DNA sequence analysis of the C3H H-2Kk and H-2Dk loci. Evolutionary relationships to H-2 genes from four other mouse strains

We generated nucleotide sequences for H-2Kk and H-2Dk from the C3H mouse, as well as for a genomic clone of H-2Db, in order to conduct an evolutionary analysis of the H-2 genes from three haplotypes, k, d, and b. H-2Kk from both the C3H and AKR strains, H-2Kd, H-2Kb, H-2Dk, H-2Ld, H-2Dd, H-2Db, and H-2Dp DNA sequences were aligned, and the alignments used to construct phylogenetic trees inferring the evolutionary relationships among the nine genes by two independent methods. Both approaches yielded trees with similar topologies. In addition, the sequence alignments revealed patterns of nucleotide substitutions which implicate both point mutation and recombination in the divergence of the H-2 genes. Future considerations for evolutionary analysis of class I genes are discussed.     

Genetic analysis of the H-2D region using a new intra-D-region recombinant mouse strain

A rare D-region recombination event which gave rise to the B10.RQDB major histocompatibility complex haplotype has been examined to ascertain the nature of the crossover and to determine which class I genes are present in the new alignment of D-region genes. Serologic analysis have shown that the B10 . RQDB major histocompatibility complex recombinant mouse inherited the H-2Dd gene from the B10.T(6R) parental line and the H-2Db gene from the B10.A(2R) parental line, representing the first example of an intra-D-region crossover resulting from an intercross. Previous molecular genetic analyses of the d and b haplotypes revealed structural diversity in the organization of their D-region gene clusters. Hence, the D region is comprised of five class I genes in the d haplotype and only one in the b haplotype. Because allelic relationships among the various D-region genes are not defined, either a homologous or nonhomologous alignment of genes has generated the RQDB crossover. Therefore, the possibility that all three D-region antigen-presenting molecules (Dd, Ld, and Db) might be encoded by the RQDB haplotype was examined. Fluorescence-activated cell sorter and cytotoxic T lymphocyte analyses revealed no detectable levels of H-2Ld cell-surface expression, confirming earlier studies with antibody-mediated cytotoxicity and immunoprecipitation. Southern blot analysis localized the recombination point to within a 1-kb region at the centromeric end of the H-2Ld gene on the B10 . T(6R) chromosome in a region of high homology to the H-2Db gene on the B10 . A(2R) chromosome. Together, these studies define the D region of the RQDB haplotype as containing the five class I genes: Dd, D2d, D3d, D4d, and Db. In addition to providing insight into rare recombination events in the D region, the B10.RQDB mouse should be a useful tool for exploring the function of D-region genes.     

Structural relationships among the H-2 D-regions of murine MHC haplotypes

The number of genes encoding functional Ag-presenting molecules in the D region of the murine MHC differs among haplotypes. For example, the H-2b D region contains a single "D/L" gene, H-2Db, whereas the d-haplotype encodes two, H-2Dd and Ld. Using D/L specific oligonucleotide probes, we have found that, as with H-2d, the q- and v-haplotypes contain two D/L genes, whereas the other haplotype examined have one. Hybridization analysis using cloned probes that map between H-2Dd and Ld revealed similar structures in each of the three haplotypes (d, q, and v) which have "duplicated" D regions. Two approaches were used to examine allelic relationships among the D/L genes. First, the 5' region of the H-2Db gene was sequenced, and found to be more similar to H-2Ld than to H-2Dd. Second, oligonucleotide probes that distinguish H-2Ld from H-2Dd revealed H-2Ld-related genes in several haplotypes, including the duplicated haplotypes H-2q and H-2v. Analogous probes specific for H-2Dd, however, did not detect similar sequences in the other haplotypes. We interpret these results to mean that the three duplicated D regions arose from a common duplication event, and share the five gene structure of the D region cluster defined in H-2d. However, subsequent events have generated sequence divergence at the D-locus.     

