Immune activation and exhaustion marker expression on T-cell subsets in ART-treated adolescents and young adults with perinatal HIV-1 infection as correlates of viral persistence.

HIV-1 infection in memory CD4+ T cells forms a latent reservoir that is a barrier to cure. Identification of immune biomarkers that correlate with HIV-1 reservoir size may aid with evaluating efficacy of HIV-1 eradication strategies, towards ART-free remission and cure. In adults living with non-perinatal HIV-1, the immune exhaustion marker PD-1 on central memory CD4+ T cells (Tcm) correlates with measures of HIV-1 reservoir size. Immune correlates of HIV-1 are less defined in adolescents and young adults with perinatal HIV-1. With multi-parameter flow cytometry, we examined immune activation (CD69, CD25, HLA-DR), and exhaustion (PD-1, TIGIT, TIM-3 and LAG-3) markers on CD4+ T cell subsets (naïve (Tn), central memory (Tcm), and the combination (Ttem) of transitional (Ttm) and effector memory (Tem) cells, in 10 adolescents and young adults living with perinatal HIV-1 (median age 15.9 years; median duration of virologic suppression 7.0 years), in whom HIV-1 reservoir size was measured with the Intact Proviral HIV-1 DNA Assay (IPDA) and an enhanced Tat/Rev limiting dilution assay (TILDA). RNA-seq was also performed on the unstimulated CD4+ T cells. The median total HIV-1 DNA concentration in memory CD4+ T cells was 211.90 copies per million CD4+ T cells. In the 7 participants with subtype B HIV-1 infection, the median intact proviral DNA load was 7.96 copies per million CD4+ T cells. Levels of HLA-DR and TIGIT on the Ttem were correlated with total HIV-1 DNA (r=0.76, p=0.015) and (r=0.72, p=0.023), respectively, but not with intact proviral load or induced reservoir size. HIV-1 DNA load was also positively correlated with transcriptional clusters associated with HLA-DR expression by RNA-seq. In contrast, PD-1 expression on Tcm was inversely correlated with total HIV-1 DNA (r=-0.67, p=0.039). Reservoir size by IPDA and TILDA were correlated (r=0.81, p=0.036). Thus, in this cohort of youths with long-standing treated perinatal infection, HLA-DR and TIGIT on Ttem were the key correlates of HIV-1 infected cell frequencies by total HIV-1 DNA, and not PD-1. Total HIV-1 DNA was negatively correlated with PD-1 expressing Tcm. These differences in longstanding perinatal HIV-1 infection compared with adult infection requires further study in larger cohorts.

Interaction of Depot Medroxyprogesterone Acetate and Tenofovir Disoproxil Fumarate/Emtricitabine on Peripheral Blood Mononuclear Cells and Cervical Tissue Susceptibility to HIV Infection and Pharmacokinetics.

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 HIV BaL ), 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.

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.

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.

Differences in inducibility of the latent HIV reservoir in perinatal and adult infection.

The HIV latent reservoir in resting memory CD4+ T cells precludes cure. Therapeutics to reactivate and eliminate this reservoir are in clinical trials in adults, but not yet in pediatric populations. We determined, ex vivo, the inducibility of the latent reservoir in perinatal infection as compared with adult infections using the Tat/rev induced limiting dilution assay (TILDA), in which a single round (12 hours) of CD4+ T cell stimulation with PMA/ionomycin maximally activates T cells and leads to proviral expression with multiply spliced HIV RNA production. Markers of immune activation and exhaustion were measured to assess interactions with inducibility. Although rates of T cell activation with PMA/ionomycin were similar, the latent reservoir in perinatal infection was slower to reactivate and of lower magnitude compared with adult infection, independent of proviral load. An enhanced TILDA with the addition of phytohemagglutin and a duration of 18 hours augmented proviral expression in perinatal but not adult infection. The baseline HLA-DR+CD4+ T cell level was significantly lower in perinatal compared with adult infections, but not correlated with induced reservoir size. These data support the hypothesis that there are differences in kinetics of latency reversal and baseline immune activation in perinatal compared with adult infections, with implications for latency reversal strategies toward reservoir clearance and remission.

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.

The effect of cellular isolation and cryopreservation on the expression of markers identifying subsets of regulatory T cells.

