Abstract Introduction Obstructive sleep apnea (OSA) has been associated with low grade systemic inflammation and greater cardiovascular risk, but the specific pathways mediating these effects are unclear. Monocyte activation is implicated in the development of cardiovascular disease in people with treated HIV infection. We sought to evaluate the impact of OSA on monocyte activation in people living with human immunodeficiency virus (HIV) infection. Methods Ten HIV seropositive participants with undetectable (<50 copies/mL) HIV viremia and at least one year of antiretroviral therapy underwent home sleep apnea testing to evaluate for the presence (apnea-hypopnea index -AHI4% ≥15 events/hour) or absence (AHI4% <5 events/hour) of OSA. Mononuclear cells were extracted and analyzed by flow cytometry. The composition of monocyte subsets (classical, intermediate, patrolling) was assessed using surface markers. Intracellular tumor necrosis factor alpha (TNFα), interleukin 1β (IL-1β), and interleukin 6 (IL-6) levels were measured by median fluorescence intensity (MFI, unit: au) and log-transformed. Differences in monocyte subset composition and intracellular cytokine levels between those with and without OSA were analyzed by t-test. Results Participants had a mean ± SD age of 61 ± 4.7 years, were 50% female, 80% Black race and had well-constituted CD4+ counts (mean ± SD: 934 ± 283 cells/ml). The mean AHI4% among participants with OSA (n=4) was 21.8 ± 4.4 events/hour whereas controls (n=6) had an AHI4% of 2.4 ± 2.0 events/hour (p-value = 0.001). There was no difference in the proportion of classical monocytes between groups (mean ± SD 83.8% ± 11.5% in participants with OSA vs. 84.9% ± 7.5% in controls, p=0.86). Intracellular TNFα was greater in monocytes from participants with OSA vs. controls (log-transformed MFI 3.49±0.20 au vs. 3.00±0.35 au, p=0.03). Additionally, there was a trend toward greater intracellular concentrations of IL-1β (mean log-transformed MFI 3.24±0.15 au vs. 2.95±0.48 au, p=0.22) and IL-6 (log-transformed MFI 3.28±0.16 au vs. 2.91±0.64 au, p=0.23), in participants with OSA vs. controls. Conclusion Pro-inflammatory cytokine levels are greater in monocytes obtained from people with treated HIV infection with OSA compared to those without OSA. Our findings suggest OSA may exacerbate chronic inflammation and cardiovascular risk in chronic HIV infection. Support (If Any) HL082610 and American Thoracic Society ASPIRE Fellowship
Myeloid-derived suppressor cells (MDSCs) were initially described more than 2 decades ago as bone marrow suppressor cells in mice with metastatic lung tumors [1]. In the past several years, there has been increasing interest in their role in cancer immune regulation [2,3]. MDSCs are a heterogeneous group of immature and progenitor myeloid cells that undergo expansion during pathologic conditions and are characterized by their strong immunosuppressive ability [4]. This is manifested as a dampening of cytotoxic reactivity of both natural and adaptive immunity, that is, natural killer (NK) and NKT cells, and CD4+ and CD8+ T cells, respectively. Unlike MDSCs in mice which can be identified using co-expression of Gr-1 and CD11b [5], a number of different markers have been used to identify this population in humans. In some cancer studies, they have been identified as lineage negative (Lin−) human leukocyte antigen DR expression negative (HLA-DR−) cells that express the common myeloid marker CD33, whereas other studies defined them as CD11b+CD14+CD15+ and HLA-DR− cells which express arginase-1 [6]. Recently Zhao et al.[7] described that specific members of the S100 protein family are expressed in MDSCs and that these may be useful markers in identifying this cell population. Apart from the phenotype, the mechanisms of suppression continue to be investigated. Some studies have attributed MDSC-suppressive ability to their expression of arginase-1 [8,9] and inducible nitric oxide synthetase (iNOS), whereas others point to the production of reactive oxygen species [10]. An important issue that is currently being studied is the interaction of MDSCs with other immune cells, particularly regulatory T cells (Tregs). MDSCs can induce the expansion or recruitment of Tregs, which can be an important mechanism for immune suppression [11–13]. In this issue of AIDS, Vollbrecht et al. describe for the first time, MDSCs, identified as the CD11b+CD14−CD33+CD15+ cell population in HIV-1 infection [14]. The cross-sectional study showed elevated frequencies of MDSCs in HIV-1-infected antiretroviral therapy (ART)-naive patients compared to healthy controls. There was a significant positive correlation, albeit modestly, to plasma HIV-1 viremia, and a modest negative correlation with CD4+ T-cell counts. The authors report a rapid drop in MDSC frequencies upon starting ART. The authors showed that isolated MDSCs inhibited the proliferation of Gag/Nef-stimulated CD8+ T cells. Moreover, the authors report a significant increase in Treg frequencies during co-incubation experiments with MDSCs, as well as a significant positive correlation with Tregs, defined as CD4+CD25+FOXP3+ T cells. The role of immunoregulatory cell populations in HIV-1 infection continues to be an important field of investigation. The results from Vollbrecht et al.'s study show a possible deleterious effect of MDSCs as they inhibit HIV-1-induced proliferation of CD8+ T cells. Yet, the immunosuppressive ability of MDSCs may be beneficial in curbing the damaging effects of persistent immune activation and consequent systemic inflammation associated with chronic HIV-1 infection. However, there are several important issues that should be considered in MDSC studies in HIV-1 infection. First, similar to studies on Tregs in HIV-1 infection, an important impediment is the lack of a specific marker(s) for MDSCs. Indeed, investigations of MDSCs in various cancers have used different combinations of markers [6]. Thus, contrasting results on MDSC frequency and function in HIV-1 infection could be affected by methodological differences in MDSC identification. Second, although there were significant modest correlations between peripheral MDSCs and plasma HIV-1 viremia and CD4+ T-cell counts in the studies of Vollbrecht et al., it will be important to evaluate MDSCs in tissues especially since it has been shown that the in-vivo suppression of MDSCs is limited to the inflammatory site [15]. Gut mucosal or lymph node specimens should be obtained to further evaluate the possible role MDSCs play in HIV-1 infection. Third, as in cancer studies, the specific mechanism of suppression that MDSCs use to inhibit HIV-1-specific immune responses, including their interaction with Tregs, should be investigated as this information will be helpful in designing HIV-1 immunotherapeutic strategies. In the same manner, these cells should be considered in the interpretation of immunotherapy studies. We have shown that the frequency of MDSCs, defined as Lin−HLA-DR−CD33+ cells, is markedly elevated in HIV-1-infected patients receiving a dendritic cell-based HIV-1 vaccine [16]. The lack of efficacy of some immune-based therapies and vaccines may be due to the expansion or the increased function of immunoregulatory cells [17], including MDSCs. This finding has also been recognized in the cancer vaccine field. In a phase I prophylactic trial using mucin 1 peptide antigen and polyinosinic-polycytidylic acid stabilized with polylysine and carboxymethylcellulose adjuvant in individuals at high risk for developing colorectal cancer, nonresponsiveness to the vaccine has been linked to an increase in the frequency of MDSCs [18]. With an increasing number of HIV-1 studies focusing on HIV-1-associated persistent immune activation and HIV-1 cure, the role of immunoregulatory cell populations become more important. As with Tregs, MDSCs may be important in decreasing levels of immune activation and systemic inflammation, but at the same time, may substantially inhibit the effects of different immunotherapeutic strategies. Also, since cytotoxic T-cell responses appear important in killing reactivated cells that harbor the latent HIV-1 provirus in HIV-1 cure studies [19], dampening the effects of MDSC suppression could be vital in purging the latent HIV-1 reservoir. This possibly is noteworthy especially since histone deacetylase inhibitors, which are used to activate HIV-1 reservoirs in these studies, have been shown in murine studies to expand MDSCs [20]. As such, it is important to further define the complicated immunoregulatory network at work in HIV-1 infection. Identification of specific pathways or interactions between these immunoregulatory cells may provide the vital key to designing highly effective HIV-1 immune-based strategies. Acknowledgements Conflicts of interest There are no conflicts of interest.
