Comparison between qPCR and RNA-seq reveals challenges of quantifying HLA expression.

Human leukocyte antigen (HLA) class I and II loci are essential elements of innate and acquired immunity. Their functions include antigen presentation to T cells leading to cellular and humoral immune responses, and modulation of NK cells. Their exceptional influence on disease outcome has now been made clear by genome-wide association studies. The exons encoding the peptide-binding groove have been the main focus for determining HLA effects on disease susceptibility/pathogenesis. However, HLA expression levels have also been implicated in disease outcome, adding another dimension to the extreme diversity of HLA that impacts variability in immune responses across individuals. To estimate HLA expression, immunogenetic studies traditionally rely on quantitative PCR (qPCR). Adoption of alternative high-throughput technologies such as RNA-seq has been hampered by technical issues due to the extreme polymorphism at HLA genes. Recently, however, multiple bioinformatic methods have been developed to accurately estimate HLA expression from RNA-seq data. This opens an exciting opportunity to quantify HLA expression in large datasets but also brings questions on whether RNA-seq results are comparable to those by qPCR. In this study, we analyze three classes of expression data for HLA class I genes for a matched set of individuals: (a) RNA-seq, (b) qPCR, and (c) cell surface HLA-C expression. We observed a moderate correlation between expression estimates from qPCR and RNA-seq for HLA-A, -B, and -C (0.2 ≤ rho ≤ 0.53). We discuss technical and biological factors which need to be accounted for when comparing quantifications for different molecular phenotypes or using different techniques.

Trans-ancestral fine-mapping of MHC reveals key amino acids associated with spontaneous clearance of hepatitis C in HLA-DQß1.

Spontaneous clearance of acute hepatitis C virus (HCV) infection is associated with single nucleotide polymorphisms (SNPs) on the MHC class II. We fine-mapped the MHC region in European (n = 1,600; 594 HCV clearance/1,006 HCV persistence) and African (n = 1,869; 340 HCV clearance/1,529 HCV persistence) ancestry individuals and evaluated HCV peptide binding affinity of classical alleles. In both populations, HLA-DQß1Leu26 (p valueMeta = 1.24 × 10-14) located in pocket 4 was negatively associated with HCV spontaneous clearance and HLA-DQß1Pro55 (p valueMeta = 8.23 × 10-11) located in the peptide binding region was positively associated, independently of HLA-DQß1Leu26. These two amino acids are not in linkage disequilibrium (r2 < 0.1) and explain the SNPs and classical allele associations represented by rs2647011, rs9274711, HLA-DQB1∗03:01, and HLA-DRB1∗01:01. Additionally, HCV persistence classical alleles tagged by HLA-DQß1Leu26 had fewer HCV binding epitopes and lower predicted binding affinities compared to clearance alleles (geometric mean of combined IC50 nM of persistence versus clearance; 2,321 nM versus 761.7 nM, p value = 1.35 × 10-38). In summary, MHC class II fine-mapping revealed key amino acids in HLA-DQß1 explaining allelic and SNP associations with HCV outcomes. This mechanistic advance in understanding of natural recovery and immunogenetics of HCV might set the stage for much needed enhancement and design of vaccine to promote spontaneous clearance of HCV infection.

A Possible Sterilizing Cure of HIV-1 Infection Without Stem Cell Transplantation.

