Luca Micci

Low levels of SIV infection in sooty mangabey central memory CD4 + T cells are associated with limited CCR5 expression

Naturally simian immunodeficiency virus (SIV)-infected sooty mangabeys do not progress to AIDS despite high-level virus replication. We previously showed that the fraction of CD4+CCR5+ T cells is lower in sooty mangabeys compared to humans and macaques. Here we found that, after in vitro stimulation, sooty mangabey CD4+ T cells fail to upregulate CCR5 and that this phenomenon is more pronounced in CD4+ central memory T cells (TCM cells). CD4+ T cell activation was similarly uncoupled from CCR5 expression in sooty mangabeys in vivo during acute SIV infection and the homeostatic proliferation that follows antibody-mediated CD4+ T cell depletion. Sooty mangabey CD4+ TCM cells that express low amounts of CCR5 showed reduced susceptibility to SIV infection both in vivo and in vitro when compared to CD4+ TCM cells of rhesus macaques. These data suggest that low CCR5 expression on sooty mangabey CD4+ T cells favors the preservation of CD4+ T cell homeostasis and promotes an AIDS-free status by protecting CD4+ TCM cells from direct virus infection.

Maintenance of intestinal Th17 cells and reduced microbial translocation in SIV-infected rhesus macaques treated with interleukin (IL)-21.

In pathogenic HIV and SIV infections of humans and rhesus macaques (RMs), preferential depletion of CD4+ Th17 cells correlates with mucosal immune dysfunction and disease progression. Interleukin (IL)-21 promotes differentiation of Th17 cells, long-term maintenance of functional CD8+ T cells, and differentiation of memory B cells and antibody-secreting plasma cells. We hypothesized that administration of IL-21 will improve mucosal function in the context of pathogenic HIV/SIV infections. To test this hypothesis, we infected 12 RMs with SIVmac239 and at day 14 post-infection treated six of them with rhesus rIL-21-IgFc. IL-21-treatment was safe and did not increase plasma viral load or systemic immune activation. Compared to untreated animals, IL-21-treated RMs showed (i) higher expression of perforin and granzyme B in total and SIV-specific CD8+ T cells and (ii) higher levels of intestinal Th17 cells. Remarkably, increased levels of Th17 cells were associated with reduced levels of intestinal T cell proliferation, microbial translocation and systemic activation/inflammation in the chronic infection. In conclusion, IL-21-treatment in SIV-infected RMs improved mucosal immune function through enhanced preservation of Th17 cells. Further preclinical studies of IL-21 may be warranted to test its potential use during chronic infection in conjunction with antiretroviral therapy.

CD4 Depletion in SIV-Infected Macaques Results in Macrophage and Microglia Infection with Rapid Turnover of Infected Cells

In rhesus macaques (RMs), experimental depletion of CD4+ T-cells prior to SIV infection results in higher viremia and emergence of CD4-independent SIV-envelopes. In this study we used the rhesus recombinant anti-CD4 antibody CD4R1 to deplete RM CD4+ T-cells prior to SIVmac251 infection and investigate the sources of the increased viral burden and the lifespan of productively infected cells. CD4-depleted animals showed (i) set-point viral load two-logs higher than controls; (ii) macrophages constituting 80% of all SIV vRNA+ cells in lymph node and mucosal tissues; (iii) substantial expansion of pro-inflammatory monocytes; (iv) aberrant activation and infection of microglial cells; and (v) lifespan of productively infected cells significantly longer in comparison to controls, but markedly shorter than previously estimated for macrophages. The net effect of CD4+ T-cell depletion is an inability to control SIV replication and a shift in the tropism of infected cells to macrophages, microglia, and, potentially, other CD4-low cells which all appear to have a shortened in vivo lifespan. We believe these findings have important implications for HIV eradication studies.

