Major Histocompatibility Complex Class I Chain-Related A and B (MICA and MICB) Gene, Allele, and Haplotype Associations With Dengue Infections in Ethnic Thais.

BACKGROUND: Major histocompatibility complex class I chain-related (MIC) A and B (MICA and MICB) are polymorphic stress molecules recognized by natural killer cells. This study was performed to analyze MIC gene profiles in hospitalized Thai children with acute dengue illness. METHODS: MIC allele profiles were determined in a discovery cohort of patients with dengue fever or dengue hemorrhagic fever (DHF) (n = 166) and controls (n = 149). A replication cohort of patients with dengue (n = 222) was used to confirm specific MICB associations with disease. RESULTS: MICA*045 and MICB*004 associated with susceptibility to DHF in secondary dengue virus (DENV) infections (odds ratio [OR], 3.22; [95% confidence interval (CI), 1.18-8.84] and 1.99 [1.07-2.13], respectively), and MICB*002 with protection from DHF in secondary DENV infections (OR, 0.41; 95% CI, .21-.68). The protective effect of MICB*002 against secondary DHF was confirmed in the replication cohort (OR, 0.43; 95% CI, .22-.82) and was stronger when MICB*002 is present in individuals also carrying HLA-B*18, B*40, and B*44 alleles which form the B44 supertype of functionally related alleles (0.29, 95% CI, .14-.60). CONCLUSIONS: Given that MICB*002 is a low expresser of soluble proteins, these data indicate that surface expression of MICB*002 with B44 supertype alleles on DENV-infected cells confer a protective advantage in controlling DENV infection using natural killer cells.

HLA Class I Supertype Associations With Clinical Outcome of Secondary Dengue Virus Infections in Ethnic Thais.

BACKGROUND: Human leukocyte antigen (HLA) supertypes are groups of functionally related alleles that present structurally similar antigens to the immune system. OBJECTIVES: To analyze HLA class I supertype associations with clinical outcome in hospitalized Thai children with acute dengue illness. METHODS: Seven hundred sixty-two patients and population-matched controls recruited predominantly in Bangkok were HLA-A and -B typed. HLA supertype frequencies were compared and tested for significant dengue disease associations using logistic regression analyses. Multivariable models were built by conducting forward stepwise selection procedures. RESULTS: In the final logistic regression model, the HLA-B44 supertype was protective against dengue hemorrhagic fever (DHF) in secondary infections (odds ratio [OR] = 0.46, 95% confidence interval [CI], .30-.72), while the HLA-A02 supertype (OR = 1.92, 95% CI, 1.30-2.83) and the HLA-A01/03 supertype (OR = 3.01, 95% CI, 1.01-8.92) were associated with susceptibility to secondary dengue fever. The B07 supertype was associated with susceptibility to secondary DHF in the univariate analysis (OR = 1.60, 95% CI, 1.05-2.46), whereas that was not retained in the final model. CONCLUSIONS: As the HLA-B44 supertype is predicted to target conserved epitopes in dengue, our results suggest that B44 supertype-restricted immune responses to highly conserved regions of the dengue proteome may protect against secondary DHF.

Distinct activation phenotype of a highly conserved novel HLA-B57-restricted epitope during dengue virus infection.

Variation in the sequence of T-cell epitopes between dengue virus (DENV) serotypes is believed to alter memory T-cell responses during second heterologous infections. We identified a highly conserved, novel, HLA-B57-restricted epitope on the DENV NS1 protein. We predicted higher frequencies of B57-NS1(26-34) -specific CD8(+) T cells in peripheral blood mononuclear cells from individuals undergoing secondary rather than primary DENV infection. However, high tetramer-positive T-cell frequencies during acute infection were seen in only one of nine subjects with secondary infection. B57-NS1(26-34) -specific and other DENV epitope-specific CD8(+) T cells, as well as total CD8(+) T cells, expressed an activated phenotype (CD69(+) and/or CD38(+)) during acute infection. In contrast, expression of CD71 was largely limited to DENV epitope-specific CD8(+) T cells. In vitro stimulation of cell lines indicated that CD71 expression was differentially sensitive to stimulation by homologous and heterologous variant peptides. CD71 may represent a useful marker of antigen-specific T-cell activation.

