Polymorphism at the ovine major histocompatibility complex class II loci

Southern hybridization analysis of the ovine major histocompatibility complex (MHC) ( MhcOvar ) class II region, using sheep-specific probes for the DQA1, DQA2, DQB and DRA loci, has revealed extensive polymorphism. DQA1 and DQAP had eight and 16 alleles respectively, DQB had six and DRA had three alleles. Little information was derived from the DRB locus owing to extensive cross-hybridization between the DRB probe and the DQB locus. Differences in allele frequency between breeds were revealed. At the DQA1 locus a null allele (DQA1-N) was observed with a frequency of between 27% and 45%, making this the most common DQA1 allele in all breeds examined. The frequency of DQA1-N homozygotes was between 11% and 18%, raising questions as to the functional significance of the DQA1 gene. Linkage analysis between the DQA1, DQA2, DQB and DRA loci did not reveal any recombination.     

HLA class II influences the immune response and antibody diversification to Ro60/Sjögren's syndrome-A: heightened antibody responses and epitope spreading in mice expressing HLA-DR molecules

Abs to Ro/SSA Ags in the sera of patients with systemic lupus erythematosus and Sj?gren's syndrome are influenced by the HLA class II genes. To investigate the role of individual HLA class II genes in immune responses to human Ro60 (hRo60), mice lacking murine class II molecules and carrying either HLA genes DR2(DRB1*1502), DR3(DRB1*0301), DQ6(DQA1*0103/DQB1*0601), or DQ8(DQA1*0301/DQB1*0302), were immunized with rhRo60. The results show that hRo60 induces strong T and B cell responses in DR2, DR3, and DQ8 mice in comparison to weaker responses in DQ6 mice. In all mice, the majority of the dominant T cell epitopes were located in the amino portion (aa 61-185) and the carboxy portion (aa 381-525) of the hRo60 molecules. In contrast, the early dominant B cell epitopes were located in the middle and carboxy portion of the hRo60 molecule (aa 281-315 and 401-538). In DR2, DR3, and DQ8 mice, the B cell epitopes subsequently spread to the amino and carboxy portion of the hRo60 molecule but were limited to the middle and carboxy portion in DQ6 mice. The DR2 and DR3 mice produced the highest titers of immunoprecipitating Abs against hRo60 and native mouse Ro60. In addition, only DR2 mice exclusively produced immunoprecipitating Abs to native mouse Ro52 and Abs to mouse La by slot blot analysis, whereas in other strains of mice Abs to mouse La were cross-reactive with the immunogen. The results of the present study demonstrate the importance of HLA class II in controlling the immune responses to the Ro-ribonucleoprotein.     

Duplicated DQ haplotypes increase the complexity of restriction element usage in cattle

The MHC of cattle encodes two distinct isotypes of class II molecules, DR and DQ. Unlike humans, cattle lack the DP locus and about half the common haplotypes express duplicated DQ genes. The number and frequency of DQA and DQB alleles means that most cattle are heterozygous. If inter- and/or intrahaplotype pairing of DQA and DQB molecules occurs, cattle carrying DQ-duplicated haplotypes may express more restriction elements than would be predicted by the number of expressed alleles. We are investigating whether duplicated haplotypes cause differences in immune response, particularly in terms of generating protective immunity. We have analyzed the Ag-presenting function of DQ molecules in two heterozygous animals, one of which carries a duplicated haplotype. We compared the class II isotype specificity of T cell clones recognizing a putative vaccinal peptide from foot-and-mouth disease virus (FMDV15). We show for the first time that bovine T cells can recognize Ag in the context of DQ molecules. We also present evidence that interhaplotype pairings of DQA and DQB molecules form functional restriction elements. Both animals showed distinct biases to usage of particular restriction elements. Mainly DQ-restricted clones were derived from the animal with duplicated DQ genes, whereas the majority of clones from the animal with a single DQ gene pair were DR restricted. Furthermore, haplotype bias was observed with both animals. These experiments show that understanding of class II chain pairing in addition to knowledge of the genotype may be important in vaccine design where effective epitope selection is essential.     

