Nutrient metabolism influences HIV-1 replication, antiviral immunity, and chronic inflammation, yet is difficult to leverage for therapeutic gain. We sought to modulate metabolism in the non-human primate model of HIV-1 by caloric restriction (CR), a modality canonically known for its antiaging benefits. Four months of 30% CR was safe and resulted in broad and systemic metabolic reprogramming in healthy adult male and female rhesus macaques. Relative to that of ad libitum- fed animals, CR lowered the frequencies of target CCR5+ CD4 T cells in the gut mucosa. Upon infection with SIV, CR reduced acute phase viremia, dampened type I interferon signaling, and overall permitted a more vigorous cycling of CD8+ T cells in lymphoid tissues. CR-induced protection from SIV was associated with a robust up-regulation of glycolysis, which supported an early reduction in viremia that ultimately waned over time. During virologic suppression with antiretroviral therapy (ART), CR significantly limited gastrointestinal (GI) immune activation, improved tricarboxylic acid cycle flux, and lowered concentrations of soluble CD14 and several TNF-related molecules in plasma. Blood SIV DNA levels however were unchanged by CR, suggesting that residual GI dysfunction and inflammation can be decoupled from viral persistence. Our findings highlight that a dietary modality can limit pathology in a primate lentiviral infection. They also reveal the robust but temporally constrained nature of glycolysis in supporting an acute antiviral response. SIGNIFICANCE:Caloric restriction (CR) is a safe dietary intervention known to confer anti-aging and health benefits across diverse animal models. However, its application in the context of infectious diseases has yielded mixed outcomes and has largely been limited to murine systems. In this study, we therefore employed CR to examine the impact of dietary modulation on SIV infection outcomes. Our findings demonstrate that CR reduced acute-phase viremia and attenuated markers of chronic inflammation following ART, effects that were associated with distinct metabolic signatures. Collectively, these findings underscore the importance of diet and nutrition in shaping chronic viral infection outcomes, such as SIV, within a clinically relevant non-human primate model.

Cytomegalovirus (CMV) is a prevalent β-herpesvirus that persists asymptomatically in immunocompetent hosts. In people with HIV-1 (PWH), CMV is associated with HIV-1 persistence and particular inflammatory-related comorbidities. The true causative role of CMV in HIV-associated pathologies, however, remains unclear given that nearly all PWH are coinfected with CMV. In this study, we examined acute phase immune and virological dynamics in cohorts of SIV-infected rhesus macaques (RMs) that were naturally seropositive or -negative for rhesus CMV (RhCMV). We observed prior to SIV, RhCMV expanded a polyclonal population of target CCR5+CD4+ T cells in gut and lymph nodes that expressed the chemotactic receptor CXCR3 and were largely not specific for RhCMV. Upon SIV infection, RhCMV+ RMs exhibited higher peak viremia and elevated levels of SIV DNA in the upper and lower intestine. Greater seeding of SIV DNA was associated with a maintenance of CCR5-expressing CD4+ T cells that were enriched within the RhCMV+ gut along a CXCR3/CXCL9 chemotactic axis. Overall, the data suggest that RhCMV can promote SIV susceptibility within a diverse, polyclonal pool of CD4+ T cells that are not entirely RhCMV specific.

