Targeting Th17 cells in HIV-1 remission/cure interventions.

Since the discovery of HIV-1, progress has been made in deciphering the viral replication cycle and mechanisms of host-pathogen interactions that has facilitated the implementation of effective antiretroviral therapies (ARTs). Major barriers to HIV-1 remission/cure include the persistence of viral reservoirs (VRs) in long-lived CD4+ T cells, residual viral transcription, and lack of mucosal immunity restoration during ART, which together fuel systemic inflammation. Recently, T helper (Th)17-polarized cells were identified as major contributors to the pool of transcriptionally/translationally competent VRs. In this review, we discuss the functional features of Th17 cells that were elucidated by fundamental immunology studies in the context of autoimmunity. We also highlight recent discoveries supporting the possibility of extrapolating this knowledge toward the identification of new putative Th17-targeted HIV-1 remission/cure strategies.

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.

Th17 cell master transcription factor RORC2 regulates HIV-1 gene expression and viral outgrowth.

Among CD4+ T cells, T helper 17 (Th17) cells are particularly susceptible to HIV-1 infection and are depleted from mucosal sites, which causes damage to the gut barrier, resulting in a microbial translocation-induced systemic inflammation, a hallmark of disease progression. Furthermore, a proportion of latently infected Th17 cells persist long term in the gastrointestinal lymphatic tract where a low-level HIV-1 transcription is observed. This residual viremia contributes to chronic immune activation. Thus, Th17 cells are key players in HIV pathogenesis and viral persistence. It is, however, unclear why these cells are highly susceptible to HIV-1 infection. Th17 cell differentiation depends on the expression of the master transcriptional regulator RORC2, a retinoic acid-related nuclear hormone receptor that regulates specific transcriptional programs by binding to promoter/enhancer DNA. Here, we report that RORC2 is a key host cofactor for HIV replication in Th17 cells. We found that specific inhibitors that bind to the RORC2 ligand-binding domain reduced HIV replication in CD4+ T cells. The depletion of RORC2 inhibited HIV-1 infection, whereas its overexpression enhanced it. RORC2 was also found to promote HIV-1 gene expression by binding to the nuclear receptor responsive element in the HIV-1 long terminal repeats (LTR). In treated HIV-1 patients, RORC2+ CD4 T cells contained more proviral DNA than RORC2- cells. Pharmacological inhibition of RORC2 potently reduced HIV-1 outgrowth in CD4+ T cells from antiretroviral-treated patients. Altogether, these results provide an explanation as to why Th17 cells are highly susceptible to HIV-1 infection and suggest that RORC2 may be a cell-specific target for HIV-1 therapy.

Daily variations of gut microbial translocation markers in ART-treated HIV-infected people.

BACKGROUND: Increased intestinal barrier permeability and subsequent gut microbial translocation are significant contributors to inflammatory non-AIDS comorbidities in people living with HIV (PLWH). Evidence in animal models have shown that markers of intestinal permeability and microbial translocation vary over the course of the day and are affected by food intake and circadian rhythms. However, daily variations of these markers are not characterized yet in PLWH. Herein, we assessed the variation of these markers over 24 h in PLWH receiving antiretroviral therapy (ART) in a well-controlled environment. METHODS: As in Canada, PLWH are predominantly men and the majority of them are now over 50 years old, we selected 11 men over 50 receiving ART with undetectable viremia for more than 3 years in this pilot study. Blood samples were collected every 4 h over 24 h before snacks/meals from 8:00 in the morning to 8:00 the next day. All participants consumed similar meals at set times, and had a comparable amount of sleep, physical exercise and light exposure. Plasma levels of bacterial lipopolysaccharide (LPS) and fungal (1→3)-ß-D-Glucan (BDG) translocation markers, along with markers of intestinal damage fatty acid binding protein (I-FABP) and regenerating islet-derived protein-3α (REG3α) were assessed by ELISA or the fungitell assay. RESULTS: Participants had a median age of 57 years old (range 50 to 63). Plasma levels of BDG and REG3α did not vary significantly over the course of the study. In contrast, a significant increase of LPS was detected between 12:00 and 16:00 (Z-score: - 1.15 ± 0.18 vs 0.16 ± 0.15, p = 0.02), and between 12:00 and 24:00 (- 1.15 ± 0.18 vs 0.89 ± 0.26, p < 0.001). The plasma levels of I-FABP at 16:00 (- 0.92 ± 0.09) were also significantly lower, compared to 8:00 the first day (0.48 ± 0.26, p = 0.002), 4:00 (0.73 ± 0.27, p < 0.001) or 8:00 on secondary day (0.88 ± 0.27, p < 0.001). CONCLUSIONS: Conversely to the fungal translocation marker BDG and the gut damage marker REG3α, time of blood collection matters for the proper evaluation for LPS and I-FABP as markers for the risk of inflammatory non-AIDS co-morbidities. These insights are instrumental for orienting clinical investigations in PLWH.

