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ObjectiveThe aim of this study was to explore the contribution of blood and colon myeloid cells to HIV persistence during antiretroviral therapy (ART).DesignLeukapheresis was collected from HIV-infected individuals with undetectable plasma viral load during ART (HIV + ART; n = 15) and viremics untreated (HIV+; n = 6). Rectal sigmoid biopsies were collected from n = 8 HIV+ART.MethodsMyeloid cells (total monocytes (Mo), CD16/CD16 Mo, CD1c dendritic cells) and CD4 T cells were isolated by magnetic-activated cell sorting (MACS) and/or fluorescence-activated cell sorting (FACS) from peripheral blood. Matched myeloid and CCR6CD4 T cells were isolated from blood and rectal biopsies by FACS. Levels of early (RU5 primers), late (Gag primers) and/or integrated HIV-DNA (Alu/HIV primers) were quantified by nested real-time PCR. Replication-competent HIV was amplified by co-culturing cells from HIV-positive individuals with CD3/CD28-activated CD4 T cells from uninfected donors.ResultsEarly/late but not integrated HIV reverse transcripts were detected in blood myeloid subsets of four out of 10 HIV+ART; in contrast, integrated HIV-DNA was exclusively detected in CD4 T cells. In rectal biopsies, late HIV reverse transcripts were detected in myeloid cells and CCR6CD4 T cells from one out of eight and seven out of eight HIV+ART individuals, respectively. Replication-competent HIV was outgrown from CD4 T cells but not from myeloid of untreated/ART-treated HIV-positive individuals.ConclusionIn contrast to CD4 T cells, blood and colon myeloid cells carry detectable HIV only in a small fraction of HIV+ART individuals. This is consistent with the documented resistance of Mo to HIV infection and the rapid turnover of Mo-derived macrophages in the colon. Future assessment of multiple lymphoid and nonlymphoid tissues is required to include/exclude myeloid cells as relevant HIV reservoirs during ART.

ObjectivesThe objective of this article is to investigate the contribution of colon and blood CD4 T-cell subsets expressing the chemokine receptor CCR6 to HIV persistence during antiretroviral therapy.DesignMatched sigmoid biopsies and blood samples (n = 13) as well as leukapheresis (n = 20) were collected from chronically HIV-infected individuals receiving antiretroviral therapy. Subsets of CD4 T cells with distinct differentiation/polarization profiles were identified using surface markers as follows: memory (TM, CD45RA), central memory (TCM; CD45RACCR7), effector (TEM/TM; CD45RACCR7), Th17 (CCR6CCR4), Th1Th17 (CCR6CXCR3), Th1 (CCR6CXCR3), and Th2 (CCR6CCR4).MethodsWe used polychromatic flow cytometry for cell sorting, nested real-time PCR for HIV DNA quantification, ELISA and flow cytometry for HIV p24 quantification. HIV reactivation was induced by TCR triggering in the presence/absence of all-trans retinoic acid.ResultsCompared with blood, the frequency of CCR6 TM was higher in the colon. In both colon and blood compartments, CCR6 TM were significantly enriched in HIV DNA when compared with their CCR6 counterparts (n = 13). In blood, integrated HIV DNA levels were significantly enriched in CCR6 versus CCR6 TCM of four of five individuals and CCR6 versus CCR6 TEM of three of five individuals. Among blood TCM, Th17 and Th1Th17 contributed the most to the pool of cells harboring integrated HIV DNA despite their reduced frequency compared with Th2, which were infected the least. HIV reactivation was induced by TCR triggering and/or retinoic acid exposure at higher levels in CCR6 versus CCR6 TM, TCM, and TEM.ConclusionCCR6 is a marker for colon and blood CD4 T cells enriched for replication-competent HIV DNA. Novel eradication strategies should target HIV persistence in CCR6CD4 T cells from various anatomic sites.

