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.     

The nucleotide sequence and comparative analysis of the H-2Dp class I H-2 gene

We present the complete nucleotide sequence and the deduced amino acid sequence of the H-2Dp class I gene. This gene, which was cloned from a B10.P genomic DNA library, encodes and intact, functional H-2Dp molecule. Comparative analysis of the Dp sequence with other class I sequences reveals both similarities and differences. This analysis also shows that these genes exhibit D region-specific, locus-specific, as well as allele-specific sequences. The H-2Dp nucleotide sequence is greater than 90% homologous to the H-2Ld and H-2Db genes and only approximately 85% homologous to the H-2Dd gene. The K region and Qa region genes are less homologous. The 3' noncoding sequences appear to be region-specific. All of the previously described D region genes, Db, Ld, and Dd, possess the B2-SINE Alu-like repetitive sequence, as does Dp. Thus, this B2 repeat is a region-specific marker present in all D region genes studied so far. The additional polyadenylation site found in the H-2Dp gene starting at nucleotide 4671, which is homologous to non-D region sequences, as well as unique protein Dp coding sequences, make this gene an interesting model for studying the evolution of polymorphism and structure/function relationships in the class I gene family.     

Slower processing, weaker beta 2-M association, and lower surface expression of H-2Ld are influenced by its amino terminus

To determine why Ld antigens are expressed on the cell surface at levels three to four times lower than Dd or Kd antigens, pulse-chase experiments were used to compare their rates of biosynthesis and processing. Electrophoresis on sodium dodecyl sulfate gradient polyacrylamide gels resolved immunoprecipitates of each of these histocompatibility complex class I molecules into a slower and faster species. During the chase period, the faster migrating species appeared to be converted to the slower migrating species in a time-dependent manner. However, the conversion of Ld from the faster to the slower migrating species proceeded significantly more slowly than did the conversion of either Dd or Kd. Endoglycosidase H sensitivity and cell surface radiolabeling were used to determine the glycosylation state and cell location of each species of Ld and Dd. The results from these experiments, along with the pulse-chase studies and cytofluorometric analyses, suggest that Ld possesses a much slower rate of processing from a faster migrating, high mannose-bearing species to a slower migrating, complex oligosaccharide-bearing species found on the cell surface. Analysis of the beta 2-microglobulin (beta 2-m) association confirmed that Ld is associated with less beta 2-m than Dd. To localize the structures on class I molecules influencing their surface expression, rate of processing, and beta 2-m association, the Ddm1 molecule was analyzed. The Ddm1 molecule of the mutant B10.D2-H-2dm1 has previously been shown to be a chimeric Dd (amino-terminal)/Ld (carboxyl-terminal) polypeptide. The surface expression, processing and beta 2-m association of Ddm1 were found to be similar to Dd rather than Ld, suggesting that each of these phenomena are influenced by protein structure in the amino terminus.     

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.     

Oxidative stress induces the expression of the major histocompatibility complex in murine tumor cells.

The effect of t-butyl hydroperoxide (t-BOOH) on the induction of the Major Histocompatibility Complex (MHC) class I genes has been studied in two cell clones (B9 and G2) of the methylcholanthrene-induced murine fibrosarcoma GR9. These two clones were selected based on their different biological and biochemical behavior specially related to their tumor induction capability when injected into a BALB/c mouse. t-BOOH (0.125 mM) induced the expression of H-2 molecules in both cell clones. In B9 cell clone, in which MHC basal expression is very low or absent, t-BOOH significantly induced H-2Kd, H-2Dd and H-2Ld molecules. In G2 cell clone the expression of MHC class I genes was also enhanced by the xenobiotic, the effect being especially significant on the H-2Ld molecule which is not expressed under basal conditions. H-2 molecules expression was accompanied by the activation of the transactivator factor NF kappa B. These results suggest that oxidative stress may modulate the antigen expression of tumor cells and thus the immune response of the host organism. Basal levels of oxidative parameters, such as anti-oxidant enzymes, malondialdehyde (MDA) and the DNA damaged base 8-hydroxy-2'-deoxyguanosine (8-OHdG), showed differences between the two fibrosarcoma cell clones.     

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.     

Cell surface expression of an in vitro recombinant class II/class I major histocompatibility complex gene product

Chimeric histocompatibility genes encoding the amino-terminal (beta 1) domain of the class II Ak beta polypeptide and the carboxy-terminal (C2, transmembrane, and intracytoplasmic) domains of either the class I H-2Ld or H-2Dd molecules were stably introduced into mouse L cells. Although both were transcribed, only 5' Ak beta/3' H-2Dd transformants had significant cell membrane expression of a 30-40 kd, heterogeneous glycoprotein containing Ak beta 1 and H-2Dd (C2) serological epitopes. These transformants had a unique pattern of reactivity with monoclonal antibodies previously identified as requiring the Ak beta 1 domain for recognition of complete I-A molecules. These results allow new insight into the structural requirements for cell surface expression of proteins and provide unique cellular reagents for the dissection of humoral and cell-mediated recognition of MHC molecules.     

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.     

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.     

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.     

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.     

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.     

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)     

Comparison of HLA class I gene sequences. Derivation of locus-specific oligonucleotide probes specific for HLA-A, HLA-B, and HLA-C genes

The major histocompatibility complex in man contains at least 20 class I genes. Included within this family are three closely linked loci with 11-47 codominant alleles that encode the classical transplantation antigens HLA-A, -B, and -C. The study of individual HLA-A, -B, and -C genes is complicated both by the high degree of sequence homology among all members of the class I gene family and by the high degree of polymorphism exhibited by HLA-A, -B, and -C genes. Identification of potential locus-specific regions suitable for use as unique probes has been limited by the small number of nucleotide sequences available for comparison. In the present study, the nucleotide sequences of two cDNA clones, designated HLA-4 and HLA-10, that encode previously unsequenced alleles of HLA-C and HLA-A genes, respectively, are compared with those of other class I genes. From these intergenic and interallelic comparisons, it was deduced that the nucleotide sequence encoding amino acids 291-299 of the transmembrane region showed sufficient divergence between loci and similarity between alleles, to be suitable for the generation of locus-specific probes. Synthetic oligonucleotides were generated and shown to be highly locus-specific in hybridization. These probes were used successfully for the quantitation of the relative amounts of mRNA transcribed in human liver from HLA-A, -B, and -C genes; they should greatly simplify future studies of restriction fragment length polymorphisms of HLA-A, -B, and -C alleles as genetic markers of disease susceptibility.