BAC/YAC contigs from the H2-M region of mouse Chr 17 define gene order as Znf173-Tctex5-Mog-D17Tu42-M3-M2

 A yeast artificial chromosome (YAC) contig from the C57BL/6 (H2 ^b ) mouse was created from the major histocompatibility complex (Mhc, H2 in mouse) class Ib subregion, H2-M. It spans approximately 1.2 megabase (Mb) pairs and unites the previous >1.5-Mb YAC contigs (Jones et al. 1995) into a single contig, which includes 21 Mhc class I genes distal to H2-T1. A bacterial artificial chromosome (BAC) contig from the 129 (H2 ^bc ) mouse, spanning approximately 600 kilobases, was also built from Znf173 (Afp, a gene for acid finger protein), through Tctex5 (t-complex testis expressed-5) and Mog (myelin oligodendrocyte glycoprotein), to H2-M2. Twenty-four sequence-tagged site (STS) markers were newly developed, and 35 markers were mapped in the YAC/BAC contigs, which define the marker order as Cen –Znf173–Tctex5 – Mog–D17Tu42–D17Mit232–H2-M3–D17Leh525–H2-M2– Tel. The gene order of Znf173 – Tctex5 – Mog – D17Tu42 is conserved between mouse and human, showing that the middle H2-M region corresponds to the subregion of the human Mhc surrounding HLA-A. Received: 25 July 1997 / Revised: 10 September 1997     

H2-Mbeta 1 and H2-Mbeta 2 heterodimers equally promote clip removal in I-A(q) molecules from autoimmune-prone DBA/1 mice

Antigen-presenting cells degrade endocytosed antigens, e.g. collagen type II, into peptides that are bound and presented to arthritogenic CD4(+) helper T cells by major histocompatibility complex (MHC) class II molecules. Efficient loading of many MHC class II alleles with peptides requires the assistance of H2-M (HLA-DM in humans), a heterodimeric MHC class II-like molecule that facilitates CLIP removal from MHC class II molecules and aids to shape the peptide repertoire presented by MHC class II to CD4(+) T cells. In contrast to the HLA-DM region in humans, the beta-chain locus is duplicated in mice, with the H2-Mb1 beta-chain distal to H2-Mb2 and the H2-Ma alpha-chain gene. H2-M alleles appear to be associated with the development of autoimmune diseases. Recent data showed that Mbeta1 and Mbeta2 isoforms are differentially expressed in isolated macrophages and B cells, respectively. The tissue expression and functional role of these heterodimers in promoting CLIP removal and peptide selection have not been addressed. We utilized the human T2 cell line, which lacks part of chromosome 6 encompassing the MHC class II and DM genes, to construct transgenic cell lines expressing the MHC class II heterodimer I-A(q) alone or in the presence of H2-Malphabeta1 or H2-Malphabeta2 heterodimers. Both H2-M isoforms facilitate the exchange of CLIP for cognate peptides on I-A(q) molecules from arthritis-susceptible DBA/1 mice and induce a conformational change in I-A(q) molecules. Moreover, I-A(q) cell-surface expression is not absolutely dependent on H2-M molecules. These data suggest that I-A(q) exhibits a high affinity for CLIP since virtually all I-A(q) molecules on T2 cells were found to be associated with CLIP in the absence of both H2-M isoforms.     

TNFB polymorphisms characterize three lineages of TNF region microsatellite haplotypes

 The human major histocompatibility complex (MHC) contains a variety of genes, many of which are highly polymorphic and of immunological importance. A database of MHC extended haplotypes was used to integrate experimental, cell line, and population data. Three alleles of the human TNF-beta (lymphotoxin-alpha) gene were identified, named TNFB ^*1SL, TNFB ^*2LL, and TNFB ^*1LS, each representing a different lineage in the evolution of TNF region haplotypes. Lower variability in the length of the associated microsatellite alleles indicates that ^*1SL characterizes the youngest of the three haplotype lineages. Microsatellite haplotypes in the two older lineages show evidence for a coevolution of alleles through concerted expansions. Genetic predispositions to high and low TNF-alpha (cachectin) responses seem to have evolved independently in more than one lineage. The literature data suggest different, or even opposite, associations concerning the regulation of TNF-alpha in macrophages and lymphoid cells. Microsatellite ud may be the most informative marker for studies of the associations of individual TNF region markers with secretion levels, immunity, and disease. Received: 10 December 1996 / Revised: 21 May 1997     

