Breadth and polyfunctionality of T cell responses to human cytomegalovirus in men who have sex with men: relationship with HIV infection and frailty.

Cytomegalovirus (CMV)-seropositive adults have large T cell responses to a wide range of CMV proteins; these responses have been associated with chronic inflammation and frailty in people with or without HIV infection. We analyzed the relationships between chronic HIV infection, frailty, and the breadth and polyfunctionality of CD4 and CD8 T cell responses to CMV. Peripheral blood mononuclear cells from 42 men (20 without HIV and 22 with virologically suppressed HIV) in the Multicenter AIDS Cohort Study (MACS) were stimulated with peptide pools spanning 19 CMV open reading frames (ORFs). As measured by flow cytometry and intracellular cytokine staining for IFN-γ, TNF-α, and IL-2, CD8 T cells from men with HIV responded to significantly more CMV ORFs than those from men without HIV. This was primarily due to a broader response to ORFs that are expressed during the late phase of CMV replication. The number of ORFs to which a participant's T cells responded was positively correlated with the sum of all that individual's T cell responses; these correlations were weaker in men with than without HIV. Polyfunctional CMV-specific CD4 responses (production of more than one cytokine) were significantly lower in men with than without HIV. Frailty status did not substantially affect the breadth or magnitude of the CMV-specific T cell responses. These results suggest that immune control of CMV infection is affected more by chronic HIV infection than by frailty. The differences between men with and without HIV were similar to those reported between young and older adults without HIV. IMPORTANCE: T cell responses to chronic cytomegalovirus (CMV) infection have significant biological and clinical implications in HIV infection and aging. Here, we systematically analyzed the breadth, magnitude, and polyfunctionality of T cell responses to multiple CMV antigens in men with and without HIV in the Multicenter AIDS Cohort Study (MACS), a longstanding study of the natural and treated history of HIV-1 infection in men who have sex with men. We found that the breadth and polyfunctionality of T cell responses to CMV were different between men with chronic, treated HIV and those without HIV. The reason for these differences is unknown, but these findings suggest that people with treated HIV may have more frequent CMV reactivation than people without HIV. Differences between people with and without HIV also resembled differences reported between young and older adults without HIV, supporting a role for the immune responses to CMV in the aging process.

Interseason waning of vaccine-induced hemagglutination inhibition antibody titers and contributing factors to pre-existing humoral immunity against influenza in community-dwelling older adults 75 years and older.

BACKGROUND: Seasonal influenza causes significant morbidity and mortality with a disproportionately high disease burden in older adults. Strain-specific hemagglutination-inhibition (HAI) antibody titer is a well-established measure of humoral immunity against influenza and pre-vaccination HAI titer is a valuable indicator of pre-existing humoral immunity at the beginning of each influenza season in highly vaccinated older adults. While vaccine-induced HAI antibody titers are known to wane over time, accurate assessment of their interseason waning has been challenging. This is because pre-vaccination HAI titers are routinely measured using current season vaccine strain antigens instead of the prior season vaccines with which individuals were immunized; as such, they do not accurately represent residual antibody titers from prior season vaccination. This study took advantage of available pre-vaccination HAI titers measured using both current and prior season vaccine strain antigens in a longitudinal influenza immunization study with participants enrolled for multiple consecutive influenza seasons from 2014 through 2017. Influenza A virus (IAV) H3N2 and influenza B virus (IBV) strains in the vaccine formula changed in 2015 and again in 2016 season. IAV H1N1 vaccine strain remained the same from 2014 through 2016 seasons, but changed in 2017. We also investigated factors contributing to pre-existing humoral immunity. RESULTS: Interseason waning of HAI titers was evident, but rates of waning varied among vaccine strains and study seasons, from 18% (p = .43) to 61% (p < .01). Rates of waning were noticeably greater when pre-vaccination HAI titers were measured by the routine approach, i.e., using current season vaccine strain antigens, from 33% (p = .12) to 83% (p < .01), adjusting for age at prior study season, sex, race, and education. This was largely because the routinely measured pre-vaccination HAI titers underrepresented residual HAI titers from prior season vaccinations. Moreover, interseason antibody waning and prior season post-vaccination HAI titers had significant and independent associations with pre-vaccination HAI titers. CONCLUSIONS: The routinely measured pre-vaccination HAI titer overestimates interseason HAI antibody waning as it underestimates residual antibody titers from prior season vaccination when virus strains in the vaccine formula change. Moreover, interseason antibody waning and prior season post-vaccination HAI titers independently contribute to pre-existing humoral immunity in this highly vaccinated, community-dwelling older adult population.