Immunogenetic surveillance of HIV/AIDS.

Evolutionary pressure by viruses is most likely responsible for the extraordinary allelic polymorphism of genes encoding class I human leukocyte antigens (HLA) and killer immunoglobulin-like receptors (KIR). Such genetic diversity has functional implications for the immune response to viruses and generates population-based variations in HLA class I allele frequencies and KIR gene profiles. The HIV-1 virus has relatively recently established itself as a major human pathogen, rapidly diversifying into a variety of phylogenetic subtypes or clades (A-G) and recombinants in different populations. HIV-1 clade C is the most common subtype in circulation accounting for 48% of all infections, followed by HIV-1 clades A and B which are responsible for 13% and 11% of infections in the current pandemic, respectively. Candidate gene studies of large cohorts of predominantly HIV-1 clade B but also clades C and A infected patients, have consistently shown significant associations between certain HLA class I alleles namely HLA-B*57, B*58, B*27, B*51 and relatively low viraemia. However, there is evidence that other associations between HLA-B*15, B*18 or B*53 and levels of HIV-1 viraemia are clade-specific. Recent genome-wide association studies of HIV-1 clade B exposed cohorts have confirmed that HLA-B, which is the most polymorphic locus in the human genome, is the major genetic locus contributing to immune control of viraemia. Moreover, the presence of natural killer cell receptors encoded by KIR-3DL1 and 3DS1 genes together with certain HLA class I alleles carrying the KIR target motif Bw4Ile80, provides an enhanced ability to control HIV-1 viraemia in some individuals. It is likely that rapid co-evolution of HIV-1 immune escape variants together with an adjustment of human immune response gene profiles has occurred in some exposed populations. Taken together, immunogenetic surveillance of HIV-1 exposed cohorts has revealed important correlates of natural immunity, which could provide a rational platform for the design and testing of future vaccines aimed at controlling the current AIDS pandemic.

Major histocompatibility complex class I-related chain A allele mismatching, antibodies, and rejection in renal transplantation.

Even when kidney allografts are well matched for human leukocyte antigen (HLA) and anti-HLA antibodies are not detected, graft rejection can still occur. There is evidence that some patients who lose their graft have antibodies specific for major histocompatibility complex (MHC) class I-related chain A (MICA) antigens. We investigated whether mismatching MICA alleles associates with MICA antibody production and graft rejection or dysfunction. MICA and HLA antibody screening in 442 recipients was performed, and specificities were confirmed in a subgroup of 227 recipients using single-antigen multiplex technology. For assignment of MICA antibody specificity, we used three independent assays. In addition, MICA alleles of 227 recipients and donors were determined by DNA sequencing. In all, 17 patients (7.5%) had MICA antibodies, and 13 patients (6%) developed MICA donor-specific antibodies (DSA). Multivariate analysis revealed MICA mismatching, as an independent significant factor associated with the presence of MICA antibodies (p = 0.009), and 14 mismatched MICA residues significantly correlated with MICA antibody production. MICA and HLA antibodies significantly associated with acute rejection (AR) and MICA DSA and HLA DSA correlated with decreased graft function by univariate and multivariate analysis. We conclude that mismatching for MICA epitopes in renal transplantation is a mechanism leading to production of MICA antibodies that associate with AR and graft dysfunction.

Risk HLA-DQA1 and PLA(2)R1 alleles in idiopathic membranous nephropathy.

