Anti-PD-1 chimeric antigen receptor T cells efficiently target SIV-infected CD4+ T cells in germinal centers.

Programmed cell death protein 1 (PD-1) is an immune checkpoint marker commonly expressed on memory T cells and enriched in latently HIV-infected CD4+ T cells. We engineered an anti-PD-1 chimeric antigen receptor (CAR) to assess the impact of PD-1 depletion on viral reservoirs and rebound dynamics in SIVmac239-infected rhesus macaques (RMs). Adoptive transfer of anti-PD-1 CAR T cells was done in 2 SIV-naive and 4 SIV-infected RMs on antiretroviral therapy (ART). In 3 of 6 RMs, anti-PD-1 CAR T cells expanded and persisted for up to 100 days concomitant with the depletion of PD-1+ memory T cells in blood and tissues, including lymph node CD4+ follicular helper T (TFH) cells. Loss of TFH cells was associated with depletion of detectable SIV RNA from the germinal center (GC). However, following CAR T infusion and ART interruption, there was a marked increase in SIV replication in extrafollicular portions of lymph nodes, a 2-log higher plasma viremia relative to controls, and accelerated disease progression associated with the depletion of CD8+ memory T cells. These data indicate anti-PD-1 CAR T cells depleted PD-1+ T cells, including GC TFH cells, and eradicated SIV from this immunological sanctuary.

HIHISIV: a database of gene expression in HIV and SIV host immune response.

In the battle of the host against lentiviral pathogenesis, the immune response is crucial. However, several questions remain unanswered about the interaction with different viruses and their influence on disease progression. The simian immunodeficiency virus (SIV) infecting nonhuman primates (NHP) is widely used as a model for the study of the human immunodeficiency virus (HIV) both because they are evolutionarily linked and because they share physiological and anatomical similarities that are largely explored to understand the disease progression. The HIHISIV database was developed to support researchers to integrate and evaluate the large number of transcriptional data associated with the presence/absence of the pathogen (SIV or HIV) and the host response (NHP and human). The datasets are composed of microarray and RNA-Seq gene expression data that were selected, curated, analyzed, enriched, and stored in a relational database. Six query templates comprise the main data analysis functions and the resulting information can be downloaded. The HIHISIV database, available at  https://hihisiv.github.io , provides accurate resources for browsing and visualizing results and for more robust analyses of pre-existing data in transcriptome repositories.

The CARD8 inflammasome dictates HIV/SIV pathogenesis and disease progression.

While CD4+ T cell depletion is key to disease progression in people living with HIV and SIV-infected macaques, the mechanisms underlying this depletion remain incompletely understood, with most cell death involving uninfected cells. In contrast, SIV infection of "natural" hosts such as sooty mangabeys does not cause CD4+ depletion and AIDS despite high-level viremia. Here, we report that the CARD8 inflammasome is activated immediately after HIV entry by the viral protease encapsulated in incoming virions. Sensing of HIV protease activity by CARD8 leads to rapid pyroptosis of quiescent cells without productive infection, while T cell activation abolishes CARD8 function and increases permissiveness to infection. In humanized mice reconstituted with CARD8-deficient cells, CD4+ depletion is delayed despite high viremia. Finally, we discovered loss-of-function mutations in CARD8 from "natural hosts," which may explain the peculiarly non-pathogenic nature of these infections. Our study suggests that CARD8 drives CD4+ T cell depletion during pathogenic HIV/SIV infections.

Macrophages: Key Cellular Players in HIV Infection and Pathogenesis.

