Gabriela Khoury

Both CD31+ and CD31- Naive CD4+ T Cells Are Persistent HIV Type 1-Infected Reservoirs in Individuals Receiving Antiretroviral Therapy

BACKGROUND Naive T cell recovery is critical for successful immune reconstitution after antiretroviral therapy (ART), but the relative contribution of CD31(+) and CD31⁻ naive T cells to immune reconstitution and viral persistence is unknown. METHODS In a cross-sectional (n = 94) and longitudinal (n = 10) study of human immunodeficiency virus (HIV)-infected patients before and after ART, we examined the ratio of CD31(+) to CD31⁻ naive CD4(+) T cells. In the longitudinal cohort we then quantified the concentration of HIV-1 DNA in each cell subset and performed single-genome amplification of virus from memory and naive T cells. RESULTS Patients receiving ART had a higher proportion of CD31(+) CD4(+) T cells than HIV-1-infected individuals naive to ART and uninfected control subjects (P < .001 and .007, respectively). After 24 months of ART, the proportion of CD31(+) naive CD4(+) T cells did not change, the concentration of HIV-1 DNA in memory CD4(+) T cells significantly decreased over time (P < .001), and there was no change in the concentration of HIV-1 DNA in CD31(+) or CD31⁻ naive CD4(+) T cells (P = .751 and .251, respectively). Single-genome amplification showed no evidence of virus compartmentalization in memory and naive T cell subsets before or after ART. CONCLUSIONS After ART, both CD31(+) and CD31⁻ naive CD4(+) T cells expand, and both subsets represent a stable, persistent reservoir of HIV-1.

Expression and reactivation of HIV in a chemokine induced model of HIV latency in primary resting CD4+ T cells

BackgroundWe recently described that HIV latent infection can be established in vitro following incubation of resting CD4+ T-cells with chemokines that bind to CCR7. The main aim of this study was to fully define the post-integration blocks to virus replication in this model of CCL19-induced HIV latency.ResultsHigh levels of integrated HIV DNA but low production of reverse transcriptase (RT) was found in CCL19-treated CD4+ T-cells infected with either wild type (WT) NL4.3 or single round envelope deleted NL4.3 pseudotyped virus (NL4.3- Δenv). Supernatants from CCL19-treated cells infected with either WT NL4.3 or NL4.3- Δenv did not induce luciferase expression in TZM-bl cells, and there was no expression of intracellular p24. Following infection of CCL19-treated CD4+ T-cells with NL4.3 with enhanced green fluorescent protein (EGFP) inserted into the nef open reading frame (NL4.3- Δnef-EGFP), there was no EGFP expression detected. These data are consistent with non-productive latent infection of CCL19-treated infected CD4+ T-cells. Treatment of cells with phytohemagluttinin (PHA)/IL-2 or CCL19, prior to infection with WT NL4.3, resulted in a mean fold change in unspliced (US) RNA at day 4 compared to day 0 of 21.2 and 1.1 respectively (p = 0.01; n = 5), and the mean expression of multiply spliced (MS) RNA was 56,000, and 5,000 copies/million cells respectively (p = 0.01; n = 5). In CCL19-treated infected CD4+ T-cells, MS-RNA was detected in the nucleus and not in the cytoplasm; in contrast to PHA/IL-2 activated infected cells where MS RNA was detected in both. Virus could be recovered from CCL19-treated infected CD4+ T-cells following mitogen stimulation (with PHA and phorbyl myristate acetate (PMA)) as well as TNFα, IL-7, prostratin and vorinostat.ConclusionsIn this model of CCL19-induced HIV latency, we demonstrate HIV integration without spontaneous production of infectious virus, detection of MS RNA in the nucleus only, and the induction of virus production with multiple activating stimuli. These data are consistent with ex vivo findings from latently infected CD4+ T-cells from patients on combination antiretroviral therapy, and therefore provide further support of this model as an excellent in vitro model of HIV latency.

CD4+ T Cells Expressing PD-1, TIGIT and LAG-3 Contribute to HIV Persistence during ART

