H-2Dd Gene Research Articles

Differential effects on T cell and NK cell development by tissue-specific expression of H-2Dd transgene

The effect of tissue-specific expression of the MHC class I molecule H-2Dd on T cell and NK cell specificity was studied in transgenic mice expressing the H-2Dd gene under the control of the mouse metallothionein-I promoter. MTD mice expressed high amounts of H-2Dd in the liver, intestine and testis, but only minute amounts in the thymus, spleen and kidney. Zinc administration resulted in a 1.5- and 8.5-fold increase in H-2Dd expression in the liver and the intestine, respectively, but did not affect expression in the other organs tested. T cell tolerance developed towards H-2Dd in MTD mice, even in the absence of zinc. In contrast, NK cell-mediated natural resistance against lymphoma grafts was not seen in MTD mice, despite zinc administration. NK cells in MTD mice also failed to develop self tolerance to H-2Dd. The lack of functional effects did not result from inability of NK cells in MTD mice to interact with H-2Dd, as down-regulation of Ly49A receptor expression was observed on liver NK cells in MTD mice. Our data reveal a difference between T cells and NK cells in their requirements for MHC class I molecules in specificity development.

Nitric oxide (NO), methylation and TIMP-1 expression in BL6 melanoma cells transfected with MHC class I genes.

We have previously found that transfection of BL6-8 melanoma cells with the H-2K, but not H-2D/L genes resulted in loss of their metastatic ability that was associated with decrease in their invasiveness and up-regulation of TIMP-1 expression. In the present study using the methylation-specific PCR (MSP) we found that lack of TIMP-1 expression in BL6-8 is associated with methylation in the TIMP-1 5' regulatory area. In the H-2Kb transfected CL8-1 melanoma cells up-regulation of TIMP-1 was in parallel with loss of TIMP-1 gene methylation. Treatment of BL6-8 with 5-azacytidine or with an inhibitor of histone deacetylase trichostatin A resulted in up-regulation of TIMP-1 expression. These results indicate that methylation and histone deacetylation play an important role in transcription repression of TIMP-1 in BL6 melanoma cells. Some data showed that nitric oxide (NO) could affect methylation and expression of various gene. Therefore we analyzed NO production in B16 melanoma cell lines with different expression of TIMP-1. We have found that B16F10 and BL6-8 melanoma cells do not express TIMP-1 and do not produce nitric oxide (NO) even after stimulation with IFN-gamma and LPS. However, BL6-8 cells transfected with H-2Kb or H-2Kd, but not H-2Dd or H-2Ld gene expressed TIMP-1 and produced NO constitutevely. NO production in these cells was further stimulated by IFN-gamma and LPS. Northern blot analysis showed that expression of iNOS was paralleled with TIMP-1 expression in the tested melanoma cells. However, NO produced by SNAP or inhibition of NO production by NMA did not affect TIMP-1 expression in the tested melanoma cells. Thus, TIMP-1 expression and NO production in BL6 melanoma cells transfected with MHC class I gene coincides but it remains unclear whether NO is responsible for the change in TIMP-1 methylation and expression.

The Regulation of Murine H-2Dd Expression by Activation Transcription Factor 1 and cAMP Response Element Binding Protein

Resistance to radiation leukemia virus (RadLV)-induced leukemia is correlated with an increase in H-2Dd expression on the thymocyte surface. It has been shown that elevated H-2Dd expression on infected thymocytes is a result of elevated mRNA transcription and that the transcriptional increase is correlated with elevated levels of a DNA binding activity, H-2 binding factor 1 (H-2 BF1), which recognizes the 5'-flanking sequence (5'-TGACGCG-3') of the H-2Dd gene. Recently, it has been shown that the activation transcription factor 1 (ATF-1) homodimer is one form of the H-2 BF1 complex. Here we demonstrate that the cAMP response element binding protein (CREB) homodimer and the heterodimer of CREB/ATF-1 also recognize the cis regulatory motif and are two additional forms of the H-2 BF1 complex. The levels of mRNA encoding ATF-1 and CREB were both increased in RadLV-infected thymocytes that showed increased levels of H-2 mRNA. Also, all three H-2 BF1 binding activities, ATF-1 homodimer, CREB homodimer, and ATF-1/CREB heterodimer, were increased in RadLV-infected thymocytes that expressed high levels of H-2Dd Ag on the cell surface. Transfection experiments demonstrated that ATF-1 and CREB activated a reporter plasmid containing the H-2 BF1 motif. These observations strongly suggest that both ATF-1 and CREB are involved in the regulation of H-2 gene expression following RadLV infection of mouse thymocytes.

