Nonlinkage of major histocompatibility complex class I and class II loci in bony fishes

 In tetrapods, the functional (classical) class I and class II B loci of the major histocompatibility complex (Mhc) are tightly linked in a single chromosomal region. In an earlier study, we demonstrated that in the zebrafish, Danio rerio, order Cypriniformes, the two classes are present on different chromosomes. Here, we show that the situation is similar in the stickleback, Gasterosteus aculeatus, order Gasterosteiformes, the common guppy, Poecilia reticulata, order Cyprinodontiformes, and the cichlid fish Oreochromis niloticus, order Perciformes. These data, together with unpublished results from other laboratories suggest that in all Euteleostei, the classical class I and class II B loci are in separate linkage groups, and that in at least some of these taxa, the class II loci are in two different groups. Since Euteleostei are at least as numerous as tetrapods, in approximately one-half of jawed vertebrates, the class I and class II regions are not linked. Received: 30 August 1999 / Revised: 20 October 1999     

A new major histocompatibility complex class I b gene expressed in the mouse blastocyst and placenta

 Because of the role major histocompatibility complex (MHC) class I b molecules may play during mouse embryonic development, we thought it would be interesting to search for additional MHC class I b molecules that might be expressed in preimplantation embryos, and in particular in the trophoblastic lineage. We therefore screened a mouse preimplantation blastocyst cDNA library for MHC class I sequences. This search led to the identification and characterization of a new MHC class I b gene, blastocyst MHC. Sequences identical to the exons and 3′ untranslated region of this gene have been found in many laboratory mouse strains, as well as in the related mouse species Mus spreciligus. The presence of this gene in mouse strains of different MHC class I haplotypes argues that blastocyst MHC is a unique, newly-described gene rather than a new allele of a previously described mouse MHC class I gene. Blastocyst MHC has the structure of an MHC class I b gene, with the six exons characteristic of T-region genes. It is linked to H2-D. The amino acid sequence encoded by this gene maintains all the features of a functional antigen-presentation domain. The blastocyst MHC gene, like the human class I b gene HLA-G, is expressed at the blastocyst stage and in the placenta, and may be the mouse analog for HLA-G. Received: 31 May 1996 / Revised: 19 August 1996     

Genomic analysis of common chimpanzee major histocompatibility complex class I genes

To investigate how MHC class I genes have changed in the approximately 5 million years since chimpanzees and humans diverged, we characterized six genomic fragments ranging in size from 5.1 to 6.1 kb, each containing the complete coding region, introns, and flanking regions of one of the following chimpanzee class I genes: Patr-A, Patr-E, Patr-F, Patr-G, Patr-H, and Patr-J. In humans, these genes are closely linked within the class I region and are representatives of three distinct functional categories of class I genes: the highly polymorphic Ia genes (HLA-A), the conserved Ib genes (HLA-E, HLA-F, and HLA-G), and the class I pseudogenes (HLA-H and HLA-J). Southern blot analysis of chimpanzee and human class I genes produced nearly identical patterns, suggesting that the organization and linkage of these genes differs little in the two species. Comparison of the chimpanzee fragment sequences with their human orthologues revealed structural conservation of these genes yet differences in their degree of functional constraint. This is apparent in the location and nature of the amino acid changes between species and the substantial differences in levels of divergence at functional and nonfunctional sites. Additionally, there is no correlation between patterns of divergence at these sites and intraspecific variation, an observation explained by either appreciable gene conversion or high levels of recombination, the latter unlikely given the observed strong linkage disequilibrium of these loci.     

The α1 and α2 domains of H-2 class I molecules interact to form unique epitopes

Mouse class I antigens are the major targets of cytolytic T lymphocytes in both major histocompatibility complex (MHC)-restricted and allogeneic responses. Considerable evidence has recently accumulated demonstrating that MHC class I molecules encoded by genes whose 1 and 2 coding exons were interchanged are not recognized by T lymphocytes specific for parental class I products. Along with the loss of T -cell reactivity, there is a loss of recognition by some, but not all monoclonal antibodies. In this communication we report that the loss of reactivity by monoclonal antibodies is accompanied by the gain of new epitopes caused by the interaction of 1 and 2 domains. These epitopes are immunodominant. They are the major determinant recognized by polyclonal antisera raised by immunization with L cells transfected with exon-shuffled class I genes. Four new monoclonal antibodies have been produced which recognize at least two separate epitopes caused by the interaction of the 1^P and 2^d domains.     

