Interaction of a dengue virus NS1-derived peptide with the inhibitory receptor KIR3DL1 on natural killer cells.

Killer immunoglobulin-like receptors (KIRs) interact with human leucocyte antigen (HLA) class I ligands and play a key role in the regulation and activation of NK cells. The functional importance of KIR-HLA interactions has been demonstrated for a number of chronic viral infections, but to date only a few studies have been performed in the context of acute self-limited viral infections. During our investigation of CD8(+) T cell responses to a conserved HLA-B57-restricted epitope derived from dengue virus (DENV) non-structural protein-1 (NS1), we observed substantial binding of the tetrameric complex to non-T/non-B lymphocytes in peripheral blood mononuclear cells (PBMC) from a long-standing clinical cohort in Thailand. We confirmed binding of the NS1 tetramer to CD56(dim) NK cells, which are known to express KIRs. Using depletion studies and KIR-transfected cell lines, we demonstrated further that the NS1 tetramer bound the inhibitory receptor KIR3DL1. Phenotypical analysis of PBMC from HLA-B57(+) subjects with acute DENV infection revealed marked activation of NS1 tetramer-binding natural killer (NK) cells around the time of defervescence in subjects with severe dengue disease. Collectively, our findings indicate that subsets of NK cells are activated relatively late in the course of acute DENV illness and reveal a possible role for specific KIR-HLA interactions in the modulation of disease outcomes.

HLA and other gene associations with dengue disease severity.

Large case control gene association studies have been performed on cohorts of dengue virus (DENV) infected patients identified in mainland Southeast Asia, South Asia and the Caribbean. Candidate genes that have shown statistically significant associations with DENV disease severity encode HLA molecules, cell receptors for IgG (FcGII), vitamin D and ICAM3 (DCSIGN or CD209), pathogen recognition molecules such as mannose binding lectin (MBL), blood related antigens including ABO and human platelet antigens (HPA1 and HPA2). In ethnic Thais with secondary infections a variety of HLA class I alleles (HLA-A 0203, 0207, A11, B 15, B 44, B 46, B 48, B 51, B 52), DCSIGN promoter polymorphisms and the AB blood group, independently associate with either susceptibility or resistance to dengue fever (DF) and the more severe dengue hemorrhagic fever (DHF). There is also evidence that some HLA associations with disease severity correlate with the DENV serotype inducing secondary infections. Taken together, there is now evidence that allelic variants of multiple gene loci involved in both acquired and innate immune responses contribute significantly to DENV disease outcome and severity. Further analysis of the genetic basis of severe DENV disease in different at risk populations may contribute to the development of new preventative and therapeutic interventions.

Two novel MICA alleles, MICA*054 and MICA*056.

We report the identification of two novel major histocompatibility complex (MHC) class I-related chain A (MICA) alleles. MICA*054 has a nucleotide substitution of A to G at position 871 (codon 268), encoding an amino acid change of serine to glycine in the alpha-3 domain. MICA*056 has a nucleotide substitution at position 758 of G to C resulting in the substitution of tryptophan for serine at codon 230, also in the alpha-3 domain.

HLA-DR and -DQ associations with melioidosis.

Melioidosis is an important infectious disease of southeast Asia caused by an intracellular bacterium, Burkholderia pseudomallei. Cellular immunity is postulated to play important roles in immunity to melioidosis that may influence the severity and clinical outcome of the disease. The present study was undertaken to investigate possible associations of melioidosis with HLA class II alleles. HLA typing of HLA-DRB1, -DQA1, and -DQB1 was performed using polymerase chain reaction and sequence-specific oligonucleotide hybridization (PCR-SSO). Seventy-nine melioidosis patients and 105 healthy, ethnically and geographically matched controls were studied. Among 24 DRB1 alleles, 7 DQA1 alleles, and 13 DQB1 alleles identified in this population, an association with melioidosis was observed with DRB1*1602 which was increased in melioidosis patients (10.1%) compared to normal controls (4.8%), p = 0.047 (odds ratio (OR) = 2.25). In addition, significant increase of DRB1*1602 allele frequency and decrease of DQA1*03 were also observed in septicemic melioidosis patients, the most severe form of the disease (p = 0.01, OR = 3.10; and p = 0.047, respectively). Furthermore, a trend of association of DRB1*0701, DQA1*0201, and DQB1*0201 with relapse cases of melioidosis was also noted. In contrast, no HLA association was observed in localized melioidosis or melioidosis with diabetes mellitus. These findings provide the suggestive evidence of an immunogenetic basis of certain aspects of melioidosis.

