Colleen S. McGary

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.

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.

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.

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.

Increased Stability and Limited Proliferation of CD4+ Central Memory T Cells Differentiate Nonprogressive Simian Immunodeficiency Virus (SIV) Infection of Sooty Mangabeys from Progressive SIV Infection of Rhesus Macaques

ABSTRACT Depletion of CD4+ central memory T (TCM) cells dictates the tempo of progression to AIDS in simian immunodeficiency virus (SIV)-infected rhesus macaques (RMs) both in the natural history of infection and in the context of vaccination. CD4+ TCM cells of sooty mangabeys (SMs), a natural host for SIV in which infection is nonpathogenic, are less susceptible to SIV infection than CD4+ TCM cells of RMs. Whether this relative protection from infection translates into increased stability of CD4+ TCM cells in natural versus nonnatural hosts has not yet been determined. Here we compared, both cross-sectionally and longitudinally, the levels of CD4+ TCM cells in a large cohort of SMs and RMs and the association between CD4+ TCM levels and the main virologic and immunologic markers of disease progression. Consistent with their lower susceptibility to infection, CD4+ TCM cells of SIV-infected SMs are lost with kinetics 20 times slower than those of SIV-infected RMs. Remarkably, the estimated length of time of SIV infection needed for CD4+ TCM cells to fall to half of their initial levels is <16 months for RMs but >17 years for SMs. Furthermore, the fraction of proliferating CD4+ TCM cells is significantly lower in SIV-infected SMs than in SIV-infected RMs, and the extent of CD4+ TCM cell proliferation is associated positively with CD4+ T cell levels in SIV-infected SMs but negatively with CD4+ T cell levels in SIV-infected RMs. Collectively, these findings identify increased stability and maintenance of the prohomeostatic role of CD4+ TCM cells as features distinguishing nonprogressive from progressive SIV infections and support the hypothesis of a direct mechanistic link between the loss of CD4+ TCM cells and disease progression. IMPORTANCE Comparison of the immunologic effects of simian immunodeficiency virus (SIV) infection on rhesus macaques (RMs), a species characterized by progression to AIDS, and natural host sooty mangabeys (SMs), a species which remains AIDS free, has become a useful tool for identifying mechanisms of human immunodeficiency virus (HIV) disease progression. One such distinguishing feature is that CD4+ central memory T (TCM) cells in SIV-infected SMs are less infected than the same cells in RMs. Here we investigated whether lower levels of infection in SMs translate into a better-preserved CD4+ TCM compartment. We found that the CD4+ TCM compartment is significantly more stable in SIV-infected SMs. Likely to compensate for this cell loss, we also found that CD4+ TCM cells increase their level of proliferation upon SIV infection in RMs but not in SMs, which mechanistically supports their preferential infectivity. Our study provides new insights into the importance of long-term maintenance of CD4+ TCM homeostasis during HIV/SIV infection.

The loss of CCR6 + and CD161 + CD4 + T-cell homeostasis contributes to disease progression in SIV-infected rhesus macaques

Although previous studies have shown that CD4+ T cells expressing CCR6 and CD161 are depleted from blood during HIV infection, the mechanisms underlying their loss remain unclear. In this study, we investigated how the homeostasis of CCR6+ and CD161+ CD4+ T cells contributes to SIV disease progression and the mechanisms responsible for their loss from circulation. By comparing SIV infection in rhesus macaques (RMs) and natural host sooty mangabeys (SMs), we found that the loss of CCR6+ and CD161+ CD4+ T cells from circulation is a distinguishing feature of progressive SIV infection in RMs. Furthermore, while viral infection critically contributes to the loss of CD161+CCR6−CD4+ T cells, a redistribution of CCR6+CD161− and CCR6+CD161+CD4+ T cells from the blood to the rectal mucosa is a chief mechanism for their loss during SIV infection. Finally, we provide evidence that the accumulation of CCR6+CD4+ T cells in the mucosa is damaging to the host by demonstrating their reduction from this site following initiation of antiretroviral therapy in SIV-infected RMs and their lack of accumulation in SIV-infected SMs. These data emphasize the importance of maintaining CCR6+ and CD161+ CD4+ T-cell homeostasis, particularly in the mucosa, to prevent disease progression during pathogenic HIV/SIV infection.

Animal models for viral infection and cell exhaustion

Purpose of reviewDespite eliciting an early antiviral T cell response, HIV-specific T cells are unable to prevent disease progression, partly because of their loss of effector functions, known as T cell exhaustion. Restoring this T cell functionality represents a critical step for regaining immunological control of HIV-1 replication, and may be fundamental for the development of a functional cure for HIV. In this context, the use of animal models is invaluable for evaluating the efficacy and mechanisms of novel therapeutics aimed at reinvigorating T cell functions. Recent findingsAlthough nonhuman primates continue to be a mainstay for studying HIV pathogenesis and therapies, recent advances in humanized mouse models have improved their ability to recapitulate the features of cell exhaustion during HIV infection. Targeting coinhibitory receptors in HIV-infected and simian immunodeficiency virus (SIV)-infected animals has resulted in viral load reductions, presumably by reinvigorating the effector functions of T cells. Additionally, studies combining programmed death-1 (PD-1) blockade with suppressive antiretroviral therapy provide further support to the use of coinhibitory receptor blockades in restoring T cell function by delaying viral load rebound upon antiretroviral therapy interruption. Future in-vivo studies should build on recent in-vitro data, supporting the simultaneous targeting of multiple regulators of cell exhaustion. SummaryIn this review, we describe the most recent advances in the use of animal models for the study of cell exhaustion following HIV/SIV infection. These findings suggest that the use of animal models is increasingly critical in translating immunotherapeutics into clinical practice.