BACKGROUND: The role of CD4(+) regulatory T cells (Tregs) and their subsets during HIV infection is controversial. Cryopreserved peripheral blood mononuclear cells (PBMC) are an important source for assessing number and function of Tregs. However, it is unknown if PBMC isolation and cryopreservation affect the expression of CD120b and CD39, markers that identify specific subsets of Tregs. METHODS: HIV-uninfected (HIV-) and -infected (HIV+) men were randomly selected from the Multicenter AIDS Cohort Study (MACS). Percentages of CD120b(+) and CD39(+) Tregs measured by flow cytometry in whole blood and in corresponding fresh and cryopreserved PBMC were compared. RESULTS: Percentages of CD120b(+) Tregs were significantly lower in a) fresh PBMC relative to whole blood, and b) freshly thawed frozen PBMC relative to fresh PBMC when the recovery of viable cryopreserved cells was low. When present, low expression of CD120b in frozen PBMC was reversible by 4h of in vitro culture. In contrast, expression of CD39 on Tregs was not affected by isolation and/or cryopreservation of PBMC, or by relative recovery of cryopreserved PBMC. These findings were unaffected by the HIV status of the donor. CONCLUSION: The data suggest that percentages of CD120b(+) Tregs and CD39(+) Tregs can be validly measured in either whole blood or PBMC (fresh and frozen) in HIV- and HIV+ men. However, for measurement of CD120b(+) Tregs one type of sample should be used consistently within a given study, and thawed frozen cells may require in vitro culture if recovery of viable cells is low.

Regulatory T Cells, Frailty, and Immune Activation in Men Who Have Sex With Men in the Multicenter AIDS Cohort Study.

BACKGROUND: Both HIV infection and frailty have been associated with chronic immune activation. One possible explanation for this chronic immune activation could be low levels of CD4(+) T regulatory cells (Tregs), which suppress immune responses. METHODS: HIV-uninfected (HIV-) and HIV-infected (HIV+) men in the Multicenter AIDS Cohort Study (MACS) were classified as frail (or nonfrail) if they expressed (or did not express) the Fried frailty phenotype at two consecutive study visits. Percentages and absolute numbers of total Tregs, and percentages of different subsets of Tregs and of activated T cells were measured by flow cytometry. The function of Tregs was measured by suppression of T-cell proliferation. RESULTS: Percentages of Tregs were higher, rather than lower, in frail men than in nonfrail men, and this difference was significant for HIV- men. Percentages of subsets of Tregs did not differ significantly by frailty status. Among HIV+ men, the suppressive function of Tregs was similar between frail and nonfrail men. Percentages of Tregs and activated T cells were negatively correlated in nonfrail men (HIV- and HIV+) and in frail HIV- men, but this correlation was strongly positive in frail HIV+ men. CONCLUSION: These data suggest that: (a) Tregs were not deficient in frail men; and (b) the immunological pathophysiology of frailty may differ by HIV status.

Late-emerging strains of HIV induce T-cell homeostasis failure by promoting bystander cell death and immune exhaustion in naïve CD4 and all CD8 T-cells.

The mechanisms involved in the decline of CD4 and CD8 T-cells that lead to HIV-induced immune dysregulation are not clearly understood. We hypothesize that late-emerging strains of HIV, such as CXCR4-tropic (X4) virions, induce T-cell homeostasis failure by promoting significantly more bystander cell death, and immune exhaustion in naïve CD4 and all CD8 T-cells, when compared to strain of HIV, such as CCR5-tropic (R5) virions, found early during the course of infection. In the reported study, inactivated X4 virions induced greater bystander cell death in sort-purified naïve CD4 T-cells compared to R5 virions, which was significant (p=0.013), and in memory CD8 T-cells, though the latter was not significant. A clearer understanding of the mechanisms involved in HIV-induced depletion of T-cell numbers and function could lead to therapies that prevent T-cell death and restore immune function. These therapies could improve current anti-retroviral and cure-related treatments by boosting the immune system's own ability to combat the virus.

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.

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.

Relationship between cytomegalovirus (CMV) IgG serology, detectable CMV DNA in peripheral monocytes, and CMV pp65(495-503)-specific CD8+ T cells in older adults.

In immunocompetent individuals, cytomegalovirus (CMV) is thought to persist in a latent state in monocytes and myeloid progenitor cells, establishing a lifelong infection. In CMV-seropositive older adults, aging has been associated with both expansion of CMV pp65(495-503)-specific CD8(+) T cell clones and shrinkage of the T cell repertoire that characterize T cell immunosenescence. In fact it has been suggested that chronic CMV infection is a driving force in age-related T cell immunosenescence. In older adults, chronic CMV infection is conventionally diagnosed by positive IgG serology which does not distinguish between past and persistent infections. To better define the relationship between chronic CMV infection and expansion of CMV pp65(495-503)-specific CD8(+) T cells, we directly assessed CMV viral DNA in monocyte-enriched peripheral blood mononuclear cells in 16 HLA-A2-positive elderly volunteers (mean age = 83 years). While all participants had positive CMV IgG serology by enzyme-linked immunosorbent assays, only nine (56%) had detectable CMV DNA by nested polymerase chain reaction. These nine individuals had significantly higher percentages of CMV pp65(495-503) tetramer-positive CD8(+) T cells (median = 1.3%) than those without detectable CMV DNA (median = 0.1%; p < 0.001). Absolute CMV IgG antibody titers did not differ between these two groups (median = 54.6 vs 44.2 EU/ml, respectively, p = 0.4). CMV IgM titers were negative for all 16 participants, suggesting that recent primary CMV infection was unlikely. These results demonstrate a strong association between the presence of CMV DNA in peripheral monocytes and the expansion of CD8(+) T cells specific for the CMV immunodominant epitope pp65(495-503). Although the sample size in this study is relatively small, these findings provide initial evidence suggesting the heterogeneity of CMV IgG-seropositive older adult population and CMV viral DNA detection in peripheral monocytes as an informative tool to better understand the relationship between chronic CMV infection and T cell immunosenescence.