Conservation of the H-2 BF1 binding motif 5' of the H-2Ds, Ks and Dq genes.

The biological consequences of radiation leukaemia virus (RadLV) infection include the stimulation of H-2 antigen expression soon after injection of the virus. Early studies demonstrated that resistance to RadLV-induced leukaemia in certain mouse strains is mediated by genes in the H-2D region of the major histocompatibility complex (MHC). Recent studies have shown that elevated H-2Dd expression on the thymocyte cell surface of resistance mouse strains results from increased mRNA transcription and is correlated with elevated levels of a DNA-binding activity that recognizes a short DNA sequence 5' of the start of transcription for the H-2Dd gene. This binding activity has been termed H-2 binding factor 1 (H-2 BF1) and is found exclusively in the thymus. In an effort to examine the H-2 genes of RadLV-susceptible mice for the presence of the H-2 BF1 binding target, we have cloned class I genes from the highly susceptible B10.S mouse strain and have identified both the Ds and the Ks genes. The entire genomic sequence for the Ds gene has been determined and is reported here. In addition, the 5' regulatory region of the previously cloned Dq gene has been sequenced; mice of the Dq haplotype are also susceptible to RadLV-induced leukaemia. In this report, we show that the H-2 BF1 DNA binding sequence is present 5' of each of these three class I genes.

Activation Transcription Factor 1 Involvement in the Regulation of Murine H-2Dd Expression

Resistance to radiation leukemia virus-induced leukemia is correlated with an increase in H-2D expression on the thymocyte surface. Recently, it has been shown that elevated H-2Dd expression on the infected thymocyte is a result of elevated mRNA transcription and that the transcriptional increase is correlated with elevated levels of a DNA binding activity, H-2 binding factor 1 (H-2 BF1), which recognizes the 5'-flanking sequences (5'-TGACGCG-3') of the H-2Dd gene. This target for transcription factor binding has been found to be identical in the 5'-regulatory region of 12 rodent class I genes, nine of which have been shown to be functional genes. Furthermore, this cis-element is found 5' of 20 primate class I genes (15 human genes), seven of which are known to be functional. Here, we demonstrate that activation transcription factor 1 (ATF-1) is one component of H-2 BF1. In addition, the levels of ATF-1 mRNA in uninfected and radiation leukemia virus-infected thymocytes parallel those of H-2Dd mRNA, and therefore, it is suggested that ATF-1 up-regulates the transcription of the H-2Dd gene after radiation leukemia virus infection of thymocytes. Transfection experiments also demonstrate that ATF-1 activates a reporter plasmid that contains the H-2 BF1 motif, but not a reporter lacking this motif. This is the first demonstration of the interaction of ATF-1 with 5'-regulatory sequences of major histocompatibility complex class I genes.

Loss of intracisternal A-type retroviral particles in BL6 melanoma cells transfected with MHC class I genes.

Electron microscopy of B16 melanoma and its sublines revealed that these cells produce numerous intracisternal A-type retroviral particles (IAPs). To identify and sequence the melanoma-associated IAPs of C57BL/6 mice, a cDNA library was constructed from IAP-producing BL6.8 cell RNA and screened using MIA14 IAP DNA as a probe. A 6-8 kb mRNA was identified that represents the full-length message for a new subfamily of IAP, termed MeIAP. The melanoma-derived IAP cDNA showed high similarity to MIA14 with major differences in the LTR. A nine base motif of the R region showed that this IAP differs from other previously sequenced IAPs. Analysis of the individual clones from BL6 melanoma revealed that IAPs were produced only in the clones that failed to express H-2Kb molecules. No IAPs were found in the melanoma clones that expressed endogenous H-2Kb. To analyse further the association between MHC class I genes and IAP production, the H-2Kb-negative clones of BL6 melanoma were transfected with various H-2 genes. Transfection of the H-2Kb or H-2Kd, but not H-2Dd or H-2Ld genes resulted in the elimination of IAPs. Northern blot analysis revealed that loss of IAPs in the H-2K gene-transfected BL6 melanoma cells was due to lack of IAP transcripts. Elimination of IAPs in the H-2Kb-positive BL6 melanoma cells was also accompanied by alterations in expression of various cellular genes and changes of their phenotypic properties.