BACKGROUND: A sterilizing cure of HIV-1 infection has been reported in 2 persons living with HIV-1 who underwent allogeneic hematopoietic stem cell transplantations from donors who were homozygous for the CCR5Δ32 gene polymorphism. However, this has been considered elusive during natural infection. OBJECTIVE: To evaluate persistent HIV-1 reservoir cells in an elite controller with undetectable HIV-1 viremia for more than 8 years in the absence of antiretroviral therapy. DESIGN: Detailed investigation of virologic and immunologic characteristics. SETTING: Tertiary care centers in Buenos Aires, Argentina, and Boston, Massachusetts. PATIENT: A patient with HIV-1 infection and durable drug-free suppression of HIV-1 replication. MEASUREMENTS: Analysis of genome-intact and replication-competent HIV-1 using near-full-length individual proviral sequencing and viral outgrowth assays, respectively; analysis of HIV-1 plasma RNA by ultrasensitive HIV-1 viral load testing. RESULTS: No genome-intact HIV-1 proviruses were detected in analysis of a total of 1.188 billion peripheral blood mononuclear cells and 503 million mononuclear cells from placental tissues. Seven defective proviruses, some of them derived from clonally expanded cells, were detected. A viral outgrowth assay failed to retrieve replication-competent HIV-1 from 150 million resting CD4+ T cells. No HIV-1 RNA was detected in 4.5 mL of plasma. LIMITATIONS: Absence of evidence for intact HIV-1 proviruses in large numbers of cells is not evidence of absence of intact HIV-1 proviruses. A sterilizing cure of HIV-1 can never be empirically proved. CONCLUSION: Genome-intact and replication-competent HIV-1 were not detected in an elite controller despite analysis of massive numbers of cells from blood and tissues, suggesting that this patient may have naturally achieved a sterilizing cure of HIV-1 infection. These observations raise the possibility that a sterilizing cure may be an extremely rare but possible outcome of HIV-1 infection. PRIMARY FUNDING SOURCE: National Institutes of Health and Bill & Melinda Gates Foundation.

Pemphigus is associated with KIR3DL2 expression levels and provides evidence that KIR3DL2 may bind HLA-A3 and A11 in vivo.

Although HLA-A3 and A11 have been reported to be ligands for KIR3DL2, evidence for any in vivo relevance of this interaction is still missing. To explore the functional importance of KIR3DL2 allelic variation, we analyzed the autoimmune disease pemphigus foliaceus, previously associated (lower risk) with activating KIR genes. KIR3DL2*001 was increased in patients (odds ratio (OR) = 2.04; p = 0.007). The risk was higher for the presence of both KIR3DL2*001 and HLA-A3 or A11 (OR = 3.76, p = 0.013), providing the first evidence that HLA-A3 and A11 may interact with KIR3DL2 in vivo. The nonsynonymous single nucleotide polymorphism 1190T (rs3745902) was associated with protection (OR = 0.52, p = 0.018). This SNP results in a threonine-to-methionine substitution. Individuals who have methionine in this position exhibit a lower percentage of KIR3DL2-positive natural killer (NK) cells and also lower intensity of KIR3DL2 on expressing natural killer cells; additionally, we show that the expression of KIR3DL2 is independent of other killer cell immunoglobulin-like receptors. Pemphigus foliaceus is a very unique complex disease strongly associated with immune-related genes. It is the only autoimmune disease known to be endemic, showing a strong correlation with environmental factors. Our data demonstrate that this relatively unknown autoimmune disease may facilitate understanding of the molecular mechanisms of KIR3DL2 ligand recognition.

Risk of human T-lymphotropic virus type I-associated diseases in Jamaica with common HLA types.

Human T-lymphotropic virus-I (HTLV-I) causes adult T-cell leukemia/lymphoma (ATL) and HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP). We postulated a higher disease risk for people with common human leukocyte antigen (HLA) types, due to a narrower immune response against viral or neoplastic antigens, compared to people with uncommon types. HLA class-I (A,B) and class-II (DRB1, DQB1) allele and haplotype frequencies in 56 ATL patients, 59 HAM/TSP patients and 190 population-based, asymptomatic HTLV-I-infected carriers were compared by logistic regression overall (score test) and with odds ratios (ORs) for common types (prevalence >50% of asymptomatic carriers) and by prevalence quartile. HTLV-I proviral load between asymptomatic carriers with common versus uncommon types was compared by t-test. ATL differed from asymptomatic carriers in overall DQB1 allele and class-I haplotype frequencies (p

Hierarchy of resistance to cervical neoplasia mediated by combinations of killer immunoglobulin-like receptor and human leukocyte antigen loci.