Effect of nutritional supplements on immune function and body weight in malnourished adults.

In the United States, approximately 5% of the population is malnourished or has low body weight, which can adversely affect immune function. Malnutrition is more prevalent in older adults and is often a result of energy imbalance from various causes. Dietary supplementation to promote positive energy balance can reverse malnutrition, but has not been assessed for its effect on immune parameters. This 8-week clinical feeding trial evaluated the effect of a commercially available, high-protein, high-energy formula on body weight and immune parameters in 30 adult volunteers with body-mass indices (BMI) <21 kg/m(2). After the intervention, participants gained a mean of 3.74 lbs and increased BMI by 0.58 kg/m(2). The intervention improved lean body mass and limited body fat accumulation. However, no clinically significant improvements in immune measures were observed. These results support the use of high-protein, high-energy supplements in the treatment of underweight/malnutrition. Further investigation utilizing feeding studies of longer duration, and/or studying severely malnourished individuals may be needed to detect an effect on immune parameters of weight gain promoted by nutritional supplements.

Nrf2 regulates an adaptive response protecting against oxidative damage following diquat-mediated formation of superoxide anion.

Mouse embryonic fibroblasts derived from Nrf2-/- mice (N0) and Nrf2+/+ mice (WT) have been used to characterize both basal and diquat (DQ)-induced oxidative stress levels and to examine Nrf2 activation during exposure to DQ-generated superoxide anion. Microarray analysis revealed that N0 cells have similar constitutive mRNA expression of genes responsible for the direct metabolism of reactive oxygen species but decreased expression of genes responsible for the production of reducing equivalents, repair of oxidized proteins and defense against lipid peroxidation, compared to WT cells. Nonetheless, the basal levels of ROS flux and oxidative damage biomarkers in WT and N0 cells were not different. Diquat dibromide (DQ), a non-electrophilic redox cycling bipyridylium herbicide, was used to generate intracellular superoxide anion. Isolated mitochondria from both cell lines exposed to DQ produced equivalent amounts of ROS, indicating a similar cellular capacity to generate ROS. However, N0 cells exposed to DQ for 24-h exhibited markedly decreased cell viability and aconitase activity as well as increased lipid peroxidation and glutathione oxidation, relative to WT cells. 2',7'-Dichlorofluorescein fluorescence was not increased in WT and N0 cells after 30-min of DQ exposure. However, increased levels of ROS were detected in N0 cells but not WT cells after 13-h of DQ treatment. Additionally, total glutathione concentrations increased in WT, but not N0 cells following a 24-h exposure to DQ. DQ exposure resulted in activation of an antioxidant response element-luciferase reporter gene, as well as induction of Nrf2-regulated genes in WT, but not N0 cells. Thus the enhanced sensitivity of N0 cells does not reflect basal differences in antioxidative capacity, but rather an impaired ability to mount an adaptive response to sustained oxidative stress.

Evaluation of a low-cost method, the Guava EasyCD4 assay, to enumerate CD4-positive lymphocyte counts in HIV-infected patients in the United States and Uganda.

OBJECTIVE: To evaluate the EasyCD4 assay, a less expensive method to enumerate CD4+ lymphocytes, in resource-limited settings. DESIGN: Cross-sectional study conducted in the United States and Uganda. METHODS: We compared CD4+ cell counts obtained on replicate samples from HIV-infected patients by the EasyCD4 assay, a microcapillary flow-based system, and by standard flow cytometry or FACSCount with linear regression and the Bland-Altman method. RESULTS: Two hundred eighteen samples were analyzed (77 in the United States and 141 in Uganda). In the United States, mean +/- SD CD4 was 697 +/- 438 cells/microL by standard flow cytometry and 688 +/- 451 cells/microL by EasyCD4. In Uganda, the mean +/- SD CD4 was 335 +/- 331 cells/microL by FACSCount and 340 +/- 327 cells/microL by EasyCD4. The 2 methods were highly correlated (US cohort, r2 = 0.97, slope = 1.0, intercept = -18; Ugandan cohort, r2 = 0.92; slope = 0.95; intercept = 23). The mean differences in CD4 cell counts were 9.0 and -4.6 cells/microL for the US and Ugandan cohorts, respectively, and they were not significant in either cohort. In the Ugandan cohort, sensitivity and specificity of the EasyCD4 for CD4 below 200 cells/microL were 90% and 98%, respectively. Positive predictive value was 96%; negative predictive value was 93%. CONCLUSIONS: Our results suggest that EasyCD4 may be used with high positive and negative predictive value in resource-limited settings.