Ongoing HIV replication while receiving combination antiretroviral therapy (cART) may reduce survival. Viremia copy-years (VCY) has shown improved mortality risk prediction over single time-point viral load measures. However, the timing of a patient's viral load history most associated with later mortality has not been studied. Here we determined the optimal duration and temporality of viral load history for predicting mortality.Survival analysis among HIV-positive men who initiated cART in the Multicenter AIDS Cohort Study (1995-2015).VCY measures were derived from area-under-the-viral load-curve. The overall VCY based upon the complete post-cART viral load history was compared with 20 VCYs derived from viral loads assessed during different shorter time periods (the most recent 1-10 years and initial 1-10 years following cART initiation) for associations with mortality.Each 10-fold increase in VCYs based on the most recent 3-8 years was significantly associated with 23-26% decrease in survival times, a magnitude of effect greater than that of the most recent viral load (16%). These associations were independent of CD4 cell count and single time-point viral loads. In addition, the degree of pre-cART immunodeficiency did not affect the mortality prognostic value of VCY based on viral loads in the most recent 3 years. Conversely, the overall VCY and VCYs based on viral loads immediately following cART initiation were not independent predictors of mortality.Among cART-treated men, VCY based upon viral loads in the recent 3 years (six viral loads) has a mortality prognostic value greater than that of the overall VCY and single time-point viral loads, making the former a more feasible measure for use.
To the Editors: Reversing T-cell exhaustion using antibodies to immune checkpoint inhibitors (ICIs) has revolutionized cancer therapy. Because T-cell exhaustion, mediated by programmed death-1/programmed death ligand-1 (PD-1/PD-L1), may be a barrier to HIV cure1,2 and CD4+ T cells expressing PD-1 are enriched for latent HIV,2–5 treatment with anti-PD-1 antibodies may provide a strategy for targeting the latent HIV reservoir. Given that elimination of latently infected cells harboring replication-competent provirus will be necessary to cure HIV infection, we initiated a phase I/IIa, double-blind, placebo-controlled, dose-escalating safety and immunotherapeutic study of 2 infusions of an anti-PD-1 antibody (cemiplimab) in virally suppressed persons with HIV (PWH). Although participants were not anticipated to receive direct benefit from this study and ICIs are associated with potentially irreversible immune-related adverse events (irAEs), feedback from PWH and the scientific and HIV communities encouraged the team to pursue this HIV cure intervention. Moreover, preliminary data6,7 provided a reasonable expectation that cemiplimab would improve HIV-specific immune responses, reverse HIV latency, and, thus, advance the field. This risk versus benefit assessment8 led to the incorporation of strict measures to limit risk to participants (eg, history of autoimmune disease was exclusionary). Four of the 5 participants enrolled were randomized to receive 0.3 mg/kg of cemiplimab at weeks 0 and 6; one participant received placebo. Possible irAEs occurred in 2 participants: CASE 1 A 50-year-old man enrolled with baseline CD4+ T-cell count of 1.957 × 109/L and normal thyroid-stimulating hormone (TSH) and free thyroxine (free T4) levels. Four weeks after the first infusion of cemiplimab (0.3 mg/kg), TSH of 0.02 µg/mL and free T4 of 2.73 ng/dL were consistent with hyperthyroidism (Table 1). Mild fatigue was the only symptom reported. Repeat laboratory tests at week 5 and consultation with an endocrinologist confirmed thyroiditis (Table 1), assessed as probably related to cemiplimab. Both TSH and free T4 normalized by week 24 without medical intervention. Fatigue resolved, and no new symptoms were reported. TABLE 1. - Laboratory Values for Participants With Possible Immune-Related AEs After Receipt of 1 Dose of 0.3 mg/kg of Cemiplimab Case 1 Study Week Screen Preentry Entry Wk 4 Wk 5 Wk 6 Wk 12 Wk 16 Wk 24 Wk 36 Wk 38 Wk 48 Study day −70 −43 0 29 36 42 92 120 176 253 267 337 Thyroid laboratory test (reference range) TSH (0.27–4.2 mIU/L) 1.91 3.91 0.02 <0.01 0.01 3.97 2.87 1.55 6.67 1.55 3.5 Thyroxine (4.5–10.9 mcg/dL) 7.1 13.4 16.7 Free T4 (0.93–1.7 ng/dL) 2.73 4.1 3.06 0.81 0.86 1.01 1.20 1.12 1.02 Thyroglobulin antibody (0.0–4.0) 80.4 TSH receptor antibody (≤122) <0.09 Case 2 Study Week Screen Preentry Entry Wk 2 Wk 2.1 Wk 2.2 Wk 4 Wk 5 Wk 6 Wk 12 Wk 16 Wk 20 Wk 24 Wk 28 Wk 36 Wk 48 Study day −64 −48 0 14 17 21 28 35 42 84 116 140 168 204 253 337 Liver laboratory test (reference range) AST (0–40 IU/L) 30 23 53 261 (G3) 192 (G2) 149 (G2) 61 50 39 27 23 