Lifetime of major histocompatibility complex class-I membrane clusters is controlled by the actin cytoskeleton

Lateral heterogeneity of cell membranes has been demonstrated in numerous studies showing anomalous diffusion of membrane proteins; it has been explained by models and experiments suggesting dynamic barriers to free diffusion, that temporarily confine membrane proteins into microscopic patches. This picture, however, comes short of explaining a steady-state patchy distribution of proteins, in face of the transient opening of the barriers. In our previous work we directly imaged persistent clusters of MHC-I, a type I transmembrane protein, and proposed a model of a dynamic equilibrium between proteins newly delivered to the cell surface by vesicle traffic, temporary confinement by dynamic barriers to lateral diffusion, and dispersion of the clusters by diffusion over the dynamic barriers. Our model predicted that the clusters are dynamic, appearing when an exocytic vesicle fuses with the plasma membrane and dispersing with a typical lifetime that depends on lateral diffusion and the dynamics of barriers. In a subsequent work, we showed this to be the case. Here we test another prediction of the model, and show that changing the stability of actin barriers to lateral diffusion changes cluster lifetimes. We also develop a model for the distribution of cluster lifetimes, consistent with the function of barriers to lateral diffusion in maintaining MHC-I clusters.     

The α2(XI) collagen gene lies within 8 kb of Pb in the proximal portion of the murine major histocompatibility complex

A number of serious hereditary disorders are now known to be associated with defective expression of collagen genes, and these findings have underscored the important and varied roles that the collagen family of genes must play during normal mammalian development. Although the activities of genes encoding the quantitatively major types of collagen are fairly well characterized, functions of the many minor types of collagen remain a matter of speculation. As a first step toward a functional analysis of type XI collagen, a member of this class of poorly understand minor collagen proteins which is expressed primarily in hyaline cartilage, we have used human probes for the gene encoding the protein's 2-subunit (COL11A2) to isolate and map homologous murine DNA sequences. Our results demonstrate that Col11a-2 is embedded within the major histocompatibility complex (MHC), within 8.4 kb of the class II pseudogene locus, Pb, and confirm that human and murine 2(XI) collagen genes are located in very similar genomic environments. The conserved location of these genes raises the possibility that type XI collagen genes may contribute to one or more of the diverse hereditary disorders known to be linked to the MHC in mouse and human.     

Evolution of introns and exons of class II major histocompatibility complex genes of vertebrates

 The phylogenetic relationships and patterns of nucleotide substitution were compared for introns and exons of class II major histocompatibility complex (MHC) genes in three datasets: human DRB1, human DQA1, and cyprinid fish DAB1. In both human DRB1 and cyprinid DAB1, there was strong evidence that recombination events between alleles have occurred in such a way that intron and exon sequences of a given allele do not necessarily share the same evolutionary history. In the case of human DRB1, recombination was found to have homogenized intron 1 and intron 2 sequences relative to exon 2 sequences within lineages of alleles but not between lineages. As a result, mean divergence times of intron sequences are much more recent than those of exonic sequences. Thus, the divergence time of DRB1 introns cannot be used to date that of exons in the same alleles, and the hypothesis that most human DRB1 polymorphism is of very recent origin is not supported. Received: 5 September 1999 / Revised: 30 December 1999     

Autophagy facilitates major histocompatibility complex class I expression induced by IFN-γ in B16 melanoma cells

The reduction or loss of MHC-I antigen surface expression in human and murine tumor cells is partly attributable to the dysregulation of various components of the MHC-I antigen-processing machinery. Accumulating evidence suggests that autophagy, besides its vital role in maintaining the cellular homeostasis, plays an important role in MHC-II surface expression. Here, we report that autophagy is a negative regulator of MHC-I antigen expression in B16 melanoma cells; however, in the presence of IFN-γ, it is converted to a positive regulator. We show that autophagy not only participates in the degradation of MHC-I antigen but also plays a role in the generation of MHC-I-binding peptides. For these two processes, IFN-γ interferes with MHC-I antigen degradation, rather than affecting peptide generation. Using B16 melanoma mouse model, we further show that autophagy may enhance the cytolysis of CTL to melanoma cells at the early stage of melanoma, but impairs the cytolysis at the late stage. Such different consequences may be explained by the different levels of IFN-γ during tumor progression. Taken together, our findings demonstrate that autophagy is involved in the regulation of MHC-I antigen expression, through which autophagy can play different roles in tumor immunity.     

