Differentiation of IL-17−Producing Effector and Regulatory Human T Cells from Lineage-Committed Naive Precursors This information is current as of June 27, 2014.

Frances Mercer, Alka Khaitan, Lina Kozhaya, Judith A. Aberg and Derya Unutmaz

Supplementary Material

http://www.jimmunol.org/content/suppl/2014/06/22/jimmunol.130293 6.DCSupplemental.html

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994. Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.

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J Immunol published online 23 June 2014 http://www.jimmunol.org/content/early/2014/06/22/jimmun ol.1302936

Published June 23, 2014, doi:10.4049/jimmunol.1302936 The Journal of Immunology

Differentiation of IL-17–Producing Effector and Regulatory Human T Cells from Lineage-Committed Naive Precursors Frances Mercer,*,1 Alka Khaitan,†,‡,2 Lina Kozhaya,*,2 Judith A. Aberg,‡,x and Derya Unutmaz*,x,{

R

egulatory T cells (Tregs) mediate immunological tolerance, curbing autoimmunity and overexuberant immune responses. Manipulation of Treg responses and numbers in inflammatory disorders, cancer, and transplantation settings is a highly sought-after therapeutic strategy (1–3). It is now clear that Tregs are a phenotypically and functionally heterogeneous subset, which can suppress a wide range of immune responses (4, 5). Of particular interest, some Tregs can produce the inflammatory cytokine IL-17A (6–8), which we refer to as IL-17+ Tregs. Recent studies suggest that IL-17+ Tregs may also have pathogenic potential (7–9), emphasizing the need for a better understanding of Treg subspecialization. However, the precursor populations and signals that lead to functionally diverse Treg subsets are not yet fully elucidated. Thymus-derived, or “natural” Tregs, express both the FOXP3 and HELIOS transcription factors (10–15). In vitro, natural Tregs can differentiate and expand from CD25+ naive T cells (TNreg), whereas conventional T cells arise from CD252 naive T cells (TN)

*Department of Microbiology, New York University School of Medicine, New York, NY 10016; †Department of Pediatrics, New York University School of Medicine, New York, NY 10016; ‡Division of Infectious Diseases, New York University School of Medicine, New York, NY 10016; xDepartment of Medicine, New York University School of Medicine, New York, NY 10016; and {Department of Pathology, New York University School of Medicine, New York, NY 10016 1 Current address: Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA. 2

A.K. and L.K. contributed equally to this work.

Received for publication October 31, 2013. Accepted for publication May 19, 2014. This work was supported by National Institutes of Health Grants R01AI065303 and R21AI087973 (to D.U.), as well as by National Institutes of Health Training Grant 5T32AI007647 (to F.M.). Address correspondence and reprint requests to Dr. Derya Unutmaz, Department of Microbiology, New York University School of Medicine, Smilow Research Center, 522 First Avenue, New York, NY 10016. E-mail address: [email protected] The online version of this article contains supplemental material. Abbreviations used in this article: DC, dendritic cell; ROR, retinoic acid–related orphan receptor; TN, CD252 naive T cell; TNreg, CD25+ naive T cell; Treg, regulatory T cell. Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302936