Although cells of the myeloid lineages, including tissue macrophages and conventional dendritic cells, were rapidly recognized, in addition to CD4+ T lymphocytes, as target cells of HIV-1, their specific roles in the pathophysiology of infection were initially largely neglected. However, numerous studies performed over the past decade, both in vitro in cell culture systems and in vivo in monkey and humanized mouse animal models, led to growing evidence that macrophages play important direct and indirect roles as HIV-1 target cells and in pathogenesis. It has been recently proposed that macrophages are likely involved in all stages of HIV-1 pathogenesis, including virus transmission and dissemination, but above all, in viral persistence through the establishment, together with latently infected CD4+ T cells, of virus reservoirs in many host tissues, the major obstacle to virus eradication in people living with HIV. Infected macrophages are indeed found, very often as multinucleated giant cells expressing viral antigens, in almost all lymphoid and non-lymphoid tissues of HIV-1-infected patients, where they can probably persist for long period of time. In addition, macrophages also likely participate, directly as HIV-1 targets or indirectly as key regulators of innate immunity and inflammation, in the chronic inflammation and associated clinical disorders observed in people living with HIV, even in patients receiving effective antiretroviral therapy. The main objective of this review is therefore to summarize the recent findings, and also to revisit older data, regarding the critical functions of tissue macrophages in the pathophysiology of HIV-1 infection, both as major HIV-1-infected target cells likely found in almost all tissues, as well as regulators of innate immunity and inflammation during the different stages of HIV-1 pathogenesis.

SIV Infection Is Associated with Transient Acute-Phase Steatosis in Hepatocytes In Vivo.

Metabolic-dysfunction-associated fatty liver disease (MAFLD) is a major cause of morbidity and mortality in HIV-infected individuals, even those receiving optimal antiretroviral therapy. Here, we utilized the SIV rhesus macaque model and advanced laparoscopic techniques for longitudinal collection of liver tissue to elucidate the timing of pathologic changes. The livers of both SIV-infected (N = 9) and SIV-naïve uninfected (N = 8) macaques were biopsied and evaluated at four time points (weeks -4, 2, 6, and 16-20 post-infection) and at necropsy (week 32). SIV DNA within the macaques' livers varied by over 4 logs at necropsy, and liver SIV DNA significantly correlated with SIV RNA in the plasma throughout the study. Acute phase liver pathology (2 weeks post-infection) was characterized by evidence for fat accumulation (microvesicular steatosis), a transient elevation in both AST and cholesterol levels within the serum, and increased hepatic expression of the PPARA gene associated with cholesterol metabolism and beta oxidation. By contrast, the chronic phase of the SIV infection (32 weeks post-infection) was associated with sinusoidal dilatation, while steatosis resolved and concentrations of AST and cholesterol remained similar to those in uninfected macaques. These findings suggest differential liver pathologies associated with the acute and chronic phases of infection and the possibility that therapeutic interventions targeting metabolic function may benefit liver health in people newly diagnosed with HIV.

SHIV-C109p5 NHP induces rapid disease progression in elderly macaques with extensive GI viral replication.

CCR5-tropic simian/human immunodeficiency viruses (SHIV) with clade C transmitted/founder envelopes represent a critical tool for the investigation of HIV experimental vaccines and microbicides in nonhuman primates, although many such isolates lead to spontaneous viral control post infection. Here, we generated a high-titer stock of pathogenic SHIV-C109p5 by serial passage in two rhesus macaques (RM) and tested its virulence in aged monkeys. The co-receptor usage was confirmed before infecting five geriatric rhesus macaques (four female and one male). Plasma viral loads were monitored by reverse transcriptase-quantitative PCR (RT-qPCR), cytokines by multiplex analysis, and biomarkers of gastrointestinal damage by enzyme-linked immunosorbent assay. Antibodies and cell-mediated responses were also measured. Viral dissemination into tissues was determined by RNAscope. Intravenous SHIV-C109p5 infection of aged RMs leads to high plasma viremia and rapid disease progression; rapid decrease in CD4+ T cells, CD4+CD8+ T cells, and plasmacytoid dendritic cells; and wasting necessitating euthanasia between 3 and 12 weeks post infection. Virus-specific cellular immune responses were detected only in the two monkeys that survived 4 weeks post infection. These were Gag-specific TNFα+CD8+, MIP1ß+CD4+, Env-specific IFN-γ+CD4+, and CD107a+ T cell responses. Four out of five monkeys had elevated intestinal fatty acid binding protein levels at the viral peak, while regenerating islet-derived protein 3α showed marked increases at later time points in the three animals surviving the longest, suggesting gut antimicrobial peptide production in response to microbial translocation post infection. Plasma levels of monocyte chemoattractant protein-1, interleukin-15, and interleukin-12/23 were also elevated. Viral replication in gut and secondary lymphoid tissues was extensive.IMPORTANCESimian/human immunodeficiency viruses (SHIV) are important reagents to study prevention of virus acquisition in nonhuman primate models of HIV infection, especially those representing transmitted/founder (T/F) viruses. However, many R5-tropic SHIV have limited fitness in vivo leading to many monkeys spontaneously controlling the virus post acute infection. Here, we report the generation of a pathogenic SHIV clade C T/F stock by in vivo passage leading to sustained viral load set points, a necessity to study pathogenicity. Unexpectedly, administration of this SHIV to elderly rhesus macaques led to extensive viral replication and fast disease progression, despite maintenance of a strict R5 tropism. Such age-dependent rapid disease progression had previously been reported for simian immunodeficiency virus but not for R5-tropic SHIV infections.