82 35 62 54 54 ALT (0–44 IU/L) 23 21 58 287 (G3) 272 (G2) 238 (G2) 93 (G1) 48 31 19 20 66 41 65 49 56 Total bilirubin (0.0–1.2 mg/dL) 0.8 0.3 0.3 0.4 1 0.8 0.5 0.6 0.4 0.2 0.4 0.3 0.4 0.4 0.5 0.7 Prothrombin time (10.2–12.8 s) 11.1 10.7 11 (Bold Type = Outside Reference Range)G, grade. CASE 2 A 57-year-old man with baseline CD4+ T-cell count of 0.911 × 109/L had normal aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels at screening. Just before the first infusion of cemiplimab (0.3 mg/kg), asymptomatic grade 1 elevations in AST and ALT levels were observed (Table 1). Routine safety assessment 2 weeks after the first infusion revealed asymptomatic grade 3 elevations in AST and ALT levels (Table 1). On further questioning, the participant reported acetaminophen (500 mg × 1) and alcohol use (6 beer and 2 whiskey drinks) the evening before the week 2 visit. Hepatology consultation revealed no autoimmune etiology or hepatic synthetic dysfunction but elicited chronic alcohol use. The pattern of the hepatic enzyme elevations and their slow resolution were deemed inconsistent with acute alcohol toxicity and, therefore, judged to be possibly related to cemiplimab. Elevated AST and ALT levels resolved 35 days postinfusion without intervention. Liver biopsy was not pursued, given the participant's asymptomatic course and gradual improvement without intervention. This significantly limited definitive assessment of causality due to drug-induced liver injury versus immune-related hepatitis versus the contribution of acute or chronic alcohol use. Per protocol-specified management of suspected irAEs, the second infusion at week 6 was held for both participants. A detailed, unblinded review of safety data from both cases by the independent Safety Monitoring Committee (SMC) was triggered and all study infusions held. Because of the probability of one irAE and the possibility of a second irAE, the SMC recommended halting accrual of additional study participants and holding further cemiplimab infusions. Of note, 2 participants who received 2 cemiplimab infusions before the occurrence of these events remained asymptomatic without laboratory abnormalities. All 4 cemiplimab-treated participants completed the study with no further irAEs or other safety events through 48 weeks after first cemiplimab infusion. The irAEs similar to these 2 cases are well described with other ICIs and frequently managed in cancer patients receiving this immunotherapy,9,10 although the resolution of the participant's thyroid abnormality in this study has not been commonly described. The irAEs can occur after a single infusion, although typically associated with higher doses, and as early as 14 days postinfusion. Given the lack of anticipated direct benefit to study participants and the frequency of possible/probable irAEs (2 of 4 participants) at the lowest dose of study drug, the study was closed to accrual. Of note, ICIs have shown an acceptable risk–benefit profile in PWH treated for cancer in previous studies.11 Whether well-suppressed HIV infection in otherwise healthy individuals without cancer contributed to risk of irAEs in this study remains unknown. The reduction or elimination of latent HIV reservoirs in PWH receiving suppressive antiretroviral therapy will likely require a combination of multiple therapeutic modalities including interventions that enhance HIV-1–specific immune responses to clear or contain these cells when activated to express replication-competent virus. Strategies to reverse HIV-specific immune exhaustion and target latently infected cells must be tested. These may require more targeted PD-1 blockade than that obtained with systemic administration of antibodies, coupled with a better understanding of risks for immune-mediated adverse events, to pursue studies of ICIs in otherwise healthy, virologically suppressed PWH. Our experience underscores the value of the multiple, carefully considered steps to minimize risk to study participants built into this study. These included engagement with representatives of the PWH community before, during, and after the study, highly restrictive entry criteria, active participation of physician investigators in the informed consent process, written assessment of understanding to document the adequacy of the informed consent process, frequent safety visits and phone contact with participants after each infusion, a 6-week observation period between infusions for safety assessment, and a detailed, predetermined toxicity management plan incorporating rapid review by our SMC in response to suspected irAEs. This study underscores the potential challenges of translating successful immunotherapeutic interventions from the high morbidity/mortality cancer field to otherwise healthy virologically suppressed PWH.
