Yuko Yuki

Differential microRNA regulation of HLA-C expression and its association with HIV control

The HLA-C locus is distinct relative to the other classical HLA class I loci in that it has relatively limited polymorphism, lower expression on the cell surface, and more extensive ligand–receptor interactions with killer-cell immunoglobulin-like receptors. A single nucleotide polymorphism (SNP) 35 kb upstream of HLA-C (rs9264942; termed −35) associates with control of HIV, and with levels of HLA-C messenger RNA transcripts and cell-surface expression, but the mechanism underlying its varied expression is unknown. We proposed that the −35 SNP is not the causal variant for differential HLA-C expression, but rather is marking another polymorphism that directly affects levels of HLA-C. Here we show that variation within the 3′ untranslated region (UTR) of HLA-C regulates binding of the microRNA hsa-miR-148 to its target site, resulting in relatively low surface expression of alleles that bind this microRNA and high expression of HLA-C alleles that escape post-transcriptional regulation. The 3′ UTR variant associates strongly with control of HIV, potentially adding to the effects of genetic variation encoding the peptide-binding region of the HLA class I loci. Variation in HLA-C expression adds another layer of diversity to this highly polymorphic locus that must be considered when deciphering the function of these molecules in health and disease.

Influence of HLA-C expression level on HIV control

Thwarting HIV Multiple genome-wide association studies have revealed that human leukocyte antigen (HLA) genes of the major histocompatibility complex locus have the strongest impact on HIV. In particular, a single-nucleotide polymorphism 35 base pairs upstream of HLA-C shows significant association with viral load and protection against HIV. How HLA-C mediates these effects is unknown. Apps et al. (p. 87) now demonstrate that increasing surface expression of HLA-C is associated with reduced viral load and reduced rate of progression to low CD4+ T cell counts in African and European Americans. High HLA-C expression likely promoted improved HIV control through a more effective cytotoxic CD8+ T cell response. In contrast to HIV infection, high HLA-C expression was associated with a higher risk of the inflammatory bowel disease, Crohns disease. Increased levels of human leukocyte antigen C are associated with control of HIV infection but increased susceptibility to Crohn’s disease. A variant upstream of human leukocyte antigen C (HLA-C) shows the most significant genome-wide effect on HIV control in European Americans and is also associated with the level of HLA-C expression. We characterized the differential cell surface expression levels of all common HLA-C allotypes and tested directly for effects of HLA-C expression on outcomes of HIV infection in 5243 individuals. Increasing HLA-C expression was associated with protection against multiple outcomes independently of individual HLA allelic effects in both African and European Americans, regardless of their distinct HLA-C frequencies and linkage relationships with HLA-B and HLA-A. Higher HLA-C expression was correlated with increased likelihood of cytotoxic T lymphocyte responses and frequency of viral escape mutation. In contrast, high HLA-C expression had a deleterious effect in Crohn’s disease, suggesting a broader influence of HLA expression levels in human disease.

Marked Epitope- and Allele-Specific Differences in Rates of Mutation in Human Immunodeficiency Type 1 (HIV-1) Gag, Pol, and Nef Cytotoxic T-Lymphocyte Epitopes in Acute/Early HIV-1 Infection

ABSTRACT During acute human immunodeficiency virus type 1 (HIV-1) infection, early host cellular immune responses drive viral evolution. The rates and extent of these mutations, however, remain incompletely characterized. In a cohort of 98 individuals newly infected with HIV-1 subtype B, we longitudinally characterized the rates and extent of HLA-mediated escape and reversion in Gag, Pol, and Nef using a rational definition of HLA-attributable mutation based on the analysis of a large independent subtype B data set. We demonstrate rapid and dramatic HIV evolution in response to immune pressures that in general reflect established cytotoxic T-lymphocyte (CTL) response hierarchies in early infection. On a population level, HLA-driven evolution was observed in ∼80% of published CTL epitopes. Five of the 10 most rapidly evolving epitopes were restricted by protective HLA alleles (HLA-B*13/B*51/B*57/B*5801; P = 0.01), supporting the importance of a strong early CTL response in HIV control. Consistent with known fitness costs of escape, B*57-associated mutations in Gag were among the most rapidly reverting positions upon transmission to non-B*57-expressing individuals, whereas many other HLA-associated polymorphisms displayed slow or negligible reversion. Overall, an estimated minimum of 30% of observed substitutions in Gag/Pol and 60% in Nef were attributable to HLA-associated escape and reversion events. Results underscore the dominant role of immune pressures in driving early within-host HIV evolution. Dramatic differences in escape and reversion rates across codons, genes, and HLA restrictions are observed, highlighting the complexity of viral adaptation to the host immune response.

