Cytomegalovirus Infection Facilitates the Costimulation of CD57+CD28- CD8 T Cells in HIV Infection and Atherosclerosis via the CD2-LFA-3 Axis.

CD8 T cells are emerging as important mediators in atherosclerosis and cardiovascular disease (CVD). Immune activation may play a particular role in people with HIV (PWH) who are at an increased risk of CVD, even after controlling for known CVD risk factors. Latent CMV infection is associated with increased CVD risk for both PWH and people without HIV, and human CMV-specific CD4 and CD8 T cells are enriched for an immunosenescent phenotype. We previously showed that CMV coinfection in PWH promotes vascular homing and activation of inflammatory CD4 T cells through the CD2-LFA-3 axis. However, the role of CD2/LFA3 costimulation of CD8 T cells in PWH with CMV has yet to be described. In the present study, we demonstrate that CD2 expression on CX3CR1+CD57+CD28- inflammescent CD8 T cells is increased on cells from CMV-seropositive PWH. In vitro CD2/LFA-3 costimulation enhances TCR-mediated activation of these inflammatory CD8 memory T cells. Finally, we show that LFA-3 is highly expressed in aortas of SIV-infected rhesus macaques and in atherosclerotic plaques of people without HIV. Our findings are consistent with a model in which CMV infection enhances CD2 expression on highly proinflammatory CD8 T cells that can then be stimulated by LFA-3 expressed in the vasculature, even in the absence of CD28 costimulation. This model, in which CMV infection exacerbates toxic cytokine and granzyme production by CD8 T cells within the vasculature, highlights a potential therapeutic target in atherosclerosis development and progression, especially for PWH.

CD8+ T-Cell-Derived Tumor Necrosis Factor Can Induce Tissue Factor Expression on Monocytes.

Circulating CD8+ T cells and monocytes are activated during human immunodeficiency virus (HIV) infection and colocalize in the aortas of simian immunodeficiency virus-infected nonhuman primates. We hypothesized that CD8+ T cells could exert a proatherosclerotic effect via paracrine actions on monocytes. We found that T-cell receptor-stimulated CD8+ T cells induce monocytes to express tissue factor, a potent activator of coagulation. Tumor necrosis factor was both necessary and sufficient for this effect.

Altered Maturation Status and Possible Immune Exhaustion of CD8 T Lymphocytes in the Peripheral Blood of Patients Presenting With Acute Coronary Syndromes.

OBJECTIVE: Inflammation in response to oxidized lipoproteins is thought to play a key role in acute coronary syndromes (ACS), but the pattern of immune activation has not been fully characterized. We sought to perform detailed phenotypic and functional analysis of CD8 T lymphocytes from patients presenting with ACS to determine activation patterns and potential immunologic correlates of ACS. APPROACH AND RESULTS: We used polychromatic flow cytometry to analyze the cytokine production profiles of naïve, effector, and memory CD8 T cells in patients with ACS compared with control subjects with stable coronary artery disease. ACS was associated with an altered distribution of circulating CD8(+) T-cell maturation subsets with reduced proportions of naïve cells and expansion of effector memory cells. ACS was also accompanied by impaired interleukin-2 production by phenotypically naïve CD8 T cells. These results were validated in a second replication cohort. Naïve CD8 cells from patients with ACS also had increased expression of programmed cell death-1, which correlated with interleukin-2 hypoproduction. In vitro, stimulation of CD8 T cells with oxidized low-density lipoprotein was sufficient to cause programmed cell death-1 upregulation and diminished interleukin-2 production by naïve CD8 T cells. CONCLUSIONS: In this exploratory analysis, naïve CD8(+) T cells from patients with ACS show phenotypic and functional characteristics of immune exhaustion: impaired interleukin-2 production and programmed cell death-1 upregulation. Exposure to oxidized low-density lipoprotein recapitulates these features in vitro. These data provide evidence that oxidized low-density lipoprotein could play a role in immune exhaustion, and this immunophenotype may be a biomarker for ACS.