Nutrient metabolism influences HIV-1 replication, immunity, and chronic inflammation, yet it is challenging to target therapeutically. We hypothesized that modulating metabolism via caloric restriction (CR), a safe, anti-aging modality with anti-inflammatory effects could improve SIV disease outcome in rhesus macaques (RMs). Healthy, non-obese, Indian RMs (n = 6) were restricted to 30% fewer daily calories than their ad-libitum (AL) intake. After 3 months of CR, they were infected with SIVmac239, left untreated for 28 days, and then given ART for 11 months while maintaining CR. They were compared to age-, gender-, and weight-matched AL-fed RMs (n = 11). CR led to an approximately 1 log reduction in acute-phase viremia compared to the AL cohort, along with significantly greater memory CD8 T cell proliferative response at prominent tissue sites of SIV replication. Plasma metabolomics showed that CR induced a predominant glycolytic signature and 3 metabolites: spermidine, glucose-6-phosphate, and ADP strongly predicted lower plasma SIV AUC. Ex vivo glycolytic flux and mTORC1 activity were also higher in PBMCs and memory CD8 T cells at acute SIV, respectively. 11 months post-ART, CR cohort exhibited reduced levels of HLA-DR+ CD4 and CD8 memory T cells in the colon and lower plasma levels of the gut permeability marker sCD14. Improved gut health in CR was associated with elevated TCA-cycle intermediates. Thus, the data shows that CR has a profoundly positive impact on SIV pathogenesis. NIH R01 AI67644-01A1; NIH R21 AI74901-01; NIH R21 OD031229. Viral Immunology (VIR)

The persistence of the latent viral reservoir is the main hurdle to curing HIV-1 infection. SIV infection of non-human primates (NHPs), namely Indian-origin rhesus macaques, is the most relevant and widely used animal model to evaluate therapies that seek to eradicate HIV-1. The utility of a model ultimately rests on how accurately it can recapitulate human disease, and while reservoirs in the NHP model behave quantitatively very similar to those of long-term suppressed persons with HIV-1 (PWH) in the most salient aspects, recent studies have uncovered key nuances at the clonotypic level that differentiate the two in qualitative terms. In this review, we will highlight differences relating to proviral intactness, clonotypic structure, and decay rate during ART between HIV-1 and SIV reservoirs and discuss the relevance of these distinctions in the interpretation of HIV-1 cure strategies. While these, to some degree, may reflect a unique biology of the virus or host, distinctions among the proviral landscape in SIV are likely to be shaped significantly by the condensed timeframe of NHP studies. ART is generally initiated earlier in the disease course, and animals are virologically suppressed for shorter periods before receiving interventions. Because these are experimental variables dictated by the investigator, we offer guidance on study design for cure-related studies performed in the NHP model. Finally, we highlight the case of GS-9620 (Vesatolimod), an antiviral TLR7 agonist tested in multiple independent pre-clinical studies in which virological outcomes may have been influenced by study-related variables.

Hyperglycemia, and exacerbation of pre-existing deficits in glucose metabolism, are manifestations of the post-acute sequelae of SARS-CoV-2. Our understanding of metabolic decline after acute COVID-19 remains unclear due to the lack of animal models. Here, we report a non-human primate model of metabolic post-acute sequelae of SARS-CoV-2 using SARS-CoV-2 infected African green monkeys. Using this model, we identify a dysregulated blood chemokine signature during acute COVID-19 that correlates with elevated and persistent hyperglycemia four months post-infection. Hyperglycemia also correlates with liver glycogen levels, but there is no evidence of substantial long-term SARS-CoV-2 replication in the liver and pancreas. Finally, we report a favorable glycemic effect of the SARS-CoV-2 mRNA vaccine, administered on day 4 post-infection. Together, these data suggest that the African green monkey model exhibits important similarities to humans and can be utilized to assess therapeutic candidates to combat COVID-related metabolic defects.

ABSTRACT Human and simian immunodeficiency viruses (HIV and SIV) are lentiviruses that reverse transcribe their RNA genome with subsequent integration into the genome of the target cell. How progressive infection and administration of antiretrovirals (ARVs) longitudinally influence the transcriptomic and epigenetic landscape of particular T cell subsets, and how these may influence the genetic location of integration are unclear. Here, we use RNAseq and ATACseq to study the transcriptomics and epigenetic landscape of longitudinally sampled naïve and memory CD4+ and CD8+ T cells in two species of non-human primates prior to SIV infection, during chronic SIV infection, and after administration of ARVs. We find that SIV infection leads to significant alteration to the transcriptomic profile of all T cell subsets that are only partially reversed by administration of ARVs. Epigenetic changes were more apparent in animals with longer periods of untreated SIV infection and correlated well with changes in corresponding gene expression. Known SIV integration sites did not vary due to SIV status but did contain more open chromatin in rhesus macaque memory T cells, and the expression of proteasome-related genes at the pre-SIV timepoint correlated with subsequent viremia. IMPORTANCE Chronic inflammation during progressive human and simian immunodeficiency virus (HIV and SIV) infections leads to significant co-morbidities in infected individuals with significant consequences. Antiretroviral (ARV)-treated individuals also manifest increased levels of inflammation which are associated with increased mortalities. These data will help guide rational development of modalities to reduce inflammation observed in people living with HIV and suggest mechanisms underlying lentiviral integration site preferences.