H2-A polymorphism contributes to H2-Eβ-mediated protection in collagen-induced arthritis

 Collagen-induced arthritis (CIA) is an animal model of auto-immune inflammatory polyarthritis which has features similar to rheumatoid arthritis (RA). Much like RA, susceptibility to mouse CIA is influenced by the major histocompatibility complex (MHC) and is restricted to the H2 haplotypes q and r. In previous experiments, we have found that the introduction of an H2-Eb ^d transgene in H2-A^q CIA-susceptible mice was able to protect these mice against disease development. More recently, we have proposed that the polymorphism of the first domain of the Eβ molecule modulates this protection, and that the presentation of a peptide from the third hypervariable region of the Eβ chain by the H2-A^q molecule plays an important role in this mechanism. In the present report, we investigated whether the H2-E-mediated protection is H2-A^q-specific and whether the source of collagen has any influence. While the source of collagen had no effect on the protection, our results showed that the H2-E molecule failed to protect B10.RIII (H2^r) mice against CIA. Further, the H2 haplotype r exerted a negative effect on the Eβ^d-mediated protection in H2-A^q-restricted disease. Our results provide additional proof that self-MHC-derived peptides, such as Eβ peptides, may play an important role in the T-cell repertoire education and/or modulation of the T-cell response in the periphery. Received: 29 May 1996 / Revised: 26 June 1996     

Allelic variants of the human MHC class I chain-related B gene (MICB)

 The human major histocompatibility complex (MHC) is located within a 4 megabase segment on chromosome 6p21.3. Recently, a highly divergent MHC class I chain-related gene family, MIC was identified within the class I region. The MICA and MICB genes in this family have unique patterns of tissue expression. The MICA gene is highly polymorphic, with more than 20 alleles identified to date. To elucidate the extent of MICB allelic variations, we sequenced exons 2 (α1), 3 (α2), 4 (α3), and 5 (transmembrane) as well as introns 2 and 4 of this gene in 46 HLA homozygous B-cell lines. We report the identification of eleven alleles based on seven non-synonymous, two synonymous, and four intronic nucleotide variations. Interestingly, one allele has a nonsense mutation resulting in a premature termination codon in the α2 domain. Thus, MICB appears to have fewer alleles than MICA, not unlike the allelic ratio between the HLA-C and -B loci. A preliminary linkage analysis of the MICB alleles with those of the closely located MICA and HLA-B genes revealed no conspicuous linkage disequilibrium between them, implying the presence of a potential recombination hotspot between the MICB and MICA genes. Received: 16 April 1997 / Revised: 19 May 1997     

Allelic repertoire of the humanMHC class IMICA gene

 The hallmark of the classical major histocompatibility complex (MHC) class I molecules is their astonishing level of polymorphism, a characteristic not shared by the nonclassical MHC class I genes. A distinct family of MHC class I genes has been recently identified within the human MHC class I region. The MICA (MHC class I chain-related A) gene in this family is a highly divergent member of the MHC class I family and has a unique pattern of tissue expression. We have sequenced exons encoding the extracellular α1, α2, and α3 domains of the MICA gene from twenty HLA homozygous typing cell lines and four unrelated individuals. We report the identification of eleven new alleles defined by a total of twenty-two amino acid substitutions. Thus, the total number of MICA alleles is sixteen. Interestingly, a tentative superimposition of MICA variable residues on the HLA-A2 structure reveals a unique pattern of distribution, concentrated primarily on the outer edge of the MICA putative antigen binding cleft, apparently bordering an invariant ligand binding site. Received: 13 May 1996 / Revised: 29 May 1996     