(16–18). Tregs with suppressive capacity can be “induced” from conventional CD252 TN cells through TGF-b signaling or ectopic expression of FOXP3 (1). However, FOXP3 is also expressed transiently upon TCR activation in the presence of TGF-b, and it does not confer suppressive ability (19–21), thus confounding the discrimination and analysis of Treg subsets ex vivo. Additionally, TGF-b is crucial for the differentiation of both Treg and Th17 subsets in a concentration-dependent manner (22), and the Treg and Th17 developmental pathways are putatively linked (23). However, to what extent Treg and Th17 lineages share a common precursor or lineage-commitment program is not yet clear. Human Th17 cells, defined by secretion of IL-17, differentiate from TN cells in the presence IL-1b, IL-6, and low concentrations of TGF-b; IL-23 is also required for expansion and maintenance of Th17 cells (24). The Th17 lineage specialization depends on the induction of transcription factor retinoic acid–related orphan receptor (ROR) gt (25), and all Th17 cells also express chemokine receptor CCR6 for their localization (24). Th17 cells are crucial for host defense against fungal and extracellular bacterial pathogens but are also implicated in the pathology of several autoimmune diseases (24, 26). However, the function and role of IL-17+ Treg cells in health and disease remain unclear. In this study, we sought to determine how human IL-17+ Tregs differentiate from precursor naive cells. We determined that a portion of the TNregs expressing CCR6 have a predetermined capacity to differentiate into IL-17+ Tregs in vitro. Remarkably, a small portion of TN and TNreg cells expressing CCR6 also have a propensity to develop into Th17 cells. The IL-17+ Tregs differentiated from CCR6+ TNregs could be partly discriminated from their conventional Th17 counterparts by their suppressive activity and surface phenotype, which resembled memory Tregs. Furthermore, we show that the IL-17+ Treg compartment is selectively reduced in a subset of HIV-infected individuals with suppressed viral loads through antiretroviral treatment. Taken together, these findings establish a framework to dissect the developmental program of naive cells poised to differentiate into Th17 or IL-17+ Tregs, and they suggest potential therapeutic strategies to reconstitute these populations after perturbation in diseases such as HIV infection.

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A subset of human regulatory T cells (Tregs) secretes IL-17 and thus resembles Th17 effector cells. How IL-17+ Tregs differentiate from naive precursors remains unclear. In this study, we show that IL-17–producing T cells can differentiate from CCR6+ naive T cell precursors in the presence of IL-2, IL-1b, TGF-b, and IL-23. CCR6+ naive T cells are present in adult peripheral and umbilical cord blood and in both conventional T naive and FOXP3+ naive Treg subsets. IL-17+ cells derived from CCR6+ naive Tregs (referred to as IL-17+ Tregs) express FOXP3 but not HELIOS, another Treg-associated transcription factor, and these cells display suppressor capacity and a surface phenotype resembling memory Tregs. Remarkably, the IL-17+ Treg compartment was preferentially reduced relative to the canonical Th17 and Treg compartments in a subset of HIV+ subjects, suggesting a specific perturbation of this subset during the course of disease. Our findings that CCR6+ naive precursors contain a predetermined reservoir to replenish IL-17–secreting cells may have implications in balancing the Th17 and IL-17+ Treg compartments that are perturbed during HIV infection and potentially in other inflammatory diseases. The Journal of Immunology, 2014, 193: 000–000.

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Materials and Methods

Treg suppression assay

Cell purification and activation

Resting naive CD4+ cells (targets) were labeled with CFSE (Invitrogen) and plated at 3 3 104 cells/well in 96-well U-bottom plates. Suppressor cells were added at various ratios, and cells were activated using DCs (generated as described above) at a DC/target cell ratio of 1:10 and antiCD3 Ab (American Type Culture Collection clone OKT-3) at 10–100 ng/ml. All cells were washed at least twice in complete media to remove any cytokines. CFSE dilution was measured on a BD LSR II flow cytometer 4 and 5 d later.

Blood samples were obtained from anonymous consenting healthy donors as buffy coats (New York Blood Center). PBMCs were isolated from umbilical cord blood or from adult peripheral blood using Ficoll-Paque PLUS gradient (GE Healthcare). CD4 + T cells were isolated using a Dynal CD4 positive isolation kit (Invitrogen) and further sorted into different naive and memory subsets using a BD FACSAria (BD Biosciences). Monocyte-derived dendritic cells (DCs) were generated from CD14+ cells as previously described (27). Purified cells were cultured in RPMI 1640 media (Life Technologies, Carlsbad, CA) containing 10% FBS (Atlanta Biologicals, Lawrenceville, GA) as previously described (27). To activate cells for expansion in vitro and in experiments other than suppression assays, anti-CD3– and anti-CD28–coated beads (Invitrogen) were used at a bead/cell ratio of 1:4 in media containing IL-2 (27).