Platelet and Monocyte Activation After Transcatheter Aortic Valve Replacement (POTENT-TAVR): A Mechanistic Randomized Trial of Ticagrelor Versus Clopidogrel.

Background: Inflammation and thrombosis are often linked mechanistically and are associated with adverse events after transcatheter aortic valve replacement (TAVR). High residual platelet reactivity (HRPR) is especially common when clopidogrel is used in this setting, but its relevance to immune activation is unknown. We sought to determine whether residual activity at the purinergic receptor P2Y12 (P2Y12) promotes prothrombotic immune activation in the setting of TAVR. Methods: This was a randomized trial of 60 patients (enrolled July 2015 through December 2018) assigned to clopidogrel (300mg load, 75mg daily) or ticagrelor (180mg load, 90 mg twice daily) before and for 30 days following TAVR. Co-primary endpoints were P2Y12-dependent platelet activity (Platelet Reactivity Units; VerifyNow) and the proportion of inflammatory (cluster of differentiation [CD] 14+/CD16+) monocytes 1 day after TAVR. Results: Compared to clopidogrel, those randomized to ticagrelor had greater platelet inhibition (median Platelet Reactivity Unit [interquartile range]: (234 [170.0-282.3] vs. 128.5 [86.5-156.5], p < 0.001), but similar inflammatory monocyte proportions (22.2% [18.0%-30.2%] vs. 25.1% [22.1%-31.0%], p = 0.201) 1 day after TAVR. Circulating monocyte-platelet aggregates, soluble CD14 levels, interleukin 6 and 8 levels, and D-dimers were also similar across treatment groups. HRPR was observed in 63% of the clopidogrel arm and was associated with higher inflammatory monocyte proportions. Major bleeding events, pacemaker placement, and mortality did not differ by treatment assignment. Conclusions: Residual P2Y12 activity after TAVR is common in those treated with clopidogrel but ticagrelor does not significantly alter biomarkers of prothrombotic immune activation. HRPR appears to be an indicator (not a cause) of innate immune activation in this setting.

Sex modifies the association between HIV and coronary artery disease among older adults in Uganda.

INTRODUCTION: Little is known about the epidemiology of coronary artery disease (CAD) in sub-Saharan Africa, where the majority of people living with HIV (PLHIV) live. We assessed the association of HIV with CAD and explored relationships with monocyte activation in sex-stratified analyses of older PLHIV and people without HIV (PWOH) in Uganda. METHODS: The Ugandan Study of HIV effects on the Myocardium and Atherosclerosis (mUTIMA) follows 100 PLHIV on antiretroviral therapy (ART) and 100 age- and sex-matched PWOH controls in Kampala, Uganda; all >45 years of age with >1 cardiovascular disease risk factor. At the year 2 exam (2017-2019), 189 participants had available coronary calcium score and 165 had coronary CT angiography (CCTA) for this analysis. A subset of participants (n = 107) had both CCTA and fresh whole blood flow cytometry for monocyte phenotyping. RESULTS: Median age was 57.8 years and 63% were females. Overall, 88% had hypertension, 37% had diabetes and 4% were smokers. Atherosclerotic cardiovascular disease (ASCVD) risk was modestly higher for PWOH, but not statistically significant (median 10-year ASCVD risk 7.2% for PLHIV vs. 8.6% for PWOH, p = 0.09). Median duration of ART was 12.7 years and 86% had suppressed viral load. Despite a high prevalence of risk factors, only 34/165 (21%, 95% CI 15-28%) had any coronary plaque. After adjustment for ASCVD risk score, HIV status was not associated with CAD (OR 0.55, 95% CI 0.23-1.30) but was associated with more severe CAD (segment severity score>3) among those with disease (OR 10.9, 95% CI 1.67-70.45). Females had a trend towards higher odds of CAD among PLHIV (OR 4.1, 95% CI 0.4-44.9), but a trend towards lower odds of CAD among PWOH (OR 0.30; 95% CI 0.07-1.3; HIV*sex interaction p = 0.019). CAD was positively correlated with classical monocytes (r = 0.3, p = 0.012) and negatively correlated with CX3CR1 expression (r = -0.31, p = 0.011) in PLHIV and negatively correlated with patrolling monocytes (r = -0.36, p = 0.031) and tissue factor expression (r = -0.39, p = 0.017) in PWOH. CONCLUSIONS: Our results suggest that HIV may be associated more with severity rather than the presence of CAD in Uganda. Sex differences in the HIV effect suggest that tailored CAD prevention strategies may be required in this setting.