Genomic organization of a mouse MHC class II region including theH2-M andLmp2 loci

The region encompassing theMa, Mb1, Mb2, andLmp2 genes of the mouse class II major histocompatibility complex (MHC) was sequenced. Since this region contains clusters of genes required for efficient class I and class II antigen presentation, it was interesting to search for putative additional genes in the 21 kilobase gap between theMb1 andLmp2 genes. Computer predictions of coding regions and CpG islands, exon trapping experiments, and cross-species comparison with the corresponding human sequence indicate that no additional functional gene is present in that stretch. However, computer analysis revealed the possible existence of an alternative 3 exon forMb1. Except for the fact that the mouse MHC contains twoMb genes, the genomic organization of theH2-M loci was found to be almost identical to the organization of the humanHLA-DM genes. The promoter regions of theMa andMb genes also resemble classical class II promoters, containing typical S, X, and Y boxes. Like the human genes, the threeH2-M genes displayed very limited polymorphism when we compared the cDNA sequences from six haplotypes. Finally, comparison ofDMB withMb1 andMb2, both at the genomic level and in their coding regions, suggests that theMb gene was recently duplicated, probably only in certain rodents.     

Polymorphisms in the TNF gene cluster and MHC serotypes in the West of Scotland

 We examined the distribution of polymorphic elements within the tumor necrosis factor (TNF) gene cluster in 105 unrelated individuals and determined their relationship to class I and class II major histocompatibility complex (MHC) antigens, and to the highly polymorphic microsatellites TNFa and TNFb. The data demonstrate the contribution of elements within the TNF cluster to two extended haplotypes which are recognized to straddle the MHC. The A1.B8.DR3 haplotype appears to contain the TNF alleles TNFa2, TNFb3, LT.Nco-1.B ^* 1, and TNF-308.2, while the A3.B7.DR2 haplotype is associated with the TNF alleles TNFa11, TNFb6, TNFc1, LT.Nco-1.B ^* 2, LT.AspH1.1, TNF-308.1, and TNFe1. The presence of other extended associations which covered smaller parts of the MHC was also suggested. In most cases, the associations described here were in keeping with previously described extended haplotypes which dominate the structure of the MHC, but these did not always match completely. Taken together, these data suggest that the structure of the TNF locus is well integrated into the rest of the MHC but that important ethnic differences may exist. Received: 12 June 1996 / Revised: 27 September 1996     

T-cell receptor Vbeta deletion and Valpha polymorphism are responsible for the resistance of SWR mouse to arthritis induction

 Collagen type II-induced arthritis (CIA) develops in susceptible mouse strains after intradermal injections of type II collagen (CII) in complete Freund's adjuvant (CFA). Susceptibility to CIA in mice is linked to genes of the major histocompatibility complex (MHC). Although the SWR mouse has a susceptible MHC haplotype (H2 ^q ), it is resistant to CIA. SWR exhibits at least two known immunological defects: (1) it contains a germline deletion of about 50% of T-cell receptor (TCR) Vβ-chain gene segments, and (2) SWR is deficient in complement component C5. It has been shown that T cells that express TCRVα11.1 and TCRVβ8.2 play a substantial role in the pathogenesis of arthritis in the DBA/1 mouse (H2 ^q ). We generated SWR transgenic (tg) mice to determine whether the expression of pathogenic Vα11.1 and/or Vβ8.2 transgenes would confer arthritis susceptibility. Arthritis was induced in the SWR TCRαβ tg mice, but not in SWR TCRβ tg mice. To address the role of Vα11.1 in arthritis susceptibility, we examined the allelic polymorphisms of the Tcra-V11-gene subfamily members between the arthritis susceptible DBA/1 mouse and the arthritis-resistant SWR mouse strain. The amino acid sequences of the Vα11.1 alleles differ at two positions (codons 18 and 68). Accordingly, these two amino acid changes are sufficient to allow the production of pathogenic T cells in SWR mice. This is the first demonstration of the association of a particular Tcra-V allele and arthritis susceptibility in mice. Received: 20 November 1998 / Revised: 15 February 1999     