HIV persists in a small pool of latently infected cells despite antiretroviral therapy (ART). Identifying cellular markers expressed at the surface of these cells may lead to novel therapeutic strategies to reduce the size of the HIV reservoir. We hypothesized that CD4+ T cells expressing immune checkpoint molecules would be enriched in HIV-infected cells in individuals receiving suppressive ART. Expression levels of 7 immune checkpoint molecules (PD-1, CTLA-4, LAG-3, TIGIT, TIM-3, CD160 and 2B4) as well as 4 markers of HIV persistence (integrated and total HIV DNA, 2-LTR circles and cell-associated unspliced HIV RNA) were measured in PBMCs from 48 virally suppressed individuals. Using negative binomial regression models, we identified PD-1, TIGIT and LAG-3 as immune checkpoint molecules positively associated with the frequency of CD4+ T cells harboring integrated HIV DNA. The frequency of CD4+ T cells co-expressing PD-1, TIGIT and LAG-3 independently predicted the frequency of cells harboring integrated HIV DNA. Quantification of HIV genomes in highly purified cell subsets from blood further revealed that expressions of PD-1, TIGIT and LAG-3 were associated with HIV-infected cells in distinct memory CD4+ T cell subsets. CD4+ T cells co-expressing the three markers were highly enriched for integrated viral genomes (median of 8.2 fold compared to total CD4+ T cells). Importantly, most cells carrying inducible HIV genomes expressed at least one of these markers (median contribution of cells expressing LAG-3, PD-1 or TIGIT to the inducible reservoir = 76%). Our data provide evidence that CD4+ T cells expressing PD-1, TIGIT and LAG-3 alone or in combination are enriched for persistent HIV during ART and suggest that immune checkpoint blockers directed against these receptors may represent valuable tools to target latently infected cells in virally suppressed individuals.

Persistent immune activation in chronic HIV infection: Do any interventions work?

The availability of combination antiretroviral therapy (cART) has led to substantial reduction in morbidity and mortality in HIV-infected patients; however, life expectancy remains reduced especially in HIV-infected patients who initiate cART with CD4 T-cell counts less than 200 cells/μl [1]. Increased immune activation in patients on long-term suppressive cART [2–4] has been associated with increased mortality [5,6] and both AIDS and non-AIDS-defining illnesses [7–10], suggesting that chronic immune activation may have a potential role in driving increased morbidity and mortality.

The role of naïve T-cells in HIV-1 pathogenesis: An emerging key player

Functional naïve T-cells are critical for an effective immune response to multiple pathogens. HIV leads to a significant reduction in CD4+ naïve T-cell number and impaired function and there is incomplete recovery following combination antiretroviral therapy (cART). Here we review the basic homeostatic mechanisms that maintain naïve CD4+ T-cells and discuss recent developments in understanding the impact of HIV infection on naïve CD4+ T-cells. Finally we review therapeutic interventions in HIV-infected individuals aimed at specifically enhancing recovery of naïve CD4+ T-cells.

Persistence of integrated HIV DNA in CXCR3 + CCR6 + memory CD4+ T cells in HIV-infected individuals on antiretroviral therapy.

Background: HIV latent infection can be established in vitro by treating resting CD4+ T cells with chemokines that bind to chemokine receptors (CKR), CCR7, CXCR3, and CCR6, highly expressed on T cells. Objective: To determine if CKR identify CD4+ T cells enriched for HIV in HIV-infected individuals receiving suppressive antiretroviral therapy (ART). Design: A cross-sectional study of CKR expression and HIV persistence in blood from HIV-infected individuals on suppressive ART for more than 3 years (n = 48). A subset of 20 individuals underwent leukapheresis and sorting of specific CD4+ T-cell subsets. Methods: We used flow cytometry to quantify CCR5, CCR6, CXCR3, and CXCR5 expression on CD4+ T cells. HIV persistence was quantified using real-time Polymerase Chain Reaction to detect total, integrated HIV DNA, 2-long terminal repeat circles and cell-associated unspliced (CA-US) HIV RNA in total CD4+ T cells from blood or sorted T-cell subsets. Associations between CKR and HIV persistence in CD4+ T cells in blood were determined using regression models and adjusted for current and nadir CD4+ T-cell counts. Results: The frequency of cells harbouring integrated HIV DNA was inversely associated with current CD4+ T-cell count and positively associated with CCR5+ CD4+ T cells, CXCR3+CCR6+ and CXCR3+CCR6– expression on total memory CD4+ T cells (P < 0.001, 0.048, 0.015, and 0.016, respectively). CXCR3+CCR6+ CM CD4+ T cells contained the highest amount of integrated HIV DNA and lowest ratio of CA-US HIV RNA to DNA compared to all T-cell subsets examined. Conclusion: CXCR3 and CCR6 coexpression defines a subset of CD4+ T cells that are preferentially enriched for HIV DNA in HIV-infected individuals on ART.