Killer immunoglobulin-like receptor (KIR) recognition of specific human histocompatibility leukocyte antigen (HLA) class I allotypes contributes to the array of receptor-ligand interactions that determine natural killer (NK) cell response to its target. Contrasting genetic effects of KIR/HLA combinations have been observed in infectious and autoimmune diseases, where genotypes associated with NK cell activation seem to be protective or to confer susceptibility, respectively. We show here that combinations of KIR and HLA loci also affect the risk of developing cervical neoplasia. Specific inhibitory KIR/HLA ligand pairs decrease the risk of developing neoplasia, whereas the presence of the activating receptor KIR3DS1 results in increased risk of disease, particularly when the protective inhibitory combinations are missing. These data suggest a continuum of resistance conferred by NK cell inhibition to susceptibility involving NK cell activation in the development of cervical neoplasia and underscore the pervasive influence of KIR/HLA genetic variation in human disease pathogenesis.

Prediagnostic human T lymphotropic virus type I provirus loads were highest in Jamaican children who developed seborrheic dermatitis and severe anemia.

In a recent clinical analysis of 308 Jamaican children, human T lymphotropic virus type I (HTLV-I) infection was found to be associated with significantly higher incidence rates of seborrheic dermatitis, eczema, and persistent hyperreflexia of the lower limbs and with nonsignificantly increased rates of severe anemia and abnormal lymphocytes. Results of examination of HTLV-I viral markers in the 28 HTLV-I-infected children provided virologic support for the epidemiologic associations of HTLV-I with seborrheic dermatitis and severe anemia in childhood.

Comprehensive analysis of human leukocyte antigen class I alleles and cervical neoplasia in 3 epidemiologic studies.

To comprehensively explore the relationship between human leukocyte antigen (HLA) class I alleles and cervical neoplasia, a subset of participants from 3 large US and Costa Rican cervix studies were typed for HLA class I alleles. Study subjects were women with cervical cancer or high-grade squamous epithelial lesions (HSILs; n=365) or low-grade squamous epithelial lesions (LSILs; n=275) or who were cytologically normal (control subjects; n=681). Allele-disease associations were assessed by logistic regression analysis. Consistent associations across all studies were observed for HLA-CW*0202 with a combined odds ratio of 0.53 (95% confidence interval [CI], 0.29-0.89) for cancer or HSILs and 0.58 (95% CI, 0.37-1.04) for LSILs, compared with control subjects and adjusted for study. This finding supports the hypothesis that a single allele may be sufficient to confer protection against cervical neoplasia. Given the relationship between HLA-C and its receptors on natural killer (NK) cells, a role is proposed for NK function in human papillomavirus infection and cervical neoplasia.

Chemokine receptor gene polymorphisms and risk of human T lymphotropic virus type I infection in Jamaica.

Polymorphisms of some chemokine receptor genes and their ligands are associated with susceptibility and progression of human immunodeficiency virus infection. This study assessed whether these variants are also responsible for susceptibility to infection with human T lymphotropic virus (HTLV) type I. Frequencies of CCR5-Delta 32, CCR2-64I, and SDF-1-3'A genotype among 116 HTLV-I-positive and 126 HTLV-I-negative persons of African descent in Jamaica were 1.0%, 14.9%, and 5.4%, respectively. The association of HTLV-I infection with the most common variant, CCR2-64I, was examined in 532 subjects. Thirteen (5.4%) of 241 HTLV-I-negative subjects were homozygous for CCR2-64I, versus 3 (1.0%) of 291 HTLV-I-positive subjects (P=.005). Among HTLV-I carriers, provirus load and antibody titer were not significantly different in persons with CCR2-+/64I or CCR2-+/+. These findings suggest that CCR2-64I, or alleles in linkage disequilibrium with it, may affect the risk of HTLV-I infection in a recessive manner.