Allorecognition of HLA-DP by CD4+ T cells is affected by polymorphism in its alpha chain.

Alloreactivity to HLA-DP molecules, class II heterodimers of an oligomorphic alpha and a polymorphic beta chain, is increasingly being studied due to its relevance in clinical transplantation. We hypothesized that not only polymorphisms in the peptide binding groove encoded by exon 2 of HLA-DPB1, but also in other regions of the molecule and the alpha chain, could play a role in CD4+ T cell allorecognition. To test this possibility, we comparatively investigated CD4+ T cell allorecognition, measured by upregulation of the activation marker CD137, against HLA-DPB1*13:01, *05:01, *03:01, *17:01 or their allele counter parts DPB1*107:01, *135:01, *104:01, *131:01, with identical exon 2 sequences but polymorphism in exons 1, 3 or 4, in the context of different HLA-DPA1 (DPA1) polymorphisms (DPA1*01:03 and *02:01). No significant differences in CD4+ T cell allorecognition levels could be demonstrated for any of the beyond exon 2 DPB1 variants studied. Interestingly, however, the mean fold change in CD4+ CD137+ cells was significantly higher when the target shared at least one DPA1 allele with the allogeneic stimulator, compared to a distinct DPA1 background (1.65 vs 0.23, P<0.005). Structural homology modeling suggested specific amino acid residues in the alpha chain, in particular position 31, to impact CD4+ T cell allorecognition of HLA-DP. Our data argue against a significant role of beyond exon 2 DPB1 polymorphisms for T cell alloreactivity, but show relevance of DPA1 polymorphism in this mechanism. These new findings impact HLA matching strategies in unrelated stem cell transplantation.

Frequency of HLA-DP-specific antibodies and a possible new cross-reacting group.

Clinical studies have demonstrated that HLA-DP-specific antibodies can be detrimental to a transplanted kidney. The number of patients affected is proportional to the frequency of DP antibodies. We determined the frequency of HLA-DP-specific antibodies en toto and in the absence of cross-reactive DR antibodies. Of 650 waitlisted renal patients, 271 (42%) were reactive with HLA-DP antigens in solid-phase immunoassays. Of these 271 sera, 58 (21%) were negative for reactivity with cross-reactive DR antigens, and 16 (5.9%) had no class II antibody other than DP. Eliminating sera containing DR cross-reactive antibodies reduced the frequency but not the overall strength of DP antibodies. Although most DP antibodies were not expected to yield a positive cytotoxicity crossmatch, 2 DP-specific antibodies yielded cytotoxic crossmatch tests with titers of >512. The occurrence of HLA-DP-specific antibody differed significantly between previously transplanted (62%) and nontransplanted (38%) patients, but no difference was observed among patients categorized by race or sex. One serum demonstrated strong cross-reactivity between DP and DRB1*01:03 in the absence of DR1 or DR11 reactivity. Sequence alignments were performed and a possible new cross-reactivity between DRB1*01:03 and DP2, DP9, DP10, DP13, DP16, and DP17 was defined. Two additional sera confirmed this cross-reactivity.

Functional classification of class II human leukocyte antigen (HLA) molecules reveals seven different supertypes and a surprising degree of repertoire sharing across supertypes.

Previous studies have attempted to define human leukocyte antigen (HLA) class II supertypes, analogous to the case for class I, on the basis of shared peptide-binding motifs or structure. In the present study, we determined the binding capacity of a large panel of non-redundant peptides for a set of 27 common HLA DR, DQ, and DP molecules. The measured binding data were then used to define class II supertypes on the basis of shared binding repertoires. Seven different supertypes (main DR, DR4, DRB3, main DQ, DQ7, main DP, and DP2) were defined. The molecules associated with the respective supertypes fell largely along lines defined by MHC locus and reflect, in broad terms, commonalities in reported peptide-binding motifs. Repertoire overlaps between molecules within the same class II supertype were found to be similar in magnitude to what has been observed for HLA class I supertypes. Surprisingly, however, the degree to which repertoires between molecules in the different class II supertypes also overlapped was found to be five to tenfold higher than repertoire overlaps noted between molecules in different class I supertypes. These results highlight a high degree of repertoire overlap amongst all HLA class II molecules, perhaps reflecting binding in multiple registers, and more pronounced dependence on backbone interactions rather than peptide anchor residues. This fundamental difference between HLA class I and class II would not have been predicted on the basis of analysis of either binding motifs or the sequence/predicted structures of the HLA molecules.

An ancient balanced polymorphism in a regulatory region of human major histocompatibility complex is retained in Chinese minorities but lost worldwide.