Expression of H-2Dd and H-2Ld mouse major histocompatibility antigen genes in L cells after DNA-mediated gene transfer

Among the more than 20 H-2-like genes in the BALB/c mouse genome, there are two classical transplantation antigens (H-2Dd and H-2Ld) encoded at the D-end of the major histocompatibility complex. Here we report the identification of a bacteriophage clone that encodes H-2Dd. The H-2Dd gene was identified by nucleotide sequence analysis and by characterization of the new H-2 antigen expressed when the cloned gene was introduced into mouse L cells by DNA-mediated gene transfer. The previously identified H-2Ld gene was then compared with the H-2Dd gene. The two genes appear to have the same general structure, and for the 854 nucleotides that have been compared, the two genes are 89% homologous. The H-2Ld and H-2Dd antigens expressed on mouse L cells after DNA-mediated gene transfer were examined by immunologic criteria. The stably transformed cell lines express apparently normal levels of H-2Dd and H-2Ld on the cell surface as measured by quantitative immunofluorescence by using monoclonal anti-H-2 antibodies. They synthesize H-2Dd and H-2Ld at normal rates as determined by endogenous labeling and immunoprecipitation of cell extracts. They evoke a strong specific serologic response when used to immunize C3H mice. The newly expressed antigens are able to serve as targets for alloreactive T cells. These cloned genes provide good substrates for examining the evolution of two closely linked H-2 antigen genes. Comparison of the structures of these genes provides clues to the basis for the differential expression of these antigens and their different biologic functions.     

Structural definition of a family of Ld-like molecules distributed among four of seven haplotypes compared

Comparative tryptic peptide analyses were performed on 12 different D region molecules representing seven different haplotypes. The Dd, Dq, and Dw16 regions were shown to encode multiple, antigenically distinct molecules (Dd Ld, Dq Lq Rq, and Dw16 Lw16, respectively). In addition, each of these molecules was found to have a unique primary structure, implying that they are the products of separate genes. However the previously described Rd molecule, which was identified by sequential immuno-precipitation and 2-D gel analyses, was indistinguishable from Ld by tryptic peptide mapping, implying that these two molecules may be products of the same gene. The Db, Ddx, Dk, and Dp regions were found to determine a single molecule with the reagents tested. Intra- and/or inter-haplotype comparisons of the peptide maps of each of these D region molecules revealed widely disparate structural relationships. For example, the Db, Dq, Lq, Rq, Dw16, and Lw16 molecules all showed striking homology with the Ld molecule. Members of this family share between 43 to 55% peptide homology with Ld, indicating a high conservation of primary structure (greater than 90%). However, because Dq and Dw16 region-encoded molecules show no exceptional relationship to each other, the portion of the conserved sequence is not the same for each of these Ld-like molecules. By contrast, comparisons of the Dk, Dd, Ddx, and Dp molecules with Ld or with each other revealed tryptic peptide homologies ranging from 22 to 38%, suggesting a sequence homology of 70 to 85%. When compared with the Kb molecule, each of the D region molecules showed between 21 to 36% peptide map homology (70 to 85% sequence homology). These studies indicate, therefore, that there is a family of Ld-like molecules representing several distinct haplotypes. This definition of a highly homologous family of D region molecules suggests that many D-region molecules have evolved from an Ld-like primordial gene and that in different haplotypes different portions of this prototypic structure have been maintained.     

The products of transferred H-2 genes express determinants that restrict hapten-specific cytotoxic T cells

Cell lines into which cloned H-2 genes had been introduced (i.e., transformants) were used to correlate the genes and their products that are capable of functioning as H-2 restriction elements for hapten-self-(AED and TNP) specific cytotoxic T cells (CTL). These transformants provided a unique system in which major histocompatibility restricted (MHC) T cell recognition could be examined by using cells that express only H-2Ld or only H-2Dd gene products. BALB/c (H-2d) anti AED-self CTL lysed both the H-2Ld and Dd transformants, but not parental, i.e., untransformed, cells. The AED-self lysis of the Ld and Dd transformants was shown to be specifically inhibited by anti-H-2Ld and anti H-2Dd monoclonal antibody, respectively. In contrast to these results, BALB/c anti TNP-self CTL were found to lyse readily the Dd but not Ld transformed lines, supporting reports indicating that H-2Ld-restricted TNP-self CTL could not be detected. The results of this study thus demonstrate that the cell surface products encoded by these transferred MHC class I genes contain self determinants recognized by CTL.     