Genetic interplay between HLA-C and MIR148A in HIV control and Crohn disease

Significance In the human population different HLA-C allotypes are present that have different expression levels at the cell surface. Individuals with higher expressed HLA-C allotypes demonstrate better HIV control but increased risk of Crohn disease. A microRNA, miR-148a, regulates expression of some HLA-C allotypes. We report here that this microRNA also varies in expression level between people. MIR148A variation showed significant and opposing effects on HIV viral control vs. risk of Crohn disease, specifically in subjects with HLA-C alleles that are regulated by miR-148a. These results are independent of confounding effects by other HLA loci because only HLA-C is regulated by miR-148a. Our data represent an example of gene interactions that affect immune response and thereby the risk of human disease. Variation in the 3′ untranslated region (3′UTR) of the HLA-C locus determines binding of the microRNA Hsa-miR-148a, resulting in lower cell surface expression of alleles that bind miR-148a relative to those alleles that escape its binding. The HLA-C 3′UTR variant was shown to associate with HIV control, but like the vast majority of disease associations in a region dense with causal candidates, a direct effect of HLA-C expression level on HIV control was not proven. We demonstrate that a MIR148A insertion/deletion polymorphism associates with its own expression levels, affecting the extent to which HLA-C is down-regulated, the level of HIV control, and the risk of Crohn disease only among those carrying an intact miR-148a binding site in the HLA-C 3′UTR. These data illustrate a direct effect of HLA-C expression level on HIV control that cannot be attributed to other HLA loci in linkage disequilibrium with HLA-C and highlight the rich complexity of genetic interactions in human disease.

Epigenetic Regulation of Differential HLA-A Allelic Expression Levels

MHC class I expression levels influence the strength of immune responses and represent another variable in determining outcome to disease beyond peptide binding alone. Identification of the HLA loci that vary in allelic expression levels and delineating the mechanism responsible for expression variation may provide the opportunity to modify their expression therapeutically. We have examined the expression levels of allelic lineages at the HLA-A locus in a sample of 216 European Americans using a real-time polymerase chain reaction assay, which amplifies all HLA-A lineages specifically with equal efficiency, and observed a gradient of expression that associates with HLA-A allelic lineage (R = 0.6, P = 5 × 10(-25)). DNA methylation of the HLA-A gene appears to contribute to the variation in HLA-A mRNA expression levels, as a significant inverse correlation was observed between HLA-A mRNA expression levels in untreated cells and the degree to which expression is increased after treatment of the cells with a DNA methyltransferase inhibitor (R = 0.6, P = 2.8 × 10(-6)). Further, deep-sequencing and immunoprecipitation assays revealed allelic lineage-specific methylation patterns within the HLA-A promoter region where increased DNA methylation levels correlated significantly with reduced HLA-A expression levels (R = 0.89, P = 3.7 × 10(-9)). These data demonstrate HLA-A allelic lineage-specific variation in expression levels, and DNA methylation as a likely factor in contributing to this variation.

Impact of Select Immunologic and Virologic Biomarkers on CD4 Cell Count Decrease in Patients with Chronic HIV-1 Subtype C Infection: Results from Sinikithemba Cohort, Durban, South Africa