Monocyte activation in persons living with HIV and tuberculosis coinfection.

OBJECTIVES: To characterize monocyte subsets and activation in persons living with HIV (PLWH) with tuberculosis coinfection. DESIGN: Cross-sectional study within a cohort of PLWH and HIV-uninfected participants at the Joint Clinical Research Centre in Kampala, Uganda. METHODS: Participants were at least 45 years old with at least one cardiovascular risk factor. PLWH had an HIV viral load 1000 copies/ml or less on stable antiretroviral therapy prior to cohort entry. QuantiFERON-TB testing was performed to define latent tuberculosis infection (LTBI). Prior active TB was defined by self-report and verified by medical records. Blood was stained with monocyte subset markers (CD14+, CD16), CD62p, CD69, CX3CR1, HLA-DR, and tissue factor, and examined with flow cytometry. RESULTS: One hundred and twenty-five participants (83 PLWH and 42 without HIV) were included. Median CD4+ count was 582 cells/µl in PLWH. PLWH had a higher frequency of total monocytes (4.3% vs. 3.2%; P < 0.001) and inflammatory monocyte subset (15.5% vs. 11.7%; P = 0.016) compared with HIV-uninfected individuals. No differences in the frequency of monocyte subsets were observed by TB status. Among PLWH, prior active TB was associated with increased frequency of total monocytes compared with LTBI (5.1% vs. 3.7%; P = 0.013). HLA-DR density on monocytes was three-fold higher in PLWH with LTBI or prior TB compared with PLWH without LTBI (P = 0.002). In multivariate analysis, a higher monocyte HLA-DR density remained associated with LTBI or prior TB in PLWH (log-MFI; b = 1.17; P < 0.001). CONCLUSION: Our findings indicate enhanced monocyte activation in PLWH with LTBI or prior active TB, which may contribute to the pathogenesis of noncommunicable diseases in HIV.

Altered Monocyte Phenotype in HIV-1 Infection Tends to Normalize with Integrase-Inhibitor-Based Antiretroviral Therapy.

BACKGROUND: Monocytes are increasingly implicated in the inflammatory consequences of HIV-1 disease, yet their phenotype following antiretroviral therapy (ART) initiation is incompletely defined. Here, we define more completely monocyte phenotype both prior to ART initiation and during 48 weeks of ART. METHODS: Cryopreserved peripheral blood mononuclear cells (PBMCs) were obtained at baseline (prior to ART initiation) and at weeks 12, 24, and 48 of treatment from 29 patients participating in ACTG clinical trial A5248, an open label study of raltegravir/emtricitibine/tenofovir administration. For comparison, cryopreserved PBMCs were obtained from 15 HIV-1 uninfected donors, each of whom had at least two cardiovascular risk factors. Thawed samples were stained for monocyte subset markers (CD14 and CD16), HLA-DR, CCR2, CX3CR1, CD86, CD83, CD40, CD38, CD36, CD13, and CD163 and examined using flow cytometry. RESULTS: In untreated HIV-1 infection there were perturbations in monocyte subset phenotypes, chiefly a higher frequency and density (mean fluorescence intensity-MFI) of HLA-DR (%-p = 0.004, MFI-p = .0005) and CD86 (%-p = 0.012, MFI-p = 0.005) expression and lower frequency of CCR2 (p = 0.0002) expression on all monocytes, lower CCR2 density on inflammatory monocytes (p = 0.045) when compared to the expression and density of these markers in controls' monocytes. We also report lower expression of CX3CR1 (p = 0.014) on patrolling monocytes at baseline, compared to levels seen in controls. After ART, these perturbations tended to improve, with decreasing expression and density of HLA-DR and CD86, increasing CCR2 density on inflammatory monocytes, and increasing expression and density of CX3CR1 on patrolling monocytes. CONCLUSIONS: In HIV-1 infected patients, ART appears to attenuate the high levels of activation (HLA-DR, CD86) and to increase expression of the chemokine receptors CCR2 and CX3CR1 on monocyte populations. Circulating monocyte phenotypes are altered in untreated infection and tend to normalize with ART; the role of these cells in the inflammatory environment of HIV-1 infection warrants further study.