Abstract Ectonucleotidases CD39 and CD73 are expressed on the surface of regulatory T cells (Treg) and cleave ATP to immunosuppressive adenosine. CD39 could potentially serve as a new functional marker for Treg isolation. The frequency and absolute numbers of CD4+CD39+ T cells were determined in 32 HIV+ patients and in 10 healthy donors (HD). CD4+ T cells were negatively selected by magnetic beads PBMC. CD39+ Treg cells were then isolated by biotin-conjugated anti-CD39 Abs and anti-biotin magnetic beads. T cells were phenotyped by flow cytometry for CD39+, CD73+, FOXP3+, CCR4+, CD127neg, CD49dneg, and CD121a+. CFSE-based proliferation assays and ATP hydrolysis were used to measure Treg functions. Cytokine expression in CD4+CD39+ T cells was determined by Luminex. HIV-patients on and off anti-retroviral regimen had a significantly lower absolute number of CD39+CD25+ Treg and CD39+CD25neg T cells (p<0.05) than HD. The CD4+CD39+ T cell populations isolated on immunobeads contained two cell subsets: FOXP3+CD25+ Treg hydrolyzed exogenous ATP and suppressed CD4+ T cell proliferation, which was reversed by blocking ectonucleotidase activity. In contrast, the FOXP3negCD25neg T cells were CD127+CD49d+ and expressed IFNg, TNFa, and IL-10. The presence of two distinct CD4+CD39+ cell subsets in PBMC is a general phenomenon seen in normal donors, as well as patients with HIV and cancer. This suggests that phenotypic and functional plasticity characterizes the human Treg subset.
Twelve weeks of dipyridamole increased extracellular adenosine levels and decreased T cell activation in people with human immunodeficiency virus (HIV). In this analysis, we investigated the effect of dipyridamole on HIV-specific T cell responses. We compared changes in Gag- and Env-specific T cell responses using intracellular cytokine staining, following 12 wk of dipyridamole treatment vs placebo. We evaluated whether frequencies of polyfunctional HIV-specific T cells were associated with purines in the adenosine pathway and with measures of HIV persistence and chronic inflammation. There was a significant decrease in CD4+ polyfunctional T cell responses to Gag (-62.6% vs -23.0%; P < 0.001) and Env (-56.1% vs -6.0%; P < 0.001) in the dipyridamole arm. In the dipyridamole group, lower frequencies of polyfunctional Env-specific CD4+ T cells were associated with higher plasma levels of adenosine (r = -0.85, P < 0.01) and inosine (r = -0.70, P = 0.04). Higher adenosine levels induced by dipyridamole treatment is associated with decreased HIV-specific CD4+ T cell polyfunctional responses in people with HIV on antiretroviral therapy.