The ability of H‐2Dd molecule to affect natural resistance to hemopoietic allografts is an intrinsic property shared by Ddm1 but not Ld

F1 hybrid resistance (HR) to parental bone marrow grafts is mediated by natural killer (NK) cells, and thought to be controlled by the non-class I hemopoietic histocompatibility (Hh) genes linked to the major histocompatibility complex (MHC). However, as in the in vitro NK cytotoxicity against hemopoietic targets, expression of certain class I MHC molecules does affect HR, although mechanisms underlying such an effect are not understood. In this study, we examine the relevance of the "self/non-self" property of class I molecules and the molecular domains responsible for this function. H-2b/Hh-1b lymphoma cells were transfected with class I H-2Dd or Ld gene, and its effect on the Hh-1 phenotype was examined by testing the transfectant's ability to competitively inhibit the in vivo rejection of parental H-2b/Hh-1b bone marrow grafts by irradiated F1 hybrid hosts. Multiple independent clones of transfectants show that the genomic or cDNA of the Dd gene, but not of Ld, renders the Hh-1b-positive cells incapable of inhibiting HR in F1 mice, although both genes belong to the same region of the same haplotype. The same effect could be observed not only in H-2b/d F1 mice for which Dd and Ld are self, but also in H-2b/k F1 mice for which both Dd and Ld are non-self. Thus, this function of the Dd molecule is an intrinsic property, not necessarily related to its self/non-self characteristic relative to the effector cells. Furthermore, given the nature of the assay used in this study, the results favor a "target interference" model as the underlying mechanism of the Dd effect. To locate the relevant domain(s) of the Dd molecule, mutant Ddm1 gene was tested and found to have the same effect as the non-mutant Dd. Ddm1 is a hybrid molecule between Dd and Ld, sharing with Dd only the alpha 1 domain and a portion of the alpha 2 domain. The two N-terminal domains of Ddm1 differ from those of Dd by three amino acid substitutions, two of which affect the molecules' peptide-binding properties.

Role of Major Histocompatibility Complex Class I α1/α2 Domain Polymorphism and In Vivo Expression Pattern in Tumor Resistance

The influence of major histocompatibility complex (MHC) class I molecules on target cell sensitivity and natural killer cell specificity was investigated. H-2b lymphoma cells grew progressively in syngeneic nontransgenic C57Bl/6 (B6) mice, but were rapidly rejected (within 24 h) in H-2Dd transgenic B6 mice (D8). Rejection required transgene expression in hematopoietic tissues, as revealed in experiments using transgenic mice with tissue-specific expression of the transgene into the target cells as well as bone marrow chimeric mice. The rejection was abrogated by transfection of the H-2Dd gene, providing evidence of "missing self" recognition in vivo triggered by a single class I allelic difference between host and graft. Lymphoma cells transfected with nonrelated class I molecules (H-2Dp and H-2Ld) did not lead to escape from rejection in D8 mice. Furthermore, transfection with exon shuffled constructs between H-2Dd and H-2Ld allowed mapping of the "protective" parts of H-2Dd. Only the transfectant expressing the alpha 1/alpha 2 domains of H-2Dd (the alpha 3 domain of H-2Ld) escaped elimination in D8 mice, while the transfectant expressing the opposite exon shuffled gene (the alpha 1/alpha 2 domains of H-2Ld in connection with the alpha 3 domain of H-2Dd) was rejected like the nontransfected wild type. The results showed that (a) introduction of a novel MHC class I gene in transgenic mice altered the specificity of natural killer cells, and (b) protection from transgenic NK cells required expression of the alpha 1/alpha 2 domains of H-2Dd at the target level.

Effect of MHC class-I transfection on local tumor growth and metastasis in an H-2-negative clone derived from a chemically induced fibrosarcoma.

