A man living with HIV was found to lack expression of CD16A on his natural killer (NK) cells and monocytes. Genetic analysis revealed compound heterozygous deletion of FCGR3A, the gene encoding CD16A. The case’s NK cells showed: (a) no antibody-dependent cell-mediated cytotoxicity and very low spontaneous cytotoxicity; (b) an immature phenotype marked by high expression of CD94, CD2, NKG2A, and NKG2D, and low expression of KIR2DL2 and CD57; (c) no expression of KIR3DL1 and very low expression of FcRγ; and (d) normal cytokine production. The case’s monocytes and DCs were similar phenotypically and functionally to those from the donors matched for HIV status, age, and percentage of NK cells in the peripheral blood. In contrast to previously reported people with CD16A deficiency, this man did not have a history of severe infections with herpes viruses, suggesting that other immune cells and/or immunoregulatory function of NK cells may compensate for deficiency of cytolytic NK cells.

Introduction: Highly active antiretroviral therapy (HAART) helps improve some measures of accelerated epigenetic aging in persons living with HIV (PLWH), but its overall impact on the epigenome is not fully understood.Methods: In this study, we analyzed the DNA methylation profiles of PLWH (n = 187) shortly before and approximately 2–3 years after they started HAART, as well as matched seronegative (SN) controls (n = 187), taken at two time intervals. Our aim was to identify specific CpGs and biologic pathways associated with HIV infection and initiation of HAART. Additionally, we attempted to identify epigenetic changes associated with HAART initiation that were independent of HIV-associated changes, using matched HIV seronegative (SN) controls (matched on age, hepatitis C status, and interval between visits) to identify CpGs that did not differ between PLWH and SN pre-HAART but were significantly associated with HAART initiation while being unrelated to HIV viral load. Epigenome-wide association studies (EWAS) on >850,000 CpG sites were performed using pre- and post-HAART samples from PLWH. The results were then annotated using the Genomic Regions Enrichment of Annotations Tool (GREAT).Results: When only pre- and post-HAART visits in PLWH were compared, gene ontologies related to immune function and diseases related to immune function were significant, though with less significance for PLWH with detectable HIV viral loads (>50 copies/mL) at the post-HAART visit. To specifically elucidate the effects of HAART separately from HIV-induced methylation changes, we performed EWAS of HAART while also controlling for HIV viral load, and found gene ontologies associated with transplant rejection, transplant-related diseases, and other immunologic signatures. Additionally, we performed a more focused analysis that examined CpGs reaching genome-wide significance (p < 1 × 10−7) from the viral load-controlled EWAS that did not differ between all PLWH and matched SN controls pre-HAART. These CpGs were found to be near genes that play a role in retroviral drug metabolism, diffuse large B cell lymphoma proliferation, and gastric cancer metastasis.Discussion: Overall, this study provides insight into potential biological functions associated with DNA methylation changes induced by HAART initiation in persons living with HIV.

IntroductionPersons living with HIV (PLWH) experience the early onset of age-related illnesses, even in the setting of successful human immunodeficiency virus (HIV) suppression with highly active antiretroviral therapy (HAART). HIV infection is associated with accelerated epigenetic aging as measured using DNA methylation (DNAm)-based estimates of biological age and of telomere length (TL).MethodsDNAm levels (Infinium MethylationEPIC BeadChip) from peripheral blood mononuclear cells from 200 PLWH and 199 HIV-seronegative (SN) participants matched on chronologic age, hepatitis C virus, and time intervals were used to calculate epigenetic age acceleration, expressed as age-adjusted acceleration residuals from 4 epigenetic clocks [Horvath’s pan-tissue age acceleration residual (AAR), extrinsic epigenetic age acceleration (EEAA), phenotypic epigenetic age acceleration (PEAA), and grim epigenetic age acceleration (GEAA)] plus age-adjusted DNAm-based TL (aaDNAmTL). Epigenetic age acceleration was compared for PLWH and SN participants at two visits: up to 1.5 years prior and 2–3 years after HAART (or equivalent visits). Flow cytometry was performed in PLWH and SN participants at both visits to evaluate T-cell subsets.ResultsEpigenetic age acceleration in PLWH decreased after the initiation of HAART but remained greater post-HAART than that in age-matched SN participants, with differences in medians of 6.6, 9.1, and 7.7 years for AAR, EEAA, and PEAA, respectively, and 0.39 units of aaDNAmTL shortening (all p < 0.001). Cumulative HIV viral load after HAART initiation was associated with some epigenetic acceleration (EEAA, PEAA, and aaDNAmTL), but even PLWH with undetectable HIV post-HAART showed persistent epigenetic age acceleration compared to SN participants (p < 0.001). AAR, EEAA, and aaDNAmTL showed significant associations with total, naïve, and senescent CD8 T-cell counts; the total CD4 T-cell counts were associated with AAR, EEAA, and PEAA (p = 0.04 to <0.001). In an epigenome-wide analysis using weighted gene co-methylation network analyses, 11 modules demonstrated significant DNAm differences pre- to post-HAART initiation. Of these, nine were previously identified as significantly different from pre- to post-HIV infection but in the opposite direction.DiscussionIn this large longitudinal study, we demonstrated that, although the magnitude of the difference decreases with HAART is associated with the cumulative viral load, PLWH are persistently epigenetically older than age-matched SN participants even after the successful initiation of HAART, and these changes are associated with changes in T-cell subsets.