Cells were stained in complete RPMI media or PBS plus 2% FCS and 0.1% sodium azide for 30 min at 4˚C and washed before running on a BD LSR II flow cytometer. Staining for chemokine receptors was done at room temperature for 45 min. Data were analyzed using FlowJo software (Tree Star) and gated on live cells based on the fixable viability dye eFluor 780 (eBioscience). The following Abs were used in stains and sorts: CD45RO, CCR6 (biotinylated), CD161, CD49d, CD25, GARP, CD127, HLA-A2, IL17A, IFN-g, FOXP3, HELIOS, CCR4, CD3, CD4 (BioLegend), CTLA-4 (BD Pharmingen), and IL-1R1-PE (R&D Systems). For intracellular cytokine staining, cells were activated with PMA (20 ng/ml for CD4+ T cells and 40 ng/ml for PBMCs) and ionomycin (500 ng/ml) (Sigma Aldrich) in the presence of GolgiStop protein transport inhibitor (BD Biosciences) for 4–6 h. Cells were then stained with fixable viability dye and surface markers and then fixed and permeabilized using eBioscience fixation/ permeabilization buffers according to the manufacturer’s instructions, before staining for cytokines and transcription factors. PBMCs were precultured in IL-7 (20 ng/ml) (BioLegend) for 1 d to enhance Th17 phenotype (28).

In vitro cytokine polarization assay Sorted TN cells and TNregs were activated with anti-CD3 and anti-CD28 beads and cultured in media containing IL-2 (10 ng/ml) (Chiron). The next day, IL-1b (10 ng/ml), TGF-b (10 ng/ml), and IL-23 (100 ng/ml) (R&D Systems) were added. Cells were expanded for 2 wk in media replenished for IL-2 only. For mixed-donor seeding experiments, donor A and donor B were chosen as HLA-A2+ or HLA-A22 as determined by Ab staining, and TN cells or TNregs from each donor were isolated on the same day. Five thousand cells from donor A were combined with 45,000 cells from donor B. On day 14, HLA-A2 Ab was added to the cytokine stains to determine donor origin. In IL-1R1/CD161 sorting experiments, to enhance expression of Th17 cell2 phenotype markers, T cells were precultured in IL-2, IL-7, or IL-15 (20 ng/ml; BioLegend) prior to sorting, as described (28).

Real-time PCR analysis Total RNA was isolated from flash-frozen cells using a Qiagen RNeasy Mini kit, and cDNA was generated using a high-capacity reverse transcriptase kit (Applied Biosystems). TaqMan primer/probe mixtures were purchased from Applied Biosystems for RORC (Hs01076112_m1) and b-actin (Hs99999903_ml). Samples were run on an Applied Biosystems 7300 apparatus. Data were normalized to b-actin for each sample.

Statistical analysis All statistics were done using GraphPad Prism software. A two-tailed t test was used in all figures except Fig. 5, in which the nonparametric Mann– Whitney U test and Spearman’s rank correlation were used.

Human subjects Twenty HIV-negative controls and 18 HIV-infected individuals were recruited in accordance with an Institutional Review Board–approved protocol and consent from New York University School of Medicine. All HIV+ subjects were well controlled on antiretroviral therapy with HIV viral load ,100 copies/ml. HIV-infected subjects had median (range) CD4 T cell counts of 524 (220–1281) cells/mm3 and median (range) CD4 percentages of 31% (11–44%). Uninfected controls were subjects recruited from an Institutional Review Board–approved protocol or random blood samples that were obtained from the blood bank.