BACKGROUND The extent to which immunologic and clinical biomarkers influence human immunodeficiency virus type 1 (HIV-1) infection outcomes remains incompletely characterized, particularly for non-B subtypes. On the basis of data supporting in vitro HIV-1 protein-specific CD8 T lymphocyte responses as correlates of immune control in cross-sectional studies, we assessed the relationship of these responses, along with established HIV-1 biomarkers, with rates of CD4 cell count decrease in individuals infected with HIV-1 subtype C. METHODS Bivariate and multivariate mixed-effects models were used to assess the relationship of baseline CD4 cell count, plasma viral load, human leukocyte antigen (HLA) class I alleles, and HIV-1 protein-specific CD8 T cell responses with the rate of CD4 cell count decrease in a longitudinal population-based cohort of 300 therapy-naive, chronically infected adults with baseline CD4 cell counts >200 cells/mm(3) and plasma viral loads >500 copies/mL over a median of 25 months of follow-up. RESULTS In bivariate analyses, baseline CD4 cell count, plasma viral load, and possession of a protective HLA allele correlated significantly with the rate of CD4 cell count decrease. No relationship was observed between HIV-1 protein-specific CD8 T cell responses and CD4 cell count decrease. Results from multivariate models incorporating baseline CD4 cell counts (201-350 vs >350 cells/mm(3)), plasma viral load (< or =100,000 vs >100,000 copies/mL), and HLA (protective vs not protective) yielded the ability to discriminate CD4 cell count decreases over a 10-fold range. The fastest decrease was observed among individuals with CD4 cell counts >350 cells/mm(3) and plasma viral loads >100,000 copies/mL with no protective HLA alleles (-59 cells/mm(3) per year), whereas the slowest decrease was observed among individuals with CD4 cell counts 201-350 cells/mm(3), plasma viral loads < or =100,000 copies/mL, and a protective HLA allele (-6 cells/mm(3) per year). CONCLUSIONS The combination of plasma viral load and HLA class I type, but not in vitro HIV-1 protein-specific CD8 T cell responses, differentiates rates of CD4 cell count decrease in patients with chronic subtype-C infection better than either marker alone.Background The extent to which immunologic and clinical biomarkers influence HIV outcomes remains incompletely characterized, particularly for non-B subtypes. Based on data supporting in vitro HIV protein-specific CD8 T-lymphocyte responses as correlates of immune control in cross-sectional studies, we assessed the relationship of these responses, along with established HIV biomarkers, with rates of CD4 decline in subtype-C infection.

Elevated HLA-A expression impairs HIV control through inhibition of NKG2A-expressing cells

Inhibiting natural killer cells in AIDS The human leukocyte antigen (HLA) gene complex varies enormously among individuals and helps explain individual variation in immunity to infectious diseases. Ramsuran et al. examined data from almost 10,000 HIV infections. Expression of the HLA-A and -B alleles was associated with higher viral load, reduced CD4+ T cell counts, and accelerated progression to AIDS. Higher levels of HLA-A expression increased expression of HLA-E, which blocks a specific receptor (NKG2A) on the immune cells that normally eliminate virus-infected cells. Thus, targeting NKG2A might provide a therapeutic avenue for HIV treatment. Science, this issue p. 86 Natural killer cell inhibition mediated by HLA-A, -E, and -B alleles elevates viremia and accelerates progression to AIDS. The highly polymorphic human leukocyte antigen (HLA) locus encodes cell surface proteins that are critical for immunity. HLA-A expression levels vary in an allele-dependent manner, diversifying allele-specific effects beyond peptide-binding preference. Analysis of 9763 HIV-infected individuals from 21 cohorts shows that higher HLA-A levels confer poorer control of HIV. Elevated HLA-A expression provides enhanced levels of an HLA-A–derived signal peptide that specifically binds and determines expression levels of HLA-E, the ligand for the inhibitory NKG2A natural killer (NK) cell receptor. HLA-B haplotypes that favor NKG2A-mediated NK cell licensing (i.e., education) exacerbate the deleterious effect of high HLA-A on HIV control, consistent with NKG2A-mediated inhibition impairing NK cell clearance of HIV-infected targets. Therapeutic blockade of HLA-E:NKG2A interaction may yield benefit in HIV disease.

Impact of select immunologic and virologic biomarkers on CD4 cell count decrease in patients with chronic HIV-1 subtype C infection