Alterations in Th17 Cells and Non-Classical Monocytes as a Signature of Subclinical Coronary Artery Atherosclerosis during ART-Treated HIV-1 Infection.

Cardiovascular disease (CVD) remains an important comorbidity in people living with HIV-1 (PLWH) receiving antiretroviral therapy (ART). Our previous studies performed in the Canadian HIV/Aging Cohort Study (CHACS) (>40 years-old; Framingham Risk Score (FRS) > 5%) revealed a 2-3-fold increase in non-calcified coronary artery atherosclerosis (CAA) plaque burden, measured by computed tomography angiography scan (CTAScan) as the total (TPV) and low attenuated plaque volume (LAPV), in ART-treated PLWH (HIV+) versus uninfected controls (HIV-). In an effort to identify novel correlates of subclinical CAA, markers of intestinal damage (sCD14, LBP, FABP2); cell trafficking/inflammation (CCL20, CX3CL1, MIF, CCL25); subsets of Th17-polarized and regulatory (Tregs) CD4+ T-cells, classical/intermediate/non-classical monocytes, and myeloid/plasmacytoid dendritic cells were studied in relationship with HIV and TPV/LAPV status. The TPV detection/values coincided with higher plasma sCD14, FABP2, CCL20, MIF, CX3CL1, and triglyceride levels; lower Th17/Treg ratios; and classical monocyte expansion. Among HIV+, TPV+ versus TPV- exhibited lower Th17 frequencies, reduced Th17/Treg ratios, higher frequencies of non-classical CCR9lowHLADRhigh monocytes, and increased plasma fibrinogen levels. Finally, Th17/Treg ratios and non-classical CCR9lowHLADRhigh monocyte frequencies remained associated with TPV/LAPV after adjusting for FRS and HIV/ART duration in a logistic regression model. These findings point to Th17 paucity and non-classical monocyte abundance as novel immunological correlates of subclinical CAA that may fuel the CVD risk in ART-treated PLWH.

Retinoic acid enhances HIV-1 reverse transcription and transcription in macrophages via mTOR-modulated mechanisms.

The intestinal environment facilitates HIV-1 infection via mechanisms involving the gut-homing vitamin A-derived retinoic acid (RA), which transcriptionally reprograms CD4+ T cells for increased HIV-1 replication/outgrowth. Consistently, colon-infiltrating CD4+ T cells carry replication-competent viral reservoirs in people with HIV-1 (PWH) receiving antiretroviral therapy (ART). Intriguingly, integrative infection in colon macrophages, a pool replenished by monocytes, represents a rare event in ART-treated PWH, thus questioning the effect of RA on macrophages. Here, we demonstrate that RA enhances R5 but not X4 HIV-1 replication in monocyte-derived macrophages (MDMs). RNA sequencing, gene set variation analysis, and HIV interactor NCBI database interrogation reveal RA-mediated transcriptional reprogramming associated with metabolic/inflammatory processes and HIV-1 resistance/dependency factors. Functional validations uncover post-entry mechanisms of RA action including SAMHD1-modulated reverse transcription and CDK9/RNA polymerase II (RNAPII)-dependent transcription under the control of mammalian target of rapamycin (mTOR). These results support a model in which macrophages residing in the intestine of ART-untreated PWH contribute to viral replication/dissemination in an mTOR-sensitive manner.