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     

A phylogenetic analysis of cattle DRB3 alleles with a deletion of codon 65

 One of the most common cattle major histocompatibility complex DRB3 alleles, ^* 0201, includes a deletion of codon 65 encoding one residue in the α-helical chain. The mutation is functionally interesting and is likely to influence peptide binding. Exon 2 of two additional del65 alleles, ^* 3301 and ^* 4101, have now been sequenced with the aim to investigate the evolutionary relationship of this allelic group. Despite a fairly large genetic distance between the three alleles (11–17 nucleotide substitutions causing 8–11 amino acid substitutions) we found clear indications of a common ancestry. The α-helical region was very similar or identical among the alleles whereas the β-strand region was quite divergent. The results indicated that interallelic recombination has contributed to the diversification of the del65 group. Deletion of codon 65 has also been found in a roe deer DRB1 allele and a cattle DQB3 allele. Sequence comparisons of the cattle and roe deer DRB del65 alleles refuted the possibility of a trans-species persistence of a del65 allelic lineage but the two species may share a short ancestral sequence motif including del65. In addition to del65, the cattle DQB3 allele did not show any striking sequence similarities to the DRB alleles. Received: 20 March 1997 / Revised: 17 June 1997     

Unique peptide binding characteristics of the disease-associated DQ(α1*0501, β1*0201) vs the non-disease-associated DQ(α1*0201, β1*0202) molecule

 To understand the dominant association of celiac disease (CD) with the presence of HLA-DQ(α1^*0501, β1^*0201), the peptide binding characteristics of this molecule were compared with that of the structurally similar, but non-CD-associated DQ(α1^*0201, β1^*0202) molecule. First, naturally processed peptides were acid-extracted from immuno-affinity-purified DQ molecules of both types. Both molecules contained the Ii-derived CLIP sequence and a particular fragment of the major histocompatibility complex (MHC) class I α chain. Use of truncated analogues of these two peptides in cell-free peptide binding assays indicated that identical peptide frames are used for binding to the two DQ2 molecules. Detailed substitution analysis of the MHC class I peptide revealed identical side chain requirements for the anchor residues at p6 and p7. At p1, p4, and p9, however, polar substitutions (such as N, Q, G, S, and T) were less well tolerated in the case of the DQ(α1^*0201, β1^*0202) molecule. The most striking difference between the two DQ molecules is the presence of an additional anchor residue at p3 for the DQ(α1^*0201, β1^*0202) molecule, whereas this residue was found not to be specifically involved in binding of peptides to DQ(α1^*0501, β1^*0201). Similar results were obtained applying substitution analysis of the CLIP sequence. Molecular modelling of the DQ2 proteins complexed with the MHC class I and CLIP peptide corresponds well with the binding data. The results suggest that both CLIP and the MHC class I peptide bind DQ(α1^*0501, β1^*0201) and DQ(α1^*0201, β1^*0202) in a DR-like fashion, following highly similar binding criteria. This detailed characterization of unique peptide binding properties of the CD-associated DQ(α1^*0501, β1^*0201) molecule should be helpful in the identification of CD-inducing epitopes. Received: 21 March 1997 / Revised: 28 May 1997     

MHCDB: database of the human MHC (release 2)

 The second release of the human major histocompatibility complex (MHC) database is now publicly available. It contains an updated physical map and considerably more genomic sequence. cDNA sequences of all current alleles are accessible as individual sequence entries. The variability of different genes is displayed graphically as static and dynamic images accessible from the database. Known disease-serotype associations have also been incorporated, together with data from the MHCPEP database of eluted peptides. Received: 13 February 1996 / Revised: 12 August 1996     