HIV-1 Myeloid Reservoirs - Contributors to Viral Persistence and Pathogenesis.

PURPOSE OF REVIEW: HIV reservoirs are the main barrier to cure. CD4+ T cells have been extensively studied as the primary HIV-1 reservoir. However, there is substantial evidence that HIV-1-infected myeloid cells (monocytes/macrophages) also contribute to viral persistence and pathogenesis. RECENT FINDINGS: Recent studies in animal models and people with HIV-1 demonstrate that myeloid cells are cellular reservoirs of HIV-1. HIV-1 genomes and viral RNA have been reported in circulating monocytes and tissue-resident macrophages from the brain, urethra, gut, liver, and spleen. Importantly, viral outgrowth assays have quantified persistent infectious virus from monocyte-derived macrophages and tissue-resident macrophages. The myeloid cell compartment represents an important target of HIV-1 infection. While myeloid reservoirs may be more difficult to measure than CD4+ T cell reservoirs, they are long-lived, contribute to viral persistence, and, unless specifically targeted, will prevent an HIV-1 cure.

CD20 CAR T cells safely and reversibly ablate B cell follicles in a non-human primate model of HIV persistence.

Chimeric antigen receptor (CAR) T cell therapies have demonstrated immense clinical success for B cell and plasma cell malignancies. We tested their impact on the viral reservoir in a macaque model of HIV persistence, comparing the functions of CD20 CAR T cells between animals infected with simian/human immunodeficiency virus (SHIV) and uninfected controls. We focused on the potential of this approach to disrupt B cell follicles (BCFs), exposing infected cells for immune clearance. In SHIV-infected animals, CAR T cells were highly functional, with rapid expansion and trafficking to tissue-associated viral sanctuaries, including BCFs and gut-associated lymphoid tissue (GALT). CD20 CAR T cells potently ablated BCFs and depleted lymph-node-associated follicular helper T (TFH) cells, with complete restoration of BCF architecture and TFH cells following CAR T cell contraction. BCF ablation decreased the splenic SHIV reservoir but was insufficient for effective reductions in systemic viral reservoirs. Although associated with moderate hematologic toxicity, CD20 CAR T cells were well tolerated in SHIV-infected and control animals, supporting the feasibility of this therapy in people living with HIV with underlying B cell malignancies. Our findings highlight the unique ability of CD20 CAR T cells to safely and reversibly unmask TFH cells within BCF sanctuaries, informing future combinatorial HIV cure strategies designed to augment antiviral efficacy.

Increased cAMP-PKA signaling pathway activation is involved in up-regulation of CTLA-4 expression in CD4+ T cells in acute SIVmac239-infected Chinese rhesus macaques.