Identification of aryl hydrocarbon receptor as a barrier to HIV-1 infection and outgrowth in CD4+ T cells.

The aryl hydrocarbon receptor (AhR) regulates Th17-polarized CD4+ T cell functions, but its role in HIV-1 replication/outgrowth remains unknown. Genetic (CRISPR-Cas9) and pharmacological inhibition reveal AhR as a barrier to HIV-1 replication in T cell receptor (TCR)-activated CD4+ T cells in vitro. In single-round vesicular stomatitis virus (VSV)-G-pseudotyped HIV-1 infection, AhR blockade increases the efficacy of early/late reverse transcription and subsequently facilitated integration/translation. Moreover, AhR blockade boosts viral outgrowth in CD4+ T cells of people living with HIV-1 (PLWH) receiving antiretroviral therapy (ART). Finally, RNA sequencing reveals genes/pathways downregulated by AhR blockade in CD4+ T cells of ART-treated PLWH, including HIV-1 interactors and gut-homing molecules with AhR-responsive elements in their promoters. Among them, HIC1, a repressor of Tat-mediated HIV-1 transcription and a tissue-residency master regulator, is identified by chromatin immunoprecipitation as a direct AhR target. Thus, AhR governs a T cell transcriptional program controlling viral replication/outgrowth and tissue residency/recirculation, supporting the use of AhR inhibitors in "shock and kill" HIV-1 remission/cure strategies.

A Blood Immunological Signature of Subclinical Coronary Artery Atherosclerosis in People Living with HIV-1 Receiving Antiretroviral Therapy.

Cardiovascular disease (CVD) remains an important co-morbidity in people living with HIV-1 (PLWH) receiving antiretroviral therapy (ART). Our previous studies performed on the Canadian HIV/Aging Cohort Study (CHACS) (>40 years-old; Framingham Risk Score (FRS) >5%), revealed a 2-3-fold increase in non-calcified coronary artery atherosclerosis (CAA) plaque burden, measured by Computed tomography angiography scan (CTAScan) as total (TPV) and low attenuated plaque volume (LAPV) in ART-treated PLWH (HIV+) versus uninfected controls (HIV-). In an effort to identify novel correlates of subclinical CAA, markers of intestinal damage (sCD14, LBP, FABP2); cell trafficking/inflammation (CCL20, CX3CL1, MIF, CCL25); subsets of Th17-polarized and regulatory (Tregs) CD4 + T-cells, classical/intermediate/non-classical monocytes, and myeloid/plasmacytoid dendritic cells, were studied in relationship with HIV and TPV/LAPV status. The TPV detection/values coincided with higher plasma sCD14, FABP2, CCL20, MIF, CX3CL1 and triglyceride levels, lower Th17/Treg ratios, and classical monocyte expansion. Among HIV + , TPV + versus TPV - exhibited lower Th17 frequencies, reduced Th17/Treg ratios, higher frequencies of non-classical CCR9 low HLADR high monocyte, and increased plasma fibrinogen levels. Finally, Th17/Treg ratios and non-classical CCR9 low HLADR high monocyte frequencies remained associated with TPV/LAPV after adjusting for FRS and HIV/ART duration in a logistic regression model. These findings point to Th17 paucity and non-classical monocyte abundance as novel immunological correlates of subclinical CAA that may fuel the CVD risk in ART-treated PLWH.

Near full-length HIV sequencing in multiple tissues collected postmortem reveals shared clonal expansions across distinct reservoirs during ART.