HLA tapasin independence: broader peptide repertoire and HIV control.

Human leukocyte antigen (HLA) class I allotypes vary in their ability to present peptides in the absence of tapasin, an essential component of the peptide loading complex. We quantified tapasin dependence of all allotypes that are common in European and African Americans (n = 97), which revealed a broad continuum of values. Ex vivo examination of cytotoxic T cell responses to the entire HIV-1 proteome from infected subjects indicates that tapasin-dependent allotypes present a more limited set of distinct peptides than do tapasin-independent allotypes, data supported by computational predictions. This suggests that variation in tapasin dependence may impact the strength of the immune responses by altering peptide repertoire size. In support of this model, we observed that individuals carrying HLA class I genotypes characterized by greater tapasin independence progress more slowly to AIDS and maintain lower viral loads, presumably due to increased breadth of peptide presentation. Thus, tapasin dependence level, like HLA zygosity, may serve as a means to restrict or expand breadth of the HLA-I peptide repertoire across humans, ultimately influencing immune responses to pathogens and vaccines.

Mycoplasma genitalium Infection Is Not Associated With Genital Tract Inflammation Among Adolescent and Young Adult Women in Baltimore, Maryland.

BACKGROUND: Mycoplasma genitalium (MG) is a prevalent sexually transmitted infection, but little is known about the associated inflammatory signatures in the genital tract of adolescents and young adult women. METHODS: Adolescents and young adult women aged 13 to 24 years were recruited. Demographic information, sexual behavior history, and medical history were collected. Vaginal swab samples were tested for MG, Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, bacterial vaginosis, and measurement of 13 cytokines, chemokines, and antimicrobial proteins. Vaginal cytokine concentrations were compared by MG infection status. The strength of associations between multiple factors and MG infection was evaluated. RESULTS: Of 215 participants, 16.7% (95% confidence interval [CI], 12.0%-22.4%) had MG infection. Inflammation was not associated with MG infection (P > 0.05). M. genitalium infection was associated with C. trachomatis infection (adjusted prevalence ratio [aPrR], 3.02; 95% CI, 1.69-5.39), bisexual behavior in the past 3 months (aPrR, 2.07; 95% CI, 1.18-3.64), genitourinary symptoms (aPrR, 2.06; 95% CI, 1.22-3.49), and self-reported Black race (aPrR, 3.53; 95% CI, 1.11-11.18). CONCLUSIONS: Higher levels of genital tract cytokines were not associated with MG infection. C. trachomatis infection, bisexual behavior, self-reported Black race, and genitourinary symptoms were associated with an increased likelihood of MG infection.

Longitudinal association of cytokine-producing CMV-specific T cells with frailty in HIV-infected and -uninfected men who have sex with men.

BACKGROUND: Chronic cytomegalovirus (CMV) infection has been postulated as a driver of chronic inflammation that has been associated with frailty and other age-related conditions in both HIV-infected (HIV+) and -uninfected (HIV-) people. METHODS: To study the T cell response to CMV as a predictor of onset and maintenance of frailty, baseline CMV-specific T cell responses of 42 men (20 HIV-, 22 HIV+; 21 frail, 21 nonfrail) in the Multicenter AIDS Cohort Study (MACS) were assessed by flow cytometric analysis of cytokine production (IFN-γ, TNF-⍺, and IL-2) in response to overlapping peptide pools spanning 19 CMV open reading frames. The Fried frailty phenotype was assessed at baseline and semiannually thereafter. Times to transition into or out of frailty were compared by tertiles of percentages of cytokine-producing T cells using Kaplan-Meier estimators and the exact log-rank test. RESULTS: Over a median follow-up of 6.5 (interquartile range: 2) years, faster onset of frailty was significantly predicted by higher (HIV- men) or lower (HIV+ men) percentages of CD4 T cells producing only IFN-γ (IFN-γ-single-producing (SP)), and by lower percentages of IFN-γ-, TNF-⍺-, and IL-2-triple-producing CD8 T cells (HIV- men). Greater maintenance of frailty was significantly predicted by lower percentages of both these T cell subsets in HIV- men, and by lower percentages of IFN-γ-SP CD4 T cells in HIV+ men. The antigenic specificity of IFN-γ-SP CD4 T cells was different between HIV- and HIV+ nonfrail men, as were the correlations between these cells and serum inflammatory markers. CONCLUSIONS: In this pilot study, percentages of CMV-specific T cells predicted the onset and maintenance of frailty in HIV- and HIV+ men. Predictive responses differed by HIV status, which may relate to differential control of CMV reactivation and inflammation by anti-CMV T cell responses.