Characterization of the Treg and Th17 compartments of human T cells Tregs are generally identified by resting expression of CD25 and FOXP3 (1). Human Tregs can also be subdivided into TNreg and Treg subsets based on expression of CD45RO (Fig. 1A) (16–18). Recently, concomitant expression of HELIOS and FOXP3 in humans and HELIOS and Foxp3 in mice was suggested as a specific marker of bona fide Tregs (12, 29). Indeed, we observed significant heterogeneity within FOXP3+ Tregs with regard to expression of HELIOS (Fig. 1B), in line with previous reports (12, 15). We found that only about half of the memory FOXP3+ HELIOS+ T cells expressed CD25, whereas within TN cells .90% of FOXP3+HELIOS+ cells were CD25+ (Supplemental Fig. 1A, 1B). Similarly, the total FOXP3+HELIOS+ within memory T cells was typically twice the percentage of the CD25bright subset (Supplemental Fig. 1C), suggesting that at least about half of all memory Tregs were low for CD25 expression. As such, to ensure that CD25low Tregs were not excluded from our analysis, we used FOXP3 and HELIOS as markers to define human Treg subsets throughout the study. To further characterize the previously described IL-17+ Treg subset (6, 30), we determined expression of HELIOS and effector cytokines on T cells derived from healthy human blood. Among CCR6+ T cells, which contain all IL-17+ cells, similar levels of IL-17, IL-22, and IFN-g were found in FOXP3+HELIOS2 cells compared with the FOXP32HELIOS2 subset. However, FOXP3+ HELIOS+ cells did not secrete any of these cytokines (Fig. 1C, 1D). Differentiation of human IL-17+ Tregs It was recently reported that IL-17–producing cells preferentially arise from TNregs in the presence of the Th17-polarizing cytokines IL-1b, IL-23, and TGF-b (31). Using this cytokine polarization protocol (Fig. 2A) we differentiated and expanded IL-17– secreting cells from highly purified TNreg precursors (Fig. 2B, 2C). Similar to ex vivo analysis, in vitro–generated FOXP3+IL17+ cells did not express HELIOS (Fig. 2D, 2E). Although we noticed significant donor-to-donor variability in deriving IL-17+ Tregs from TNreg precursors, each individual displayed consistent potential to produce the same relative amount when the experiment was performed on cells purified from blood of the same donor collected at different time points (Supplemental Fig. 2A). As previously shown, expanded TN cells also expressed FOXP3, which is induced by TGF-b during T cell activation (32, 33). Because expanded TNregs contained both FOXP32 and FOXP3+HELIOS2 cells among the canonical HELIOS+FOXP3+ Tregs, we designed an experimental approach to rule out that TNreg cultures could be providing an in trans polarization signal to a small amount of “contaminant” non-TNregs therein. We therefore intentionally seeded FOXP32 TN cells into TNreg polarization cultures. To ensure identification of the seeded cells at the end of the expansion, we used two donors in each experiment, one for seeded and the other for a driver population, which were discerned by presence or absence of MHC HLA-A2 expression.

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FACS staining and analysis

Results

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We mixed cells at a 1:10 seed/driver ratio and stimulated them in the presence of polarizing cytokines as in Fig. 2A. We found that IL-17 was produced only from seeded cells with a TNreg origin, even when they were mixed at 1:10 ratio with TN cells (Fig. 2F), and that seeded TN cells did not differentiate into IL-17–secreting cells, even in the presence of TNregs (Fig. 2G). Thus, TNregs have a preferential propensity to differentiate into IL-17–secreting cells. IL-17+ Tregs are derived from CCR6+ naive precursors We next asked whether there was a specific subset within TNregs that was poised for IL-17+ Treg differentiation. We first assessed the previously reported IL-1R1 and CD161 as potential markers for IL-17+ Treg precursors (34–36). Although sorting cells based on these markers did enhance IL-17–secreting cell differentiation (Supplemental Fig. 2B), when CCR6+ cells were excluded, IL1R1+CD161+ cells did not give rise to IL-17+ Tregs (Supplemental Fig. 2C). Therefore, we sorted the CCR6+ cells present within both the adult TN and TNreg cell compartment (Fig. 3A). Almost all CCR6+ TNregs expressed FOXP3, and ∼75% were also HELIOS+, very similar to CCR62 TNregs, whereas CD252CCR6+ TN cells were FOXP32 (Supplemental Fig. 2D). Importantly, CCR6+ TN or TNreg cells also expressed higher levels of RORC compared with CCR62 TN or TNreg cells (Supplemental Fig. 2E). Although RORC expression within resting CCR6+ TN cells

