Levels of HIV-1 persistence on antiretroviral therapy are not associated with markers of inflammation or activation

Antiretroviral therapy (ART) reduces levels of HIV-1 and immune activation but both can persist despite clinically effective ART. The relationships among pre-ART and on-ART levels of HIV-1 and activation are incompletely understood, in part because prior studies have been small or cross-sectional. To address these limitations, we evaluated measures of HIV-1 persistence, inflammation, T cell activation and T cell cycling in a longitudinal cohort of 101 participants who initiated ART and had well-documented sustained suppression of plasma viremia for a median of 7 years. During the first 4 years following ART initiation, HIV-1 DNA declined by 15-fold (93%) whereas cell-associated HIV-1 RNA (CA-RNA) fell 525-fold (>99%). Thereafter, HIV-1 DNA levels continued to decline slowly (5% per year) with a half-life of 13 years. Participants who had higher HIV-1 DNA and CA-RNA before starting treatment had higher levels while on ART, despite suppression of plasma viremia for many years. Markers of inflammation and T cell activation were associated with plasma HIV-1 RNA levels before ART was initiated but there were no consistent associations between these markers and HIV-1 DNA or CA-RNA during long-term ART, suggesting that HIV-1 persistence is not driving or driven by inflammation or activation. Higher levels of inflammation, T cell activation and cycling before ART were associated with higher levels during ART, indicating that immunologic events that occurred well before ART initiation had long-lasting effects despite sustained virologic suppression. These findings should stimulate studies of viral and host factors that affect virologic, inflammatory and immunologic set points prior to ART initiation and should inform the design of strategies to reduce HIV-1 reservoirs and dampen immune activation that persists despite ART

T-cell responses targeting HIV Nef uniquely correlate with infected cell frequencies after long-term antiretroviral therapy

HIV-specific CD8+ T-cell responses limit viral replication in untreated infection. After the initiation of antiretroviral therapy (ART), these responses decay and the infected cell population that remains is commonly considered to be invisible to T-cells. We hypothesized that HIV antigen recognition may persist in ART-treated individuals due to low-level or episodic protein expression. We posited that if persistent recognition were occurring it would be preferentially directed against the early HIV gene products Nef, Tat, and Rev as compared to late gene products, such as Gag, Pol, and Env, which have higher barriers to expression. Using a primary cell model of latency, we observed that a Nef-specific CD8+ T-cell clone exhibited low-level recognition of infected cells prior to reactivation and robust recognition shortly thereafter. A Gag-specific CD8+ T-cell clone failed to recognized infected cells under these conditions, corresponding with a lack of detectable Gag expression. We measured HIV-specific T-cell responses in 96 individuals who had been suppressed on ART for a median of 7 years, and observed a significant, direct correlation between cell-associated HIV DNA levels and magnitudes of IFN-γ-producing Nef/Tat/Rev-specific T-cell responses. This correlation was confirmed in an independent cohort (n = 18). Correlations were not detected between measures of HIV persistence and T-cell responses to other HIV antigens. The correlation with Nef/Tat/Rev-specific T-cells was attributable to Nef-specific responses, the breadth of which also correlated with HIV DNA levels. These results suggest that ongoing Nef expression in ART-treated individuals drives preferential maintenance and/or expansion of T-cells reactive to this protein, implying sensing of infected cells by the immune system. The direct correlation, however, suggests that recognition does not result in efficient elimination of infected cells. These results raise the possibility that enhancing the cytolytic activity of Nef-specific T-cells may lead to reductions in infected cell frequencies, even in the absence of therapeutic latency reversal

Clonal expansions of CD8+ T cells with IL-10 secreting capacity occur during chronic Mycobacterium tuberculosis infection.

