Lymph node dendritic cells harbor inducible replication-competent HIV despite years of suppressive ART.

Although gut and lymph node (LN) memory CD4 T cells represent major HIV and simian immunodeficiency virus (SIV) tissue reservoirs, the study of the role of dendritic cells (DCs) in HIV persistence has long been limited to the blood due to difficulties to access lymphoid tissue samples. In this study, we show that LN migratory and resident DC subpopulations harbor distinct phenotypic and transcriptomic profiles. Interestingly, both LN DC subpopulations contain HIV intact provirus and inducible replication-competent HIV despite the expression of the antiviral restriction factor SAMHD1. Notably, LN DC subpopulations isolated from HIV-infected individuals treated for up to 14 years are transcriptionally silent but harbor replication-competent virus that can be induced upon TLR7/8 stimulation. Taken together, these results uncover a potential important contribution of LN DCs to HIV infection in the presence of ART.

A scalable workflow to test "shock and kill" therapeutic approaches against the HIV-1 latent reservoir in blood cells ex vivo.

Integrated HIV-1 DNA persists in cells of people living with HIV during antiretroviral treatment, but its quantification is hindered by its rarity. Here, we present an optimized protocol to evaluate "shock and kill" therapeutic strategies, including both the latency reactivation ("shock") and elimination of infected cells ("kill") stages. We describe steps for the sequential use of nested PCR-based assays and viability sorting to allow for scalable and rapid screening of candidate therapeutics in patient-derived blood cells. For complete details on the use and execution of this protocol, please refer to Shytaj et al..1.

Active PD-L1 incorporation within HIV virions functionally impairs T follicular helper cells.

The limited development of broadly neutralizing antibodies (BnAbs) during HIV infection is classically attributed to an inadequate B-cell help brought by functionally impaired T follicular helper (Tfh) cells. However, the determinants of Tfh-cell functional impairment and the signals contributing to this condition remain elusive. In the present study, we showed that PD-L1 is incorporated within HIV virions through an active mechanism involving p17 HIV matrix protein. We subsequently showed that in vitro produced PD-L1high but not PD-L1low HIV virions, significantly reduced Tfh-cell proliferation and IL-21 production, ultimately leading to a decreased of IgG1 secretion from GC B cells. Interestingly, Tfh-cell functions were fully restored in presence of anti-PD-L1/2 blocking mAbs treatment, demonstrating that the incorporated PD-L1 proteins were functionally active. Taken together, the present study unveils an immunovirological mechanism by which HIV specifically exploits the regulatory potential of PD-L1 to suppress the immune system during the course of HIV infection.

Glycolysis downregulation is a hallmark of HIV-1 latency and sensitizes infected cells to oxidative stress.

HIV-1 infects lymphoid and myeloid cells, which can harbor a latent proviral reservoir responsible for maintaining lifelong infection. Glycolytic metabolism has been identified as a determinant of susceptibility to HIV-1 infection, but its role in the development and maintenance of HIV-1 latency has not been elucidated. By combining transcriptomic, proteomic, and metabolomic analyses, we here show that transition to latent HIV-1 infection downregulates glycolysis, while viral reactivation by conventional stimuli reverts this effect. Decreased glycolytic output in latently infected cells is associated with downregulation of NAD+ /NADH. Consequently, infected cells rely on the parallel pentose phosphate pathway and its main product, NADPH, fueling antioxidant pathways maintaining HIV-1 latency. Of note, blocking NADPH downstream effectors, thioredoxin and glutathione, favors HIV-1 reactivation from latency in lymphoid and myeloid cellular models. This provides a "shock and kill effect" decreasing proviral DNA in cells from people living with HIV/AIDS. Overall, our data show that downmodulation of glycolysis is a metabolic signature of HIV-1 latency that can be exploited to target latently infected cells with eradication strategies.

Lymph node migratory dendritic cells modulate HIV-1 transcription through PD-1 engagement.

T-follicular helper (Tfh) cells, co-expressing PD-1 and TIGIT, serve as a major cell reservoir for HIV-1 and are responsible for active and persistent HIV-1 transcription after prolonged antiretroviral therapy (ART). However, the precise mechanisms regulating HIV-1 transcription in lymph nodes (LNs) remain unclear. In the present study, we investigated the potential role of immune checkpoint (IC)/IC-Ligand (IC-L) interactions on HIV-1 transcription in LN-microenvironment. We show that PD-L1 (PD-1-ligand) and CD155 (TIGIT-ligand) are predominantly co-expressed on LN migratory (CD1chighCCR7+CD127+) dendritic cells (DCs), that locate predominantly in extra-follicular areas in ART treated individuals. We demonstrate that TCR-mediated HIV production is suppressed in vitro in the presence of recombinant PD-L1 or CD155 and, more importantly, when LN migratory DCs are co-cultured with PD-1+/Tfh cells. These results indicate that LN migratory DCs expressing IC-Ls may more efficiently restrict HIV-1 transcription in the extra-follicular areas and explain the persistence of HIV transcription in PD-1+/Tfh cells after prolonged ART within germinal centers.