Tetramer-guided epitope mapping: rapid identification and characterization of immunodominant CD4+ T cell epitopes from complex antigens

T cell responses to Ags involve recognition of selected peptide epitopes contained within the antigenic protein. In this report, we describe a new approach for direct identification of CD4+ T cell epitopes of complex Ags that uses human class II tetramers to identify reactive cells. With a panel of 60 overlapping peptides covering the entire sequence of the VP16 protein, a major Ag for HSV-2, we generated a panel of class II MHC tetramers loaded with peptide pools that were used to stain peripheral lymphocytes of an HSV-2 infected individual. With this approach, we identified four new DRA1*0101/DRB1*0401- and two DRA1*0101/DRB1*0404-restricted, VP16-specific epitopes. By using tetramers to sort individual cells, we easily obtained a large number of clones specific to these epitopes. Although DRA1*0101/DRB1*0401 and DRA1*0101/DRB1*0404 are structurally very similar, nonoverlapping VP16 epitopes were identified, illustrating high selectivity of individual allele polymorphisms within common MHC variants. This rapid approach to detecting CD4+ T cell epitopes from complex Ags can be applied to any known Ag that gives a T cell response.     

IgG and IgG2 antibodies from cattle naturally infected with Anaplasma marginale recognize the recombinant vaccine candidate antigens VirB9, VirB10, and elongation factor-Tu

Anaplasma marginale is an important vector-borne rickettsia of ruminants in tropical and subtropical regions of the world. Immunization with purified outer membranes of this organism induces protection against acute anaplasmosis. Previous studies, with proteomic and genomic approach identified 21 proteins within the outer membrane immunogen in addition to previously characterized major surface protein1a-5 (MSP1a-5). Among the newly described proteins were VirB9, VirB10, and elongation factor-Tu (EF-Tu). VirB9, VirB10 are considered part of the type IV secretion system (TFSS), which mediates secretion or cell-to-cell transfer of macromolecules, proteins, or DNA-protein complexes in Gram-negative bacteria. EF-Tu can be located in the bacterial surface, mediating bacterial attachment to host cells, or in the bacterial cytoplasm for protein synthesis. However, the roles of VirB9, VirB10, and TFSS in A. marginale have not been defined. VirB9, VirB10, and EF-Tu have not been explored as vaccine antigens. In this study, we demonstrate that sera of cattle infected with A. marginale, with homologous or heterologous isolates recognize recombinant VirB9, VirB10, and EF-Tu. IgG2 from naturally infected cattle also reacts with these proteins. Recognition of epitopes by total IgG and by IgG2 from infected cattle with A. marginale support the inclusion of these proteins in recombinant vaccines against this rickettsia.     

A novel HURRAH protocol reveals high numbers of monomorphic MHC class II loci and two asymmetric multi-locus haplotypes in the Père David's deer

The Père David's deer is a highly inbred, but recovered, species, making it interesting to consider their adaptive molecular evolution from an immunological perspective. Prior to this study, genomic sequencing was the only method for isolating all functional MHC genes within a certain species. Here, we report a novel protocol for isolating MHC class II loci from a species, and its use to investigate the adaptive evolution of this endangered deer at the level of multi-locus haplotypes. This protocol was designated "HURRAH" based on its various steps and used to estimate the total number of MHC class II loci. We confirmed the validity of this novel protocol in the giant panda and then used it to examine the Père David's deer. Our results revealed that the Père David's deer possesses nine MHC class II loci and therefore has more functional MHC class II loci than the eight genome-sequenced mammals for which full MHC data are currently available. This could potentially account at least in part for the strong survival ability of this species in the face of severe bottlenecking. The results from the HURRAH protocol also revealed that: (1) All of the identified MHC class II loci were monomorphic at their antigen-binding regions, although DRA was dimorphic at its cytoplasmic tail; and (2) these genes constituted two asymmetric functional MHC class II multi-locus haplotypes: DRA1*01 ～ DRB1 ～ DRB3 ～ DQA1 ～ DQB2 (H1) and DRA1*02 ～ DRB2 ～ DRB4 ～ DQA2 ～ DQB1 (H2). The latter finding indicates that the current members of the deer species have lost the powerful ancestral MHC class II haplotypes of nine or more loci, and have instead fixed two relatively weak haplotypes containing five genes. As a result, the Père David's deer are currently at risk for increased susceptibility to infectious pathogens.     