The exact role of CD8(+) T cells during Mycobacterium tuberculosis (Mtb) infection has been heavily debated, yet it is generally accepted that CD8(+) T cells contribute to protection against Mtb. In this study, however, we show that the Mtb-susceptible CBA/J mouse strain accumulates large numbers of CD8(+) T cells in the lung as infection progresses, and that these cells display a dysfunctional and immunosuppressive phenotype (PD-1(+), Tim-3(+), CD122(+)). CD8(+) T cell expansions from the lungs of Mtb-infected CBA/J mice were also capable of secreting the immunosuppressive cytokine interleukin-10 (IL-10), although in vivo CD8(+) T cell depletion did not significantly alter Mtb burden. Further analysis revealed that pulmonary CD8(+) T cells from Mtb-infected CBA/J mice were clonally expanded, preferentially expressing T cell receptor (TcR) Vβ chain 8 (8.2, 8.3) or Vβ 14. Although Vβ8(+) CD8(+) T cells were responsible for the majority of IL-10 production, in vivo depletion of Vβ8(+) did not significantly change the outcome of Mtb infection, which we hypothesize was a consequence of their dual IL-10/IFN-γ secreting profiles. Our data demonstrate that IL-10-secreting CD8(+) T cells can arise during chronic Mtb infection, although the significance of this T cell population in tuberculosis pathogenesis remains unclear

Surface phenotype of pulmonary T cells in CBA/J and C57BL/6 mice.

<p>C57BL/6 and CBA/J mice were infected with an aerosolized dose of <i>Mtb</i> and at various timepoints post-infection lungs were removed and processed for flow cytometry. Absolute numbers of IFN-γ⁺ CD4⁺ (<b>a</b>) or CD8⁺ (<b>b</b>) T cells after 4 hr <i>ex vivo</i> stimulation with anti-CD3/CD28/GolgiSTOP, with representative flow plots at day 150 post-infection. Absolute numbers of CD8⁺ T cells expressing CD69 (<b>c</b>), Tim3 (<b>d</b>), or PD-1 (<b>e</b>) after <i>Mtb</i> infection. (<b>f</b>) Absolute number of CD8⁺ T cells expressing both PD-1 and CD122. Data representative of at least two independent experiments with 4 mice per group per timepoint. * p<0.05, ** p<0.01, *** p<0.001 as obtained by Student's <i>t</i> test. (<b>c, d</b>) ⁺ p<0.05, ⁺⁺ p<0.01, ⁺⁺⁺ p<0.001 obtained by two-way analysis of variance comparing only CBA/J mice across all timepoints.</p

IL-10 production by CD8⁺ T cells from CBA/J mice.

<p>C57BL/6 and CBA/J mice were infected with an aerosolized dose of <i>Mtb</i> and at various times post-infection lungs were removed and cell populations were purified. Spot-forming units (SFU) representing the absolute number of IL-10⁺CD8⁺ T cells (<b>a</b>) or CD8^neg T cells (<b>b</b>) per lung after 72 hr culture with anti-CD3/CD28. (<b>c</b>) Supernatants from (a) were analyzed for IFN-γ levels by ELISA. (<b>d</b>) SFU per lung of CBA/J IL-10⁺ CD8⁺ or CD8^neg T cells cultured with <i>Mtb</i>-infected BMDCs for 72 hr. Data representative of two independent experiments with 4 mice per group per timepoint, * p<0.05, ** p<0.01, *** p<0.001 as obtained by Student's <i>t</i> test.</p

CD8⁺ T cell depletion in CBA/J mice.

<p>CBA/J mice were infected with an aerosolized dose of <i>Mtb</i> and from day 90–120 were treated weekly with depletion antibody via intraperitoneal injection then sacrificed at day 125 post-infection. (<b>a</b>) Lungs of anti-CD8⁺ T cell depleted or control mice were homogenized and plated on 7H11 plates and CFU enumerated after 21 days. Absolute number of total CD4⁺ T cells (<b>b</b>) or IFN-γ⁺CD4⁺ T cells (<b>c</b>) after CD8⁺ T cell depletion as determined by flow cytometry. (<b>d</b>) Levels of pulmonary IL-10 after CD8⁺ T cell depletion as determined by ELISA. (<b>e</b>) Lungs of anti-Vβ8 depleted or control mice were homogenized and plated onto 7H11 plates and CFU enumerated. Control group  =  no treatment and isotype control. Results representative of at least two independent experiments with 5–10 mice per group. Depletion resulted in 95% reduction in cell number, as determined by flow cytometry. * p<0.05, ** p<0.01, *** p<0.001 as obtained by Student's <i>t</i> test.</p

Vβ TcR expression in CBA/J and C57BL/6 mice.