BACKGROUND: Idiopathic membranous nephropathy is a major cause of the nephrotic syndrome in adults, but its etiologic basis is not fully understood. We investigated the genetic basis of biopsy-proven cases of idiopathic membranous nephropathy in a white population. METHODS: We performed independent genomewide association studies of single-nucleotide polymorphisms (SNPs) in patients with idiopathic membranous nephropathy from three populations of white ancestry (75 French, 146 Dutch, and 335 British patients). The patients were compared with racially matched control subjects; population stratification and quality controls were carried out according to standard criteria. Associations were calculated by means of a chi-square basic allele test; the threshold for significance was adjusted for multiple comparisons (with the Bonferroni method). RESULTS: In a joint analysis of data from the 556 patients studied (398 men), we identified significant alleles at two genomic loci associated with idiopathic membranous nephropathy. Chromosome 2q24 contains the gene encoding M-type phospholipase A(2) receptor (PLA(2)R1) (SNP rs4664308, P=8.6×10(-29)), previously shown to be the target of an autoimmune response. Chromosome 6p21 contains the gene encoding HLA complex class II HLA-DQ alpha chain 1 (HLA-DQA1) (SNP rs2187668, P=8.0×10(-93)). The association with HLA-DQA1 was significant in all three populations (P=1.8×10(-9), P=5.6×10(-27), and P=5.2×10(-36) in the French, Dutch, and British groups, respectively). The odds ratio for idiopathic membranous nephropathy with homozygosity for both risk alleles was 78.5 (95% confidence interval, 34.6 to 178.2). CONCLUSIONS: An HLA-DQA1 allele on chromosome 6p21 is most closely associated with idiopathic membranous nephropathy in persons of white ancestry. This allele may facilitate an autoimmune response against targets such as variants of PLA2R1. Our findings suggest a basis for understanding this disease and illuminate how adaptive immunity is regulated by HLA.

Memory CD8+ T cells from naturally acquired primary dengue virus infection are highly cross-reactive.

Cross-reactive memory T cells induced by primary infection with one of the four serotypes of dengue virus (DENV) are hypothesized to have an immunopathological function in secondary heterologous DENV infection. To define the T-cell response to heterologous serotypes, we isolated HLA-A(*)1101-restricted epitope-specific CD8(+) T-cell lines from primary DENV-immune donors. Cell lines exhibited marked cross-reactivity toward peptide variants representing the four DENV serotypes in tetramer binding and functional assays. Many clones responded similarly to homologous and heterologous serotypes with striking cross-reactivity between the DENV-1 and DENV-3 epitope variants. In vitro-stimulated T-cell lines consistently revealed a hierarchical induction of MIP-1ß>degranulation>tumor necrosis factor α (TNFα)>interferon-γ (IFNγ), which depended on the concentration of agonistic peptide. Phosphoflow assays showed peptide dose-dependent phosphorylation of ERK1/2, which correlated with cytolysis, degranulation, and induction of TNFα and IFNγ, but not MIP-1ß production. This is the first study to show significant DENV serotype-cross-reactivity of CD8(+) T cells after naturally acquired primary infection. We also show qualitatively different T-cell receptor signaling after stimulation with homologous and heterologous peptides. Our data support a model whereby the order of sequential DENV infections influences the immune response to secondary heterologous DENV infection, contributing to varying disease outcomes.

Cross-reactivity and expansion of dengue-specific T cells during acute primary and secondary infections in humans.

Serotype-cross-reactive memory T cells responding to secondary dengue virus (DENV) infection are thought to contribute to disease. However, epitope-specific T cell responses have not been thoroughly compared between subjects with primary versus secondary DENV infection. We studied CD8(+) T cells specific for the HLA-A*1101-restricted NS3(133) epitope in a cohort of A11(+) DENV-infected patients throughout acute illness and convalescence. We compared the expansion, serotype-cross-reactivity, and activation of these cells in PBMC from patients experiencing primary or secondary infection and mild or severe disease by flow cytometry. Our results show expansion and activation of DENV-specific CD8(+) T cells during acute infection, which are predominantly serotype-cross-reactive regardless of DENV infection history. These data confirm marked T cell activation and serotype-cross-reactivity during the febrile phase of dengue; however, A11-NS3(133)-specific responses did not correlate with prior antigenic exposure or current disease severity.

Meiotic recombination generates rich diversity in NK cell receptor genes, alleles, and haplotypes.