Human immunodeficiency virus-1 (HIV-1) infection can cause chronic activation, exhaustion, and anergy of the immune system. Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is an immune checkpoint molecule, which plays an important role in immune homeostasis and disease. CTLA-4 expression is elevated in HIV-1-infected patients and is associated with disease progression. However, the mechanism controlling expression of CTLA-4 in HIV-1 infection is poorly characterized. In this study, we used a SIV-infected Chinese rhesus macaque (ChRM) model to explore CTLA-4 expression in SIV infection. Results showed that SIV infection significantly increased CTLA-4 expression in all T cell subsets, especially central memory T cells. CTLA-4+CD4+ T cell frequency was significantly associated with disease progression markers. Activation of the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling pathway regulated CTLA-4 expression in CD4+T cells, as confirmed by stimulation with dibutyryl cyclic adenosine monophosphate, forskolin, and 3-isobutyl-1-methylxanthine, and inhibition with H-89 ex vivo. Simultaneously, cAMP concentration in PBMCs and PKA activity in both PBMCs and CD4+ T cells were increased in acute SIV-infected ChRMs, accompanied by an increase in adenylate cyclase 6 expression and a decrease in cAMP-phosphodiesterase 3A (PDE3A), PDE4B, and PDE5A expression in PBMCs. In addition, selective inhibition of PDE4B and PDE5A activity enhanced CTLA-4 expression in CD4+ T cells. These results suggest that SIV infection alters cAMP metabolism and increases cAMP-PKA signaling pathway activation, which up-regulates the expression of CTLA-4 in acute SIVmac239-infected ChRMs. Thus, regulation of the cAMP-PKA signaling pathway may be a potential strategy for the restoration of T cell function and therapy for AIDS.

More than the Infinite Monkey Theorem: NHP Models in the Development of a Pediatric HIV Cure.

PURPOSE OF REVIEW: An HIV cure that eliminates the viral reservoir or provides viral control without antiretroviral therapy (ART) is an urgent need in children as they face unique challenges, including lifelong ART adherence and the deleterious effects of chronic immune activation. This review highlights the importance of nonhuman primate (NHP) models in developing an HIV cure for children as these models recapitulate the viral pathogenesis and persistence. RECENT FINDINGS: Several cure approaches have been explored in infant NHPs, although knowledge gaps remain. Broadly neutralizing antibodies (bNAbs) show promise for controlling viremia and delaying viral rebound after ART interruption but face administration challenges. Adeno-associated virus (AAV) vectors hold the potential for sustained bNAb expression. Therapeutic vaccination induces immune responses against simian retroviruses but has yet to impact the viral reservoir. Combining immunotherapies with latency reversal agents (LRAs) that enhance viral antigen expression should be explored. Current and future cure approaches will require adaptation for the pediatric immune system and unique features of virus persistence, for which NHP models are fundamental to assess their efficacy.

Fusion peptide-directed antibodies: Humoral armor against HIV-1 infection.

Infused neutralizing antibodies to the fusion peptide of the HIV envelope glycoprotein protected macaques from mucosal viral challenge (Pegu et al.).

Antibodies targeting the fusion peptide on the HIV envelope provide protection to rhesus macaques against mucosal SHIV challenge.

The fusion peptide (FP) on the HIV-1 envelope (Env) trimer can be targeted by broadly neutralizing antibodies (bNAbs). Here, we evaluated the ability of a human FP-directed bNAb, VRC34.01, along with two vaccine-elicited anti-FP rhesus macaque mAbs, DFPH-a.15 and DF1W-a.01, to protect against simian-HIV (SHIV)BG505 challenge. VRC34.01 neutralized SHIVBG505 with a 50% inhibitory concentration (IC50) of 0.58 µg/ml, whereas DF1W-a.01 and DFPH-a.15 were 4- or 30-fold less potent, respectively. VRC34.01 was infused into four rhesus macaques at a dose of 10 mg/kg and four rhesus macaques at a dose of 2.5 mg/kg. The animals were intrarectally challenged 5 days later with SHIVBG505. In comparison with all 12 control animals that became infected, all four animals infused with VRC34.01 (10 mg/kg) and three out of four animals infused with VRC34.01 (2.5 mg/kg) remained uninfected. Because of the lower potency of DF1W-a.01 and DFPH-a.15 against SHIVBG505, we infused both Abs at a higher dose of 100 mg/kg into four rhesus macaques each, followed by SHIVBG505 challenge 5 days later. Three of four animals that received DF1W-a.01 were protected against infection, whereas all animals that received DFPH-a.15 were protected. Overall, the protective serum neutralization titers observed in these animals were similar to what has been observed for other bNAbs in similar SHIV infection models and in human clinical trials. In conclusion, FP-directed mAbs can thus provide dose-dependent in vivo protection against mucosal SHIV challenges, supporting the development of prophylactic vaccines targeting the HIV-1 Env FP.