'Ovar-Mhc' - ovine major histocompatibility complex: structure and gene polymorphisms

The major histocompatibility complex (MHC) in sheep, Ovar-Mhc, is poorly characterised, when compared to other domestic animals. However, its basic structure is similar to that of other mammals, comprising class I, II and III regions. Currently, there is evidence for the existence of four class I loci. The class II region is better characterised, with evidence of one DRA, four DRB (one coding and three non-coding), one DQA1, two DQA2, and one each of the DQB1, DQB2, DNA, DOB, DYA, DYB, DMA, and DMB genes in the region. The class III region is the least characterised, with the known presence of complement cascade (C4, C2 and Bf), TNFalpha and CYP21 genes. Products of the class I and II genes, MHC molecules, play a pivotal role in antigen presentation required for eliciting immune responses against invading pathogens. Several studies have focused on polymorphisms of Ovar-Mhc genes and their association with disease resistance. However, more research emphasis is needed on characterising the remaining Ovar-Mhc genes and developing simplified and cost-effective methods to score gene polymorphisms. Haplotype screening, employing multiple markers rather than single genes, would be more meaningful in MHC-disease association studies, as it is well known that most of the MHC loci are tightly linked, exhibiting very little recombination. This review summarises the current knowledge of the structure of Ovar-Mhc and polymorphisms of genes located in the complex.     

Characterization of a Mycobacterium tuberculosis peptide that is recognized by human CD4+ and CD8+ T cells in the context of multiple HLA alleles

The secreted Mycobacterium tuberculosis 10-kDa culture filtrate protein (CFP)10 is a potent T cell Ag that is recognized by a high percentage of persons infected with M. tuberculosis. We determined the molecular basis for this widespread recognition by identifying and characterizing a 15-mer peptide, CFP10(71-85), that elicited IFN-gamma production and CTL activity by both CD4(+) and CD8(+) T cells from persons expressing multiple MHC class II and class I molecules, respectively. CFP10(71-85) contained at least two epitopes, one of 10 aa (peptide T1) and another of 9 aa (peptide T6). T1 was recognized by CD4(+) cells in the context of DRB1*04, DR5*0101, and DQB1*03, and by CD8(+) cells of A2(+) donors. T6 elicited responses by CD4(+) cells in the context of DRB1*04 and DQB1*03, and by CD8(+) cells of B35(+) donors. Deleting a single amino acid from the amino or carboxy terminus of either peptide markedly reduced IFN-gamma production, suggesting that they are minimal epitopes for both CD4(+) and CD8(+) cells. As far as we are aware, these are the shortest microbial peptides that have been found to elicit responses by both T cell subpopulations. The capacity of CFP10(71-85) to stimulate IFN-gamma production and CTL activity by CD4(+) and CD8(+) cells from persons expressing a spectrum of MHC molecules suggests that this peptide is an excellent candidate for inclusion in a subunit antituberculosis vaccine.     

Cloning of the bovine major histocompatibility complex class II genes

Class II genes of the bovine major histocompatibility complex (MHC) have been cloned from a genomic library. The library was constructed in the bacteriophage lambda vector EMBL3 and comprises approximately 10 times the equivalent of the haploid genome. Half the library was screened with the human DQA, DQB, DRA and DRB cDNA probes. Of the 100 positively hybridizing phage clones, 37 were eventually fully characterized and mapped by means of Southern blot analysis. The exons encoding the first, second and transmembrane domain of all different A and B genes were subcloned and mapped in more detail. These analyses showed that these 37 clones were derived from five different A and 10 different B genes. The hybridization studies indicate that we have cloned and mapped two DQA genes, one DRA gene, two other A genes, four DQB genes, three DRB genes and three other B genes. Since the library was made from a heterozygous animal, this would suggest that there are at least one DQA, one DRA one other undefined A, two DQB, two DRB and one or two other undefined B genes in the haploid genome of Holstein Friesian cattle.     