Natural killer (NK) cells contribute to the essential functions of innate immunity and reproduction. Various genes encode NK cell receptors that recognize the major histocompatibility complex (MHC) Class I molecules expressed by other cells. For primate NK cells, the killer-cell immunoglobulin-like receptors (KIR) are a variable and rapidly evolving family of MHC Class I receptors. Studied here is KIR3DL1/S1, which encodes receptors for highly polymorphic human HLA-A and -B and comprises three ancient allelic lineages that have been preserved by balancing selection throughout human evolution. While the 3DS1 lineage of activating receptors has been conserved, the two 3DL1 lineages of inhibitory receptors were diversified through inter-lineage recombination with each other and with 3DS1. Prominent targets for recombination were D0-domain polymorphisms, which modulate enhancer function, and dimorphism at position 283 in the D2 domain, which influences inhibitory function. In African populations, unequal crossing over between the 3DL1 and 3DL2 genes produced a deleted KIR haplotype in which the telomeric "half" was reduced to a single fusion gene with functional properties distinct from its 3DL1 and 3DL2 parents. Conversely, in Eurasian populations, duplication of the KIR3DL1/S1 locus by unequal crossing over has enabled individuals to carry and express alleles of all three KIR3DL1/S1 lineages. These results demonstrate how meiotic recombination combines with an ancient, preserved diversity to create new KIR phenotypes upon which natural selection acts. A consequence of such recombination is to blur the distinction between alleles and loci in the rapidly evolving human KIR gene family.

TNF and LTA gene, allele, and extended HLA haplotype associations with severe dengue virus infection in ethnic Thais.

Severe dengue virus (DENV) infection is characterized by a cascade of cytokine production, including the production of tumor necrosis factor-alpha (TNF-alpha) and lymphotoxin-alpha (LT-alpha). We have analyzed a variety of polymorphisms in the TNF and LTA genes of 435 ethnic Thais who had subclinical DENV infection, primary or secondary dengue fever (DF), or primary or secondary dengue hemorrhagic fever (DHF). The TNF -238A polymorphism marking the TNF-4,LTA-3 haplotype occurred in a significantly greater number of patients with secondary DHF (20 [15.2%] of 132) than patients with secondary DF (7 [4.1%] of 169) (P < .001; P corrected by use of Bonferroni adjustment, .022; odds ratio, 4.13 [95% confidence interval, 1.59-11.17]). In a subset of patients, the LTA-3 haplotype was associated with in vivo intracellular production of LT-alpha and TNF-alpha during the acute viremic phase of infection. Two extended human major histocompatibility complex (MHC) haplotypes containing TNF-4 and LTA-3, together with HLA-B48, HLA-B57, and HLA-DPB1*0501, were detected only in patients with secondary DHF. These observations indicate that polymorphism in functionally distinct MHC-encoded proteins contributes to the risk of developing severe secondary DENV infection and warrants further investigation.

Unusual selection on the KIR3DL1/S1 natural killer cell receptor in Africans.

Interactions of killer cell immunoglobulin-like receptors (KIRs) with major histocompatibility complex (MHC) class I ligands diversify natural killer cell responses to infection. By analyzing sequence variation in diverse human populations, we show that the KIR3DL1/S1 locus encodes two lineages of polymorphic inhibitory KIR3DL1 allotypes that recognize Bw4 epitopes of protein">HLA-A and HLA-B and one lineage of conserved activating KIR3DS1 allotypes, also implicated in Bw4 recognition. Balancing selection has maintained these three lineages for over 3 million years. Variation was selected at D1 and D2 domain residues that contact HLA class I and at two sites on D0, the domain that enhances the binding of KIR3D to HLA class I. HLA-B variants that gained Bw4 through interallelic microconversion are also products of selection. A worldwide comparison uncovers unusual KIR3DL1/S1 evolution in modern sub-Saharan Africans. Balancing selection is weak and confined to D0, KIR3DS1 is rare and KIR3DL1 allotypes with similar binding sites predominate. Natural killer cells express the dominant KIR3DL1 at a high frequency and with high surface density, providing strong responses to cells perturbed in Bw4 expression.

HIV-1 diversity versus HLA class I polymorphism.

HIV-1 is rapidly diversifying in African, Asian and Caucasoid populations, which in parallel display extensive polymorphism of genes encoding class I human leukocyte antigens (HLA). Immune responses mediated by HLA class I molecules are imprinting mutations in HIV-1, which in turn affects HIV-1 diversity. Intra- and inter-ethnic studies have shown reproducible HLA class I allele, haplotype and supertype associations with HIV-1 infection and the development of AIDS (HIV/AIDS). In Caucasoids and Africans, HLA-B57 and related alleles of the B58 supertype associate with low viraemia, delayed onset of AIDS and, possibly, cytotoxic T lymphocyte (CTL)-driven attenuation of HIV-1. In HIV-1-exposed but uninfected Southeast Asians, HLA-A11 has been associated with CTL responses directed against HIV-1 Nef. HLA-A11 displays unique peptide-binding properties and is recognized by natural killer cells utilizing the inhibitory killer Ig-like receptor 3DL2 in a peptide-dependent manner.