The coding regions of many of the major histocompatibility complex (MHC) (human leukocyte antigen [HLA] in humans) molecules are believed to be subject to balancing selection. But it is less certain whether the regulatory regions of such coding sequences are also subject to the same type of selection. Here, we studied the polymorphism of the regulatory regions of the HLA-DPA1 and HLA-DPB1 genes among ethnic minorities in southwestern China. Phylogenetic analysis revealed two deep clades >10 million years old. There is almost complete linkage disequilibrium between the regulatory and coding regions of HLA-DPA1, which hints at coadaptive balancing selection on the entire region. Thus, the molecular mechanism of balancing selection in MHC may involve expression modulation in addition to coding-region polymorphisms. Although the frequency of clade II is >30% in some ethnic minorities, it decreases to <5% among southern Han Chinese and vanishes among Europeans. As suspected, some ancient balanced polymorphisms, lost in major populations, still exist in isolated ethnicities. These isolated populations may thus contribute disproportionately to the total diversity of modern humans.

In silico identification of supertypes for class II MHCs.

The development of epitope-based vaccines, which have wide population coverage, is greatly complicated by MHC polymorphism. The grouping of alleles into supertypes, on the basis of common structural and functional features, addresses this problem directly. In the present study we applied a combined bioinformatics approach, based on analysis of both protein sequence and structure, to identify similarities in the peptide binding sites of 2225 human class II MHC molecules, and thus define supertypes and supertype fingerprints. Two chemometric techniques were used: hierarchical clustering using three-dimensional Comparative Similarity Indices Analysis fields and nonhierarchical k-means clustering using sequence-based z-descriptors. An average consensus of 84% was achieved, i.e., 1872 of 2225 class II molecules were classified in the same supertype by both techniques. Twelve class II supertypes were defined: five DRs, three DQs, and four DPs. The HLA class II supertypes and their fingerprints given in parenthesis are DR1 (Trp(9beta)), DR3 (Glu(9beta), Gln(70beta), and Gln/Arg(74beta)), DR4 (Glu(9beta), Gln/Arg(70beta), and Glu/Ala(74beta)), DR5 (Glu(9beta), Asp(70beta)), and DR9 (Lys/Gln(9beta)); DQ1 (Ala/Gly(86beta)), DQ2 (Glu(86beta), Lys(71beta)), and DQ3 (Glu(86beta), Thr/Asp(71beta)); DPw1 (Asp(84beta) and Lys(69beta)), DPw2 (Gly/Val(84beta) and Glu(69beta)), DPw4 (Gly/Val(84beta) and Lys(69beta)), and DPw6 (Asp(84beta) and Glu(69beta)). Apart from the good agreement between known binding motifs and our classification, several new supertypes, and corresponding thematic binding motifs, were also defined.

[The association between HLA typing and different subtypes of Guillain Barré syndrome].

OBJECTIVE: To study the relationship between the susceptibility to two subtypes of Guillain-Barre syndrome(GBS) AIDP and AMAN and the frequency of HLA-class I and II alleles as well as to approach the characteristics of AIDP and AMAN patients in immune genetics. METHODS: A case control research was done on 31 AIDP and 33 AMAN and 132 health individuals. DNA was extracted from peripheral blood leucocytes by improved fast salting out. HLA genes were typed with DNA-based technology and PCR-sequence specific primers (PCR-SSP) method. RESULTS: HLA-A33 frequency of HLA-class I, and DR16 and DQ5 frequencies of HLA-class II showed a significant increase in AIDP group as compared with the control. Relative risk (RR) was 6.13, 8.28 and 3.47 respectively. Corrected probability (Pc) was 0.011, 0.014 and 0.025(P < 0.05). HLA-B15,B35 frequency of HLA-class I showed a significant increase in patients with AMAN as compared with the controls,RR was 4.09 and 7.08 respectively, Pc was 0.015 and 0.000 8 respectively. CONCLUSIONS: HLA-A33, DR15 and DQ5 may have association with susceptibility to AIDP; HLA-B15, B35 may have association with susceptibility to AMAN. HLA-class II genes were not found to have any association with AMAN.

Identification of a novel DPB1* allele (DPB1*6901) and the occurrence of HLA haplotypes that extend to DPB1.

The sequence of a novel DPB1 allele, DPB1*6901, observed in a Caucasian bone marrow donor phenotype HLA A2; Cw*0501,*1601; B*4402, *4403; DRB1*0401; *07; DQB1*02; *0301; DPB1*0401; *6901, is described. The sequence is consistent with that previously described for DPB1*0601 with the exception of codon 69. The sequence at this codon is consistent with that previously observed only in the DPB1*1101 and *1501 alleles. It is suggested that DPB1*6901 may have arisen as a result of a recombination event occurring between codons 58 and 64 between DPB1*0601 and DPB1*1101. The sequence of DPB1*1501 from codon 64 is not consistent with DPB1*6901. A linkage disequilibria analysis that examined 212 potential bone marrow recipients in which HLA-A to DQ haplotypes had been established by family studies showed linkage disequilibrium between HLA-B, DRB1 and DPB1 in some haplotypes and not others.

High-resolution HLA class I typing in the CEPH families: analysis of linkage disequilibrium among HLA loci.