Host immunity associated with spontaneous suppression of viremia in therapy-naïve young rhesus macaques following neonatal SHIV infection.

IMPORTANCE: Despite the advent of highly active anti-retroviral therapy, people are still dying from HIV-related causes, many of whom are children, and a protective vaccine or cure is needed to end the HIV pandemic. Understanding the nature and activation states of immune cell subsets during infection will provide insights into the immunologic milieu associated with viremia suppression that can be harnessed via therapeutic strategies to achieve a functional cure, but these are understudied in pediatric subjects. We evaluated humoral and adaptive host immunity associated with suppression of viremia in rhesus macaques infected soon after birth with a pathogenic SHIV. The results from our study provide insights into the immune cell subsets and functions associated with viremia control in young macaques that may translate to pediatric subjects for the design of future anti-viral strategies in HIV-1-infected infants and children and contribute to an understudied area of HIV-1 pathogenesis in pediatric subjects.

Mathematical modeling indicates that regulatory inhibition of CD8+ T cell cytotoxicity can limit efficacy of IL-15 immunotherapy in cases of high pre-treatment SIV viral load.

Immunotherapeutic cytokines can activate immune cells against cancers and chronic infections. N-803 is an IL-15 superagonist that expands CD8+ T cells and increases their cytotoxicity. N-803 also temporarily reduced viral load in a limited subset of non-human primates infected with simian immunodeficiency virus (SIV), a model of HIV. However, viral suppression has not been observed in all SIV cohorts and may depend on pre-treatment viral load and the corresponding effects on CD8+ T cells. Starting from an existing mechanistic mathematical model of N-803 immunotherapy of SIV, we develop a model that includes activation of SIV-specific and non-SIV-specific CD8+ T cells by antigen, inflammation, and N-803. Also included is a regulatory counter-response that inhibits CD8+ T cell proliferation and function, representing the effects of immune checkpoint molecules and immunosuppressive cells. We simultaneously calibrate the model to two separate SIV cohorts. The first cohort had low viral loads prior to treatment (≈3-4 log viral RNA copy equivalents (CEQ)/mL), and N-803 treatment transiently suppressed viral load. The second had higher pre-treatment viral loads (≈5-7 log CEQ/mL) and saw no consistent virus suppression with N-803. The mathematical model can replicate the viral and CD8+ T cell dynamics of both cohorts based on different pre-treatment viral loads and different levels of regulatory inhibition of CD8+ T cells due to those viral loads (i.e. initial conditions of model). Our predictions are validated by additional data from these and other SIV cohorts. While both cohorts had high numbers of activated SIV-specific CD8+ T cells in simulations, viral suppression was precluded in the high viral load cohort due to elevated inhibition of cytotoxicity. Thus, we mathematically demonstrate how the pre-treatment viral load can influence immunotherapeutic efficacy, highlighting the in vivo conditions and combination therapies that could maximize efficacy and improve treatment outcomes.

Intradermal but not intramuscular modified vaccinia Ankara immunizations protect against intravaginal tier2 simian-human immunodeficiency virus challenges in female macaques.

Route of immunization can markedly influence the quality of immune response. Here, we show that intradermal (ID) but not intramuscular (IM) modified vaccinia Ankara (MVA) vaccinations provide protection from acquisition of intravaginal tier2 simian-human immunodeficiency virus (SHIV) challenges in female macaques. Both routes of vaccination induce comparable levels of serum IgG with neutralizing and non-neutralizing activities. The protection in MVA-ID group correlates positively with serum neutralizing and antibody-dependent phagocytic activities, and envelope-specific vaginal IgA; while the limited protection in MVA-IM group correlates only with serum neutralizing activity. MVA-ID immunizations induce greater germinal center Tfh and B cell responses, reduced the ratio of Th1 to Tfh cells in blood and showed lower activation of intermediate monocytes and inflammasome compared to MVA-IM immunizations. This lower innate activation correlates negatively with induction of Tfh responses. These data demonstrate that the MVA-ID vaccinations protect against intravaginal SHIV challenges by modulating the innate and T helper responses.