Validation of Preamplification to Improve Quantification of Cytomegalovirus DNA Using Droplet Digital Polymerase Chain Reaction.

Subclinical cytomegalovirus (CMV) replication is associated with strong cellular immune response and chronic inflammation, which could contribute to aging-related conditions such as cardiovascular disease and frailty. However, because of very low levels of CMV DNA present in people with chronic CMV infection, it has been difficult to explore the virologic and immunologic mechanisms of chronic low-level CMV infection and a sensitive method to monitor CMV replication is needed. Droplet digital PCR (ddPCR) has been shown to have higher precision and reproducibility than real-time quantitative PCR (qPCR) in quantifying low levels of CMV DNA, but it is not always sensitive enough for this purpose. Through rigorous validation experiments, we demonstrated that sensitivity and precision of quantification of very low levels of CMV DNA by ddPCR can be significantly increased by preamplification of samples with 10-20 cycles of conventional PCR, especially when testing CMV DNA in the presence of cellular DNA. With preamplification, we could reliably quantify down to two copies of CMV DNA, as opposed to five copies without preamplification. Further studies are needed to determine if ddPCR with preamplification can facilitate mechanistic studies of the characteristics and consequences of chronic CMV infection in aging adults.

Cell-Specific Requirements for STAT Proteins and Type I IFN Receptor Signaling Discretely Regulate IL-24 and IL-10 Expression in NK Cells and Macrophages.

Il10 forms a cytokine cluster with Il19, Il20, and Il24 in a conserved region of chromosome 1. The latter genes are in the IL-20 subfamily of IL-10-related cytokines and, although they are not as well studied their biologic actions and expression patterns, seem to have little in common with IL-10. IL-24, like IL-10, however, is uniquely expressed in T cells and is a signature gene of the Th2 lineage, which suggests they could be coregulated in certain cell types. Little is known about other cellular sources of IL-24. We investigated IL-24 and IL-10 expression in murine macrophages and NK cells, and found that although they are coexpressed under most stimulation conditions, IL-24 and IL-10 are controlled by distinct, cell type-specific pathways. In bone marrow-derived macrophages, optimal IL-24 expression required LPS+IL-4 costimulation and STAT6 but was independent of type I IFN receptor signaling and STAT4. Conversely, LPS-induced IL-10 was independent of IL-4/STAT6 and STAT4 but, consistent with other reports, required type I IFN receptor signaling for optimal expression. Remarkably, NK-specific IL-24 (but not IL-10) expression was dependent on both type I IFN receptor signaling and STAT4. Induction of IL-24 expression was accompanied by cell-specific recruitment of STAT6 and STAT4 to multiple sites that we identified within Il24, which mediated STAT-dependent histone modifications across the gene. Collectively, our results indicate that despite being coexpressed, IL-10 and IL-24 are independently regulated by different type I IFN receptor signaling pathways in innate immune cells and provide insight into the mechanisms that fine-tune cell type-specific gene expression within the Il10 cluster.

Relationship Between T-Cell Responses to CMV, Markers of Inflammation, and Frailty in HIV-uninfected and HIV-infected Men in the Multicenter AIDS Cohort Study.

