Background: Chronic HIV is associated with CD4+ and CD8+ T-cells bearing higher frequency of negative checkpoint receptors (NCRs). CD4+ T-cells expressing NCRs contribute to HIV persistence during antiretroviral therapy (ART). We have previously reported that HIV-infected patients had lower mitochondrial Complex I activity compared with HIV-negative controls. We report associations between NCR expression in T-cells and mitochondrial oxidative phosphorylation (OXPHOS) in peripheral blood mononuclear cells (PBMCs) among chronically HIV-infected patients on ART.
Methods: The Hawaii Aging with HIV cohort enrolled patients with documented HIV infection, age≥40 years old, and on stable ART ≥3 months. Multiparametric flow cytometry was performed on cryopreserved PBMCs to quantitate the percentages of CD4+ and CD8+ T-cells expressing exhaustion markers (PD-1/TIM-3/TIGIT). Spearman''s correlations were used to identify cross-sectional associations between NCR-expressing T-cells and previously assessed mitochondrial Complex I (NADH dehydrogenase) and IV (cytochrome c oxidase) activities in PBMCs.
Results: Of 43 HIV+ patients, median age was 51 years, current CD4 count 518.0 cells/uL, and nadir CD4 count 93.5 cells/uL. Majority (88.4%) were male and 83.7% had undetectable plasma HIV RNA< 50 copies/ml. Four patients (9%) were on zidovudine. Higher CD4 count was associated with higher Complex I (rho=0.33, p=0.01) and Complex IV activities (rho=0.43, p=0.005). Higher NCRs in T-cells correlated with lower Complex I and IV activities (Table). In multivariate linear regression analyses adjusted for age, zidovudine use, and undetectable HIV RNA, Complex I activity was significantly associated with TIGIT+CD4+ (β=-0.35, p=0.04), TIGIT+PD1+CD4+ (β=-0.35, p=0.04), and TIGIT+TIM3+CD4+ T-cells (β=-0.41, p=0.02). Complex IV activity was associated with TIGIT+CD4+ (β=-0.38, p=0.02), TIGIT+PD1+CD4+ (β=-0.41,p=0.02), TIGIT+TIM3+CD4+ (β=-0.49, p=0.003), and TIM3+PD1+CD4+ T-cells (β=-0.37,p=0.04).
Conclusions: OXPHOS activities were decreased in HIV-infected individuals on ART and correlated with disease severity, as assessed by CD4 count. Higher NCRs on CD4 T-cells were associated with decreased OXPHOS activities in PBMCs. Further studies on the relationship between NCR-expressing CD4 T-cells and immunometabolism are warranted to understand their role in HIV persistence.

Negative checkpoint receptors (%)Complex I (optical density (OD)/µg of protein×103)Complex IV (OD/µg of protein×103)
TIM3+CD8+ T-cells-0.20 (p=0.21)-0.34 (p=0.03)
TIGIT+TIM3+CD8+ T-cells-0.16 (p=0.31)-0.32 (p=0.04)
TIGIT+CD4+ T-cells-0.33 (p=0.03)-0.35 (p=0.02)
TIGIT+PD1+CD4+ T-cells-0.35 (p=0.03)-0.36 (p=0.02)
TIM3+PD1+CD4+ T-cells-0.31 (p=0.05)-0.29 (p=0.06)
TIGIT+TIM3+CD4+ T-cells-0.40 (p=0.009)-0.42 (p=0.006)
[Spearman correlation between negative checkpoint receptors in T-cells and mitochondrial oxidative phosphorylation activity in PBMCs]

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