A human-specific motif facilitates CARD8 inflammasome activation after HIV-1 infection.

Inflammasomes are cytosolic innate immune complexes that assemble upon detection of diverse pathogen-associated cues and play a critical role in host defense and inflammatory pathogenesis. Here, we find that the human inflammasome-forming sensor CARD8 senses HIV-1 infection via site-specific cleavage of the CARD8 N-terminus by the HIV protease (HIV-1PR). HIV-1PR cleavage of CARD8 induces pyroptotic cell death and the release of pro-inflammatory cytokines from infected cells, processes regulated by Toll-like receptor stimulation prior to viral infection. In acutely infected cells, CARD8 senses the activity of both de novo translated HIV-1PR and packaged HIV-1PR that is released from the incoming virion. Moreover, our evolutionary analyses reveal that the HIV-1PR cleavage site in human CARD8 arose after the divergence of chimpanzees and humans. Although chimpanzee CARD8 does not recognize proteases from HIV or simian immunodeficiency viruses from chimpanzees (SIVcpz), SIVcpz does cleave human CARD8, suggesting that SIVcpz was poised to activate the human CARD8 inflammasome prior to its cross-species transmission into humans. Our findings suggest a unique role for CARD8 inflammasome activation in response to lentiviral infection of humans.

Opioid abuse and SIV infection in non-human primates.

Human immunodeficiency virus (HIV) and drug abuse are intertwined epidemics, leading to compromised adherence to combined antiretroviral therapy (cART) and exacerbation of NeuroHIV. As opioid abuse causes increased viral replication and load, leading to a further compromised immune system in people living with HIV (PLWH), it is paramount to address this comorbidity to reduce the NeuroHIV pathogenesis. Non-human primates are well-suited models to study mechanisms involved in HIV neuropathogenesis and provide a better understanding of the underlying mechanisms involved in the comorbidity of HIV and drug abuse, leading to the development of more effective treatments for PLWH. Additionally, using broader behavioral tests in these models can mimic mild NeuroHIV and aid in studying other neurocognitive diseases without encephalitis. The simian immunodeficiency virus (SIV)-infected rhesus macaque model is instrumental in studying the effects of opioid abuse on PLWH due to its similarity to HIV infection. The review highlights the importance of using non-human primate models to study the comorbidity of opioid abuse and HIV infection. It also emphasizes the need to consider modifiable risk factors such as gut homeostasis and pulmonary pathogenesis associated with SIV infection and opioid abuse in this model. Moreover, the review suggests that these non-human primate models can also be used in developing effective treatment strategies for NeuroHIV and opioid addiction. Therefore, non-human primate models can significantly contribute to understanding the complex interplay between HIV infection, opioid abuse, and associated comorbidities.

Downregulation of CCR5 on brain perivascular macrophages in simian immunodeficiency virus-infected rhesus macaques.

BACKGROUND: C-C chemokine receptor 5 (CCR5) is a major coreceptor for Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) cell entry; however, its role in brain pathogenesis is largely understudied. Thus, we sought to examine cell type-specific protein expression of CCR5 during SIV infection of the brain. METHODS: We examined occipital cortical tissue from uninfected rhesus macaques and SIV-infected animals with or without encephalitis using immunohistochemistry and immunofluorescence microscopy to determine the number and distribution of CCR5-positive cells. RESULTS: An increase in the number of CCR5+ cells in the brain of SIV-infected animals with encephalitis was accounted for by increased CD3+CD8+ cells expressing CCR5, but not by increased CCR5+ microglia or perivascular macrophages (PVMs), and a concurrent decrease in the percentage of CCR5+ PVMs was observed. Levels of CCR5 and SIV Gag p28 protein expression were examined on a per-cell basis, and a significant, negative relationship was established indicating decreased CCR5 expression in productively infected cells. While investigating the endocytosis-mediated CCR5 internalization as a mechanism for CCR5 downregulation, we found that phospho-ERK1/2, an indicator of clathrin-mediated endocytosis, was colocalized with infected PVMs and that macrophages from infected animals showed significantly increased expression of clathrin heavy chain 1. CONCLUSIONS: These findings show a shift in CCR5-positive cell types in the brain during SIV pathogenesis with an increase in the number of CCR5+ CD8 T cells, and downregulated CCR5 expression on infected PVMs, likely through ERK1/2-driven, clathrin-mediated endocytosis.