Background: Both aging and treated human immunodeficiency virus (HIV)-infected populations exhibit low-level chronic immune activation of unknown etiology, which correlates with morbidity and mortality. Cytomegalovirus (CMV) infection is common in both populations, but its relation to immune activation is unknown. Methods: T cells from men who have sex with men (22 virologically suppressed HIV+, 20 HIV-) were stimulated with peptides spanning 19 CMV open reading frames, and intracellular cytokine responses were assessed. Soluble and cellular inflammatory markers were assessed by multiplex electrochemiluminescence and flow cytometry, respectively. Frailty was assessed by the Fried criteria. Results: All men had responses to CMV. Proportions of CMV-responsive T cells correlated strongly (r ≥ 0.6 or ≤ -0.6; P < .05) with immunologic markers, depending on donor HIV and frailty status. Markers significantly correlated in some groups after adjustment for multiple comparisons included interferon-γ, tumor necrosis factor-α, interleukin-6, and several chemokines in serum, and the proportion of activated T cells. The magnitude of the CD4 IL-2 response significantly predicted onset of frailty in HIV- nonfrail men, but not in HIV+ nonfrail men. Conclusions: T-cell responses to CMV may strongly influence chronic immune activation in HIV-uninfected and virologically suppressed HIV-infected men, and may predict frailty in HIV-uninfected men.

Multiplex assay reliability and long-term intra-individual variation of serologic inflammatory biomarkers.

BACKGROUND: Circulating cytokines, chemokines, and soluble cytokine receptors can serve as biomarkers of inflammation and immune dysregulation. Good reliability of multiplex platforms, which allow for simultaneous, comprehensive biomarker assessment, is critical for their utility in epidemiologic studies. We examined the reliability of the Meso-Scale Discovery (MSD) platform to simultaneously quantitate 15 cytokines and chemokines and the Luminex platform (R&D Systems) to quantitate 5 soluble receptors and 2 chemokines and cytokines and evaluated long-term within-person correlation of these biomarkers. METHODS: The detectability and reliability of these assay systems were assessed using the same external controls across plates and archived sera from 250 HIV- men in the Multicenter AIDS Cohort Study. Using up to four visits per person from 1984 to 2009, age-adjusted intraclass correlation coefficients (ICC) of biomarkers with >80% detectability (CCL11, CXCL8, CXCL10, CCL2, CCL4, CCL13, CCL17, CXCL13, IL-10, IL-12p70, IL-6, TNF-α, BAFF, sCD14, sCD27, sgp130, sIL-2Rα, and sTNF-R2) were obtained using linear mixed models. RESULTS: Most biomarkers were detectable in 80% of control samples; IFN-γ, GM-CSF, and IL-2 were undetectable in >20% of samples. Among the HIV-uninfected men, most biomarkers showed fair to strong within-person correlation (ICC>0.40) up to 15years. The ICC for CXCL8 was good in the short term but decreased with increasing time between visits, becoming lower (ICC<0.40) after 8years. CONCLUSIONS: These multiplexed assays showed acceptable reliability for use in epidemiologic research, despite some technical variability and limitations in cytokine quantitation. Most biomarkers displayed moderate-to-excellent intra-individual variability over the long term, suggesting their utility in prospective studies investigating etiologic associations with diverse chronic conditions.

LILRB2 interaction with HLA class I correlates with control of HIV-1 infection.

Natural progression of HIV-1 infection depends on genetic variation in the human major histocompatibility complex (MHC) class I locus, and the CD8+ T cell response is thought to be a primary mechanism of this effect. However, polymorphism within the MHC may also alter innate immune activity against human immunodeficiency virus type 1 (HIV-1) by changing interactions of human leukocyte antigen (HLA) class I molecules with leukocyte immunoglobulin-like receptors (LILR), a group of immunoregulatory receptors mainly expressed on myelomonocytic cells including dendritic cells (DCs). We used previously characterized HLA allotype-specific binding capacities of LILRB1 and LILRB2 as well as data from a large cohort of HIV-1-infected individuals (N = 5126) to test whether LILR-HLA class I interactions influence viral load in HIV-1 infection. Our analyses in persons of European descent, the largest ethnic group examined, show that the effect of HLA-B alleles on HIV-1 control correlates with the binding strength between corresponding HLA-B allotypes and LILRB2 (p = 10(-2)). Moreover, overall binding strength of LILRB2 to classical HLA class I allotypes, defined by the HLA-A/B/C genotypes in each patient, positively associates with viral replication in the absence of therapy in patients of both European (p = 10(-11)-10(-9)) and African (p = 10(-5)-10(-3)) descent. This effect appears to be driven by variations in LILRB2 binding affinities to HLA-B and is independent of individual class I allelic effects that are not related to the LILRB2 function. Correspondingly, in vitro experiments suggest that strong LILRB2-HLA binding negatively affects antigen-presenting properties of DCs. Thus, we propose an impact of LILRB2 on HIV-1 disease outcomes through altered regulation of DCs by LILRB2-HLA engagement.