BackgroundTh17 cells are permissive to HIV-1 infection and their depletion from the gut of infected individuals leads to microbial translocation, a major cause for non-AIDS co-morbidities. Most recent evidence supports the contribution of long-lived Th17 cells to HIV persistence during antiretroviral therapy (ART). However, the identity of long-lived Th17 cells remains unknown.ResultsHere, we performed an in-depth transcriptional and functional characterization of four distinct Th17 subsets and investigated their contribution to HIV reservoir persistence during ART. In addition to the previously characterized CCR6(+)CCR4(+) (Th17) and CCR6(+)CXCR3(+) (Th1Th17) subsets, we reveal the existence of two novel CCR6(+) subsets, lacking (double negative, CCR6(+)DN) or co-expressing CXCR3 and CCR4 (double positive, CCR6(+)DP). The four subsets shared multiple Th17-polarization markers, a fraction of cells proliferated in response to C. albicans, and exhibited lineage commitment and plasticity when cultured under Th17 and Th1 conditions, respectively. Of note, fractions of CCR6(+)DN and Th17 demonstrated stable Th17-lineage commitment under Th1-polarization conditions. Among the four subsets, CCR6(+)DN expressed a unique transcriptional signature indicative of early Th17 development (IL-17F, STAT3), lymph-node homing (CCR7, CD62L), follicular help (CXCR5, BCL6, ASCL2), and self-renewal (LEFI, MYC, TERC). Cross sectional and longitudinal studies demonstrated that CCR6(+)DN cells were the most predominant CCR6(+) subset in the blood before and after ART initiation; high frequencies of these cells were similarly observed in inguinal lymph nodes of individuals receiving long-term ART. Importantly, replication competent HIV was isolated from CCR6(+)DN of ART-treated individuals.ConclusionsTogether, these results provide new insights into the functional heterogeneity of Th17-polarized CCR6(+)CD4(+) T-cells and support the major contribution of CCR6(+)DN cells to HIV persistence during ART.

Interleukin 32 (IL-32) is a potent multi-isoform proinflammatory cytokine, which is upregulated in people with HIV (PWH) and is associated with cardiovascular disease (CVD) risk. However, the impact of IL-32 isoforms on CD4 T-cell cardiotropism, a mechanism potentially contributing to heart inflammation, remains unknown. Here we show that IL-32 isoforms β and γ induce the generation of CCR4+CXCR3+ double positive (DP) memory CD4 T-cell subpopulation expressing the tyrosine kinase receptor c-Met, a phenotype associated with heart-homing of T cells. Our ex vivo studies on PWH show that the frequency of DP CD4 T cells is significantly higher in individuals with, compared to individuals without, subclinical atherosclerosis and that DP cells from antiretroviral-naive and treated individuals are highly enriched with HIV DNA. Together, these data demonstrate that IL-32 isoforms have the potential to induce heart-homing of HIV-infected CD4 T cells, which may further aggravate heart inflammation and CVD in PWH. [ABSTRACT FROM AUTHOR]

HIV-1 Nef protein down-regulates several cell surface receptors through its interference with the cell sorting and trafficking machinery. Here we demonstrate for the first time the ability of Nef to down-regulate cell surface expression of the negative immune modulator CTLA-4. Down-regulation of CTLA-4 required the Nef motifs DD175, EE155 and LL165, all known to be involved in vesicle trafficking. Disruption of the lysosomal functions by pH-neutralizing agents prevented CTLA-4 down-regulation by Nef, demonstrating the implication of the endosomal/lysosomal compartments in this process. Confocal microscopy experiments visualized the co-localization between Nef and CTLA-4 in the early and recycling endosomes but not at the cell surface. Overall, our results provide a novel mechanism by which HIV-1 Nef interferes with the surface expression of the negative regulator of T cell activation CTLA-4. Down-regulation of CTLA-4 may contribute to the mechanisms by which HIV-1 sustains T cell activation, a critical step in viral replication and dissemination.