The MHC class I molecule H-2Dp inhibits murine NK cells via the inhibitory receptor Ly49A.

MHC class I molecules strongly influence the phenotype and function of mouse NK cells. NK cell-mediated lysis is prevented through the interaction of Ly49 receptors on the effector cell with appropriate MHC class I ligands on the target cell. In addition, host MHC class I molecules have been shown to modulate the in vivo expression of Ly49 receptors. We have previously reported that H-2Dd and H-2Dp MHC class I molecules are able to protect (at the target cell level) from NK cell-mediated lysis and alter the NK cell specificity (at the host level) in a similar manner, although the mechanism behind this was not clear. In this study, we demonstrate that the expression of both H-2Dd and H-2Dp class I molecules in target cells leads to inhibition of B6 (H-2b)-derived Ly49A+ NK cells. This inhibition could in both cases be reversed by anti-Ly49A Abs. Cellular conjugate assays showed that Ly49A-expressing cells indeed bind to cells expressing H-2Dp. The expression of Ly49A and Ly49G2 receptors on NK cells was down-regulated in H-2Dp-transgenic (B6DP) mice compared with nontransgenic B6 mice. However, B6DP mice expressed significantly higher levels of Ly49A compared with H-2Dd-transgenic (D8) mice. We propose that both H-2Dd and H-2Dp MHC class I molecules can act as ligands for Ly49A.     

The DNA sequence of the H-2kb gene: evidence for gene conversion as a mechanism for the generation of polymorphism in histocompatibilty antigens

We have determined the DNA sequence of the H-2Kb gene of the C57B1/10 mouse. Comparison of this sequence with that of the allelic H-2Kd shows surprisingly that the exons have accumulated more mutations than their introns. Moreover, many of these changes in the exons are clustered in short regions or hot spots. Additional comparison of these sequences with the H-2Ld and H-2Db sequences shows that, in several cases, the altered sequence generated at the hot spot is identical to the corresponding region of a non-allelic H-2 gene. The clustered changes are responsible for 60% of the amino acid differences between the H-2Kb and H-2Kd genes and suggest that micro-gene conversion events occurring within the exons and involving only tens of nucleotides are an important mechanism for the generation of polymorphic differences between natural H-2 alleles.     

Cytotoxic T lymphocyte recognition of class I H-2 antigens after DNA-mediated gene transfer

Cytotoxic T lymphocyte (CTL) recognition sites on class I major histocompatibility complex molecules have been investigated by several laboratories by using cloned genes expressed on mouse L cells by DNA-mediated gene transfer. Recombinant genes, constructed by restriction endonuclease treatment of cloned H-2Dd and Ld genes and exchange of the N and C1 exons (exon shuffling) have provided an additional tool. These hybrid H-2 molecules expressed on L cells have been used as targets to achieve more precise localization of site(s) recognized by allospecific and virus-specific CTLs. CTL systems were chosen that limit recognition to either the Dd or Ld alloantigen or to virus and Dd or Ld complexes. Using this approach, we were able to map essential restricting site(s) to the N and/or C1 domains. Additional evidence is presented that the cytoplasmic tail of H-2 may be involved in interactions with some viral antigens and effect the formation of an immunogenic complex.     