GR9 is a chemically induced fibrosarcoma composed of clones with different H-2 class-I expression. These clones differ with respect to local growth and spontaneous metastasis. The B9 clone (H-2 negative) is highly tumorigenic (local growth) but of low metastatic potential (spontaneous metastasis assay). We have analyzed the effect that transfection of H-2Dd and H-2Kd genes on this clone have upon local growth, lung colonization after i.v. injection and ability to form spontaneous metastases. The results showed that the effect on local growth of transfection of the Kd-gene was stronger than that of the Dd gene. In addition, B9 co-transfected with H-2Kd and Dd genes showed the highest immunogenic properties in syngeneic BALB/c mice. Interestingly, the pSV2-neo transfected clone gave almost the same result as that obtained with Dd transfection. Lung colonization after i.v. injection of the different clones (experimental metastasis), paralleled the results obtained for local growth: the number of lung nodules followed the cadence KdDd less than Kd less than Dd less than pSV2. Survival of mice was always inversely correlated with local growth, e.g., all mice injected with 5 x 10(5) B9 H-2KdDd transfected cells survived. In contrast, no mice injected with the B9 control did. These differences were abrogated in irradiated and nude BALB/c mice. Finally, all transfected clones remained non-metastatic in a spontaneous metastasis assay, behaving as the control, non-transfected B9 cells.

A novel DNA binding activity is elevated in thymocytes expressing high levels of H-2Dd after radiation leukemia virus infection.

Resistance to radiation leukemia virus-induced leukemia is mediated by gene(s) in the H-2D region of the MHC; a clear correlation exists between disease resistance and increased H-2Dd expression on the thymocyte surface. We have investigated the molecular basis for this stimulation of H-2Dd class I expression. Elevated H-2 mRNA and H-2 transcription are demonstrated in the infected thymocytes as compared to normal thymocytes indicating that the elevation of H-2 surface expression is the result of transcriptional activation. Gel mobility assays performed with nuclear extracts of normal and infected thymocytes and sequences 5' of the H-2Dd gene show that specific binding occurs with both extracts; the binding differs both quantitatively and qualitatively, however. DNase I protection analysis detects a protein binding site that is protected only by extracts from infected cells. The protected region contains a sequence similar to the AP-1 consensus sequence. Gel shift competition assays and UV photo-cross-linking to an oligonucleotide containing this sequence demonstrate that specific binding of an H-2 binding factor 1 occurs and that this factor is not the AP-1 binding complex. This novel binding factor, activated in vivo, might also be involved in the normal regulation of H-2 gene expression by recognizing the highly conserved binding sequence (TGACGCG) found in the 5' flanking region of many MHC class I genes. This is the first demonstration of the parallel stimulation of a DNA binding activity and increased transcription occurring in thymocytes after infection with a leukemogenic retrovirus.

Developmental failure of chimeric embryos expressing high levels of H-2Dd transplantation antigens.

The absence of expression of class I products of the major histocompatibility complex at early stages of development is thought to play a key role in maternal tolerance of the fetal allograft. To test this, we developed a strategy that would allow us to describe the consequences of overexpression of the H-2Dd transplantation antigen in the developing embryo. A construct containing the H-2Dd gene under control of the human beta-actin promoter was transfected into pluripotent embryonic stem (ES) cells. Particularly in this case, since overexpression of major histocompatibility complex class I gene products may profoundly affect embryonic development, an important advantage of the ES cell system is the ability to analyze gene expression and study effects on cell growth and differentiation in vitro. ES cells do not constitutively express beta 2-microglobulin. Consistent with this, H-2Dd H chains expressed by ES cell transformants were not associated with beta 2-microglobulin or transported to the cell surface. Significant levels of beta 2-microglobulin and H-2Dd membrane glycoproteins were expressed following differentiation in vitro. H-2Dd-transfected ES cells gave rise to a wide range of differentiated cell types, and there was no evidence to suggest that expression of the introduced H-2Dd gene affects the differentiation abilities of ES cells in vitro. When introduced into blastocysts, H-2Dd-transfected ES cells extensively contribute to embryonic and extraembryonic tissues, but this results in the failure of chimeric conceptuses at midgestation. Considering that transgenic chimeras cannot be rescued by transfer into syngeneic foster females, it seems likely that nonimmunological mechanisms are responsible for these prenatal lethalities.