Hyperglycemia, and exacerbation of pre-existing deficits in glucose metabolism, are major manifestations of the post-acute sequelae of SARS-CoV-2 (PASC). Our understanding of lasting glucometabolic disruptions after acute COVID-19 remains unclear due to the lack of animal models for metabolic PASC. Here, we report a non-human primate model of metabolic PASC using SARS-CoV-2 infected African green monkeys (AGMs). Using this model, we have identified a dysregulated chemokine signature and hypersensitive T cell population during acute COVID-19 that correlates with elevated and persistent hyperglycemia four months post-infection. This persistent hyperglycemia correlates with elevated hepatic glycogen, but there was no evidence of long-term SARS-CoV-2 replication in the liver and pancreas. Finally, we report a favorable glycemic effect of the SARS-CoV-2 mRNA vaccine, administered on day 4 post-infection. Together, these data suggest that the AGM metabolic PASC model exhibits important similarities to human metabolic PASC and can be utilized to assess therapeutic candidates to combat this syndrome.

IntroductionSevere COVID-19 results initially in pulmonary infection and inflammation. Symptoms can persist beyond the period of acute infection, and patients with Post-Acute Sequelae of COVID (PASC) often exhibit a variety of symptoms weeks or months following acute phase resolution including continued pulmonary dysfunction, fatigue, and neurocognitive abnormalities. We hypothesized that dysregulated NAD metabolism contributes to these abnormalities. MethodsRNAsequencing of lungs from transgenic mice expressing human ACE2 (K18-hACE2) challenged with SARS-CoV-2 revealed upregulation of NAD biosynthetic enzymes, including NAPRT1, NMNAT1, NAMPT, and IDO1 6 days post-infection. ResultsOur data also demonstrate increased gene expression of NAD consuming enzymes: PARP 9,10,14 and CD38. At the same time, SIRT1, a protein deacetylase (requiring NAD as a cofactor and involved in control of inflammation) is downregulated. We confirmed our findings by mining sequencing data from lungs of patients that died from SARS-CoV-2 infection. Our validated findings demonstrating increased NAD turnover in SARS-CoV-2 infection suggested that modulating NAD pathways may alter disease progression and may offer therapeutic benefits. Specifically, we hypothesized that treating K18-hACE2 mice with nicotinamide riboside (NR), a potent NAD precursor, may mitigate lethality and improve recovery from SARS-CoV-2 infection. We also tested the therapeutic potential of an anti- monomeric NAMPT antibody using the same infection model. Treatment with high dose anti-NAMPT antibody resulted in significantly decreased body weight compared to control, which was mitigated by combining HD anti-NAMPT antibody with NR. We observed a significant increase in lipid metabolites, including eicosadienoic acid, oleic acid, and palmitoyl carnitine in the low dose antibody + NR group. We also observed significantly increased nicotinamide related metabolites in NR treated animals. DiscussionOur data suggest that infection perturbs NAD pathways, identify novel mechanisms that may explain some pathophysiology of CoVID-19 and suggest novel strategies for both treatment and prevention.