Inflammatory Biomarkers and Mortality Risk Among HIV-Suppressed Men: A Multisite Prospective Cohort Study.

BACKGROUND: Human immunodeficiency virus (HIV)-induced inflammation and immune activation persist after initiation of combination antiretroviral therapy (cART) and HIV suppression and may contribute to mortality risks that exceed those in HIV-uninfected populations, though associations are unclear. METHODS: In the prospective Multicenter AIDS Cohort Study, comprising men who have sex with men from Baltimore, Chicago, Los Angeles, and Pittsburgh, concentrations of 24 biomarkers of inflammation and immune activation were measured in stored serum from HIV-positive men obtained after cART-induced HIV suppression between 1996 and 2009. The outcome was nonaccidental death, with follow-up until 2014. We used Cox proportional hazards models to test whether biomarker concentrations predict time from HIV suppression to death and adjusted for multiple tests. Exploratory factor analysis (EFA) was employed to identify groupings of biomarkers that predict mortality risk. RESULTS: Of 670 men followed up from HIV suppression, 54 died by the end of 2013. After adjustment for age, CD4(+) cell count, hepatitis B or C virus infection, and smoking, concentrations in the highest quartile of 4 biomarkers were significantly associated with mortality risk after controlling the false discovery rate at 5%: interleukin (IL) 6 (hazard ratio, 3.54; 95% confidence interval, 2.06-6.10), soluble IL 2Rα (3.29, 1.85-5.85), soluble CD14 (2.67, 1.55-4.61), and chemokine (CXC motif) ligand 13 (CXCL13; 2.26; 1.29-3.95). EFA yielded 2 biomarker groupings that were independent predictors of mortality risk. CONCLUSIONS: Despite having undetectable HIV RNA levels during cART, men with higher concentrations of several biomarkers (particularly IL 6, soluble IL 2Rα, soluble CD14, and CXCL13) had higher hazards of long-term mortality. Correlations observed among biomarker concentrations may represent underlying inflammatory processes that contribute to mortality risk.

The association of medication use with clearance or persistence of oral HPV infection.

PURPOSE: Persistent oral human papillomavirus (HPV) infection increases risk for oropharyngeal carcinoma, and people living with HIV have higher rates of oral HPV infection and related cancers. Some prescription medications have immunomodulatory effects, but the impact of medication use on oral HPV natural history is unknown. METHODS: Scope® oral rinse-and-gargle samples were collected semi-annually from 1,666 participants and tested for 37 types of oral HPV DNA using PCR; 594 HPV-infected participants with 1,358 type-specific oral HPV infections were identified. Data were collected on recent (past 6 months) use of medications. The relationship between medication use and oral HPV clearance was evaluated using Wei-Lin-Weissfeld regression, adjusting for biologic sex, prevalent versus incident infection, age, HIV status and CD4+ T cell count. RESULTS: Out of 11 medications examined, oral HPV clearance was significantly reduced in participants reporting recent use of antipsychotics (HR 0.75, 95% CI 0.57-0.99), anxiolytics/sedatives (HR 0.78, 95% CI 0.63-0.96) and antidepressants (HR 0.82, 95% CI 0.67-0.999). Among antipsychotics users, effect modification by HIV status was observed, with reduced clearance in HIV-infected (HR 0.67, 95% CI 0.49-0.91), but not HIV-uninfected participants (p-interaction = 0.009). After adjusted analysis, antipsychotic use remained significantly associated with reduced oral HPV clearance overall (aHR 0.75, 95% CI 0.57-0.99), and when restricted to only HIV-infected participants (aHR 0.66, 95% CI 0.48-0.90). After adjustment, anxiolytic/sedative use and antidepressant use were no longer significantly associated with reduced oral HPV clearance. CONCLUSIONS: Some medications were associated with decreased oral HPV clearance, most notably antipsychotic medications. These medications are prescribed for conditions that may have immunomodulating effects, so characteristics of underlying illness may have partially contributed to reduced oral HPV clearance.