ObjectiveMild forms of HIV-associated neurocognitive disorders (HAND) remain prevalent in the era of combination antiretroviral therapy (cART). Although elevated lipopolysaccharide (LPS) and immune activation are implicated in HAND pathogenesis, relationships of LPS and inflammatory markers to mild forms of HAND or impairment in specific cognitive domains are unknown. To examine these relationships, we compared plasma soluble CD14 (sCD14), CCL2, and LPS levels with neurocognitive test scores in a cART era cohort.MethodsWe analyzed plasma from HIV+ subjects (n = 97) with nadir CD4 counts

: Introduction: Guidelines regarding antiretroviral therapy (ART) initiation in HIV infection have varied over time, with the 2015 World Health Organization recommendation suggesting ART initiation at the time of diagnosis regardless of CD4 T‐cell counts. Herein, we investigated the influence of socio‐demographic and clinical factors in addition to time trends on early ART initiation among participants of the Montreal Primary HIV Infection Study. Methods: The Montreal Primary HIV Infection Study is a prospective cohort established in three community medical centres (CMCs) and two university medical centres (UMCs). Recently diagnosed HIV‐infected adults were categorized as receiving early (vs. delayed) ART if ART was initiated within 180 days of the baseline visit. Associations between early ART initiation and socio‐demographic, socio‐economic and behavioural information were examined. Independent associations of factors linked with early ART initiation were determined using multivariable binary logistic regression analysis. Results: A total of 348 participants had a documented date of HIV acquisition of Conclusions: Early ART initiation during primary HIV infection was associated with diminished biological prognostic factors and calendar time mirroring evolution of treatment guidelines. In addition, socio‐economic factors such as having a paid employment, contribute to early ART initiation in the context of universal access to care in Canada., Introduction Human immunodeficiency virus (HIV) affects over 36.7 million people worldwide. Nearly, half of those infected with HIV remain untreated, increasing their risks for acquired immune deficiency syndrome (AIDS), onward [...]

Viral replication and microbial translocation from the gut to the blood during HIV infection lead to hyperimmune activation, which contributes to the decline in [CD4.sup.+] T cell numbers during HIV infection. Programmed death-1 (PD-1) and interleukin-10 (IL-10) are both upregulated during HIV infection. Blocking interactions between PD-1 and programmed death ligand-1 (PD-L1) and between IL-10 and IL-10 receptor (IL-10R) results in viral clearance and improves T cell function in animal models of chronic viral infections. Here we show that high amounts of microbial products and inflammatory cytokines in the plasma of HIV-infected subjects lead to upregulation of PD-1 expression on monocytes that correlates with high plasma concentrations of IL-10. Triggering of PD-1 expressed on monocytes by PD-L1 expressed on various cell types induced IL-10 production and led to reversible [CD4.sup.+] T cell dysfunction. We describe a new function for PD-1 whereby microbial products inhibit T cell expansion and function by upregulating PD-1 levels and IL-10 production by monocytes after binding of PD-1 by PD-L1., Clearance of viral infections requires potent T cell responses. However, in HIV and other chronic viral infections, viral persistence (1,2) leads to the decay of [CD4.sup.+] T cells and their [...]

HIV persists in a reservoir of latently infected [CD4.sup.+] T cells in individuals treated with highly active antiretroviral therapy (HAART). Here we identify central memory ([T.sub.CM]) and transitional memory ([T.sub.TM]) [CD4.sup.+] T cells as the major cellular reservoirs for HIV and find that viral persistence is ensured by two different mechanisms. HIV primarily persists in [T.sub.CM] cells in subjects showing reconstitution of the [CD4.sup.+] compartment upon HAART. This reservoir is maintained through T cell survival and low-level antigen-driven proliferation and is slowly depleted with time. In contrast, proviral DNA is preferentially detected in [T.sub.TM] cells from aviremic individuals with low [CD4.sup.+] counts and higher amounts of interleukin-7-mediated homeostatic proliferation, a mechanism that ensures the persistence of these cells. Our results suggest that viral eradication might be achieved through the combined use of strategic interventions targeting viral replication and, as in cancer, drugs that interfere with the self renewal and persistence of proliferating memory T cells., Treatment of HIV infection has markedly reduced the death rate from AIDS and improved the quality of life of HIV-infected individuals (1). However, complete eradication of HIV with antiretroviral drugs [...]