African green monkeys (AGMs), Chlorocebus pygerythrus, are a natural host for a lentivirus related to HIV, SIV. SIV-infected AGMs rarely progress to AIDS despite robust viral replication. Though multiple mechanisms are involved, a primary component is the animals' ability to downregulate CD4 expression on mature CD4+ Th cells, rendering these cells resistant to infection by SIV. These CD8αα+ T cells retain functional characteristics of CD4+ Th cells while simultaneously acquiring abilities of cytotoxic CD8αβ+ T cells. To determine mechanisms underlying functional differences between T cell subsets in AGMs, chromatin accessibility in purified populations was determined by assay for transposase-accessible chromatin sequencing. Differences in chromatin accessibility alone were sufficient to cluster cells by subtype, and accessibility at the CD4 locus reflected changes in CD4 expression. DNA methylation at the CD4 locus also correlated with inaccessible chromatin. By associating accessible regions with nearby genes, gene expression was found to correlate with accessibility changes. T cell and immune system activation pathways were identified when comparing regions that changed accessibility from CD4+ T cells to CD8αα+ T cells. Different transcription factor binding sites are revealed as chromatin accessibility changes, and these differences may elicit downstream changes in differentiation. This comprehensive description of the epigenetic landscape of AGM T cells identified genes and pathways that could have translational value in therapeutic approaches recapitulating the protective effects CD4 downregulation.

Invariant natural killer T-lymphocytes (iNKT) are unique immunomodulatory innate T cells with an invariant TCRα recognizing glycolipids presented on MHC class-I-like CD1d molecules. Activated iNKT rapidly secrete pro-and anti-inflammatory cytokines, potentiate immunity, and modulate inflammation. Here, we report the effects of in vivo iNKT activation in Mauritian-origin cynomolgus macaques by a humanized monoclonal antibody, NKTT320, that binds to the invariant region of the iNKT TCR. NKTT320 led to rapid iNKT activation, increased polyfunctionality, and elevation of multiple plasma analytes within 24 hours. Flow cytometry and RNA-Seq confirmed downstream activation of multiple immune subsets, enrichment of JAK/STAT and PI3K/AKT pathway genes, and upregulation of inflammation-modulating genes. NKTT320 also increased iNKT frequency in adipose tissue and did not cause iNKT anergy. Our data indicate that NKTT320 has a sustained effect on in vivo iNKT activation, potentiation of innate and adaptive immunity, and resolution of inflammation, which supports its future use as an immunotherapeutic.

Rationale: Pulmonary vascular endotheliitis, perivascular inflammation, and immune activation are observed in COVID-19 patients. While the initial SARS-CoV-2 infection mainly infects lung epithelial cells, whether it also infects endothelial cells (ECs) and to what extent SARS-CoV-2-mediated pulmonary vascular endotheliitis is associated with immune activation remain to be determined. Methods: To address these questions, we studied SARS-CoV-2-infected K18-hACE2 (K18) mice, a severe COVID-19 mouse model, as well as lung samples from SARS-CoV-2-infected nonhuman primates (NHP) and patient deceased from COVID-19. We used immunostaining, RNAscope, and electron microscopy to analyze the organs collected from animals and patient. We conducted bulk and single cell (sc) RNA-seq analyses, and cytokine profiling of lungs or serum of the severe COVID-19 mice. Results: We show that SARS-CoV-2-infected K18 mice develop severe COVID-19, including progressive body weight loss and fatality at 7 days, severe lung interstitial inflammation, edema, hemorrhage, perivascular inflammation, systemic lymphocytopenia, and eosinopenia. Body weight loss in K18 mice correlated with the severity of pneumonia, but not with brain infection. We also observed endothelial activation and dysfunction in pulmonary vessels evidenced by the up-regulation of VCAM1 and ICAM1 and the downregulation of VE-cadherin. We detected SARS-CoV-2 in capillary ECs, activation and adhesion of platelets and immune cells to the vascular wall of the alveolar septa, and increased complement deposition in the lungs, in both COVID-19-murine and NHP models. We also revealed that pathways of coagulation, complement, K-ras signaling, and genes of ICAM1 and VCAM1 related to EC dysfunction and injury were upregulated, and were associated with massive immune activation in the lung and circulation. Conclusion: Together, our results indicate that SARS-CoV-2 causes endotheliitis via both infection and infection-mediated immune activation, which may contribute to the pathogenesis of severe COVID-19 disease.