Rosuvastatin reduces vascular inflammation and T-cell and monocyte activation in HIV-infected subjects on antiretroviral therapy.

BACKGROUND: Despite suppressive antiretroviral therapy (ART), increased levels of immune activation persist in HIV-infected subjects. Statins have anti-inflammatory effects and may reduce immune activation in HIV disease. METHODS: Stopping Atherosclerosis and Treating Unhealthy bone with RosuvastatiN in HIV (SATURN-HIV) is a randomized, double-blind placebo-controlled trial assessing the effect of rosuvastatin (10 mg daily) on markers of cardiovascular risk and immune activation in ART-treated patients. T-cell activation was measured by expression of CD38, HLA-DR, and PD1. Monocyte activation was measured with soluble markers (sCD14 and sCD163) and by enumeration of monocyte subpopulations and tissue factor expression. Markers of systemic and vascular inflammation and coagulation were also measured. SATURN-HIV is registered on clinicaltrials.gov (identifier: NCT01218802). RESULTS: Rosuvastatin, compared with placebo, reduced sCD14 (-10.4% vs 0.5%, P = 0.006), lipoprotein-associated phospholipase A2 (-12.2% vs -1.7%, P = 0.0007), and IP-10 (-27.5 vs -8.2%, P = 0.03) levels after 48 weeks. The proportion of tissue factor-positive patrolling (CD14CD16) monocytes was also reduced by rosuvastatin (-41.6%) compared with placebo (-18.8%, P = 0.005). There was also a greater decrease in the proportions of activated (CD38HLA-DR) T cells between the arms (-38.1% vs -17.8%, P = 0.009 for CD4 cells, and -44.8% vs -27.4%, P = 0.003 for CD8 cells). CONCLUSIONS: Forty-eight weeks of rosuvastatin treatment reduced significantly several markers of inflammation and lymphocyte and monocyte activation in ART-treated subjects.

Oxidized LDL Levels Are Increased in HIV Infection and May Drive Monocyte Activation.

BACKGROUND: HIV infection is associated with increased cardiovascular risk, and this risk correlates with markers of monocyte activation. We have shown that HIV is associated with a prothrombotic monocyte phenotype, which can be partially mitigated by statin therapy. We therefore explored the relationship between oxidized low-density lipoprotein (oxLDL) particles and monocyte activation. METHODS: We performed phenotypic analysis of monocytes using flow cytometry on fresh whole blood in 54 patients with HIV and 24 controls without HIV. Plasma levels of oxLDL, soluble CD14, IL-6, and soluble CD163 were measured by enzyme-linked immunosorbent assay. In vitro experiments were performed using flow cytometry. RESULTS: Plasma levels of oxLDL were significantly increased in HIV infection compared with controls (60.1 units vs. 32.1 units, P < 0.001). Monocyte expression of the oxLDL receptors, CD36 and Toll-like receptor 4, was also increased in HIV. OxLDL levels correlated with markers of monocyte activation, including soluble CD14, tissue factor expression on inflammatory monocytes, and CD36. In vitro stimulation with oxLDL, but not to low-density lipoprotein, resulted in expansion of inflammatory monocytes and increased monocyte expression of tissue factor, recapitulating the monocyte profile we find in HIV disease. CONCLUSIONS: OxLDL may contribute to monocyte activation, and further study in the context of HIV disease is warranted.