HLA-A, -B, -DRB1, -DQA1, and -DQB1 polymorphism in Thais.

In this study we examined HLA-A, -B, -DRB1, -DQA1, and -DQB1, gene allele, and haplotype frequencies in two ethnic Thai populations. We compared these frequencies to the known HLA class I and II allele profiles of non-Thai mainland and insular Southeast (SE) Asians. HLA-A locus gene and allele frequencies, are comparatively homogeneous in both Thai and non-Thai SE Asians. In contrast, HLA-B; -DRB1, -DQA1, and -DQB1 gene and allele frequencies, show more ethnic and geographic variation in SE Asians. Conserved haplotypes, or combinations of linked HLA class I and II alleles were detected in Thais, but at relatively low frequencies. It would appear that ethnic Thais, reflect an admixture of peoples from both the northern mainland and southern island groups of SE Asia.

HLA-DPB1 polymorphism in the Thais of Southeast Asia.

In this study we examined DPB1 allele frequencies in five ethnic Thai populations resident in different regions of Thailand and neighboring countries. In contrast to other Asian and Pacific populations such as the Japanese, Chinese, Korean, and Papua New Guineans, where DPB1*0501 has consistently been shown to be the most frequent allele, NE Thais and Thai-Khmers demonstrate a prevalence of DPB1*1301. Comparison of DPB1 allele frequencies in the Thais of SE Asia, with known frequencies in the Chinese and Japanese populations of E Asia, would appear to confirm previous calculations of genetic divergence between these Oriental populations.

A comparison of molecular HLA-DR and DQ allele profiles forming DR51-, DR52-, and DR53-related haplotypes in five ethnic Thai populations from mainland southeast Asia.

Using PCR-SSOP typing we have deduced the composition and frequency of HLA-DRB1, -DRB3, -DRB4, -DRB5, -DQA1, and -DQB1 alleles present in DR51-, DR52-, and DR53-related haplotypes, in 519 individuals representative of five ethnic Thai populations recruited in central, northeastern and northern Thailand. In total, we have unequivocally detected at varying frequencies, 17 DR51-related haplotypes, 24 DR52 haplotypes, and 12 DR53 haplotypes in the study groups. We document evidence of north-south gradients of DR51-related haplotypes, whereby the overall frequency of DR51-containing haplotypes is relatively more common in the northern Thai groups. Similarly, within DR53-related haplotypes the frequency of DRB1*0901-containing haplotypes increases in the more northerly groups, and an inverse effect was observed with DRB1*0701-containing haplotypes that were relatively more common in the northeastern and central Thais. We have also compared the class II haplotype profiles of the Thais with the equivalent profiles reported in other non-Thai ethnic groups from mainland and insular SE Asia. One DR51-related haplotype DRB1*1502x, DRB5*0102x, DQA1*0101/4, DQB1*0501, would appear to be characteristic of Thai populations, as it was the most common DR2 haplotype in all five study groups and is also prevalent in other mainland southeast Asians, but is much less evident in the more northern populations of eastern Asia or China.

HIV-specific cytotoxic T lymphocytes, HLA-A11, and chemokine-related factors may act synergistically to determine HIV resistance in CCR5 delta32-negative female sex workers in Chiang Rai, northern Thailand.

Understanding how highly HIV-exposed individuals remain HIV uninfected may be useful for HIV vaccine design and development of new HIV prevention strategies. To elucidate mechanisms associated with resistance to HIV infection, immunologic and genetic factors were examined in 14 HIV-exposed but persistently seronegative (HEPS) female sex workers from Chiang Rai, northern Thailand and in ethnically matched, HIV-positive (n = 9) and HIV-negative women (n = 9). The HEPS women were identified in a study of commercial sex workers who had an HIV-1 incidence of 20.3 per 100 person-years. A high frequency of HLA-A11 was observed in HEPS women (86%) compared with northern Thai controls (56%). HIV-specific cytotoxic T lymphocyte (CTL) lytic responses were detected in cryopreserved peripheral blood mononuclear cells (PBMCs), using HLA-A-matched subtype E HIV-1 peptides in four of seven (57%) HEPS women, eight of eight HIV-positive women, and zero of nine HIV-negative unexposed controls (p = 0.019 HEPS women vs. HIV-negative controls). CTL lysis levels were low, but responses were detected to peptides from Nef, Pol, Gag, and Env. Nef responses predominated in HEPS women. Compared with controls, HEPS women tended to have higher frequencies of CCR5 promotor 59402GG and SDF-1 3'UTR 801A genotypes known to influence HIV transmission or course of disease. HEPS women also had higher levels of spontaneous RANTES production by PBMCs than other groups. Each of these factors could potentially contribute to HIV resistance. As most HEPS women had one or more of these factors, they may prevent HIV infection synergistically by blocking HIV cell entry, delaying its dissemination, or killing HIV-infected cells.