The relationship between lipid levels in plasma and inflammatory indices is complex and fatty meals alter plasma inflammatory markers in people with diabetes. There is interest in monitoring the effects of interventions on plasma inflammatory and coagulation elements in people with HIV, as they have been linked to risk for morbid outcomes and HIV persistence. Understanding the effects of feeding and time of specimen acquisition is important for the correct scheduling of clinical sampling.We examined the effects of feeding on plasma inflammatory, coagulation and homeostatic indices among 24 non-diabetic people with HIV, with controlled viraemia and on antiretroviral therapy after fasting and then 1, 3 and 6 hours after ingesting a fatty meal, and also approximately 1 week later after fasting and after an isocaloric non-fatty meal. Plasma levels of IL-6, IL-7, IP-10, sCD14, sCD163, sTNFrII and D-dimer were monitored by immunoassay.Fasting levels of all markers obtained approximately 1 week apart were significantly correlated (P<0.001). Mild alterations in plasma concentrations of inflammatory markers were observed after feeding but geometric means varied more than 10% from baseline for only IL-6 and IL-7. Meal type was differentially associated with changes in plasma levels for IL-7 only. Antiretroviral treatment regimen, body mass index and changes in plasma triglyceride levels were not linked to post-feeding changes in these biomarkers.These plasma inflammatory, coagulation and homeostatic indices are relatively stable at fasting and are only minimally affected by feeding or time of day. These findings will aid in the monitoring of inflammatory and homeostatic indices that may contribute to control of HIV expression and its persistence.
Tenofovir disoproxil fumarate (TDF) can cause kidney damage, but current clinical tests are insensitive for detecting toxicity. Among 884 HIV-infected men enrolled in the Multicenter AIDS Cohort Study, we measured urine biomarkers specific for tubular damage (interleukin-18, kidney injury molecule-1, procollagen type III N-terminal propeptide) and albuminuria. In adjusted analyses, each year of TDF exposure was independently associated with 3.3% higher interleukin-18 (95% CI: 0.8% to 5.8%), 3.4% higher kidney injury molecule-1 (1.1% to 5.7%), and 3.1% higher procollagen type III N-terminal propeptide (0.8% to 5.5%), but not with albuminuria (2.8%; -0.6% to 6.2%). Biomarkers of tubular damage may be more sensitive than albuminuria for detecting toxicity from TDF and other medications.
People living with HIV (PLWH) experience increased vulnerability to premature aging and inflammation-associated comorbidities, even when HIV replication is suppressed by antiretroviral therapy (ART). However, the factors associated with this vulnerability remain uncertain. In the general population, alterations in the N-glycans on IgGs trigger inflammation and precede the onset of aging-associated diseases. Here, we investigate the IgG N-glycans in cross-sectional and longitudinal samples from 1214 women and men, living with and without HIV. PLWH exhibit an accelerated accumulation of pro-aging-associated glycan alterations and heightened expression of senescence-associated glycan-degrading enzymes compared to controls. These alterations correlate with elevated markers of inflammation and the severity of comorbidities, potentially preceding the development of such comorbidities. Mechanistically, HIV-specific antibodies glycoengineered with these alterations exhibit a reduced ability to elicit anti-HIV Fc-mediated immune activities. These findings hold potential for the development of biomarkers and tools to identify and prevent premature aging and comorbidities in PLWH.
Abstract Antiretroviral therapies (ART) durably suppress HIV replication to undetectable levels – however, infection persists in the form of long-lived reservoirs of infected cells with integrated proviruses, that re-seed systemic replication if ART is interrupted. A central tenet of our current understanding of this persistence is that infected cells are shielded from immune recognition and elimination through a lack of antigen expression from proviruses. Efforts to cure HIV infection have therefore focused on reactivating latent proviruses to enable immune-mediated clearance, but these have yet to succeed in driving reductions in viral reservoirs. Here, we revisited the question of whether HIV reservoirs are predominately immunologically silent from a new angle, by querying the dynamics of HIV-specific T-cell responses over long-term ART for evidence of ongoing recognition of HIV-infected cells. We show that T-cell responses to autologous reservoir viruses persist over years, and that the maintenance of HIV-Nef-specific responses was uniquely associated with residual frequencies of infected cells. These responses disproportionately exhibited a cytotoxic, effector functional profile, indicative of recent in vivo recognition of HIV-infected cells. These results indicate substantial visibility of the HIV reservoir to T-cells on stable ART, presenting both opportunities and challenges for the development of therapeutic approaches to curing HIV infection.