Defective assembly of class I major histocompatibility complex molecules in an embryonic cell line

Developmentally regulated expression of the products of the major histocompatibility complex (MHC) is thought to play a key role in maternal tolerance of the fetal allograft. Here we analyze a cell line (EE2H3), derived from early post-implantation-stage mouse embryos, that is defective for MHC class I assembly. To follow expression of a single well-defined class I product, we introduced the H-2Dd gene under control of the human beta-actin promoter. We found that the transfected EE2H3 cells expressed abundant levels of H-2Dd heavy chains and beta 2-microglobulin protein, but only small amounts of H-2Dd surface protein. Surface expression was rescued by the addition of an appropriate antigenic peptide, or by culturing the cells at low temperature. The phenotype exhibited by EE2H3 is thus remarkably similar to that described for class I-negative somatic cell variants selected using antibodies and complement. However, a striking difference was that surface expression in H-2Dd-transfected EE2H3 cells was markedly enhanced in response to treatment with interferon. Thus, we have identified a novel class I assembly-defective cell line. Considering that EE2H3 was established from primary cultures of mouse embryo cells without immunoselection, and is therefore likely to represent a cell population normally present in post-implantation-stage embryos, these findings raise the possibility that expression of class I surface antigens during early development may in part be controlled post-translationally at the level of MHC class I assembly.

Biochemical analysis of a novel H-2 class I-like glycoprotein expressed in five AKR-(Gross virus) derived spontaneous T cell leukemias.

The H-2 class I Ag profiles of five spontaneous AKR (H-2K) Gross virus leukemic cell lines were analyzed. A novel H-2 class I, "alloantigen"-like glycoprotein was immunoprecipitated and isolated from all the tumor cell lines using an H-2Dd-specific mAb 35-5-8. The novel Ag was also recognized in vitro by anti-H-2Dd-specific CTL. In addition, DNA from all the thymomas, but not the DNA from normal adult AKR thymic cells showed a transcribed gene detectable with an H-2Dd-specific oligonucleotide probe. The molecular profile of the novel antigen was further studied by two-dimensional gel electrophoresis and analyzed by a computer based image analyzer system and reverse-phase HPLC tryptic peptide mapping. Its molecular pattern was different from the syngeneic H-2Kk, H-2Dk, and the allogeneic H-2Dd gene products. The two-dimensional gel pattern of the novel H-2 class I molecule had a different overall structure reflected in isoelectric point, number, and distribution of polypeptide spots. The tryptic peptide map analysis showed six peaks exclusively identified with the novel Ag. The calculated degree of homology with the corresponding H-2Dd, H-Dk, and H-Kk peptides was 41, 56, and 51%, respectively. In addition, an unusual cell surface distribution of the novel Ag was observed in most of the leukemic lines. The removal of sialic acid residues by neuraminidase treatment facilitated the detection of the allodeterminants by anti-H-2Dd-specific mAb and CTL. Furthermore, we showed that in one AKR tumor line, 424, there is a close association of the novel Ag with the syngeneic class I molecules. Prior preclearance of the syngeneic class I molecules revealed the presence of the H-2Dd-like allospecificity. The genetic and molecular relationship between the expression of this novel class I-like glycoprotein and the recently sequenced Q5 gene is under current investigation.

Expression of mutant H-2d proteins encoded by class I genes which alternatively process the 5' end of their transcripts.

Mutation of the 3' splice sites bordering exon 2 of the H-2Dd and H-2Kd genes generated alternatively spliced transcripts when the constructs were transfected into L cells (J. Immunol. 143:1018). The H-2Dd transcripts contained an additional 84 nucleotides derived from the first intervening sequence, whereas 60 extra bases were included in the H-2Kd mRNA. Proteins derived from these transcripts were recognized by mAb. Moreover, both Ag served as recognition elements for CTL, and the mutant H-2Kd molecule functioned as a restricting element for an Ag peptide. As a result of alternative splicing, the mutant proteins should have additional residues at their NH2 termini to increase their lengths by 28 (Dd) or 20 (Kd) amino acids. Immunoprecipitation and analysis on SDS-PAGE demonstrated that the mutant H-2Kd molecule was indeed larger than the normal H-2Kd protein, but the mutant and wild-type H-2Dd Ag were the same size. In addition, treatment of H-2Dd mutant and normal Ag with N-glycanase produced molecules of equal size, demonstrating that the mutant protein was completely glycosylated. Limited amino acid sequencing of this Ag indicated that it was normal H-2Dd. Therefore, before its transfer to the cell surface, post-translational modifications remove the additional NH2-terminal residues of the mutant Dd but not Kd protein.

Mutation of 3' splice sites in two different class I genes results in different usage of cryptic splice sites.