was ∼10-fold lower compared with memory CCR6+ cells (Supplemental Fig. 2E), after TCR activation and expansion of CCR6+ TN and TNreg subsets in polarization conditions, their RORC expression became comparable to memory T cells (Supplemental Fig. 2F). Additionally, to ascertain whether these CCR6+ naive cells were recent migrants from the thymus, we determined their numbers in umbilical cord blood (Fig. 3B). Although this population was relatively small, it was consistently present in healthy donors and had fewer numbers in cord blood (Fig. 3C). We then compared the differentiation of naive CCR6+ and CCR62 T cells into IL-17+ cells using the polarization scheme described above (Fig. 2A). We found that only CCR6+ cells, but not CCR62 cells, were capable of differentiating into IL-17+ cells, regardless of whether they were purified from adult TN cells, adult TNregs, or umbilical cord blood naive cells (Fig. 3D, 3E). However, CCR6+ TN cells sorted from the adult blood displayed increased capacity to differentiate into IL-17+ cells (Fig. 3F) relative to CCR6+ naive cells sorted from the cord blood (Fig. 3G). This finding suggests that the CCR6+ TN and TNreg cell subsets may be both developmentally and environmentally regulated, which is also consistent with significant adult donor-to-donor variation (Supplemental Fig. 2A). Although we observed that FOXP3+IL-17+ cells were derived from both CCR6+ TN and TNreg populations, we hypothesized that only CCR6+ TNregs give rise to bona fide IL-17+ Tregs. To address this question, we compared both in vitro–differentiated

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FIGURE 1. Defining IL-17–secreting human Treg subsets. CD4+ T cells were sorted from PBMCs and stained with Abs against CD25, CD45RO (surface), and FOXP3/HELIOS (intracellular). (A) Surface phenotype and (B) FOXP3/HELIOS expression within CD25 and CD45RO populations are shown. (C) CD4+ cells cultured overnight in IL-2–containing media were stimulated with PMA/ionomycin and then intracellularly stained for IL-17, IL-22, and IFN-g within FOXP3/ HELIOS populations of memory CCR6+ cells. (D) Percentages of cytokine-expressing cells from several donors are shown.

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FIGURE 2. In vitro generation of IL-17–producing cells from TNregs. (A) Polarization scheme with TN cells or TNregs. (B) Representative FOXP3/ HELIOS expression in day 14 polarization cultures. (C) Averages of IL-17 production from TN cells or TNregs cultured in IL-2, compared with polarizing cytokines. The data are averages of three donors from three independent experiments. (D) Representative intracellular IL-17/IFN-g within FOXP3/HELIOS gated populations shown in (B). (E) IL-17 expression in polarized TNregs gated as shown in (B) and (C) for multiple donors and independent experiments. (F) TN cells were seeded at a 1:10 ratio with TN cells or TNregs from a different donor, with discrete HLA-A2 phenotype, and polarized as shown in (A). (G) TNregs were seeded with different donor TN cells or TNregs, as in (F), and polarized as shown in (A). Data in (F) and (G) are levels of IL-17 produced in gated seed donor cells. Three donors per independent experiment are shown. **p , 0.005.

subsets to ex vivo Tregs. We first performed in vitro suppression assays, the gold standard for Treg function, and found that our TNreg-derived cells were similarly suppressive to ex vivo CD45RO+ memory Tregs (Fig. 4A). Because it was not technically

feasible to determine the suppressive capacity of only IL-17– secreting cells within our bulk cultures, we assessed a set of Treg surface markers (1, 37, 38) on these cells. We found that FOXP3+ IL-17+ cells generated from the CCR6+ TNreg subset, but not