Interaction of alpha 1 with alpha 2 region in class I MHC proteins contributes determinants recognized by antibodies and cytotoxic T cells

The structure-function relationship of individual coding regions of class I mouse major histocompatibility complex proteins was studied by a combination of recombinant DNA, gene transfer techniques, and serologic and functional characterization. To examine the role of alpha 1 and alpha 2 regions in antibody and CTL recognition, the third exon of H-2Dd, Kd, and Ld transplantation antigen genes was replaced by the homologous coding region of the Qa-2-coded class I gene, Q6. We have chosen to carry out the exon shuffling experiments between these two different types of class I genes, because they are structurally similar and did not evolve to carry out identical functions. Therefore, it is less likely that the hybrid proteins will fortuitously recreate alpha 1-alpha 2 controlled functionally important determinants. The replacement of H-2 alpha 2 coding region with its Q6 counterpart had different effects on the expression of the three genes. The mutant H-2Dd gene transfected into L cells was expressed at high levels and retained several of the serologic determinants found on parental H-2Dd and Q6 domains. The serologic epitopes on the mutant H-2Kd-transfected cells were detectable at very low levels, whereas the product of the mutant H-2Ld gene could not be identified at all. Analysis of cells transfected with mutant H-2Dd gene with alloreactive and minor antigen(s)-restricted cytotoxic T cells indicated that the hybrid proteins lost the ability to be recognized by T cells. Our data suggest that cytotoxic T cells recognize conformational determinants composed of amino acids from alpha 1 and alpha 2 regions. Alternatively, it could be proposed that T cell recognition sites located in a single alpha 1 or alpha 2 protein region are susceptible to distortion upon alpha 1-alpha 2 interactions. Such susceptibility to conformational changes of the amino-terminal domain of transplantation antigens could be of functional importance for H-2-restricted antigen presentation.     

Differential lack of class I H-2d antigen expression by sublines of the BALB/c S49 T cell lymphoma

Five different sublines of the BALB/c murine S49.1 T cell lymphoma were found to exhibit distinct patterns of absence of detectable H-2d class I major histocompatibility antigen expression. The results were demonstrated and verified by a) the generation of H-2Kd-, H-2Dd,Ld-, and H-2Ld-specific cytotoxic T lymphocytes that were assayed on S49.1 target cell lines, b) antibody-mediated cytotoxicity with the use of anti-H-2d monoclonal reagents, and c) flow microfluorometry. The five lines investigated were S49.1, T-25, T-25ADH, Thy-1-, and 100/0. None of these lines expressed detectable levels of Ld. S49.1 expressed both Kd and Dd, T-25 and T-25ADH expressed Dd but not Kd or Ld, Thy-1- expressed Kd but not Dd or Ld, and 100.0 did not express any detectable amounts of Kd, Dd, or Ld. These results indicate that K and D (and L) antigens can be expressed independently of each other and suggest that expression of class I antigens is controlled in a locus-specific manner.     

Analysis of hybrid H-2D and L antigens with reciprocally mismatched aminoterminal domains: functional T cell recognition requires preservation of fine structural determinants

Studies of immune recognition of hybrid class I antigens expressed on transfected cells have revealed an apparent general requirement that the N(alpha 1) and C1(alpha 2) domains be derived from the same gene in order to preserve recognition by virus-specific H-2-restricted and allospecific T cells. One exception has been the hybrid DL antigen in which the N domain of H-2Ld has been replaced by that of H-2Dd. Cells bearing this molecule serve as targets for some virus and allospecific CTL. Because cells expressing the reciprocal hybrid LD (N domain of H-2Dd replaced by that of H-2Ld) antigen have not been available, it has not been possible to evaluate whether this exception stemmed from the relatedness of H-2Ld and H-2Dd or whether the DL antigen fortuitously preserved some function of the parent molecule as a rare exception. To assess this question, and to evaluate the contribution of the N and C1 domains of H-2Ld and H-2Dd to serologic and T cell recognition, we have constructed the reciprocal chimeric gene pLD (the N exon of H-2Ld substituted for that of H-2Dd), introduced this into mouse L cells by DNA-mediated gene transfer, and analyzed the expressed product biochemically, serologically, and functionally. Transformant L cells expressing either LD or DL antigens were both reactive with a number of anti-H-2Ld or anti-H-2Dd N/C1-specific monoclonal antibodies, indicating the preservation in the hybrid molecules of determinants controlled by discrete domains. Mab binding was generally greater with cells expressing hybrid DL antigen than with those transformants expressing LD molecules. Moreover, the amount of beta 2M associated with DL antigens was more than that associated with LD. Cells expressing hybrid DL antigens were recognized as targets by bulk and cloned allospecific anti-H-2Dd and anti-H-2Ld CTL, whereas cells expressing LD molecules were not recognized by any of the T cells tested. VSV-specific H-2Ld-restricted CTL failed to lyse VSV-infected targets expressing either DL or LD. These results indicate that T cell reactivity of cells expressing the DL hybrid antigen is an exception to the observed general requirement for class I antigens to possess matched N and C1 domains for functional T cell recognition by T cells restricted to parental antigens.(ABSTRACT TRUNCATED AT 400 WORDS)     