Paucity of IL-21–producing CD4+ T cells is associated with Th17 cell depletion in SIV infection of rhesus macaques

IL-21 regulates Th17 cell homeostasis, enhances the differentiation of memory B cells and antibody-secreting plasma cells, and promotes the maintenance of CD8(+) T-cell responses. In this study, we investigated the phenotype, function, and frequency of blood and intestinal IL-21-producing cells in nonhuman primates that are hosts of progressive (rhesus macaques [RMs]) and nonprogressive (sooty mangabeys [SMs]) SIV infection. We found that, in both species, memory CD4(+)CD95(+)CCR6(-) T cells are the main IL-21 producers, and that only a small fraction of CD4(+)IL-21(+) T cells produce IL-17. During chronic SIV infection of RMs, CD4(+)IL-21(+) T cells were significantly depleted in both blood and rectal mucosa, with the extent of this depletion correlating with the loss of Th17 cells. Furthermore, treatment with IL-21 increased the in vivo levels of Th17 cells in SIV-infected RMs. In contrast, normal levels of CD4(+)IL-21(+) T cells were found in SIV-infected SMs. Collectively, these data indicate that depletion of IL-21-producing CD4(+) T cells distinguishes progressive from nonprogressive SIV infection of RMs and SMs, and suggest that depletion of CD4(+)IL-21(+) T cells is involved in the preferential loss of Th17 cells that is associated with SIV disease progression. Further preclinical studies of IL-21 as a potential immunotherapeutic agent for HIV infection may be warranted.

Interleukin-21 combined with ART reduces inflammation and viral reservoir in SIV-infected macaques.

Despite successful control of viremia, many HIV-infected individuals given antiretroviral therapy (ART) exhibit residual inflammation, which is associated with non-AIDS-related morbidity and mortality and may contribute to virus persistence during ART. Here, we investigated the effects of IL-21 administration on both inflammation and virus persistence in ART-treated, SIV-infected rhesus macaques (RMs). Compared with SIV-infected animals only given ART, SIV-infected RMs given both ART and IL-21 showed improved restoration of intestinal Th17 and Th22 cells and a more effective reduction of immune activation in blood and intestinal mucosa, with the latter maintained through 8 months after ART interruption. Additionally, IL-21, in combination with ART, was associated with reduced levels of SIV RNA in plasma and decreased CD4(+) T cell levels harboring replication-competent virus during ART. At the latest experimental time points, which were up to 8 months after ART interruption, plasma viremia and cell-associated SIV DNA levels remained substantially lower than those before ART initiation in IL-21-treated animals but not in controls. Together, these data suggest that IL-21 supplementation of ART reduces residual inflammation and virus persistence in a relevant model of lentiviral disease and warrants further investigation as a potential intervention for HIV infection.

Divergent CD4+ T Memory Stem Cell Dynamics in Pathogenic and Nonpathogenic Simian Immunodeficiency Virus Infections

Recent studies have identified a subset of memory T cells with stem cell-like properties (TSCM) that include increased longevity and proliferative potential. In this study, we examined the dynamics of CD4+ TSCM during pathogenic SIV infection of rhesus macaques (RM) and nonpathogenic SIV infection of sooty mangabeys (SM). Whereas SIV-infected RM show selective numeric preservation of CD4+ TSCM, SIV infection induced a complex perturbation of these cells defined by depletion of CD4+CCR5+ TSCM, increased rates of CD4+ TSCM proliferation, and high levels of direct virus infection. The increased rates of CD4+ TSCM proliferation in SIV-infected RM correlated inversely with the levels of central memory CD4+ T cells. In contrast, nonpathogenic SIV infection of SM evidenced preservation of both CD4+ TSCM and CD4+ central memory T cells, with normal levels of CD4+ TSCM proliferation, and lack of selective depletion of CD4+CCR5+ TSCM. Importantly, SIV DNA was below the detectable limit in CD4+ TSCM from 8 of 10 SIV-infected SM. We propose that increased proliferation and infection of CD4+ TSCM may contribute to the pathogenesis of SIV infection in RM.

