close evolutionary relationship of L and D loci.
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Analysis of the heterogeneity of the mouse H-2K, D, and L gene products.
The detection and partial characterization of the major histocompatibility antigens encoded by the three H-2 loci, K, D, and L were performed by analyzing immunoprecipitates from detergent-solubilized radiolabeled cells on two-dimensional gels. Each locus was found to code for a population of proteins heterogeneous in both charge and m.w. Only part of the heterogeneity was shown to be due to sialylation as demonstrated by shifts in mobility after neuraminidase treatment of antigen preparations. The 2-D gel analysis of the H-2K, H-2D, and H-2L antigens of the d haplotype shows that each constitutes a distinct series of products.
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Structural relation of murine "third locus" (H-2L) major histocompatibility antigens to the products of H-2K and H-2D loci.
The primary structures of the allelic products of H-2L, the murine "third locus" major histocompatibility antigen, were compared with the structures of the H-2K and H-2D antigens by tryptic-peptide analysis. The H-2Ld,H-2Lq, H-2Dd, H-2Dq and H-2Kd molecules were radiolabeled and isolated from spleen cell detergent lysates with specific alloantisera. Comparisons of the tryptic maps of the H-2L, H-2D, and H-2K molecules of the H-2d haplotype demonstrated that H-2Ld molecules were as different from H-2Dd molecules as H-2Dd molecules were from H-2Kd molecules. In contrast, the H-2Lq and H-2Dq molecules were found to be extremely similar and a second peptide separation method was required to discern structural differences between the 2 molecules. Comparisons of the allelic products H-2Ld and H-2Lq indicated possible structural differences despite the lack of a known serologic difference. These results, while raising questions about the genetic evolution of the H-2L locus relative to the H-2K and H-2D loci, demonstrate that the structure of the H-2L molecule is distinct from that of the H-2K and H-2D molecules. These structural analyses further substantiate the functional studies that define the H-2L molecule as the "third" major histocompatibility molecule of the mouse.
Alleles↗
Cross-reactivity among the products of three nonallelic H-2 loci, H-2Ld, H-2Dq, and H-2Kk.
Cross-reactions of the products of three nonallelic H-2 loci, H-2K, H-2D, and H-2L, have been studied by serological, immunochemical, and cellular immune techniques. Immunoprecipitation experiments revealed a determinant shared between Ld and Dq molecules but absent on Dd molecules, By cytotoxic screening of sera from individual mice an exceptional anti-Ld serum was found which cross-reacted with an H-2k antigen. Genetic mapping studies suggested that the basis of this activity was an Ld-Kk serological cross-reaction. Cytotoxic T cells generated to Ld alloantigens were also found to recognize a cross-reacting Kk determinant. These studies therefore suggest that L, D, and K are three structurally homologous cell surface glycoproteins that have evolved from a common primordial gene.
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Two new recombinant H-2 haplotypes, one of which juxtaposes Kb and Ik alleles.
Two new recombinant H-2 haplotypes have been detected and established as congenic resistant lines on the C57BL/10 background. On the basis of serologic testing and immunoprecipitation analyses, the sublocus composition of the first recombinant haplotype, H-2bq1 (B10.MBR) has been shown to be KbIkDq, and that of the second recombinant, H-2sq3 (B10.SQR) to be KsIsSsDq. The occurrence of the Kb I-Ak juxtaposition after a recombination between H-2b and H-2m contrasts with the almost uniform failure to observe H-2b/H-2k recombinants in previous studies. This finding and the occurrence of a second recombination event involving the same chromosome soon after the first in our studies may imply that recombination within H-2 is not generally a random event. The B10.MBR line has proved useful in the production of specific anti-H-2Kb and anti-I-Ab antisera previously quite difficult to obtain contamination by anti-Ia or anti-H-2K antibodies, respectively.
Alleles↗
Involvement of H-2L gene products in virus-immune T-cell recognition. Evidence for an H-2L-restricted T-cell response.
The H-2L locus is closely linked to H-2D and codes for antigenic specificities present on a 45,000 mol wt glycoprotein that is distinct from the molecule which bears the D region private specificity. It was found that BALB/c-H-2db mice, which lack detectable cell-surface H-2L gene products, were able to generate influenza- and vaccinia-immune cytotoxic T cells which lyse D region-compatible target cells, although they have been reported to be incapable of making a similar response to ectromelia virus (7). Thus, the lack of H-2L antigenic specificities does not produce a general loss of responsiveness for other viruses even when a highly cross-reactive pox virus (vaccinia) was studied. Antisera-blocking experiments utilizing sera specific for either L or D molecules indicated that BALB/c mice generate influenza virus-immune cytotoxic T-cell subsets which independently recognize H-2L and H-2D gene products in association with viral antigens. These results are the first indication that products of the H-2L locus can operate analogously to H-2K/D gene products in virus-immune T-cell recognition.
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Alloantigens determined by a second D region locus elicit a strong in vitro cytotoxic response.
The newly defined L antigen (previously designated D') has been studied with the CML assay. Balb/c-H-2db mice, which carry a loss mutation in the D region, were found to generate anti-L cytotoxic effector cells when stimulated with wild-type (BALB/c) cells. When the target cells were treated with an antiserum against L, cytolysis by BALB/c-H-2db anti-BALB/c effectors was completely blocked. However, an antiserum against D, the antigen that bears the D region private specificity, had no effect on the anti-L cytotoxic response. In a control CML experiment that was specific for the D antigen, the antiserum to L had no effect, whereas the antiserum to D blocked completely. These results indicate that the L and D antigens are recognized in a CML response as separate entities on the cell surface. In an allogeneic response to the products of the D region, cytotoxic effectors were generated that were specific for L in addition to those specific for D. This conclusion was supported by blocking experiments with either antisera or unlabeled targets. These functional studies thus extend previous co-capping and immunochemical studies, and further support the hypothesis that L is a third transplantation antigen similar to the K and D antigens.
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Isolation and antigenic characterization of the product of a third polymorphic H-2 locus, H-2L.
The serology, immunochemistry, and genetics of the product(s) of a third H-2 locus, H-2L (previously designated D') have been studied by using an antiserum raised in BALB/c H-2db mutant mice against tissues from the wild type strain, BALB/c. Genetic mapping studies and sequential immunoprecipitation experiments both indicate that this antiserum reacts specifically with L molecules. These results imply that an H-2L product is antigenically undetectable in BALB/c-H-2db mice and that the lesion in this mutant is confined to the H-2L and not the H-2D locus. Two new specificities, H-2.64 and H-2.65, are defined by the reactivity of anti-L serum on allogeneic cells, and the strain distribution of these specificities suggests the existence of at least three H-2L alleles. This third H-2 locls is therefore polymorphic and in view of this and other similarities to the H-2K and H-2D loci, it must be considered in any evolutionary models dealing with the origin of multiple subloci of the major histocompatibility complex.
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