SNP haplotypes and allele frequencies show evidence for disruptive and balancing selection in the human leukocyte receptor complex.

The human leukocyte receptor complex (LRC) of Chromosome 19q13.4 encodes polymorphic and highly homologous genes that are expressed by cells of the immune system and regulate their function. There is an enormous diversity at the LRC, most particularly the variable number of killer cell immunoglobulin-like receptor (KIR) genes. KIR have been associated with several disease processes due to their interaction with polymorphic human leukocyte antigen class I molecules. We have assessed haplotype compositions, linkage disequilibrium patterns and allele frequencies in two Caucasoid population samples (n=54, n=100), using a composite of single-nucleotide polymorphism (SNP) markers and high-resolution, allele-specific molecular genotyping. Particular KIR loci segregated with SNP and other markers, forming two blocks that were separated by a region with a greater history of recombination. The KIR haplotype composition and allele frequency distributions were consistent with KIR having been subject to balancing selection (Watterson's F: P=0.001). In contrast, there was a high inter-population heterogeneity measure for the LRC-encoded leukocyte immunoglobulin-like receptor A3 (LILRA3), indicating pathogen-driven disruptive selection (Wright's FST=0.32). An assessment of seven populations representative of African, Asian and Caucasoid ethnic groups (total n=593) provided little evidence for long-range LRC haplotypes. The different natural selection pressures acting on each locus may have contributed to a lack of linkage disequilibrium between them.

SNP haplotypes and allele frequencies show evidence for disruptive and balancing selection in the human leukocyte receptor complex.

The human leukocyte receptor complex (LRC) of Chromosome 19q13.4 encodes polymorphic and highly homologous genes that are expressed by cells of the immune system and regulate their function. There is an enormous diversity at the LRC, most particularly the variable number of killer cell immunoglobulin-like receptor (KIR) genes. KIR have been associated with several disease processes due to their interaction with polymorphic human leukocyte antigen class I molecules. We have assessed haplotype compositions, linkage disequilibrium patterns and allele frequencies in two Caucasoid population samples (n=54, n=100), using a composite of single-nucleotide polymorphism (SNP) markers and high-resolution, allele-specific molecular genotyping. Particular KIR loci segregated with SNP and other markers, forming two blocks that were separated by a region with a greater history of recombination. The KIR haplotype composition and allele frequency distributions were consistent with KIR having been subject to balancing selection (Watterson's F: P=0.001). In contrast, there was a high inter - population heterogeneity measure for the LRC-encoded leukocyte immunoglobulin-like receptor A3 (LILRA3), indicating pathogen-driven disruptive selection (Wright's FST=0.32). An assessment of seven populations representative of African, Asian and Caucasoid ethnic groups (total n=593) provided little evidence for long-range LRC haplotypes. The different natural selection pressures acting on each locus may have contributed to a lack of linkage disequilibrium between them.

DNA sequence variation and molecular genotyping of natural killer leukocyte immunoglobulin-like receptor, LILRA3.

Leukocyte immunoglobulin-like receptors (LILRs) resemble killer cell immunoglobulin-like receptors (KIR) in structure and function and the KIR and LILR gene families form the major part of the leukocyte receptor cluster (LRC) of human chromosome 19q13.4. Unlike KIR, the LILR gene clusters do not vary in gene number. However, some individuals lack expression of LILRA3. This null allele has a 6.7-kb deletion, which encompasses the first six translated exons. This haplotype enabled unambiguous direct sequencing of LILRA3 alleles using genomic DNA from individuals heterozygous for the deletion. We have performed nucleotide sequencing of a 2.5-kb region within LILRA3 and identified eight bi-allelic substitutions, four of which were non-synonymous. Two from four previously identified LILRA3 cDNA sequences were confirmed and a further six alleles characterised, of which four will encode unique peptides. At least one of the polymorphic positions identified (encoding residue 84 of the first Ig domain) is likely to directly influence ligand binding. A PCR-SSP molecular genotyping system was developed and used to describe a panel of 172 Caucasoid individuals from South-East England. Six alleles were present in this group but they were unevenly distributed, as three alleles accounted for 88% of the studied chromosomes.