Lymph-Node-Based CD3+ CD20+ Cells Emerge from Membrane Exchange between T Follicular Helper Cells and B Cells and Increase Their Frequency following Simian Immunodeficiency Virus Infection.

CD4+ T follicular helper (TFH) cells are key targets for human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) replication and contribute to the virus reservoir under antiretroviral therapy (ART). Here, we describe a novel CD3+ CD20+ double-positive (DP) lymphocyte subset, resident in secondary lymphoid organs of humans and rhesus macaques (RMs), that appear predominantly after membrane exchange between TFH and B cells. DP lymphocytes are enriched in cells displaying a TFH phenotype (CD4+ PD1hi CXCR5hi), function (interleukin 21 positive [IL-21+]), and gene expression profile. Importantly, expression of CD40L upon brief in vitro mitogen stimulation identifies, by specific gene-expression signatures, DP cells of TFH-cell origin versus those of B-cell origin. Analysis of 56 RMs showed that DP cells (i) significantly increase following SIV infection, (ii) are reduced after 12 months of ART in comparison to pre-ART levels, and (iii) expand to a significantly higher frequency following ART interruption. Quantification of total SIV-gag DNA on sorted DP cells from chronically infected RMs showed that these cells are susceptible to SIV infection. These data reinforce earlier observations that CD20+ T cells are infected and expanded by HIV infection, while suggesting that these cells phenotypically overlap activated CD4+ TFH cells that acquire CD20 expression via trogocytosis and can be targeted as part of therapeutic strategies aimed at HIV remission. IMPORTANCE The HIV reservoir is largely composed of latently infected memory CD4+ T cells that persist during antiretroviral therapy and constitute a major barrier toward HIV eradication. In particular, CD4+ T follicular helper cells have been demonstrated as key targets for viral replication and persistence under ART. In lymph nodes from HIV-infected humans and SIV-infected rhesus macaques, we show that CD3+ CD20+ lymphocytes emerge after membrane exchange between T cells and B cells and are enriched in phenotypic, functional, and gene expression profiles found in T follicular helper cells. Furthermore, in SIV-infected rhesus macaques, these cells expand following experimental infection and after interruption of ART and harbor SIV DNA at levels similar to those found in CD4+ T cells; thus, CD3+ CD20+ lymphocytes are susceptible to SIV infection and can contribute to SIV persistence.

Allogeneic immunity clears latent virus following allogeneic stem cell transplantation in SIV-infected ART-suppressed macaques.

Allogeneic hematopoietic stem cell transplantation (alloHSCT) from donors lacking C-C chemokine receptor 5 (CCR5Δ32/Δ32) can cure HIV, yet mechanisms remain speculative. To define how alloHSCT mediates HIV cure, we performed MHC-matched alloHSCT in SIV+, anti-retroviral therapy (ART)-suppressed Mauritian cynomolgus macaques (MCMs) and demonstrated that allogeneic immunity was the major driver of reservoir clearance, occurring first in peripheral blood, then peripheral lymph nodes, and finally in mesenteric lymph nodes draining the gastrointestinal tract. While allogeneic immunity could extirpate the latent viral reservoir and did so in two alloHSCT-recipient MCMs that remained aviremic >2.5 years after stopping ART, in other cases, it was insufficient without protection of engrafting cells afforded by CCR5-deficiency, as CCR5-tropic virus spread to donor CD4+ T cells despite full ART suppression. These data demonstrate the individual contributions of allogeneic immunity and CCR5 deficiency to HIV cure and support defining targets of alloimmunity for curative strategies independent of HSCT.