ABSTRACT 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.

Supplementary Tables 1 and 2 from B-Cell Stimulatory Cytokines and Markers of Immune Activation Are Elevated Several Years Prior to the Diagnosis of Systemic AIDS–Associated Non-Hodgkin B-Cell Lymphoma

Risk of prostate cancer excluding cases diagnosed within 2 and 5 years comparing highest quartilea of cytokine concentration with lowest quartile_a of concentration, CLUE II.

PDF file - 106K, Spearman's correlation coefficients calculated for each time-window, separately in cases and controls; Association Between CXCL13 and CXCR5 tagSNPs and CXCL13 Serum Levels; Association between CXCL13 serum levels and HIV-associated non-Hodgkin lymphoma risk at three time-windows, stratified by SNP genotypes.

PDF - 123K, The risk of developing AIDS-NHL is elevated for those individuals who have higher levels of inflammatory biomarkers prior to AIDS-NHL diagnosis, excluding those who received cART.

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.

PDF - 122K, Geometrical mean serum values and confidence intervals of IL-5, IL-6, IL-8, IL-10, IL-17F, IL-20, IL-27, IFN-γ, IP-10, LIF, neopterin, sCD40L, TNFα, VEGF, κ and λ for HIV+ individuals who later developed AIDS-NHL and for HIV+ controls (p-values calculated with Student t-tests). Fraction of detectable values (shaded area) of IL-1β, IL-2, IL-4, IL-12, IL-17A, IL-23, IL-31, GM-CSF, and SDF-1α for HIV+ individuals who later developed AIDS-NHL and for HIV+ controls (p-values calculated using a chi-squared test).

The pathophysiology of erythroderma is heterogeneous, with many cases of unknown etiology and variable treatment response. We thus sought to create an immunophenotyping platform able to identify differential biomarkers in various subtypes of erythroderma to guide the selection of targeted therapeutics. We report an index case of a 68-year-old male with erythroderma who failed multiple systemic therapies. We utilized single cell flow cytometry analysis to identify unique immunopathogenic signatures in the peripheral blood of the index patient as compared to other erythrodermic patients with pityriasis rubra pilaris (PRP) and Sézary syndrome (SS), and healthy controls. The index patient's unique flow cytometry findings from peripheral blood mononuclear cells (PBMCs) included a greater than 10-fold increase in interleukin (IL)-13 (p=0.0009) and IL-17 (p=0.002) producing γVδ2T-cells. Flow cytometry of whole blood revealed 2 to 3-fold increases in basophils (p=0.009) and activated platelets (p=0.017). Single-cell peripheral blood immunophenotyping also identified unique pathogenic features of both PRP and SS patients, differentiating between erythroderma subtypes. The cutaneous immune profile of the index patient matched with peripheral blood findings, as whole genome sequencing and immunofluorescence staining of the index patient's skin biopsies demonstrated mutations in IL-13, IL-4, and IL-17 signaling pathways and increased expression of IL-4R (p=0.043), IL-13 (p=0.004), and IL-17R (p=0.014), respectively. Subsequent targeted intervention in the index patient via dual secukinumab and dupilumab resulted in complete disease control and reduction of immunopathogenic cell populations, including γVδ2T-cells, basophils, and activated platelets. This study highlights a minimally invasive peripheral blood-based immunophenotyping platform that may provide a novel personalized medicine approach to the management of inflammatory skin diseases.