To determine the pattern of alternative splicing at the 5' end of class I genes, the 3' splice sites bordering exon 2 of the H-2Dd and H-2Kd genes were mutated from AG to GG (H-2Dd) or CG (H-2Kd). The mutant genes were transfected into L cells, and RNA from clones expressing these Ag was used for analysis by RNase and S1 nuclease mapping techniques. The first intervening sequence of both class I genes contains several potential 3' splice acceptor sites. However, a clear preference for only one site was detected in each of the H-2Dd and H-2Kd mRNA. Examination of the endogenous H-Dd and H-2Kd class I transcripts in normal murine tissues and in tumors demonstrated that the alternatively spliced mRNAs were produced, but at a low frequency. Infection of transfected L cells or tumor lines with vesicular stomatitis virus altered the level of differentially spliced message in these cells.

Analysis of D2d: a D-region class I gene.

The mouse major histocompatibility complex is composed of several genes arranged into the K, D, Qa, and Tla regions. The D region of the BALB/c mouse includes genes D2d, D3d, and D4d, in addition to H-2Dd and H-2Ld. We have determined the DNA sequence of the D2d gene and compared it with the known sequences of several class I genes. The exon/intron structure of the D2d gene is similar to other class I genes. It also contains similar 5' regulatory elements. A frameshift occurs in exon seven, resulting in a gene product with a truncated cytoplasmic tail. To examine the surface expression of the D2d molecule, we generated an exon-shuffled construct containing the promoter and exons 1-3, encoding the signal peptide, alpha 1, and alpha 2 external domains of the D2d gene linked to exons 4-8, encoding the alpha 3, transmembrane and cytoplasmic domains, of the H-2Dd gene. The construct was transfected into mouse L cells, and a protein was detected at the cell surface by a monoclonal antibody (mAb) specific for the alpha 3 domain of H-2Dd, as well as by other class I-specific mAbs. Although D2d is expressed at low levels, it may be a functional class I gene that most probably evolved from a Qa region gene.

Substitution at residue 227 of H–2 class I molecules abrogates recognition by CD8-dependent, but not CD8-independent, cytotoxic T lymphocytes

The CD8 (Lyt 2) molecule is a phenotypic marker for T lymphocytes that recognize and react with major histocompatibility complex (MHC) class I molecules. Antibody blocking experiments and gene transfection studies indicate that CD8 binds to a determinant on MHC class I molecules on the target cells, facilitating interaction between effector T lymphocytes and the target cell. The CD8 molecule may also be involved in transmembrane signalling during T-cell activation. The existence of CD8- cytotoxic T lymphocytes (CTL) and class I-reactive CTL that are not inhibited by antibody to CD8 suggests that at least some CTL do not require the CD8 molecule to interact with and lyse target cells. We have recently demonstrated that cells transfected with an H-2Dd gene that carries a mutation at residue 227 are not killed by primary CTL8. Here we show that although this mutation abrogates recognition by primary CTL, it does not affect recognition by CD8-independent CTL, suggesting that residue 227 of class I molecules might contribute to a determinant that is the ligand of the CD8 molecule.

Regulation of gene expression by interferons: control of H-2 promoter responses.

The magnitude of the response to interferons and the requirement for individual elements in the promoter of the H-2Dd gene were shown to be cell-specific and dependent on the type of interferon used. Three DNA sequences in the promoter were found to bind murine nuclear factors. Two of these sequences are in functionally defined enhancer regions and also bind to the transcription factor AP-1. The third sequence is part of the region involved in interferon regulation and is homologous to the enhancer element of the interferon beta gene. A model for interferon regulation of H-2 promoters is 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.

A single amino acid substitution in the alpha 3 domain of an H-2 class I molecule abrogates reactivity with CTL.

We previously described a somatic cell expressing a variant H-2Dd molecule that did not serve as a target for alloreactive anti-Dd CTL. The mutant cell line had been isolated by its failure to express a serological epitope present on the H-2Dd alpha 3 domain. In the present study the alpha 3 domain of the Dd molecule of this somatic cell variant was sequenced and a single nucleotide change resulting in a glutamic acid to lysine substitution at residue 227 was identified. This change was reproduced in the cloned H-2Dd gene by oligonucleotide-directed mutagenesis. Cells transfected with this mutant gene were not killed by anti-H-2Dd CTL. Because previous studies using hybrid H-2 class I molecules had established that the alpha 3 domain does not express allele-specific determinants recognized by CTL, our results raise the possibility that residues in the alpha 3 domain of H-2 class I molecules are critical for CTL recognition and constitute a conserved (or monomorphic) determinant recognized by CTL.