Elastase inhibition by the C-terminal domains of α-crystallin and small heat-shock protein

α-Crystallin, an abundant eye-lens protein and a stress protein in other tissues, shows structural and functional similarities with the small heat-shock proteins. One of the properties in common is the inhibition of elastase. We now report that the separated subunits of α-crystallin, αA and αB,also exhibit elastase inhibition, whereas phosphorylation of these subunits apparently has no influence on the inhibitory capacity. Furthermore, for both αA-crystallin and mouse HSP25 the putative C-terminal structural domain, comprising the major region of homology between these proteins, is sufficient to give elastase inhibition. With database search no homology could be found between the three proteins under investigation and any of the known consensus sequences of proteinase inhibitor families.     

The helper T-cell repertoire of mice expressing class II major histocompatibility complex β chains in the absence of α chains

 Mutant mice generated by disrupting the H2-Aa ^b major histocompatibility complex (Mhc) gene are demonstrated here to express Aβ^b chains in the absence of α chains. These mice possess a CD4⁺ helper T cell (Th) repertoire that uses predominantly the Vβ7 T-cell antigen receptor (Tcr) segment for recognition of any protein antigen presented by the α-free Aβ molecule. As an alloantigen, the Aα-free Aβ molecule is recognized very poorly by T cells from a series of class II disparate mouse strains, indicating that it is grossly different from normal α/β heterodimers. Indeed, molecular modeling suggests a β/β homodimer arrangement with an altered geometry of the Tcr contact area. Interestingly, the mutant mice exhibit normal alloreactivity, without a restricted Vβ usage, toward a series of foreign α/β class II heterodimers, although their T cells developed in the absence of such heterodimers. Thus, the complementarity of Tcr to normal α/β heterodimers, and thereby also alloreactivity, appears to be an ontogeny independent (i. e., germline-encoded) feature. Received: 30 September 1996 / Revised: 18 October 1996     

Genetic polymorphism of the swine major histocompatibility complex (SLA) class I genes, SLA-1, -2 and -3

In order to identify and characterize genetic polymorphism of the swine major histocompatibility complex (Mhc: SLA) class I genes, RT-PCR products of the second and third exons of the three SLA classical class I genes, SLA-1, SLA-2 and SLA-3 were subjected to nucleotide determination. These analyses allowed the identification of four, eight and seven alleles at the SLA-1, SLA-2 and SLA-3 loci, respectively, from three different breeds of miniature swine and one mixed breed. Among them, 12 alleles were novel. Construction of a phylogenetic tree using the nucleotide sequences of those 19 alleles indicated that the SLA-1 and -2 genes are more closely related to each other than to SLA-3. Selective forces operating at single amino acid sites of the SLA class I molecules were analyzed by the Adaptsite Package program. Ten positive selection sites were found at the putative antigen recognition sites (ARSs). Among the 14 positively selected sites observed in the human MHC (HLA) classical class I molecules, eight corresponding positions in the SLA class I molecules were inferred as positively selected. On the other hand, four amino acids at the putative ARSs were identified as negatively selected in the SLA class I molecules. These results suggest that selective forces operating in the SLA class I molecules are almost similar to those of the HLA class I molecules, although several functional sites for antigen and cytotoxic T-lymphocyte recognition by the SLA class I molecules may be different from those of the HLA class I molecules.The DNA sequence data reported in this paper have been submitted to the DDBJ, EMBL and GenBank nucleotide databases and have been assigned the accession numbers, AB105379, AB105380, AB105381, AB105382, AB105383, AB105384, AB105385, AB105386, AB105388, AB105389, AB105390 and AB105391     

Treatment of ovarian cancer cell lines with 5-aza-2′-deoxycytidine upregulates the expression of cancer-testis antigens and class I major histocompatibility complex-encoded molecules