Background: Depot medroxyprogesterone acetate (DMPA) is a widely used contraceptive method. HIV pre-exposure prophylaxis with emtricitabine and tenofovir disoproxil fumarate (F/TDF) is highly effective in reducing HIV acquisition in women. We sought to determine the impact of DMPA on F/TDF pharmacokinetics and pharmacodynamics. Methods: Twelve healthy premenopausal cisgender women were enrolled and each completed 4 sequential conditions: (1) baseline, (2) steady-state F/TDF alone, (3) steady-state F/TDF + DMPA, and (4) DMPA alone. Assessments included clinical, pharmacokinetic, viral infectivity (ex vivo challenge of peripheral blood mononuclear cells by X4- and R5-tropic green fluorescent protein pseudoviruses and cervical tissue by HIVBaL), endocrine, immune cell phenotyping, and renal function. Results: Compared with baseline, F/TDF (± DMPA) significantly decreased both %R5- and X4-infected CD4 T cells and F/TDF + DMPA decreased cervical explant p24 (all P < 0.05). The %R5- and X4-infected CD4 T cells were higher during DMPA alone than during F/TDF periods and lower than baseline (not statistically significant). Cervical explant p24 fell between baseline and F/TDF values (not statistically significant). There were neither statistically significant differences in F/TDF pharmacokinetics, including total or renal clearance of either antiviral drug, nor changes in glomerular filtration rate with the addition of DMPA. There were few immune cell phenotypic differences across conditions. Conclusions: F/TDF decreased HIV infection in both challenge assays, whereas DMPA alone did not enhance HIV infection in either challenge assay. DMPA did not alter F/TDF pharmacokinetics or renal function.

[This corrects the article DOI: 10.1016/j.isci.2022.104488.].

Post-vaccination cytokine levels from 256 young adults who subsequently suffered breakthrough influenza infections were compared with matched controls. Modulation within the immune system is important for eliciting a protective response, and the optimal response differs according to vaccine formulation and delivery. For both inactivated influenza vaccine (IIV) and live attenuated influenza vaccines (LAIV) lower levels of IL-8 were observed in post-vaccination sera. Post-vaccination antibody levels were higher and IFN-γ levels were lower in IIV sera compared to LAIV sera. Subjects who suffered breakthrough infections after IIV vaccination had higher levels of sCD25 compared to the control group. There were differences in LAIV post-vaccination interleukin levels for subjects who subsequently suffered breakthrough infections, but these differences were masked in subjects who received concomitant vaccines. Wide variances, sex-based differences and confounders such as concomitant vaccines thwart the establishment of specific cytokine responses as a correlate of protection, but our results provide real world evidence that the status of the immune system following vaccination is important for successful vaccination and subsequent protection against disease.

Background: Epigenetic aging is accelerated in tissues of persons living with HIV (PLWH) and may underlie the early onset of age-related illnesses. This study examines the rate-of-change in epigenetic age in PLWH following HIV infection but before HAART, using archived longitudinal samples from the Multicenter AIDS Cohort Study.Methods: DNA was isolated from cryopreserved peripheral blood mononuclear cells from 101 men living with HIV, with baseline visit <2.5 years after HIV seroconversion (Visit 1) and follow-up visit <1.5 years before the initiation of HAART (Visit 2), and 100 HIV-uninfected men matched on age and visits with comparable time intervals. DNA methylation (DNAm) age was estimated for five clocks (Pan-tissue, Extrinsic, Phenotypic, Grim, and Skin & Blood age), and a DNAm-based estimate of telomere length (DNAmTL). Multivariate linear regression models were used to examine baseline factors associated with rate-of-aging, defined as (DNAm age visit 2–DNAm age visit 1)/(age visit 2–age visit 1).Results: Epigenetic age increased approximately twice as fast in PLWH as uninfected controls (Pan-tissue, Extrinsic, and Phenotypic clocks). Shortening of DNAmTL was nearly 3-fold faster in PLWH than controls. Faster rate-of-aging was associated with HIV status (Pan-Tissue, Extrinsic, Phenotypic, and DNAmTL), white race (Extrinsic, DNAmTL), higher cumulative HIV viral load (Grim), and lower baseline DNAm age (Phenotypic, Skin & Blood).Conclusion: Epigenetic rates-of-aging were significantly faster for untreated PLWH. Our findings expand on the important impact of HIV infection on biologic aging, both in elevating epigenetic age and increasing the rate-of-aging in the years following infection.

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. 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. 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. 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.

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.

It is now recognized that to fully understand the role of host genetic variation on susceptibility to HIV-1 infection, investigations must be extended to African populations. We sought to determine if genetic variation in IL10 are associated with HIV-1 infection in a West African cohort in Mali. HIV-infected and -uninfected individuals were genotyped for three common single nucleotide polymorphisms (SNPs) located at positions -592 (C/A), -819 (C/T), and -1082 (G/A) of the IL10 promoter. We found that the ATA haplotype, which has been previously associated with low IL-10 expression, was the most represented in the cohort. Although we observed a trend toward an increased frequency of ATA/ATA carriage in HIV-infected compared with -uninfected individuals, the difference was not statistically significant. Similarly, individual IL10 SNPs were not significantly enriched in the HIV-infected group, suggesting that IL10 genetic variants are not associated with HIV-1 in this West African cohort from Mali.

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.

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.