<p>C57BL/6 and CBA/J mice were infected with an aerosol dose of <i>Mtb</i> and at various timepoints post-infection lungs were removed and processed for flow cytometry. Percentages of CD8⁺ (<b>a</b>) or CD4⁺ (<b>b</b>) T cells expressing specific Vβ TcRs at day 120 post-infection. Percentages of CD8⁺ T cells expressing Vβ8 (<b>c</b>) or Vβ14 (<b>d</b>) TcR over the course of <i>Mtb</i> infection. (<b>e</b>) Percentages of Vβ8⁺ or Vβ14⁺ CD69⁺CD8⁺ pulmonary T cells over time. (<b>f</b>) Vβ8⁺ and Vβ8^neg T cells from day 120 post-infection were cultured for 72 hr with anti-CD3/CD28 and IL-10 SFU determined by ELISpot. (<b>g</b>) Supernatants from (a) were analyzed for IFN-γ levels by ELISA. Data representative of at least two independent experiments with 4 mice per group per timepoint. * p<0.05, ** p<0.01, *** p<0.001 as obtained by Student's <i>t</i> test.</p

Blockade of the PD-1 axis alone is not sufficient to activate HIV-1 virion production from CD4+ T cells of individuals on suppressive ART.

Blockade of the programmed cell death protein/ligand 1 (PD-1/PD-L1) pathway with monoclonal antibodies (mAb) is now commonly used for cancer immunotherapy and has therapeutic potential in chronic viral infections including HIV-1. PD-1/PD-L1 blockade could augment HIV-1-specific immune responses and reverse HIV-1 latency, but the latter effect has not been clearly shown. We tested the ability of the human anti-PD-L1 mAb BMS-936559 and the human anti-PD-1 mAb nivolumab to increase HIV-1 virion production ex vivo from different peripheral blood mononuclear cell populations obtained from donors on suppressive antiretroviral therapy (ART). Fresh peripheral blood mononuclear cells (PBMC), CD8-depleted PBMC, total CD4+ T cells, and resting CD4+ T cells were purified from whole blood of HIV-1-infected donors and cultured in varying concentrations of BMS-936559 (20, 5, or 1.25μg/mL) or nivolumab (5 or 1.25μg/mL), with or without anti-CD3/CD28 stimulatory antibodies. Culture supernatants were assayed for virion HIV-1 RNA by qRT-PCR. Ex vivo exposure to BMS-936559 or nivolumab, with or without anti-CD3/CD28 stimulation, did not consistently increase HIV-1 virion production from blood mononuclear cell populations. Modest (2-fold) increases in virus production were observed in a subset of donors and in some cell types but were not reproducible in longitudinal samples. Cell surface expression of PD-1 and PD-L1 were not associated with changes in virus production. Ex vivo blockade of the PD-1 axis alone has limited effects on HIV-1 latency

Associations of HIV persistence, cigarette smoking, inflammation, and pulmonary dysfunction in people with HIV on antiretroviral therapy

We aimed to investigate the relationship between measures of HIV persistence with antiretroviral therapy (ART) and cigarette smoking, systemic markers of inflammation, and pulmonary function. Retrospective study of 82 people with HIV (PWH) on ART for a median of 6.9 years (5.6-7.8) and plasma HIV RNA levels &lt;50 copies/mL. HIV DNA and cell-associated HIV RNA (CA-RNA) were measured in peripheral blood mononuclear cells (PBMC) and plasma HIV RNA was measured by single-copy assay (SCA). Plasma levels of 17 inflammatory mediators were measured by Bio-Plex, and standard pulmonary function tests (PFT) were performed in all participants. Median age was 52 years and 41% were women. Most had preserved CD4+ T cell counts (median (IQR) 580 (361-895) cells/mm3). Median plasma HIV RNA was 1.3 (0.7-4.6) copies/mL, and median levels of HIV DNA and CA-RNA in PBMC were 346 (140-541) copies and 19 (3.7-49) copies per 1 million PBMC, respectively. HIV DNA was higher in smokers than in nonsmokers (R = 0.3, P &lt; 0.05), and smoking pack-years positively correlated with HIV DNA and CA-RNA (R = 0.3, P &lt; 0.05 and R = 0.4, P &lt; 0.01, respectively). HIV DNA, CA-RNA, and plasma HIV RNA were not significantly associated with any measure of pulmonary function or inflammation. Cigarette smoking was associated with HIV DNA and CA-RNA levels in blood, but measures of HIV persistence were not associated with pulmonary function or inflammation