Replicative fitness of transmitted HIV-1 drives acute immune activation, proviral load in memory CD4+ T cells, and disease progression

Significance HIV infection is associated with elevated inflammation and aberrant cellular immune activation. Indeed, the activation status of an HIV-infected individual is often more predictive of disease trajectory than viral load. Here, we highlight the importance of the replicative fitness of the transmitted viral variant in driving an early inflammatory state, characterized by T-cell activation and immune dysfunction. This impact on T-cell homeostasis is independent of protective host immune response genes and viral load. Highly replicating transmitted variants were also significantly more efficient at infecting memory CD4+ T cells, a population important for maintaining the latent viral reservoir. Together, these data provide a mechanism whereby viral replicative fitness acts as a major determinant of disease progression and persistence. HIV-1 infection is characterized by varying degrees of chronic immune activation and disruption of T-cell homeostasis, which impact the rate of disease progression. A deeper understanding of the factors that influence HIV-1–induced immunopathology and subsequent CD4+ T-cell decline is critical to strategies aimed at controlling or eliminating the virus. In an analysis of 127 acutely infected Zambians, we demonstrate a dramatic and early impact of viral replicative capacity (vRC) on HIV-1 immunopathogenesis that is independent of viral load (VL). Individuals infected with high-RC viruses exhibit a distinct inflammatory cytokine profile as well as significantly elevated T-cell activation, proliferation, and CD8+ T-cell exhaustion, during the earliest months of infection. Moreover, the vRC of the transmitted virus is positively correlated with the magnitude of viral burden in naive and central memory CD4+ T-cell populations, raising the possibility that transmitted viral phenotypes may influence the size of the initial latent viral reservoir. Taken together, these findings support an unprecedented role for the replicative fitness of the founder virus, independent of host protective genes and VL, in influencing multiple facets of HIV-1–related immunopathology, and that a greater focus on this parameter could provide novel approaches to clinical interventions.

Loss of Function of Intestinal IL-17 and IL-22 Producing Cells Contributes to Inflammation and Viral Persistence in SIV-Infected Rhesus Macaques.

In HIV/SIV-infected humans and rhesus macaques (RMs), a severe depletion of intestinal CD4+ T-cells producing interleukin IL-17 and IL-22 associates with loss of mucosal integrity and chronic immune activation. However, little is known about the function of IL-17 and IL-22 producing cells during lentiviral infections. Here, we longitudinally determined the levels and functions of IL-17, IL-22 and IL-17/IL-22 producing CD4+ T-cells in blood, lymph node and colorectum of SIV-infected RMs, as well as how they recover during effective ART and are affected by ART interruption. Intestinal IL-17 and IL-22 producing CD4+ T-cells are polyfunctional in SIV-uninfected RMs, with the large majority of cells producing four or five cytokines. SIV infection induced a severe dysfunction of colorectal IL-17, IL-22 and IL-17/IL-22 producing CD4+ T-cells, the extent of which associated with the levels of immune activation (HLA-DR+CD38+), proliferation (Ki-67+) and CD4+ T-cell counts before and during ART. Additionally, Th17 cell function during ART negatively correlated with residual plasma viremia and levels of sCD163, a soluble marker of inflammation and disease progression. Furthermore, IL-17 and IL-22 producing cell frequency and function at various pre, on, and off-ART experimental points associated with and predicted total SIV-DNA content in the colorectum and blood. While ART restored Th22 cell function to levels similar to pre-infection, it did not fully restore Th17 cell function, and all cell types were rapidly and severely affected—both quantitatively and qualitatively—after ART interruption. In conclusion, intestinal IL-17 producing cell function is severely impaired by SIV infection, not fully normalized despite effective ART, and strongly associates with inflammation as well as SIV persistence off and on ART. As such, strategies able to preserve and/or regenerate the functions of these CD4+ T-cells central for mucosal immunity are critically needed in future HIV cure research.