The HLA region on the short arm of chromosome 6 (6p21.3) contains the most polymorphic coding sequences in the human genome. High-resolution DNA-based HLA typing of population samples of the polymorphic class I loci, HLA-A, -B, and -C has only recently become feasible. Here, we report molecular HLA typing on family-based samples of European origin (the CEPH repository), which demonstrated very high polymorphism, with 20 A alleles, 38 B alleles and 19 C alleles in the sample of 248 independent haplotypes. In general, allele frequency distributions are consistently more even (lower observed homozygosity statistic) than expected from a past of selective neutrality suggesting a history of balancing selection. This was also true for the class II loci, DRB1, DQA1 and DQB1 in these samples, but not for the DPA1 and DPB1 loci, whose allelic frequency distributions were more skewed (higher observed homozygosity statistic) than expected under a neutral model. Although linkage disequilibrium is a prominent feature across the HLA region, only 19% of the eight locus haplotypes were sampled more than once. The relative age of some of the B alleles could be inferred from the pattern of B-C haplotypic associations. We suggest that the observed patterns of linkage disequilibrium reflect the operation of selection on nearly all HLA alleles.

Four new DP alleles identified in a study of 500 unrelated bone marrow donor-recipient pairs.

HLA-DP genotyping of 500 donor recipient pairs in a retrospective analysis sponsored by the National Marrow Donor Program (NMDP) identified four new DP alleles, two DPB1 and two DPA1. DNA sequencing confirmed that DPB1*8001 and *8101, each found in a single individual, are novel combinations of previously described sequence motifs in the six variable regions of DPB1. DPA1*02014, found in two individuals, is identical to DPA1*02011 except for a novel silent substitution, a G to A transition at the third position of codon 14. DPA1*01032, found in one individual, is identical to DPB1*01031 except for a silent G to A transition at the third position of codon 20. The identification of these novel alleles brings the total number of reported DPB1 alleles to 85 and DPA1 alleles to 15.

A novel strategy for unambiguous DPB1 typing.

A DPB1 typing method is described that assigns DPB1 alleles into six groups based on polymorphism at amino acid positions 8-9 and 84-87 using sequence-specific priming (SSP). The results obtained allow the selection of primers for a subsequent sequence-specific oligonucleotide (SSO) hybridization procedure which permits DPB1 alleles to be analysed separately in a heterozygote individual. This has greatly reduced the occurrence of typing reaction patterns consistent with multiple combinations of DPB1 alleles seen in other DPB1 typing methods.

Response to hepatitis B vaccine: multiple HLA genes are involved.

The mechanism underlying the impaired immune response to hepatitis B vaccines in up to 10% of healthy subjects is not known. An increased incidence of poor responsiveness in subjects with HLA-DR3+ or -DR7+ haplotypes has been documented, suggesting that HLA-DR-linked genes may regulate the human response to hepatitis B surface antigen. However, not all HLA-DR3+ and/or -DR7+ individuals are poor responders, and subjects with identical HLA-DR haplotypes sometimes display totally divergent antibody responses to vaccination. HLA class II DNA typing was performed in well and poorly responding hepatitis B vaccine recipients and we analyzed the role of the single HLA-DR, -DP, and -DQ molecules and of their associated (interaction) haplotypes in the response to hepatitis B vaccination. Statistical analysis revealed that HLA-DRB1*010*, -DR5, -DPB1*040*, -DQB1*0301, and -DQB1*0501 were more abundant in good responders, whereas HLA-DRB1*07, -DPB1*1101, and -DQB1*020* were associated with poor response, with DQB1*020* showing the strongest association with poor responsiveness. We further investigated whether there were interactions between the HLA factors contributing to poor responsiveness. We show here that HLA-DPB1*02 was negatively associated with responsiveness when it occurred in association with haplotype DRB1*0701/DRB4*0101-DQB1*020*, and DRB4*0101 was negatively associated with responsiveness when it occurred in association with haplotype DRB1*0301/DRB3*0101-DQB1*020*. Our results indicate that the immune response to hepatitis B vaccine is largely determined by HLA-DR, -DP, and -DQ genes and that interaction between HLA molecules that are not in linkage disequilibrium contributes to poor responsiveness.

HLA-DRB1, -DQB1 and -DPB1 polymorphism in the Slovak population.

HLA-DRB1, -DQB1 and -DPB1 allele frequencies were investigated in a sample of the Slovak population by PCR-SSP and PCR-RFLP methods. The most frequent DRB1 alleles were DRB1*1101-5 (0.2038), DRB1*0701-2 (0.1423), and DRB1*1501-2 (0.1231). The most rare alleles found were DRB1*0901 (0.0038), and DRB1*1201 (0.015). The most common DQB1 alleles were DQB1*0301 (0.2448), DQB1*0201 (0.2098), and DQB1*0501 (0.1119), respectively. The alleles with the least occurrence rate were DQB1*0601 (0.0035) and DQB1*0401 (0.007). The most common DPB1 alleles found were DPB1*0401 (0.4329), DPB1*0402 (0.2089), and DPB1*0201 (0.1438), respectively. The least frequent alleles were DPB1*0601, *1101, and *1501 (0.0034). Allele frequencies found in our study were compared to those in Czech, Austrian, and German populations. No statistically significant differences were observed.