A comparison of HLA-DR and -DQ allele and haplotype frequencies in Trinidadian populations of African, South Asian, and mixed ancestry.

Using polymerase chain reaction-sequence-specific primer (PCR-SSP) typing, this study determined the frequency of human leukocyte antigen (HLA) DR- and -DQ alleles and haplotypes in individuals of African (n = 75), South Asian (n = 98), and mixed (n = 102) ancestry from the Caribbean island of Trinidad. We detected 19 different haplotypes containing DRB3, 8 containing DRB4, 6 containing DRB5, and 6 different haplotypes without DRB3/4/5 genes. Twenty-nine haplotypes were identified in Africans, 24 in the South Asians, and 32 in the mixed group. We detected significant differences between the populations, principally at the DQA1 and DQB1 loci, although the allele frequency for DRB1*0901 was highest in the Africans (p(c) < 0.05). Trinidad African and mixed groups were generally more diverse than the South Asians and displayed a wider range of DRB1-DQB1 associations; DQB1*02 and DQB1*0301 each associated with five to six different DRB1 alleles in the Africans and mixed group but only two in South Asians. In the Africans and the mixed group, DQB1*04 was found with DRB1*0302 and DRB1*04, but only with DRB1*08 in the South Asians. Trinidad Africans revealed consistencies with populations in Western, Central, and Northern Africa, but differed considerably from individual populations on the African continent. Trinidad South Asians displayed similar allele frequencies and associations to other populations from Northern India.

High resolution molecular phototyping of MICA and MICB alleles using sequence specific primers.

Major histocompatibility complex (MHC) class I chain-related genes, MICA and MICB, are located centromeric to human leukocyte antigen B (HLA-B) on chromosome 6. In response to stress stimuli, MIC is expressed on epithelial, endothelial and fibroblast cells, but not lymphocytes and has been demonstrated to ligate the natural killer (NK) cell receptor, NKG2D. Nucleotide sequences of MICA and MICB are highly polymorphic and several methods have been established to identify these polymorphisms, including sequence-based typing and sequence-specific oligonucleotide probing. In this study we have developed a high-resolution polymerase chain reaction-sequence-specific primer (PCR-SSP) phototyping scheme that detects all WHO-recognized MICA alleles and all 12 MICB alleles. Our method will also recognize a MICA deletion haplotype and distinguish between MICA alleles with different binding affinities for NKG2D, encoded by a non-synonymous nucleotide substitution in codon 129. Furthermore, our scheme targets almost 90% of the dimorphic codon positions in exons 2, 3, and 4, which result in non-synonymous amino acid changes. This method can be used to determine MIC allele frequencies within different populations, as well as investigate MIC associations in cohorts of patients with autoimmune and infectious diseases and explore the impact of MIC on the survival of solid organ and stem cell transplants.

T cell responses to an HLA-B*07-restricted epitope on the dengue NS3 protein correlate with disease severity.

Dengue hemorrhagic fever (DHF), the severe manifestation of dengue virus (DV) infection characterized by plasma leakage, is more common in secondary DV infections in previously infected individuals and is associated with high levels of immune activation. To determine the Ag specificity of this immune response, we studied the response to an HLA-B*07-restricted T cell epitope, residues 221-232 of the DV NS3 protein, in 10 HLA-B*07(+) Thai children who were studied during and after acute DV infections. Peptide-specific T cells were detected in 9 of 10 subjects. The frequency of peptide-specific T cells was higher in subjects who had experienced DHF than in those who had experienced DF. We also detected peptide-specific T cells in PBMC obtained at the time of the acute DV infection in 2 of 5 subjects. These data suggest that the NS3 (221-232) epitope is an important target of CD8(+) T cells in secondary DV infection and that the activation and expansion of DV-specific T cells is greater in subjects with DHF than in those with dengue fever. These findings support the hypothesis that activation of DV-specific CD8(+) T cells plays an important role in the pathogenesis of DHF.