BACKGROUND The extent to which immunologic and clinical biomarkers influence human immunodeficiency virus type 1 (HIV-1) infection outcomes remains incompletely characterized, particularly for non-B subtypes. On the basis of data supporting in vitro HIV-1 protein-specific CD8 T lymphocyte responses as correlates of immune control in cross-sectional studies, we assessed the relationship of these responses, along with established HIV-1 biomarkers, with rates of CD4 cell count decrease in individuals infected with HIV-1 subtype C. METHODS Bivariate and multivariate mixed-effects models were used to assess the relationship of baseline CD4 cell count, plasma viral load, human leukocyte antigen (HLA) class I alleles, and HIV-1 protein-specific CD8 T cell responses with the rate of CD4 cell count decrease in a longitudinal population-based cohort of 300 therapy-naive, chronically infected adults with baseline CD4 cell counts >200 cells/mm(3) and plasma viral loads >500 copies/mL over a median of 25 months of follow-up. RESULTS In bivariate analyses, baseline CD4 cell count, plasma viral load, and possession of a protective HLA allele correlated significantly with the rate of CD4 cell count decrease. No relationship was observed between HIV-1 protein-specific CD8 T cell responses and CD4 cell count decrease. Results from multivariate models incorporating baseline CD4 cell counts (201-350 vs >350 cells/mm(3)), plasma viral load (< or =100,000 vs >100,000 copies/mL), and HLA (protective vs not protective) yielded the ability to discriminate CD4 cell count decreases over a 10-fold range. The fastest decrease was observed among individuals with CD4 cell counts >350 cells/mm(3) and plasma viral loads >100,000 copies/mL with no protective HLA alleles (-59 cells/mm(3) per year), whereas the slowest decrease was observed among individuals with CD4 cell counts 201-350 cells/mm(3), plasma viral loads < or =100,000 copies/mL, and a protective HLA allele (-6 cells/mm(3) per year). CONCLUSIONS The combination of plasma viral load and HLA class I type, but not in vitro HIV-1 protein-specific CD8 T cell responses, differentiates rates of CD4 cell count decrease in patients with chronic subtype-C infection better than either marker alone.Background The extent to which immunologic and clinical biomarkers influence HIV outcomes remains incompletely characterized, particularly for non-B subtypes. Based on data supporting in vitro HIV protein-specific CD8 T-lymphocyte responses as correlates of immune control in cross-sectional studies, we assessed the relationship of these responses, along with established HIV biomarkers, with rates of CD4 decline in subtype-C infection.

Impact of select immunologic and virologic biomarkers on CD4 decline in chronic HIV-1 subtype C infection: Results from Sinikithemba longitudinal population-based cohort, Durban, South Africa

BACKGROUND The extent to which immunologic and clinical biomarkers influence human immunodeficiency virus type 1 (HIV-1) infection outcomes remains incompletely characterized, particularly for non-B subtypes. On the basis of data supporting in vitro HIV-1 protein-specific CD8 T lymphocyte responses as correlates of immune control in cross-sectional studies, we assessed the relationship of these responses, along with established HIV-1 biomarkers, with rates of CD4 cell count decrease in individuals infected with HIV-1 subtype C. METHODS Bivariate and multivariate mixed-effects models were used to assess the relationship of baseline CD4 cell count, plasma viral load, human leukocyte antigen (HLA) class I alleles, and HIV-1 protein-specific CD8 T cell responses with the rate of CD4 cell count decrease in a longitudinal population-based cohort of 300 therapy-naive, chronically infected adults with baseline CD4 cell counts >200 cells/mm(3) and plasma viral loads >500 copies/mL over a median of 25 months of follow-up. RESULTS In bivariate analyses, baseline CD4 cell count, plasma viral load, and possession of a protective HLA allele correlated significantly with the rate of CD4 cell count decrease. No relationship was observed between HIV-1 protein-specific CD8 T cell responses and CD4 cell count decrease. Results from multivariate models incorporating baseline CD4 cell counts (201-350 vs >350 cells/mm(3)), plasma viral load (< or =100,000 vs >100,000 copies/mL), and HLA (protective vs not protective) yielded the ability to discriminate CD4 cell count decreases over a 10-fold range. The fastest decrease was observed among individuals with CD4 cell counts >350 cells/mm(3) and plasma viral loads >100,000 copies/mL with no protective HLA alleles (-59 cells/mm(3) per year), whereas the slowest decrease was observed among individuals with CD4 cell counts 201-350 cells/mm(3), plasma viral loads < or =100,000 copies/mL, and a protective HLA allele (-6 cells/mm(3) per year). CONCLUSIONS The combination of plasma viral load and HLA class I type, but not in vitro HIV-1 protein-specific CD8 T cell responses, differentiates rates of CD4 cell count decrease in patients with chronic subtype-C infection better than either marker alone.Background The extent to which immunologic and clinical biomarkers influence HIV outcomes remains incompletely characterized, particularly for non-B subtypes. Based on data supporting in vitro HIV protein-specific CD8 T-lymphocyte responses as correlates of immune control in cross-sectional studies, we assessed the relationship of these responses, along with established HIV biomarkers, with rates of CD4 decline in subtype-C infection.