Host factors associated with serologic inflammatory markers assessed using multiplex assays.

Chronic systemic inflammation contributes to the development of adverse health conditions, yet the influence of fixed and modifiable risk factors on many serologic biomarkers of inflammation remains largely unknown. Serum concentrations of twenty-three biomarkers, including C-reactive protein (CRP), cytokines (CXCL11, CXCL8, CXCL10, CCL2, CCL13, CCL4, CCL17, CXCL13, IL-10, IL-12p70, IL-6, TNF-α, IL-2, IFN-γ, IL-1ß, GM-CSF, BAFF), and soluble immune receptors (sCD14, sIL-2Rα, sCD27, sgp130, sTNF-R2) were measured longitudinally using multiplexed immunometric assays in 250 HIV-uninfected men followed in the Multicenter AIDS Cohort Study (1984-2009). Generalized gamma regression was used to determine the statistical significance of factors associated with each biomarker. After accounting for age, race, and education, and for analysis of multiple biomarkers, higher concentrations of specific individual biomarkers were significantly (P<0.002) associated with hypertension, obesity, hepatitis C infection, stimulant use, and diabetes and lower concentrations with hypercholesterolemia. These associations should be taken into account in epidemiological studies of these biomarkers, and may provide potential targets for disease prevention and treatment.

Association of serum cytokines with oral HPV clearance.

BACKGROUND: Initial studies suggest higher serum levels of some pro-inflammatory cytokines may be associated with decreased cervical human papillomavirus (HPV) clearance. However, the relationship of cytokines with oral HPV clearance has not been explored. METHODS: From 2010 to 2014, oral rinse and serum samples were collected semi-annually from 1601 adults. Oral rinse samples were tested for HPV DNA using PCR. Based on oral HPV results, 931 serum samples were selected for cytokine evaluation to include a roughly equal number of prevalent (n=307), incident (n=313), and no oral HPV infections (n=311). Electrochemiluminescence multiplex assays were used to determine the concentrations of IL-6, IL-8, TNF-α, IFN-γ, IL-1ß, IL-2, IL-4, IL-10, IL-12 and IL-13. The relationship between serum cytokine concentrations (categorized into quartiles) and oral HPV clearance was evaluated with Wei-Lin-Weissfeld regression models, adjusting for HPV infection type (prevalent vs. incident), age, HIV status, and CD4 T cell count. RESULTS: Higher TNF-α concentration was associated with decreased clearance in men (highest vs. lowest quartile, adjusted hazard ratio [aHR]=0.52, 95% CI=0.34-0.79) and women (aHR=0.76, 95% confidence interval [CI]=0.55-1.04), with stronger associations in men than women (p-interaction=0.049). Higher IL-2 concentration was associated with reduced clearance in men (aHR=0.69, 95% CI=0.50-0.95), but not women (p-interaction=0.058). Results were similar within CD4 T cell strata (CD4⩾500 or CD4<500 cells/µl) among HIV-infected participants. No other cytokines were associated with clearance. CONCLUSION: High serum TNF-α is associated with reduced clearance of oral HPV infection.

Distinct assembly profiles of HLA-B molecules.