Targeting antigen to bone marrow stromal cell-2 expressed by conventional and plasmacytoid dendritic cells elicits efficient antigen presentation

Bone marrow stromal cell‐2 (BST‐2) has major roles in viral tethering and modulation of interferon production. Here we investigate BST‐2 as a receptor for the delivery of antigen to dendritic cells (DCs). We show that BST‐2 is expressed by a panel of mouse and human DC subsets, particularly under inflammatory conditions. The outcome of delivering antigen to BST‐2 expressed by steady state and activated plasmacytoid DC (pDC) or conventional CD8+ and CD8− DCs was determined. T‐cell responses were measured for both MHC class I (MHCI) and MHC class II (MHCII) antigen presentation pathways in vitro. Delivering antigen via BST‐2 was compared with that via receptors DEC205 or Siglec‐H. We show that despite a higher antigen load and faster receptor internalisation, when antigen is delivered to steady state or activated pDC via BST‐2, BST‐2‐targeted activated conventional DCs present antigen more efficiently. Relative to DEC205, BST‐2 was inferior in its capacity to deliver antigen to the MHCI cross‐presentation pathway. In contrast, BST‐2 was superior to Siglec‐H at initiating either MHCI or MHCII antigen presentation. In summary, BST‐2 is a useful receptor to target with antigen, given its broad expression pattern and ability to access both MHCI and MHCII presentation pathways with relative efficiency.

Ex vivo response to histone deacetylase (HDAC) inhibitors of the HIV long terminal repeat (LTR) derived from HIV-infected patients on antiretroviral therapy.

Histone deacetylase inhibitors (HDACi) can induce human immunodeficiency virus (HIV) transcription from the HIV long terminal repeat (LTR). However, ex vivo and in vivo responses to HDACi are variable and the activity of HDACi in cells other than T-cells have not been well characterised. Here, we developed a novel assay to determine the activity of HDACi on patient-derived HIV LTRs in different cell types. HIV LTRs from integrated virus were amplified using triple-nested Alu-PCR from total memory CD4+ T-cells (CD45RO+) isolated from HIV-infected patients prior to and following suppressive antiretroviral therapy. NL4-3 or patient-derived HIV LTRs were cloned into the chromatin forming episomal vector pCEP4, and the effect of HDACi investigated in the astrocyte and epithelial cell lines SVG and HeLa, respectively. There were no significant differences in the sequence of the HIV LTRs isolated from CD4+ T-cells prior to and after 18 months of combination antiretroviral therapy (cART). We found that in both cell lines, the HDACi panobinostat, trichostatin A, vorinostat and entinostat activated patient-derived HIV LTRs to similar levels seen with NL4-3 and all patient derived isolates had similar sensitivity to maximum HDACi stimulation. We observed a marked difference in the maximum fold induction of luciferase by HDACi in HeLa and SVG, suggesting that the effect of HDACi may be influenced by the cellular environment. Finally, we observed significant synergy in activation of the LTR with vorinostat and the viral protein Tat. Together, our results suggest that the LTR sequence of integrated virus is not a major determinant of a functional response to an HDACi.

HIV persistence: Chemokines and their signalling pathways

Latently infected resting CD4+ T cells are the major barrier to curing HIV. We have recently demonstrated that chemokines, which bind to the chemokine receptors CCR7, CXCR3 and CCR6, facilitate efficient HIV nuclear localisation and integration in resting CD4+ T cells, leading to latency. As latently infected cells are enriched in lymphoid tissues, where chemokines are highly concentrated, this may provide a mechanism for the generation of latently infected cells in vivo. Here we review the role of chemokines in HIV persistence; the main signalling pathways that are involved; and how these pathways may be exploited to develop novel strategies to reduce or eliminate latently infected cells.

Human Immunodeficiency Virus Persistence and T-Cell Activation in Blood, Rectal, and Lymph Node Tissue in Human Immunodeficiency Virus–Infected Individuals Receiving Suppressive Antiretroviral Therapy

Background Immune activation and inflammation remain elevated in human immunodeficiency virus (HIV)-infected individuals receiving antiretroviral therapy (ART) and may contribute to HIV persistence. Methods Using flow cytometry expression of CD38, HLA-DR and PD-1 were measured in blood (n = 48), lymph node (LN; n = 9), and rectal tissue (n = 17) from virally suppressed individuals. Total and integrated HIV DNA, 2-LTR circles, and cell-associated unspliced HIV RNA were quantified. Results CD4+ T cells from rectal tissue had a higher frequency of integrated HIV DNA compared with blood (4.26 fold-change in DNA; 95% confidence interval [CI] = 2.61-7.00; P < .001) and LN (2.32 fold-change in DNA; 95% CI = 1.22-4.41; P = .01). In rectal tissue, there were positive associations between integrated HIV DNA with PD-1+ CD4+ T-cells (1.44 fold-change in integrated HIV DNA per 10-unit increase in PD-1+ CD4+ T cells; 95% CI = 1.01-2.05; P = .045) and CD38+HLA-DR+ CD8+ T cells (1.40 fold-change in integrated HIV DNA per 1-unit increase in CD38+HLA-DR+ CD8+ T cells; 95% CI = 1.05-1.86; P = .02). Both associations were independent of current and nadir CD4+ T-cell counts. Conclusions During ART, rectal tissue is an important reservoir for HIV persistence with a high frequency of activated CD4+ and CD8+ T cells. PD-1 may represent a marker of HIV persistence in rectal tissue.