HIV persists in tissues during antiretroviral therapy (ART), but the relative contribution of different anatomical compartments to the viral reservoir in humans remains unknown. We performed an extensive characterization of HIV reservoirs in two men who donated their bodies to HIV cure research and who had been on suppressive ART for years. HIV DNA is detected in all tissues, with large variations across anatomical compartments and between participants. Intact HIV genomes represent 2% and 25% of all proviruses in the two participants and are mainly detected in secondary lymphoid organs, with the spleen and mediastinal lymph nodes harboring intact viral genomes in both individuals. Multiple copies of identical HIV genomes are found in all tissues, indicating that clonal expansions are common in anatomical sites. The majority (>85%) of these expanded clones are shared across multiple tissues. These findings suggest that infected cells expand, migrate, and possibly circulate between anatomical sites.

Overt IL-32 isoform expression at intestinal level during HIV-1 infection is negatively regulated by IL-17A.

OBJECTIVES: Untreated HIV infection was previously associated with IL-32 overexpression in gut/intestinal epithelial cells (IEC). Here, we explored IL-32 isoform expression in the colon of people with HIV (PWH) receiving antiretroviral therapy (ART) and IL-32 triggers/modulators in IEC. DESIGN: Sigmoid colon biopsies (SCB) and blood were collected from ART-treated PWH (HIV + ART; n = 17; mean age: 56 years; CD4+ cell counts: 679 cells/µl; time on ART: 72 months) and age-matched HIV-uninfected controls (HIVneg; n = 5). The IEC line HT-29 was used for mechanistic studies. METHODS: Cells from SCB and blood were isolated by enzymatic digestion and/or gradient centrifugation. HT-29 cells were exposed to TLR1-9 agonists, TNF-α, IL-17A and HIV. IL-32α/ß/γ/D/ε/θ and IL-17A mRNA levels were quantified by real-time RT-PCR. IL-32 protein levels were quantified by ELISA. RESULTS: IL-32ß/γ/ε isoform transcripts were detectable in the blood and SCB, with IL-32ß mRNA levels being predominantly expressed in both compartments and at significantly higher levels in HIV + ART compared to HIVneg. IL-17A transcripts were only detectable in SCB, with increased IL-17A levels in HIVneg compared with HIV + ART and negatively correlated with IL-32ß mRNA levels. IL-32ß/γ/ε isoform mRNA were detected in HT-29 cells upon exposure to TNF-α, Poly I:C (TLR3 agonist), Flagellin (TLR-5 agonist) and HIV. IL-17A significantly decreased both IL-32 ß/γ/ε mRNA and cell-associated IL-32 protein levels induced upon TNF-α and Poly I:C triggering. CONCLUSION: We document IL-32 isoforms abundant in the colon of ART-treated PWH and reveal the capacity of the Th17 hallmark cytokine IL-17A to attenuate IL-32 overexpression in a model of inflamed IEC.

IL-17A reprograms intestinal epithelial cells to facilitate HIV-1 replication and outgrowth in CD4+ T cells.

The crosstalk between intestinal epithelial cells (IECs) and Th17-polarized CD4+ T cells is critical for mucosal homeostasis, with HIV-1 causing significant alterations in people living with HIV (PLWH) despite antiretroviral therapy (ART). In a model of IEC and T cell co-cultures, we investigated the effects of IL-17A, the Th17 hallmark cytokine, on IEC ability to promote de novo HIV infection and viral reservoir reactivation. Our results demonstrate that IL-17A acts in synergy with TNF to boost IEC production of CCL20, a Th17-attractant chemokine, and promote HIV trans-infection of CD4+ T cells and viral outgrowth from reservoir cells of ART-treated PLWH. Importantly, the Illumina RNA-sequencing revealed an IL-17A-mediated pro-inflammatory and pro-viral molecular signature, including a decreased expression of type I interferon (IFN-I)-induced HIV restriction factors. These findings point to the deleterious features of IL-17A and raise awareness for caution when designing therapies aimed at restoring the paucity of mucosal Th17 cells in ART-treated PLWH.