Reconstitution of Intestinal CD4 and Th17 T Cells in Antiretroviral Therapy Suppressed HIV-Infected Subjects: Implication for Residual Immune Activation from the Results of a Clinical Trial

Introduction During HIV infection the severe depletion of intestinal CD4+ T-cells is associated with microbial translocation, systemic immune activation, and disease progression. This study examined intestinal and peripheral CD4+ T-cell subsets reconstitution under combined antiretroviral therapy (cART), and systemic immune activation markers. Methods This longitudinal single-arm pilot study evaluates CD4+ T cells, including Th1 and Th17, in gut and blood and soluble markers for inflammation in HIV-infected individuals before (M0) and after eight (M8) months of cART. From January 2010 to December 2011, 10 HIV-1 naïve patients were screened and 9 enrolled. Blood and gut CD4+ T-cells subsets and cellular immune activation were determined by flow-cytometry and plasma soluble CD14 by ELISA. CD4+ Th17 cells were detected in gut biopsies by immunohistochemistry. Microbial translocation was measured by limulus-amebocyte-lysate assay to detect bacterial lipopolysaccharide (LPS) and PCR Real Time to detect plasma bacterial 16S rDNA. Results Eight months of cART increased intestinal CD4+ and Th17 cells and reduced levels of T-cell activation and proliferation. The magnitude of intestinal CD4+ T-cell reconstitution correlated with the reduction of plasma LPS. Importantly, the magnitude of Th17 cells reconstitution correlated directly with blood CD4+ T-cell recovery. Conclusion Short-term antiretroviral therapy resulted in a significant increase in the levels of total and Th17 CD4+ T-cells in the gut mucosa and in decline of T-cell activation. The observation that pre-treatment levels of CD4+ and of CD8+ T-cell activation are predictors of the magnitude of Th17 cell reconstitution following cART provides further rationale for an early initiation of cART in HIV-infected individuals. Trial Registration ClinicalTrials.gov NCT02097381

CTLA-4+PD-1− Memory CD4+ T Cells Critically Contribute to Viral Persistence in Antiretroviral Therapy-Suppressed, SIV-Infected Rhesus Macaques

&NA; Antiretroviral therapy (ART) suppresses viral replication in HIV‐infected individuals but does not eliminate the reservoir of latently infected cells. Recent work identified PD‐1+ follicular helper T (Tfh) cells as an important cellular compartment for viral persistence. Here, using ART‐treated, SIV‐infected rhesus macaques, we show that CTLA‐4+PD‐1− memory CD4+ T cells, which share phenotypic markers with regulatory T cells, were enriched in SIV DNA in blood, lymph nodes (LN), spleen, and gut, and contained replication‐competent and infectious virus. In contrast to PD‐1+ Tfh cells, SIV‐enriched CTLA‐4+PD‐1− CD4+ T cells were found outside the B cell follicle of the LN, predicted the size of the persistent viral reservoir during ART, and significantly increased their contribution to the SIV reservoir with prolonged ART‐mediated viral suppression. We have shown that CTLA‐4+PD‐1− memory CD4+ T cells are a previously unrecognized component of the SIV and HIV reservoir that should be therapeutically targeted for a functional HIV‐1 cure. Graphical Abstract Figure. No caption available. HighlightsCTLA‐4+PD‐1− memory CD4+ T cells are enriched in SIV DNA across multiple tissuesPersistently infected CTLA‐4+PD‐1− T cells localize outside the follicle on ARTCTLA‐4+PD‐1− T cells, which share Treg cell features, harbor replication‐competent virusSeeding of CTLA‐4+PD‐1− memory CD4+ T cells predicts viral persistence during ART &NA; HIV persists in T follicular helper cells within the lymph node during antiretroviral therapy, but decays with time. McGary et al. identify the persistence of replication‐competent SIV and HIV outside the lymph node follicle in a unique subset of CTLA‐4+PD‐1− memory CD4+ T cells that share features with regulatory T cells.