T cell receptor Vbeta gene usage in Thai children with dengue virus infection.

T lymphocyte activation during dengue is thought to contribute to the pathogenesis of dengue hemorrhagic fever (DHF). We examined the T cell receptor Vbeta gene usage by a reverse transcriptase-polymerase chain reaction assay during infection and after recovery in 13 children with DHF and 13 children with dengue fever (DF). There was no deletion of specific Vbeta gene families. We detected significant expansions in usage of single Vbeta families in six subjects with DHF and three subjects with DF over the course of infection, but these did not show an association with clinical diagnosis, viral serotype, or HLA alleles. Differences in Vbeta gene usage between subjects with DHF and subjects with DF were of borderline significance. These data suggest that the differences in T cell activation in DHF and DF are quantitative rather than qualitative and that T cells are activated by conventional antigen(s) and not a viral superantigen.

Identification of genetic susceptibility loci for insulin-dependent diabetes in Sudan.

In this study we report, for the first time, the molecular analysis of HLA-DR and DQ gene frequencies in a large cohort of well-characterized type 1 (insulin-dependent) diabetes mellitus (IDDM) patients (n = 72), and ethnically matched controls (n = 59) collected in sub-Saharan Africa. High molecular mass DNA was prepared and analysed in Southern blots and by oligonucleotide typing. We have shown a strong positive association between IDDM and the Asp 57- DQB1 allele *0201 (DQw2). A rare DR4, DQw2 haplotype was also identified at high frequency in the IDDM cohort. We can now confirm that the association between Asp 57- DQB1 alleles and IDDM, previously reported in ethnically diverse cohorts collected in Western Europe, North America, and South Asia, is also present in an IDDM cohort collected in Africa.

HLA class II allele frequencies in northern Thais (Kamphaeng Phet).

The polymorphism of HLA class II genes (HLA-DRB1, -DQA1, -DQB1 and -DPB1) was investigated in 97 normal Northern Thais (NT) from Kamphaeng Phet province using PCR-SSO typing. Allele frequencies (AF) have been determined. DRB1*1202 (17.5%), DRB1*1502 (16.5%), DQA1*0101 (25.8%), DQA1*0102 (21.7%), DQB1*0502 (22.7%), DPB1*0501 (23.2%) and DPB1*1301 (22.7%) showed the highest frequencies in each locus. These results were more similar to those observed in Present-day Thais (PDT) and Central Thais (CT) than Northern Thais from Chiang Mai (CM) and Dai Lue (DL). However, the data presented in this population study should be useful in many fields, such as anthropology, organ transplantation, disease susceptibility and evolutionary genetics.

Does the polymorphism of MHC class II promoters matter?

The promoters of genes of the major histocompatibility complex vary not only because of linkage disequilibrium with their coding sequences but also, we argue, because of natural selection that acts particularly strongly on MHC II gene promoters. Thus, the promoter of H2Eb varies more than that of H2K, to an extent that cannot be accounted for by coding variation, and the same applies to HLA.DRB1 in comparison with H2D. We discuss how transduction by lentivirus vectors followed by adoptive transfer of monoclonal T cells could be used to test the functional activity of variant mouse promoters in vivo, and how homologous recombination in suitable cell lines might provide a short cut to obtaining promoter knock-ins.

MICA and MICB genes: can the enigma of their polymorphism be resolved?

The human MHC class I chain-related genes (MICA and MICB) are located within the HLA class I region of chromosome 6. Their organization, expression and products differ considerably from classical HLA class I genes. MIC proteins are considered to be markers of "stress" in the epithelia, and act as ligands for cells expressing a common activatory natural killer-cell receptor (NKG2D). Molecular models are now available for the MICA protein, both bound and complexed with NKG2D. MICA molecules appear to be highly flexible and polymorphic, although the functional relevance and implications of their polymorphism have yet to be fully discerned.

Null cell immunoregulation in SLE.

Fresh normal T cells do not lyse MDA-157 target cells. Normal null cells, cultured for 4 days with MDA-157 stimulators, and then mixed overnight with fresh normal T cells, induce cytotoxicity on MDA-157 targets and suppressor activity in the T-cell acceptor population. With the same normal acceptor T cells, MDA-157 activated null cells from 13/18 patients with Systemic Lupus Erythematosus (SLE), unlike activated cells from a disease control population, induce little or no T-cell cytotoxicity or suppression. These results provide further evidence for abnormal null cell function in SLE.