Pharmacological Inhibition of PPARy Boosts HIV Reactivation and Th17 Effector Functions, While Preventing Progeny Virion Release and de novo Infection.

The frequency and functions of Th17-polarized CCR6+RORyt+CD4+ T cells are rapidly compromised upon HIV infection and are not restored with long-term viral suppressive antiretroviral therapy (ART). In line with this, Th17 cells represent selective HIV-1 infection targets mainly at mucosal sites, with long-lived Th17 subsets carrying replication-competent HIV-DNA during ART. Therefore, novel Th17-specific therapeutic interventions are needed as a supplement of ART to reach the goal of HIV remission/cure. Th17 cells express high levels of peroxisome proliferator-activated receptor gamma (PPARy), which acts as a transcriptional repressor of the HIV provirus and the rorc gene, which encodes for the Th17-specific master regulator RORyt. Thus, we hypothesized that the pharmacological inhibition of PPARy will facilitate HIV reservoir reactivation while enhancing Th17 effector functions. Consistent with this prediction, the PPARy antagonist T0070907 significantly increased HIV transcription (cell-associated HIV-RNA) and RORyt-mediated Th17 effector functions (IL-17A). Unexpectedly, the PPARy antagonism limited HIV outgrowth from cells of ART-treated people living with HIV (PLWH), as well as HIV replication in vitro. Mechanistically, PPARy inhibition in CCR6+CD4+ T cells induced the upregulation of transcripts linked to Th17-polarisation (RORyt, STAT3, BCL6 IL-17A/F, IL-21) and HIV transcription (NCOA1-3, CDK9, HTATIP2). Interestingly, several transcripts involved in HIV-restriction were upregulated (Caveolin-1, TRIM22, TRIM5α, BST2, miR-29), whereas HIV permissiveness transcripts were downregulated (CCR5, furin), consistent with the decrease in HIV outgrowth/replication. Finally, PPARy inhibition increased intracellular HIV-p24 expression and prevented BST-2 downregulation on infected T cells, suggesting that progeny virion release is restricted by BST-2-dependent mechanisms. These results provide a strong rationale for considering PPARy antagonism as a novel strategy for HIV-reservoir purging and restoring Th17-mediated mucosal immunity in ART-treated PLWH.

HIV-1 is rarely detected in blood and colon myeloid cells during viral-suppressive antiretroviral therapy.

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

Host MicroRNAs-221 and -222 Inhibit HIV-1 Entry in Macrophages by Targeting the CD4 Viral Receptor.

Macrophages are heterogeneous immune cells with distinct origins, phenotypes, functions, and tissue localization. Their susceptibility to HIV-1 is subject to variations from permissiveness to resistance, owing in part to regulatory microRNAs. Here, we used RNA sequencing (RNA-seq) to examine the expression of >400 microRNAs in productively infected and bystander cells of HIV-1-exposed macrophage cultures. Two microRNAs upregulated in bystander macrophages, miR-221 and miR-222, were identified as negative regulators of CD4 expression and CD4-mediated HIV-1 entry. Both microRNAs were enhanced by tumor necrosis factor alpha (TNF-α), an inhibitor of CD4 expression. MiR-221/miR-222 inhibitors recovered HIV-1 entry in TNF-α-treated macrophages by enhancing CD4 expression and increased HIV-1 replication and spread in macrophages by countering TNF-α-enhanced miR-221/miR-222 expression in bystander cells. In line with these findings, HIV-1-resistant intestinal myeloid cells express higher levels of miR-221 than peripheral blood monocytes. Thus, miR-221/miR-222 act as effectors of the antiviral host response activated during macrophage infection that restrict HIV-1 entry.

HIV persists in CCR6+CD4+ T cells from colon and blood during antiretroviral therapy.

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