MHC class I polymorphisms are known to influence outcomes in a number of infectious diseases, cancers, and inflammatory diseases. Human MHC class I H chains are encoded by the HLA-A, HLA-B, and HLA-C genes. These genes are highly polymorphic, with the HLA-B locus being the most variable. Each HLA class I protein binds to a distinct set of peptide Ags, which are presented to CD8(+) T cells. HLA-disease associations have been shown in some cases to link to the peptide-binding characteristics of individual HLA class I molecules. In this study, we show that polymorphisms at the HLA-B locus profoundly influence the assembly characteristics of HLA-B molecules and the stabilities of their peptide-deficient forms. In particular, dependence on the assembly factor tapasin is highly variable, with frequent occurrence of strongly tapasin-dependent or independent allotypes. Several polymorphic HLA-B residues located near the C-terminal end of the peptide are key determinants of tapasin-independent assembly. In vitro refolded forms of tapasin-independent allotypes assemble more readily with peptides compared to tapasin-dependent allotypes that belong to the same supertype, and, during refolding, reduced aggregation of tapasin-independent allotypes is observed. Paradoxically, in HIV-infected individuals, greater tapasin-independent HLA-B assembly confers more rapid progression to death, consistent with previous findings that some HLA-B allotypes shown to be tapasin independent are associated with rapid progression to multiple AIDS outcomes. Together, these findings demonstrate significant variations in the assembly of HLA-B molecules and indicate influences of HLA-B-folding patterns upon infectious disease outcomes.

A genome-wide association study of resistance to HIV infection in highly exposed uninfected individuals with hemophilia A.

Human genetic variation contributes to differences in susceptibility to HIV-1 infection. To search for novel host resistance factors, we performed a genome-wide association study (GWAS) in hemophilia patients highly exposed to potentially contaminated factor VIII infusions. Individuals with hemophilia A and a documented history of factor VIII infusions before the introduction of viral inactivation procedures (1979-1984) were recruited from 36 hemophilia treatment centers (HTCs), and their genome-wide genetic variants were compared with those from matched HIV-infected individuals. Homozygous carriers of known CCR5 resistance mutations were excluded. Single nucleotide polymorphisms (SNPs) and inferred copy number variants (CNVs) were tested using logistic regression. In addition, we performed a pathway enrichment analysis, a heritability analysis, and a search for epistatic interactions with CCR5 Δ32 heterozygosity. A total of 560 HIV-uninfected cases were recruited: 36 (6.4%) were homozygous for CCR5 Δ32 or m303. After quality control and SNP imputation, we tested 1 081 435 SNPs and 3686 CNVs for association with HIV-1 serostatus in 431 cases and 765 HIV-infected controls. No SNP or CNV reached genome-wide significance. The additional analyses did not reveal any strong genetic effect. Highly exposed, yet uninfected hemophiliacs form an ideal study group to investigate host resistance factors. Using a genome-wide approach, we did not detect any significant associations between SNPs and HIV-1 susceptibility, indicating that common genetic variants of major effect are unlikely to explain the observed resistance phenotype in this population.

17ß-estradiol protects females against influenza by recruiting neutrophils and increasing virus-specific CD8 T cell responses in the lungs.

UNLABELLED: 17ß-Estradiol (E2) treatment limits the pathology associated with pulmonary diseases caused by pathogens, allergens, and asthma, partly by reducing the production of proinflammatory cytokines and chemokines. To test the hypothesis that E2 protects against influenza A virus (IAV) infection by altering the recruitment and activity of innate immune cells and T cells, chemokine concentrations were measured and innate and adaptive immune cells were enumerated from the lungs of E2- and placebo-treated ovariectomized female C57BL/6 mice following infection. Females treated with E2 experienced less morbidity but had similar lung virus titers to placebo-treated females. Females treated with E2 had lower induction of CCL2 but higher CCL3 and CXCL1 responses in their lungs than placebo-treated females. Pulmonary recruitment of neutrophils, NK cells, macrophages, and dendritic cells was increased following infection, but only neutrophil numbers were greater in E2-treated than placebo-treated females. Neutrophils enhance the responses of influenza virus-specific CD8 T cells to promote virus clearance and improve the outcome of infection. Total numbers of virus-specific CD8 T cells were not altered by treatment with E2, but the proportion of gamma interferon (IFN-γ)- and tumor necrosis factor alpha (TNF-α)-producing, virus-specific CD8 T cells was increased. Neutrophil depletion in E2-treated females increased morbidity, reduced pulmonary production of chemoattractants for neutrophils, and reduced IFN-γ production by virus-specific CD8 T cells. Neutrophils mediate both inflammation and tissue repair during IAV infection and are regulated by E2 to improve the outcome of influenza in females. IMPORTANCE: Severe influenza is associated with excessive inflammation that leads to tissue damage. We demonstrate that estradiol (E2) is a potent anti-inflammatory hormone that reduces the severity of influenza A virus infection in females. Treatment of female C57BL/6 mice with E2 does not affect virus replication but rather alters the production of chemokines, pulmonary recruitment of neutrophils, and the cytokine responses of virus-specific CD8 T cells to protect females against severe influenza.