PD-1 blockade potentiates HIV latency reversal ex vivo in CD4+ T cells from ART-suppressed individuals.

HIV persists in latently infected CD4+ T cells during antiretroviral therapy (ART). Immune checkpoint molecules, including PD-1, are preferentially expressed at the surface of persistently infected cells. However, whether PD-1 plays a functional role in HIV latency and reservoir persistence remains unknown. Using CD4+ T cells from HIV-infected individuals, we show that the engagement of PD-1 inhibits viral production at the transcriptional level and abrogates T-cell receptor (TCR)-induced HIV reactivation in latently infected cells. Conversely, PD-1 blockade with the monoclonal antibody pembrolizumab enhances HIV production in combination with the latency reversing agent bryostatin without increasing T cell activation. Our results suggest that the administration of immune checkpoint blockers to HIV-infected individuals on ART may facilitate latency disruption.

HCV RNA Activates APCs via TLR7/TLR8 While Virus Selectively Stimulates Macrophages Without Inducing Antiviral Responses.

The innate and adaptive immune systems fail to control HCV infection in the majority of infected individuals. HCV is an ssRNA virus, which suggests a role for Toll-like receptors (TLRs) 7 and 8 in initiating the anti-viral response. Here we demonstrate that HCV genomic RNA harbours specific sequences that initiate an anti-HCV immune response through TLR7 and TLR8 in various antigen presenting cells. Conversely, HCV particles are detected by macrophages, but not by monocytes and DCs, through a TLR7/8 dependent mechanism; this leads to chloroquine sensitive production of pro-inflammatory cytokines including IL-1ß, while the antiviral type I Interferon response is not triggered in these cells. Antibodies to DC-SIGN, a c-type lectin selectively expressed by macrophages but not pDCs or mDCs, block the production of cytokines. Novel anti-HCV vaccination strategies should target the induction of TLR7/8 stimulation in APCs in order to establish potent immune responses against HCV.

PD-1(+) and follicular helper T cells are responsible for persistent HIV-1 transcription in treated aviremic individuals.

The mechanisms responsible for the persistence of HIV-1 after many years of suppressive antiretroviral therapy (ART) have been only partially elucidated. Most of the studies investigating HIV-1 persistence have been performed with blood, although it is well known that germinal centers (GCs) within lymph nodes (LNs) serve as primary sites for HIV-1 replication. We sought to identify the memory CD4 T cell populations in blood and LNs that are responsible for the production of replication-competent and infectious HIV-1, as well as for active and persistent virus transcription in ART-treated (for 1.5-14.0 years), aviremic (<50 HIV RNA copies/ml) HIV-infected individuals. We demonstrate that LN CD4 T cells that express programmed cell death 1 (PDCD1; also known as PD-1), which are composed of about 65% T follicular helper cells as defined by the expression of the cell surface receptors CXCR5 and PD-1, are the major source of replication-competent HIV-1 and of infectious virus, as compared to any other (CXCR5(-)PD-1(-) and CXCR5(+)PD-1(-)) blood or LN memory CD4 T cell populations. LN PD-1(+) cells accounted for 46% and 96% of the total pools of memory CD4 T cells containing inducible replication-competent or infectious virus, respectively. Notably, higher levels of cell-associated HIV-1 RNA were present in LN PD-1(+) cells after long-term (up to 12 years) ART than in other memory CD4 T cell subpopulations. These results indicate that LN PD-1(+) cells are the major CD4 T cell compartment in the blood and LNs for the production of replication-competent and infectious HIV-1, and for active and persistent virus transcription in long-term-ART-treated aviremic individuals. Thus, these cells may represent a major obstacle to finding a functional cure for HIV-1 infection.

In Vitro Reactivation of Replication-Competent and Infectious HIV-1 by Histone Deacetylase Inhibitors.