Determination of glutamic acid decarboxylase 65 peptides presented by the type I diabetes-associated HLA-DQ8 class II molecule identifies an immunogenic peptide motif

Particular HLA class II allelic sequences are associated with susceptibility to type I diabetes. To understand the mechanism, knowledge of the molecular nature of the specific TCR/peptide/class II interactions involved in the disease process is required. To this end, we have introduced the diabetes-associated human class II HLA-DQ8 allele (DQA1*0301/DQB1*0302) as a transgene into mice and analyzed T cell responses restricted by this molecule to an important Ag in human diabetes, human glutamic acid decarboxylase 65. Hybridomas were used to determine the particular peptides from this Ag presented by HLA-DQ8 to T cells and to map the core minimal epitopes required for T cell stimulation. Analysis of these core epitopes reveals a motif and relevant features for peptides that are immunogenic to T cells when presented by HLA-DQ8. The major immunogenic epitopes of glutamic acid decarboxylase 65 do not contain a negatively charged residue that binds in the P9 pocket of the HLA-DQ8 molecule. PBMC from HLA-DQ8+ diabetic and nondiabetic individuals respond to these peptides, confirming that the mouse model is a useful tool to define epitopes of autoantigens that are processed by human APC and recognized by human T cells.     

Refining the rules of gliadin T cell epitope binding to the disease-associated DQ2 molecule in celiac disease: importance of proline spacing and glutamine deamidation

Celiac disease is driven by intestinal T cells responsive to proline-rich gluten peptides that often harbor glutamate residues formed by tissue transglutaminase-mediated glutamine conversion. The disease is strongly associated with the HLA variant DQ2.5 (DQA1*05, DQB1*02), and intestinal gluten-reactive T cells from DQ2.5-positive patients are uniquely restricted by this HLA molecule. In this study, we describe the mapping of two novel T cell epitopes of gamma-gliadin and the experimental identification of the DQ2.5 binding register of these and three other gamma-gliadin epitopes. The new data extend the knowledge base for understanding the binding of gluten peptides to DQ2.5. The alignment of the experimentally determined binding registers of nine gluten epitopes reveal positioning of proline residues in positions P1, P3, P6, and P8 but never in positions P2, P4, P7, and P9. Glutamate residues formed by tissue transglutaminase-mediated deamidation are found in position P1, P4, P6, P7, or P9, but only deamidations in positions P4 and P6, and rarely in P7, seem to be crucial for T cell recognition. The majority of these nine epitopes are recognized by celiac lesion T cells when presented by the related but nonassociated DQ2.2 (DQA1*0201, DQB1*02) molecule. Interestingly, the DQ2.2 presentation for most epitopes is less efficient than presentation by the DQ2.5 molecule, and this is particularly prominent for the alpha-gliadin epitopes. Contrary to previous findings, our data do not show selective presentation of DQ2.5 over DQ2.2 for gluten epitopes that carry proline residues at the P3 position.     

Cloning of the bovine major histocompatibility complex class II genes

Summary. Class II genes of the bovine major histocompatibility complex (MHC) have been cloned from a genomic library. The library was constructed in the bacteriophage Λ vector EMBL3 and comprises approximately 10 times the equivalent of the haploid genome. Half the library was screened with the human DQA, DQB, DRA and DRB cDNA probes. Of the 100 positively hybridizing phage clones, 37 were eventually fully characterized and mapped by means of Southern blot analysis. The exons encoding the first, second and transmembrane domain of all different A and B genes were subcloned and mapped in more detail. These analyses showed that these 37 clones were derived from five different A and 10 different B genes. The hybridization studies indicate that we have cloned and mapped two DQA genes, one DRA gene, two other A genes, four DQB genes, three DRB genes and three other B genes. Since the library was made from a heterozygous animal, this would suggest that there are at least one DQA, one DRA one other undefined A, two DQB, two DRB and one or two other undefined B genes in the haploid genome of Holstein Friesian cattle.     