Although many HIV cure strategies seek to expand HIV-specific CD8+ T cells to control the virus, all are likely to fail if cellular exhaustion is not prevented. A loss in stem-like memory properties (i.e., the ability to proliferate and generate secondary effector cells) is a key feature of exhaustion; little is known, however, about how these properties are regulated in human virus–specific CD8+ T cells. We found that virus-specific CD8+ T cells from humans and nonhuman primates naturally controlling HIV/SIV infection express more of the transcription factor TCF-1 than noncontrollers. HIV-specific CD8+ T cell TCF-1 expression correlated with memory marker expression and expansion capacity and declined with antigenic stimulation. CRISPR-Cas9 editing of TCF-1 in human primary T cells demonstrated a direct role in regulating expansion capacity. Collectively, these data suggest that TCF-1 contributes to the regulation of the stem-like memory property of secondary expansion capacity of HIV-specific CD8+ T cells, and they provide a rationale for exploring the enhancement of this pathway in T cell–based therapeutic strategies for HIV.

Understanding SARS-CoV-2 immune pathology is critical for the development of effective vaccines and treatments. Here, we employed unbiased serial whole-blood transcriptome profiling by weighted gene network correlation analysis (WGCNA) at pre-specified timepoints of infection to understand SARS-CoV-2-related immune alterations in a cohort of rhesus macaques (RMs) and African green monkeys (AGMs) presenting with varying degrees of pulmonary pathology. We found that the bulk of transcriptional changes occurred at day 3 post-infection and normalized to pre-infection levels by 3 weeks. There was evidence of coordination of transcriptional networks in blood (defined by WGCNA) and the nasopharyngeal SARS-CoV-2 burden as well as the absolute monocyte count. Pathway analysis of gene modules revealed prominent regulation of type I and type II interferon stimulated genes (ISGs) in both RMs and AGMs, with the latter species exhibiting a greater breadth of ISG upregulation. Notably, pathways relating to neutrophil degranulation were enriched in blood of SARS-CoV-2 infected AGMs, but not RMs. Our results elude to hallmark similarities as well as differences in the RM and AGM acute response to SARS-CoV-2 infection, and may help guide the selection of particular NHP species in modeling aspects of COVID-19 disease outcome.

Invariant natural killer T-lymphocytes (iNKT) are a unique subset of immunomodulatory innate T-cells with an invariant TCRα chain recognizing glycolipids presented on the MHC class-I-like CD1d molecule. Activated iNKT rapidly secrete pro-and anti-inflammatory cytokines, potentiate innate and adaptive immunity, and modulate inflammation. While iNKT activation by glycolipid agonists are being explored as an adjuvant, their use depends on CD1d-restricted antigen presentation. Here, we report the effects of iNKT activation by a novel humanized monoclonal antibody, NKTT320, that binds to the invariant region of the iNKT TCR. A single dose of NKTT320 led to rapid iNKT activation, increased polyfunctionality, and elevation of multiple pro-inflammatory and chemotactic plasma analytes within 24 hours in cynomolgus macaques. Flow cytometry and RNA-Seq confirmed downstream effects of NKTT320 on multiple immune cell subsets. Inflammatory response, JAK/STAT and PI3K/AKT pathway genes were enriched along with upregulation of the inflammation-modulating genes CMKLR1, ARG2 and NLRP12. Finally, NKTT320 induced iNKT trafficking to adipose tissue and did not cause iNKT anergy. Our data indicate that NKTT320 has a sustained effect on in vivo iNKT activation, potentiation of innate and adaptive immunity, and resolution of inflammation, properties that support its future application as an immunotherapeutic and vaccine adjuvant.