UNLABELLED: The existence of long-lived HIV-1-infected resting memory CD4 T cells is thought to be the primary obstacle to HIV-1 eradication. In the search for novel therapeutic approaches that may reverse HIV-1 latency, inhibitors of histone deacetylases (HDACis) have been tested to reactivate HIV-1 replication with the objective of rendering HIV-1-infected cells susceptible to elimination either by HIV-specific CD8 T cells or through virus-mediated cytopathicity. In the present study, we evaluated the efficiency of HDACis to reactivate HIV-1 replication from resting memory CD4 T cells isolated from aviremic long-term-treated HIV-1-infected subjects. We demonstrate that following prolonged/repeated treatment of resting memory CD4 T cells with HDACis, HIV-1 replication may be induced from primary resting memory CD4 T cells isolated from aviremic long-term-treated HIV-1-infected subjects. More importantly, we demonstrate that HIV-1 reactivated in the cell cultures was not only replication competent but also infectious. Interestingly, givinostat, an HDACi that has not been investigated in clinical trials, was more efficient than vorinostat, panobinostat, and romidepsin in reversing HIV-1 latency in vitro. Taken together, these results support further evaluation of givinostat as a latency-reversing agent (LRA) in aviremic long-term-treated HIV-1-infected subjects. IMPORTANCE: The major barrier to HIV cure is the existence of long-lived latently HIV-1-infected resting memory CD4 T cells. Latently HIV-1-infected CD4 T cells are transcriptionally silent and are therefore not targeted by conventional antiretroviral therapy (ART) or the immune system. In this context, one strategy to target latently infected cells is based on pharmacological molecules that may force the virus to replicate and would therefore render HIV-1-infected cells susceptible to elimination either by HIV-specific CD8 T cells or through virus-mediated cytopathicity. In this context, we developed an experimental strategy that would allow the evaluation of latency-reversing agent (LRA) efficiency in vitro using primary CD4 T cells. In the present study, we demonstrate that HDACis are potent inducers of replication-competent and infectious HIV-1 in resting memory CD4 T cells of long-term ART-treated patients and identify givinostat as the most efficient LRA tested.

A Novel Assay to Measure the Magnitude of the Inducible Viral Reservoir in HIV-infected Individuals.

BACKGROUND: Quantifying latently infected cells is critical to evaluate the efficacy of therapeutic strategies aimed at reducing the size of the long-lived viral reservoir, but the low frequency of these cells makes this very challenging. METHODS: We developed TILDA (Tat/rev Induced Limiting Dilution Assay) to measure the frequency of cells with inducible multiply-spliced HIV RNA, as these transcripts are usually absent in latently infected cells but induced upon viral reactivation. TILDA requires less than a million cells, does not require RNA extraction and can be completed in two days. FINDINGS: In suppressed individuals on ART, we found the median frequency of latently infected CD4 + T cells as estimated by TILDA to be 24 cells/million, which was 48 times more than the frequency measured by the quantitative viral outgrowth assay, and 6-27 times less than the frequencies of cells harbouring viral DNA measured by PCR-based assays. TILDA measurements strongly correlated with most HIV DNA assays. The size of the latent reservoir measured by TILDA was lower in subjects who initiated ART during the early compared to late stage of infection (p = 0.011). In untreated HIV disease, the frequency of CD4 + cells carrying latent but inducible HIV largely exceeded the frequency of actively producing cells, demonstrating that the majority of infected cells are transcriptionally silent even in the absence of ART. INTERPRETATIONS: Our results suggest that TILDA is a reproducible and sensitive approach to measure the frequency of productively and latently infected cells in clinical settings. We demonstrate that the latent reservoir represents a substantial fraction of all infected cells prior to ART initiation. RESEARCH IN CONTEXT: In this manuscript, we describe the development of a novel assay that measures the magnitude of the latent HIV reservoir, the main barrier to HIV eradication. This novel assay, termed TILDA for Tat/rev Induced Limiting Dilution Assay, requires only 10 ml of blood, does not necessitate extraction of viral nucleic acids, is highly reproducible, covers a wide dynamic range of reservoir sizes and can be completed in two days. As such, TILDA may represent an alternative to existing assays used to evaluate the efficacy of therapeutic strategies aimed at reducing the size of the latent HIV reservoir.

Profound metabolic, functional, and cytolytic differences characterize HIV-specific CD8 T cells in primary and chronic HIV infection.

Immediate-early host-virus interactions that occur during the first weeks after HIV infection have a major impact on disease progression. The mechanisms underlying the failure of HIV-specific CD8 T-cell response to persist and control viral replication early in infection are yet to be characterized. In this study, we performed a thorough phenotypic, gene expression and functional analysis to compare HIV-specific CD8 T cells in acutely and chronically infected subjects. We showed that HIV-specific CD8 T cells in primary infection can be distinguished by their metabolic state, rate of proliferation, and susceptibility to apoptosis. HIV-specific CD8 T cells in acute/early HIV infection secreted less IFN-γ but were more cytotoxic than their counterparts in chronic infection. Importantly, we showed that the levels of IL-7R expression and the capacity of HIV-specific CD8 T cells to secrete IL-2 on antigenic restimulation during primary infection were inversely correlated with the viral set-point. Altogether, these data suggest an altered metabolic state of HIV-specific CD8 T cells in primary infection resulting from hyperproliferation and stress induced signals, demonstrate the discordant function of HIV-specific CD8 T cells during early/acute infection, and highlight the importance of T-cell maintenance for viral control.