An empirical method for the prediction of T-cell epitopes

Identification of T-cell epitopes from foreign proteins is the current focus of much research. Methods using simple two or three position motifs have proved useful in epitope prediction for major histocompatibility complex (MHC) class I, but to date not for MHC class II molecules. We utilized data from pool sequence analysis of peptides eluted from two HLA-DR13 alleles to construct a computer algorithm for predicting the probability that a given sequence will be naturally processed and presented on these alleles. We assessed the ability of this method to predict know self-peptides from these DR-13 alleles, DRB1 ^*1301 and *1302, as well as an immunodominant T-cell epitope. We also compared the predictions of this scoring procedure with the measured binding affinities of a panel of overlapping peptides from hepatitis B virus surface antigen. We concluded that this method may have wide application for the prediction of T-cell epitopes for both MHC class I and class II molecules.     

Identifying DNA polymorphisms in human TCRA/D variable genes by direct sequencing of PCR products

 The T-cell receptor (TCR) is a highly variable molecule composed of two polypeptide chains that recognize antigenic peptides in the context of major histocompatibility complex (MHC) molecules. In this study, we describe a sequence-based search for germline polymorphisms in the variable (V) gene segments of the human TCRA/D locus. Thirty different V gene segments were amplified from six to eight unrelated individuals and sequenced from low melting point agarose. Twenty-seven polymorphisms were identified in 15 V gene segments. These polymorphisms are mainly single nucleotide substitutions, but an insertion/deletion polymorphism and a single dinucleotide repeat with variable length were also seen. Of the 15 sequence variations found in the coding regions, six are silent and nine encode amino acid changes. All of the amino acid changes are found at non-conserved residues, frequently in the hypervariable regions, where they may influence MHC and/or peptide recognition. Therefore, it is possible that germline variations in TCR genes could influence an individual’s immune response, and may also contribute to susceptibility to diseases such as autoimmunity. Received: 9 January 1996 / Revised: 22 February 1996     

Human retinoblastoma: in vitro differentiation and immunoglobulin superfamily antigen modulation by retinoic acid

 Suspension and attachment cultures of Y79 human retinoblastoma cells were treated with all-trans retinoic acid (RA) for up to 10 days to assess its effect on growth and cell-surface expression of immunoglobulin superfamily antigens MHC class I and class II, ICAM-1, NCAM and Thy1. RA up to 10 μM induced growth inhibition, and marked morphological differentiation with extension of prominent processes resembling neurites was seen in attachment cultures. However, above 10 μM RA produced extensive cell death. We also observed increased cell-surface expression of MHC class I, ICAM-1, NCAM and Thy1 on Y79 cells treated with 10 μM over 10 days; constitutive MHC class II expression was not apparent, nor did RA treatment appear to induce Y79 cells to express MHC class immunoreactivity. The up-modulation of cell-adhesion molecules (NCAM, ICAM-1 and Thy1) and immune recognition molecules (NCAM, ICAM-1 and MHC class I), associated with reduced growth and tumour cell differentiation, suggests that RA may have a potential role in regulating the growth and development of retinoblastoma tumours. Received: 29 August 1996 / Accepted: 16 January 1997     

High variability in the MHC class II DA beta chain of the brushtail possum (<Emphasis Type="Italic">Trichosurus vulpecula</Emphasis>)

The diversity of class II major histocompatibility complex (MHC) loci was investigated in the brushtail possum, an important marsupial pest species in New Zealand. Immunocontraception, a form of fertility control that generates an autoimmune response, is being developed as a population control method for the possum. Because the immune response is partly under genetic control, an understanding of immunogenetics in possum will be crucial to the development of immunocontraceptive vaccines. MHC molecules are critical in the vertebrate immune response. Class II MHC molecules bind and present exogenously derived peptides to T lymphocytes and may be important in the presentation of immunocontraceptives. We used polymerase chain reaction primers designed to amplify the peptide binding region of possum class II MHC genes to isolate sequences from 49 animals. We have previously described 19 novel alleles from the DAB locus in the possum (Holland et al., Immunogenetics 60:449–460, 2008). Here, we report on another 11 novel alleles isolated from possum DAB, making this the most diverse marsupial locus described so far. This high level of diversity indicates that DAB is an important MHC locus in the possum and will need to be taken into account in the design of immunocontraceptive vaccines.     