Structural and functional studies of trans-encoded HLA-DQ2.3 (DQA1*03:01/DQB1*02:01) protein molecule

MHC class II molecules are composed of one α-chain and one β-chain whose membrane distal interface forms the peptide binding groove. Most of the existing knowledge on MHC class II molecules comes from the cis-encoded variants where the α- and β-chain are encoded on the same chromosome. However, trans-encoded class II MHC molecules, where the α- and β-chain are encoded on opposite chromosomes, can also be expressed. We have studied the trans-encoded class II HLA molecule DQ2.3 (DQA1*03:01/DQB1*02:01) that has received particular attention as it may explain the increased risk of certain individuals to type 1 diabetes. We report the x-ray crystal structure of this HLA molecule complexed with a gluten epitope at 3.05 Å resolution. The gluten epitope, which is the only known HLA-DQ2.3-restricted epitope, is preferentially recognized in the context of the DQ2.3 molecule by T-cell clones of a DQ8/DQ2.5 heterozygous celiac disease patient. This preferential recognition can be explained by improved HLA binding as the epitope combines the peptide-binding motif of DQ2.5 (negative charge at P4) and DQ8 (negative charge at P1). The analysis of the structure of DQ2.3 together with all other available DQ crystal structures and sequences led us to categorize DQA1 and DQB1 genes into two groups where any α-chain and β-chain belonging to the same group are expected to form a stable heterodimer.     

Patterns of Adaptive and Neutral Diversity Identify the Xiaoxiangling Mountains as a Refuge for the Giant Panda

Genetic variation plays a significant role in maintaining the evolutionary potential of a species. Comparing the patterns of adaptive and neutral diversity in extant populations is useful for understanding the local adaptations of a species. In this study, we determined the fine-scale genetic structure of 6 extant populations of the giant panda (Ailuropoda melanoleuca) using mtDNA and DNA fingerprints, and then overlaid adaptive variations in 6 functional Aime-MHC class II genes (DRA, DRB3, DQA1, DQA2, DQB1, and DQB2) on this framework. We found that: (1) analysis of the mtDNA and DNA fingerprint-based networks of the 6 populations identified the independent evolutionary histories of the 2 panda subspecies; (2) the basal (ancestral) branches of the fingerprint-based Sichuan-derived network all originated from the smallest Xiaoxiangling (XXL) population, suggesting the status of a glacial refuge in XXL; (3) the MHC variations among the tested populations showed that the XXL population exhibited extraordinary high levels of MHC diversity in allelic richness, which is consistent with the diversity characteristics of a glacial refuge; (4) the phylogenetic tree showed that the basal clades of giant panda DQB sequences were all occupied by XXL-specific sequences, providing evidence for the ancestor-resembling traits of XXL. Finally, we found that the giant panda had many more DQ alleles than DR alleles (33∶13), contrary to other mammals, and that the XXL refuge showed special characteristics in the DQB loci, with 7 DQB members of 9 XXL-unique alleles. Thus, this study identified XXL as a glacial refuge, specifically harboring the most number of primitive DQB alleles.     

Redundancy in antigen-presenting function of the HLA-DR and -DQ molecules in the multiple sclerosis-associated HLA-DR2 haplotype

The three HLA class II alleles of the DR2 haplotype, DRB1*1501, DRB5*0101, and DQB1*0602, are in strong linkage disequilibrium and confer most of the genetic risk to multiple sclerosis. Functional redundancy in Ag presentation by these class II molecules would allow recognition by a single TCR of identical peptides with the different restriction elements, facilitating T cell activation and providing one explanation how a disease-associated HLA haplotype could be linked to a CD4+ T cell-mediated autoimmune disease. Using combinatorial peptide libraries and B cell lines expressing single HLA-DR/DQ molecules, we show that two of five in vivo-expanded and likely disease-relevant, cross-reactive cerebrospinal fluid-infiltrating T cell clones use multiple disease-associated HLA class II molecules as restriction elements. One of these T cell clones recognizes >30 identical foreign and human peptides using all DR and DQ molecules of the multiple sclerosis-associated DR2 haplotype. A T cell signaling machinery tuned for efficient responses to weak ligands together with structural features of the TCR-HLA/peptide complex result in this promiscuous HLA class II restriction.     