Induction of the H-2 D antigen during B cell activation

Mitogenic activation causes increased expression of class I Ag of the MHC in mouse B cells. The increased expression was seen in flow cytometry analysis for both K and D in k as well as d haplotypes. A more detailed molecular analysis was carried out for H-2Dd. Increased expression (10- to 20-fold) of the H-2 Dd gene was detected at both protein and messenger RNA levels, and the time course for the accumulation of H-2 Dd protein on the cell surface parallels the increase in the steady-state messenger RNA levels. The increase in H-2 Dd expression in small B cells stimulated with LPS is detectable after 10 h of culture. The present data provide molecular and serologic evidence about alterations in the expression of the H-2 Dd Ag, previously identified as a B cell activation antigen B7.2. Our results indicate a new significance for the function and regulation of the MHC during immune responses, and suggest that the class I molecules may serve some role in the B cell activation process.     

Molecular analysis of deficient class I H-2 antigen expression by mouse lung carcinoma cells

We have continued our investigations of line lung carcinoma cells to understand the molecular basis of decreased expression of class I H-2 Ag and class I Ag induction with DMSO. We show that line 1, a murine lung carcinoma cell line, has low levels of class I Ag (H-2K, D, and L) because it is deficient in both class I and beta 2-microglobulin (B2M) RNA, and that these mRNA can be coordinately induced with DMSO. Evidence presented herein also shows that IFN-gamma can induce surface expression of class I Ag and suggests that it may act through a different mechanism than DMSO in inducing class I Ag. To further evaluate the regulation of class I expression, H-2Dp genes were transfected into line 1 cells. The transfected H-2 genes appear to be constitutively expressed at much higher levels than are the endogenous class I genes because surface expression of the foreign Dp Ag on the transfectants is elevated relative to the endogenous H-2d haplotype class I Ag. Both Dp surface expression and Dp mRNA are induced after treatment with DMSO. In all the Dp transfectants, we observed higher constitutive levels of class I mRNA as well as increased constitutive levels of endogenous B2M mRNA when compared to control or untransfected line 1 cells, however, we could not correlate these constitutive levels with Dp copy number. These results suggest that the regulation of class I and B2M genes is linked and that expression of class I genes can affect the expression of B2M genes.     

Tracing the evolution of H-2 D region genes using sequences associated with a repetitive element

The class I genes in the murine MHC are genetically divided into the K, D, Qa, and T1a region subfamilies. These genes presumably arose by duplication from a common class I ancestor. Oligonucleotide probes specific for sequences associated with a moderately repetitive B2 SINE element, which is inserted into the 3' untranslated region of the H-2D and H-2L genes, were used to examine the evolutionary relationship between these classically defined D region genes (H-2D and H-2L) and the other members of the class I gene family. Hybridization analyses of recombinant cosmid and genomic DNA indicated that the D region genes separated genetically from the other members of the class I gene family 12 to 14 million years ago. The evidence suggests that during this time frame the chromosomal segment harboring the characteristic insertion became fixed in the ancestral population which gave rise to Mus domesticus. Previous studies have shown that the number of genes present in the Qa and T1a regions varies among inbred strains and among laboratory stocks of wild mice derived from more distant species on the genus Mus. No evidence was found in this study to support the hypothesis that variation in class I gene number is the result of recent duplications of the functionally defined class I genes of the D region, H-2D and H-2L.     