Purpose  To test the hypothesis that decrease in DNA methylation will increase the expression of cancer-testis antigens (CTA) and class I major histocompatibility complex (MHC)-encoded molecules by ovarian cancer cells, and thus increase the ability of these cells to be recognized by antigen-reactive CD8⁺ T cells. Methods  Human ovarian cancer cell lines were cultured in the presence or absence of varying concentrations of the DNA demethylating agent 5-aza-2′-deoxycytidine (DAC) for 3–7 days. The expression levels of 12 CTA genes were measured using the polymerase chain reaction. The protein expression levels of class I MHC molecules and MAGE-A1 were measured by flow cytometry. T cell reactivity was determined using interferon-γ ELISpot analysis. Results  DAC treatment of ovarian cancer cell lines increased the expression of 11 of 12 CTA genes tested including MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, NY-ESO-1, TAG-1, TAG-2a, TAG-2b, and TAG-2c. In contrast, DAC treatment decreased the already low expression of the MAGE-A2 gene by ovarian cancer cells, a finding not previously observed in cancers of any histological type. DAC treatment increases the expression of class I MHC molecules by the cells. These effects were time-dependent over a 7-day interval, and were dose-dependent up to 1–3 μM for CTA and up to 10 μM for class I MHC molecules. Each cell line tested had a unique pattern of gene upregulation after exposure to DAC. The enhanced expression levels increased the recognition of 2 of 3 antigens recognized by antigen-reactive CD8⁺ T cells. Conclusions  These results demonstrate the potential utility of combining DAC therapy with vaccine therapy in an attempt to induce the expression of antigens targeted by the vaccine, but they also demonstrate that care must be taken to target inducible antigens. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The major and a minor class II β-chain (B-LB ) gene flank the Tapasin gene in the B-F /B-L region of the chicken major histocompatibility complex

 We have identified the major histocompatibility complex class II β-chain (B-LB) genes present in the B-F/B-L region of the B complex of nine well-characterized lines of chickens and have cleared up much of the confusion concerning numbers and location of B-LB genes in this region. By amplifying DNA sequences between adjacent genes, we found two B-LB genes that lie on either side of Tapasin. The dominantly expressed 'major' B-LB gene in all haplotypes lies between Tapasin and RING-3, and belongs to the B-LBII family of class II β-chain genes. The poorly expressed 'minor' B-LB gene in all haplotypes lies between B-lec1 and Tapasin, and belongs either to the B-LBII family or to the previously unmapped B-LBVI family of class II β-chain genes. The data suggest that the B-LBII and B-LBVI genes are two lineages of B-LB genes and we propose that they all be termed B-LB genes. The location of a third B-LB gene in the B12 haplotype (and possibly other haplotypes as well) has yet to be determined. The structural organization and expression of the class II β-chain genes in the B-F/B-L region is similar to that of chicken class I (B-F) genes, one functional result of which is differential resistance to disease and response to vaccines. Received: 29 July 1999 / Revised: 27 September 1999     

Sequence of the pig major histocompatibility region containing the classical class I genes

A segment comprising 307,078 nucleotides of the pig major histocompatibility complex (SLA) was completely sequenced. The segment corresponded to the entire SLA classical class I-containing region of the serologically defined SLA H01 haplotype. In all, 11 genes were characterized, comprising 7 class I genes located on the centromeric part of the sequence (SLA-1, 2, 3, 4, 5, 9, and 11) and 4 ring finger-related family genes located on its telomeric part. No member of one family was intermingled with a member of the other or with any third-party gene. All class I genes except SLA-11 were similarly orientated. The SLA-1, 2, and 3 genes displayed both promoter and overall coding regions compatible with normal functions. The SLA-4, 11, and 9 genes were considered pseudogenes because they exhibited marked anomalies. Although the SLA-5 gene had a complete coding region, it displayed mutations in promoter elements which could modify its expression. The great molecular similarity observed among the class I genes extended far outside them, and resulted from segmental duplications. The ring finger genes exhibited great homology with their human counterparts. In pig, one of these genes appeared to correspond to a complete gene which in humans is probably a pseudogene. In all, the 11 genes characterized span about 20% of the total sequence. The remaining 80% consists of interspersed repeat elements. The present results, together with the sequence previously reported involving the SLA class I-related genes, open the way for a better understanding of pig MHC organization.     

Different rates of HLA class I molecule assembly which are determined by amino acid sequence in the α2 domain

Assembly of HLA class I molecules was studied using pulse-chase labeling of B-lymphoblastoid cell lines with ³⁵S-methionine, immunoprecipitation with antibodies detecting free or ₂-microglobulin-associated heavy chain and isoelectric focusing. Marked differences between the products of different class I alleles were noted. HLA-B51 assembled very inefficiently, with considerable free heavy chain still detected in an unsialated form after a four hour chase. The closely related molecule HLA-B35 was in contrast rapidly assembled, all newly synthesized heavy chain being detected in a 2m-associated, sialated form within 30 minutes. Analysis of naturally occurring variants related to HLA-B35 and HLA-B51 localized the region determining assembly efficency to the 2 domain, in which these molecules differ at eight amino acid residues. The effect was not due to a linked dominant gene, as both patterns of assembly were observed in a single cell line.