Charactreization of polymorphism within the H-2M MHC class II loci

The products of the class II-like H2-M genes of the major histocompatibility complex are required for class II antigen processing. We sequenced H2-Ma and Mb from several mouse strains to determine whether these genes are polymophic, like H2-O. Both Mb loci appear to be transcribed and are distinct from each other. Mb1 and Mb2 differ by about 11% at the nucleotide level and are most dissimilar in their second exons (corresponding to the 1 domain). Relative to the published Mb 1 ^d haplotype sequence, the products of the b, g7, f, and k2 alleles of Mb 1 from Mus musculus domesticus and the separate mouse species Mus spretus differ by only one to four amino acids. The majority of the changes occured in the second exon of Mb 1, in contrast to HLA-DMB, the human orthologue. Little polymorphism was seen for Mb 2, and Ma was invariant in all strians tested. The similarity of the g7 allele to those from other haplotypes makes it unlikely that the M class II genes play a role in the autoimmune diabetes of NOD strain mice. The M genes are regulated in a manner similar to classical class II genes, in that they are upregulated by IFN-gd in mcrophages, and to a lesser extend by IL4 in B cells. When modeled on the crystal structure of the HLA-DR1 class II molecule, nearly all of the differences between M1 and M2 affect residues facing away from the putative peptide binding groove.     

Histocompatibility antigen(s) linked to Rfp-Y (Mhc-like) genes in the chicken

 Major histocompatibility complex (Mhc) genes influencing transplantation rejections were first described in mice within the H2 complex and secondly in chickens within the B complex. In chickens, Rfp-Y haplotypes have recently been identified which contain class I and class II Mhc-like genes that assort independently of the B complex. Three Rfp-Y haplotypes have been defined in a closed breeding flock of line N chickens. In this study, progeny were obtained from line N Rfp-Y heterozygous matings to establish the role of Rfp-Y in transplantation immunity. Rfp-Y incompatibility did not induce significant one-way mixed lymphocyte responses. However, Rfp-Y-incompatible skin grafts were rejected more frequently and at a faster rate than Rfp-Y-compatible grafts by two-week-old chicks. The control Mhc B-incompatible grafts were rejected faster than the Rfp-Y-incompatible grafts; the latter were rejected at speeds that resemble rejection of minor histocompatibility antigens. We conclude that Rfp-Y class I and II Mhc-like genes are linked to the expression of minor histocompatibility antigens in chickens. Received: 21 June 1996 / Revised: 23 July 1996     

Evolution of the proteasome components

 A phylogenetic analysis of proteasome subunits revealed two major families (α and β) which originated by an ancient gene duplication prior to the divergence of archaebacteria and eukaryotes. Numerous gene duplications have subsequently occurred in eukaryotes; at least nine of these duplications were shown to have occurred prior to the divergence of animals and fungi. In mammals, two genes encoding proteasome subunits (LMP2 and LMP7) are located in the major histocompatibility complex (MHC) region and play a specific role in generation of peptides for presentation by class I MHC molecules. Phylogenetic analysis of LMP7 and related sequences from mammals and lower vertebrates indicated that this locus arose by gene duplication prior to the divergence of jawed and jawless vertebrates; the time of this duplication was estimated to have been about 600 million years ago. The evolutionary history of the proteasome subunits provides support for a model of the evolution of new gene function postulating that, after gene duplication, the proteins encoded by daughter loci can adapt to specialized functions previously performed by the product of a single generalized ancestral locus. Received: 19 August 1996 / Revised: 24 December 1996