SARS-CoV-2 infection can cause fatal inflammatory lung pathology, including thrombosis and increased pulmonary vascular permeability leading to edema and hemorrhage. In addition to the lung, cytokine storm-induced inflammatory cascade also affects other organs. SARS-CoV-2 infection-related vascular inflammation is characterized by endotheliopathy in the lung and other organs. Whether SARS-CoV-2 causes endotheliopathy by directly infecting endothelial cells is not known and is the focus of the present study. We observed 1) the co-localization of SARS-CoV-2 with the endothelial cell marker CD31 in the lungs of SARS-CoV-2-infected mice expressing hACE2 in the lung by intranasal delivery of adenovirus 5-hACE2 (Ad5-hACE2 mice) and non-human primates at both the protein and RNA levels, and 2) SARS-CoV-2 proteins in endothelial cells by immunogold labeling and electron microscopic analysis. We also detected the co-localization of SARS-CoV-2 with CD31 in autopsied lung tissue obtained from patients who died from severe COVID-19. Comparative analysis of RNA sequencing data of the lungs of infected Ad5-hACE2 and Ad5-empty (control) mice revealed upregulated KRAS signaling pathway, a well-known pathway for cellular activation and dysfunction. Further, we showed that SARS-CoV-2 directly infects mature mouse aortic endothelial cells (AoECs) that were activated by performing an aortic sprouting assay prior to exposure to SARS-CoV-2. This was demonstrated by co-localization of SARS-CoV-2 and CD34 by immunostaining and detection of viral particles in electron microscopic studies. Moreover, the activated AoECs became positive for ACE-2 but not quiescent AoECs. Together, our results indicate that in addition to pneumocytes, SARS-CoV-2 also directly infects mature vascular endothelial cells in vivo and ex vivo, which may contribute to cardiovascular complications in SARS-CoV-2 infection, including multipleorgan failure.

African green monkeys (AGMs) are natural hosts of Simian immunodeficiency virus (SIV) that post-thymically downregulate CD4 to maintain a large population of CD4−CD8aa+ virus-resistant T cells which retain T-helper functionality. AGMs can become aviremic and apparently cured of SIV by down-regulating CD4 to completion. To understand the mechanisms of this process, purified CD4+ T cells from four AGMs, closely-related Patas monkeys, and rhesus macaques were stimulated with SEB for 5 days and RNAseq was performed on divided cells induced to downregulate CD4 (AGM, Patas) and those that maintain CD4 expression (rhesus). 1,917 DEGs were revealed to be common among divided, CD4-downregulated AGM and Patas T cells, yet unique from divided rhesus CD4+ T cells. Genes well-known to be regulated in natural hosts were selectively present in this dataset, including CD4, CD8A, and CXCR6 (p= 1.27e−27, 2.68e−5, 6.72e−15, respectively). Pathway analysis of DEGs revealed proteins involved in DNA methylation to be enriched in CD4-downregulated AGM and Patas T cells (p=0.013). Inhibition of DNA methyltransferases (DNMT) with 5-aza-2 deoxycitidine inhibited CD4 downregulation in AGM CD4+ T cells induced to divide in vitro (p=0.005), indicating CD4 can be pharmacologically manipulated in natural hosts. Cytosine residues within the AGM CD4 promoter region became methylated during CD4 downregulation in vitro (p= < 0.0001), and were stably inherited in AGM CD4−CD8aa+ T cells sorted directly ex vivo. These results suggest AGMs employ epigenetic mechanisms to durably silence the CD4 gene. Manipulation of these mechanisms could provide avenues for modulating SIV/HIV-1 entry receptor expression in hosts that become progressively HIV-1/SIV-infected.

HIV infection is associated with an increase in the proportion of activated CD8(+) memory T cells (Tmem) that express CX3CR1, but how these cells are generated and maintained in vivo is unclear. We demonstrate that increased CX3CR1 expression on CD8(+) Tmem in people living with HIV (PLWH) is dependent on coinfection with human CMV, and CX3CR1(+)CD8(+) Tmem are enriched for a putatively immunosenescent CO57(+)C1328(-) phenotype. The cytokine IL-15 promotes the phenotype, survival, and proliferation of CX3CR1(+)VD57(+)CD8(+) Tmem in vitro, whereas T cell receptor stimulation leads to their death. IL-15-driven survival is dependent on STATS and Bcl-2 activity, and IL-15-induced proliferation requires STATS and mTORC1. Thus, we identify mechanistic pathways that could explain how "inflammescent" CX3CR1(+)CD57(+)CD8(+) Tmem dominate the overall memory T cell pool in CMV-seropositive PLWH and that support reevaluation of immune senescence as a nonproliferative dead end.