Insensitivity to interferon of NK cells from patients with systemic lupus erythematosus.

Natural cytotoxicity (NK) by fresh E-rosette-negative (ER-) cells from normal donors was increased after overnight incubation with purified IFN alpha and with supernatants containing IFN gamma. ER- cells from 61% of 23 patients with systemic lupus erythematosus did not show increased cytotoxicity after treatment with IFN alpha. Similarly, ER- cells from nine of 18 patients that were treated with IFN gamma-containing supernatants failed to show increased cytotoxicity. The patients who did not show enhanced cytotoxic responses to IFN had higher mean indices of disease activity than responding patients.

Analysis of HLA-DR and -DQ gene polymorphisms in Sudanese patients with type 1 (insulin-dependent) diabetes.

In this study we report for the first time, the molecular analysis of HLA-DR and -DQ gene frequencies in a large cohort of well characterized type 1 (insulin-dependent) diabetes mellitus (IDDM) patients (n = 72), and ethnically matched controls (n = 59) collected in sub-Saharan Africa. High molecular mass DNA was prepared and analyzed in Southern blots with DRB1, DQA1, and DQB1 probes. By identifying DR and DQ allele-specific restriction fragment length polymorphisms (RFLPs), we have shown a strong positive association between IDDM and the Asp 57- DQB1 allele *0201 (DQw2). A rare DR4, DQw2 haplotype was also identified at high frequency in the IDDM cohort. We can now confirm that the association between Asp 57-DQB1 alleles and IDDM, previously reported in ethnically diverse cohorts collected in Western Europe, North America, and South Asia, is also present in an IDDM cohort collected in Africa.

Towards a molecular phototyping system for allelic variants of MICA, encoded by polymorphisms in exons 2, 3 and 4 of MHC class I chain-related genes.

In this study we have developed a polymerase chain reaction-sequence-specific primer (PCR-SSP) or phototyping system to analyse the polymorphism of the human MICA gene locus. By scrutinising the reported MICA sequence variations in exons 2, 3 and 4 which encode the extracellular protein domains, we have selected and tested 20 MICA-specific primer mixes that should discriminate between the majority of homozygous and heterozygous combinations of MICA alleles 001-016. We have tested this scheme on DNA prepared from a large number of well-characterised tissue culture cell lines with previously reported MICA nucleotide sequences and found an excellent correlation with the observed PCR-SSP phototypes. We believe that this scheme can also be modified to detect new MICA alleles when they are characterised, as well as be incorporated into standard phototyping protocols to generate allele and haplotype profiles of both classical and non-classical HLA gene loci in test DNA samples.

Distribution of natural killer cell immunoglobulin-like receptor sequences in three ethnic groups.

Killer cell immunoglobulin-like receptors (KIRs) are members of a group of molecules that specifically recognize HLA class I ligands and are found on subsets of human lymphopoetic cells. The number of KIR loci can vary between individuals, resulting in a heterogeneous array of possible KIR genes. The range of observed profiles has been explained by the occurrence of two haplotype families termed A and B which can be distinguished on the basis of certain KIR sequences. Here we attempted to determine whether the frequencies of putative KIR loci and the two haplotype groups vary in three ethnically defined, healthy, and unrelated control populations, namely UK Caucasoid (n=136), Palestinian (n=105) and Thai (n=119). We molecularly typed genomic DNA for the presence of 12 putative KIR loci, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR3DL1, KIR3DL2, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, and KIR3DS1, using modified PCR sequence-specific primers. The patterns of KIR locus frequencies combined with the similar linkage disequilibrium values suggest that there was a distinction in the distribution of the two broad haplotype groups between the populations studied. The A haplotype was always the most prevalent, but the ratio of A to B varied between populations. The frequency of B haplotype was highest in the Palestinians and lowest in the Thais (Pc<0.0001).

Inhibition of proliferative and suppressor responses in the autologous mixed lymphocyte reaction by serum from patients with systemic lupus erythematosus.

Serum from patients with systemic lupus erythematosus (SLE) prevents the proliferative response of normal T cells when stimulated by autologous or allogeneic non-T cells. The abrogation of proliferation in an autologous mixed lymphocyte reaction (AMLR) with SLE serum is associated with a lack of suppressor T cell generation. Fractionation of SLE sera on sucrose gradients reveals an 18-12 S peak of Raji cell binding material. Fractions with an S value of