Properties of H-2L locus products in allogeneic and H-2 restricted, trinitrophenyl-specific cytotoxic responses.

The role of the recently defined L antigen (a second D region product) in allogeneic and TNP-specific syngeneic primary CML responses has been investigated. The lysis by anti-L specific cytotoxic effector cells was not inhibited when the target cells were pretreated with an antiserum directed against K and D, whereas an antiserum against L completely abrogated this response. Therefore, H-2L products are recognized on the target cell independently of H-2K and H-2D locus products. Both A.SW cells as well as B10 cells were found to respond to Ld alloantigens, in addition to Dd alloantigens when stimulated by cells differing only in the D region. The results of cold target blocking and antiserum inhibition experiments failed to detect cytotoxic cells with specificity of L antigens in association with TNP, under conditions in which TNP-specific effectors to K and D antigens were demonstrable. These findings suggest that there is a more limited involvement of H-2L locus products than the H-2K or H-2D locus products in the induction and specificity of these responses.     

Class I MHC genes and CD8+ T cells determine cyst number in Toxoplasma gondii infection

To determine roles of MHC class I and II genes in protection against Toxoplasma gondii, H-2 congenic and mutant mice were infected perorally with bradyzoites of T. gondii and brain cysts were enumerated 30 days later. As B10 mice (H-2b) are cyst susceptible and B10.A mice (H-2a) are cyst resistant, B10 congenic mice having the same alleles but different H-2 haplotypes were used to locate the controlling gene. Genes located at H-2L (i.e., class I genes) were found to regulate the number of brain cysts which form following peroral infection with T. gondii (p less than 0.001) with Ld being resistant and Lb being susceptible. The regulatory function of the H-2L gene product was confirmed through the study of D mutant (dm) mice. B10.D2-H-2dm1 (dm1) mice have a gain-loss mutation in Dd and Ld (i.e., recombination of Ld and Dd) and BALB/c-H-2dm2 (dm2) mice have a deletion of the Ld gene. Both these dm strains were cyst susceptible (p less than 0.001). These results provide the first direct evidence that class I genes regulate numbers of T. gondii cysts that form. In vivo ablation of CD8+ T cells with mAb YTS 169.4 converted cyst resistant B10.BAR12 mice to cyst susceptible. This result is consistent with a role for MHC restricted CD8+ cytotoxic (or suppressor) T cell regulation of cyst formation. A mutation in Ia in B6.C-H-2bm12 (bm12) mice amplified cyst numbers in susceptible mice, which is consistent with the importance of helper/inducer T cells in the induction of cytotoxic T cells. These findings are relevant to understanding the complex immunologic mechanisms that protect against T. gondii infection, development of protective preparations, and provide a conceptual basis for determining whether similar immunogenetic regulation of susceptibility is also operative in humans.     

Recognition of the alpha-1 and alpha-2 domains of H-2 molecules by allospecific cloned T cells

Previously we had shown that allospecific bulk cultures of cytolytic T lymphocytes lysed the products of cloned class I major histocompatibility genes expressed after DNA-mediated gene transfer. In these experiments, performed by using cloned allospecific T cell effectors, a T cell hybridoma, and recombinant DNA technology, we have been able to map determinants recognized by these T cell clones to the alpha-1 domain of H-2Dd and the alpha-2 domain of H-2Ld (four of eight clones). Target cells used were L cells (H-2k), expressing wild type or hybrid H-2 antigens of H-2d origin. Thus, for the first time determinants recognized by cloned T cells are found in the recombined alpha-1 and alpha-2 domains.