MAPKAP kinase 3 suppresses Ifng gene expression and attenuates NK cell cytotoxicity and Th1 CD4 T-cell development upon influenza A virus infection.

MK2 and MK3 are downstream targets of p38 and ERK1/2. They control the mRNA stability of several inflammatory cytokines, including TNF-α and IL-10. Whereas MK2 is expressed ubiquitously, the expression of MK3 is restricted to muscle, liver, and heart tissues and T and NK cells. Using Mk-deficient and wild-type (WT) mice, we demonstrated an inhibitory effect of MK3, but not of MK2, on interferon (IFN)-γ expression in T and NK lymphocytes. The results provided evidence that the inhibitory effect of MK3 is based on negative feedback phosphorylation of p38 and ERK1/2, which causes decreased binding of Stat4 to the IFN-γ promoter and reduced expression of IFN-γ mRNA and protein. Consequently, all Mk3(-/-) mice challenged with the Th1-inducing influenza A virus (IAV) survived the WT LD50 virus dose. The reduced disease severity in the Mk3(-/-) mice was accompanied by a >10-fold reduction in viral lung titer and an increase in the number of activated NK cells and enhanced Th1 activation of CD4 T cells. Thus, our data describe the protein kinase MK3 as a novel regulator of the innate and adaptive immune responses.-Köther, K., Nordhoff, C., Masemann, D., Varga, G., Bream, J. H., Gaestel, M., Wixler, V., Ludwig, S. MAPKAP kinase 3 suppresses Ifng gene expression and attenuates NK cell cytotoxicity and Th1 CD4 T-cell development upon influenza A virus infection.

Heterogeneity of CD4+ and CD8+ T-cell responses to cytomegalovirus in HIV-infected and HIV-uninfected men who have sex with men.

Studies of T-cell immunity to human cytomegalovirus (CMV) primarily reflect anti-CMV pp65 or immediate early antigen 1 (IE-1) activity. We assessed responses of T cells from human immunodeficiency virus (HIV)-negative and HIV-infected men to peptide pools spanning 19 CMV open reading frames selected because they previously correlated with total CMV-specific T-cell responses in healthy donors. Cells producing cytokines in response to pp65 or IE-1 together composed <12% and <40% of the total CD4(+) and CD8(+) T-cell responses to CMV, respectively. These proportions were generally similar regardless of HIV serostatus. Thus, analyses of total CMV-specific T-cell responses should extend beyond pp65 and IE-1 regardless of HIV serostatus.

Copy number variation of KIR genes influences HIV-1 control.

A genome-wide screen for large structural variants showed that a copy number variant (CNV) in the region encoding killer cell immunoglobulin-like receptors (KIR) associates with HIV-1 control as measured by plasma viral load at set point in individuals of European ancestry. This CNV encompasses the KIR3DL1-KIR3DS1 locus, encoding receptors that interact with specific HLA-Bw4 molecules to regulate the activation of lymphocyte subsets including natural killer (NK) cells. We quantified the number of copies of KIR3DS1 and KIR3DL1 in a large HIV-1 positive cohort, and showed that an increase in KIR3DS1 count associates with a lower viral set point if its putative ligand is present (p = 0.00028), as does an increase in KIR3DL1 count in the presence of KIR3DS1 and appropriate ligands for both receptors (p = 0.0015). We further provide functional data that demonstrate that NK cells from individuals with multiple copies of KIR3DL1, in the presence of KIR3DS1 and the appropriate ligands, inhibit HIV-1 replication more robustly, and associated with a significant expansion in the frequency of KIR3DS1+, but not KIR3DL1+, NK cells in their peripheral blood. Our results suggest that the relative amounts of these activating and inhibitory KIR play a role in regulating the peripheral expansion of highly antiviral KIR3DS1+ NK cells, which may determine differences in HIV-1 control following infection.