African green monkeys (AGMs) are natural hosts of SIV that postthymically downregulate CD4 to maintain a large population of CD4–CD8aa+ virus-resistant cells with Th functionality, which can result in AGMs becoming apparently cured of SIVagm infection. To understand the mechanisms of this process, we performed genome-wide transcriptional analysis on T cells induced to downregulate CD4 in vitro from AGMs and closely related patas monkeys and T cells that maintain CD4 expression from rhesus macaques. In T cells that downregulated CD4, pathway analysis revealed an atypical regulation of the DNA methylation machinery, which was reversible when pharmacologically targeted with 5-aza-2 deoxycytidine. This signature was driven largely by the dioxygenase TET3, which became downregulated with loss of CD4 expression. CpG motifs within the AGM CD4 promoter region became methylated during CD4 downregulation in vitro and were stably imprinted in AGM CD4–CD8aa+ T cells sorted directly ex vivo. These results suggest that AGMs use epigenetic mechanisms to durably silence the CD4 gene. Manipulation of these mechanisms could provide avenues for modulating SIV and HIV-1 entry receptor expression in hosts that become progressively infected with SIV, which could lead to novel therapeutic interventions aimed to reduce HIV viremia in vivo.

CD8+ T cell responses are necessary for immune control of simian immunodeficiency virus (SIV). However, the key parameters that dictate antiviral potency remain elusive, conceivably because most studies to date have been restricted to analyses of circulating CD8+ T cells. We conducted a detailed clonotypic, functional, and phenotypic survey of SIV-specific CD8+ T cells across multiple anatomical sites in chronically infected rhesus macaques with high (>10,000 copies/mL plasma) or low burdens of viral RNA (<10,000 copies/mL plasma). No significant differences in response magnitude were identified across anatomical compartments. Rhesus macaques with low viral loads (VLs) harbored higher frequencies of polyfunctional CXCR5+ SIV-specific CD8+ T cells in various lymphoid tissues and higher proportions of unique Gag-specific CD8+ T cell clonotypes in the mesenteric lymph nodes relative to rhesus macaques with high VLs. In addition, public Gag-specific CD8+ T cell clonotypes were more commonly shared across distinct anatomical sites than the corresponding private clonotypes, which tended to form tissue-specific repertoires, especially in the peripheral blood and the gastrointestinal tract. Collectively, these data suggest that functionality and tissue localization are important determinants of CD8+ T cell-mediated efficacy against SIV.

Although many HIV cure strategies seek to expand HIV-specific CD8+ T cells to control the virus, all are likely to fail if cellular exhaustion is not prevented. A loss in stem-like memory properties (i.e., the ability to proliferate and generate secondary effector cells) is a key feature of exhaustion; little is known, however, about how these properties are regulated in human virus-specific CD8+ T cells. We found that virus-specific CD8+ T cells from humans and non-human primates naturally controlling HIV/SIV infection express more of the transcription factor, TCF-1, than non-controllers. HIV-specific CD8+ T cell TCF-1 expression correlated with memory marker expression and proliferative capacity and declined with antigenic stimulation. CRISPR-Cas9 editing of TCF-1 in human primary T cells demonstrated a direct role in regulating expansion capacity. Collectively, these data suggest that TCF-1 controls the stem-like memory properties of HIV-specific CD8+ T cells and provides a rationale for enhancing this pathway in T cell-based therapeutic strategies for HIV. One Sentence Summary TCF-1 is highly expressed in HIV-specific CD8+ T cells from elite controllers and directly regulates human CD8+ T cell expansion capacity in response to T cell receptor stimulation.