Presentation of endogenously synthesized MHC class II-restricted epitopes by MHC class II cancer vaccines is independent of transporter associated with Ag processing and the proteasome

Cell-based vaccines consisting of invariant chain-negative tumor cells transfected with syngeneic MHC class II (MHC II) and costimulatory molecule genes are prophylactic and therapeutic agents for the treatment of murine primary and metastatic cancers. Vaccine efficacy is due to direct presentation of endogenously synthesized, MHC II-restricted tumor peptides to CD4+ T cells. Because the vaccine cells lack invariant chain, we have hypothesized that, unlike professional APC, the peptide-binding groove of newly synthesized MHC II molecules may be accessible to peptides, allowing newly synthesized MHC II molecules to bind peptides that have been generated in the proteasome and transported into the endoplasmic reticulum via the TAP complex. To test this hypothesis, we have compared the Ag presentation activity of multiple clones of TAP-negative and TAP-positive tumor cells transfected with I-Ak genes and the model Ag hen egg white lysozyme targeted to the endoplasmic reticulum or cytoplasm. Absence of TAP does not diminish Ag presentation of three hen egg white lysozyme epitopes. Likewise, cells treated with proteasomal and autophagy inhibitors are as effective APC as untreated cells. In contrast, drugs that block endosome function significantly inhibit Ag presentation. Coculture experiments demonstrate that the vaccine cells do not release endogenously synthesized molecules that are subsequently endocytosed and processed in endosomal compartments. Collectively, these data indicate that vaccine cell presentation of MHC II-restricted endogenously synthesized epitopes occurs via a mechanism independent of the proteasome and TAP complex, and uses a pathway that overlaps with the classical endosomal pathway for presentation of exogenously synthesized molecules.     

Mutations in the<Emphasis Type="Italic"> HLA</Emphasis> class II genes leading to loss of expression of HLA-DR and HLA-DQ in diffuse large B-cell lymphoma

Loss of expression of human leukocyte antigen (HLA) class II molecules on tumor cells affects the onset and modulation of the immune response through lack of activation of CD4+ T lymphocytes. Previously, we showed that the frequent loss of expression of HLA class II in diffuse large B-cell lymphoma (DLBCL) of the testis and the central nervous system (CNS) is mainly due to homozygous deletions in the HLA region on chromosome band 6p21.3. A minority of cases showed hemizygous deletions or mitotic recombination, implying that mutation of the remaining copy of the class II genes might be involved. Here, we studied three DLBCLs with loss of HLA-DQ expression for mutations in the DQB1 and DQA1 genes and three tumors with loss of HLA-DR expression for mutations in the DRB1 and DRA genes. In one case, a point mutation in exon 2 of the DQB1 gene, leading to the formation of a stop codon, was detected at position 47. In a second case, a stop codon was found at position 11 due to a deletion of 19 bp in exon 1 of the DRA gene. No mutations were found in the promoter sequences of the DRA, DQA1 and DQB1 genes. We conclude that both homozygous deletions and hemizygous deletions or mitotic recombination with mutations of the remaining allele may lead to loss of expression of the HLA class II genes, which is comparable to the mechanisms affecting HLA class I expression in solid cancers.     

Staining of celiac disease-relevant T cells by peptide-DQ2 multimers.

Gluten-specific T cells in the small intestinal mucosa are thought to play a central role in the pathogenesis of celiac disease (CD). The vast majority of these T cells recognize gluten peptides when presented by HLA-DQ2 (DQA1*05/DQB1*02), a molecule which immunogenetic studies have identified as conferring susceptibility to CD. We have previously identified and characterized three DQ2-restricted gluten epitopes that are recognized by intestinal T cells isolated from CD patients, two of which are immunodominant. Because almost all of the gluten epitopes are restricted by DQ2, and because we have detailed knowledge of several of these epitopes, we chose to develop peptide-DQ2 tetramers as a reagent to further investigate the role of these T cells in CD. In the present study, stable soluble DQ2 was produced such that it contained leucine zipper dimerization motif and a covalently coupled peptide. We have made four different peptide-DQ2 staining reagents, three containing the gluten epitopes and one containing a DQ2-binding self-peptide that provides a negative control for staining. We show in this study that peptide-DQ2 when adhered to plastic specifically stimulates T cell clones and that multimers comprising these molecules specifically stain peptide-specific T cell clones and lines. Interestingly, T cell activation caused severe reduction in staining intensities obtained with the multimers and an Ab to the TCR. The problem of TCR down-modulation must be taken into consideration when using class II multimers to stain T cells that may have been recently activated in vivo.