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from CCR6+ TN cells (Fig. 4B), closely resembled ex vivo–isolated IL-17+ Tregs in expression of CD25, IL-1R1, CCR4, CTLA-4, and CD49d when compared with FOXP32IL-17+ cells within the same cultures (Fig. 4C). Similarly, the FOXP3+ but IL-172 cells expanded and polarized from CCR6+ TNregs expressed Treg markers not seen on FOXP32 cells. However, among CCR6+ TN cells, there was no difference between FOXP3+IL-172 and FOXP32IL-172 cells within the same cultures (Supplemental Fig. 3). Taken together, these results corroborate the notion that IL-17+ Tregs mostly develop from CCR6+ TNreg precursors. IL-17+ Tregs are decreased in an HIV-infected cohort The homeostasis of Treg and Th17 populations during HIV infection is of keen interest because both are integral to proper immune function in the gut mucosa, and both subsets have been reported to be perturbed at different stages of the infection (39, 40). Given our findings that IL-17+ Tregs derive from a predetermined naive precursor, we asked whether this subset differed in HIV+ subjects compared with HIV2 controls. We profiled PBMCs from a subset of HIV-infected individuals who were on antiretroviral

therapy and had very low or undetectable viremia. HIV+ patients and HIV2 controls were analyzed for T cells that express Treg markers and/or secrete Th17 cytokines. We found that there was a specific and significant increase in total HELIOS+FOXP3+ Tregs in HIV+ subjects (Fig. 5A), whereas FOXP3+HELIOS2 T cells did not differ from healthy controls (Fig. 5B). Thus, as a proportion of total FOXP3+ T cells, the HELIOS2 subset was significantly lower in HIV+ subjects (Supplemental Fig. 4A). Importantly, whereas the percentage of CCR6+ memory T cells that were IL17+IFN-g2 were not different in HIV+ subjects compared with healthy controls (Fig. 5C), the IL-17+ Tregs (defined as FOXP3+ IL-17+IFN-g2 T cells) were greatly reduced among this cohort of HIV+ individuals (Fig. 5D). Additionally, the proportion of FOXP3+IL-17+ cells within the Th17 compartment was also significantly decreased in HIV+ subjects compared with controls (Supplemental Fig. 4B). Remarkably, the proportion of FOXP3+ HELIOS2 T cells in the total FOXP3+ compartment positively correlated with the proportion of IL-17+ Tregs within the total IL-17+ compartment in HIV+ subjects but not in healthy controls (Fig. 5E, 5F). These data suggest a functional association between

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FIGURE 3. A subset of TN and TNreg cells that express CCR6 develop into IL-17 producers. CCR6 surface expression was assessed in total CD4+ T cells, along with CD25 and CD45RO, for gating on Treg populations as shown in Fig. 1A. Representative CCR6 expression versus CD45RO expression is shown for each subset of (A) adult blood or (B) umbilical cord blood. (C) Multiple donors/independent stainings are shown. (D) Day 14 cultures of polarized CCR6+ or CCR62 cells were restimulated with PMA/ionomycin in the presence of GolgiStop and stained for FOXP3 and HELIOS. (E) Populations gated as in (D) were assessed for IL-17 and IFN-g production. (F) Multiple donors/independent experiments shown for representative plots for adult blood in (E). (G) Multiple donors/independent experiments shown for representative plots for umbilical cord blood in (E). **p , 0.005, ****p , 0.00005.

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Treg and IL-17+ Treg perturbation during HIV disease and identify a specific “immunological hole” of IL-17+ Tregs in some of the infected individuals.

Discussion In this study, we show that CCR6+ human TN and TNreg cells selectively differentiate into IL-17–secreting cells; CCR6+ TNreg precursors are lineage-committed toward IL-17+ Tregs, whereas CCR6+ TN cells differentiate into conventional Th17 cells. Corroborating these findings, the in vitro–generated human IL-17+ Tregs phenotypically and functionally resemble endogenous memory IL-17+ Tregs. These findings may reconcile previously conflicting reports about whether the origin of IL-17 producers is CD25+ or CD252 T cells (31, 41, 42). We demonstrate that both types of naive cells may be differentiated to produce IL-17, but only from the respective CCR6+ fractions. IL-17 producers within the human TN cell differentiation cultures were probably overlooked (31) because ,1% of human TN cells express CCR6 (Fig. 3A) (43). Additionally, the two previous studies that reported Th17 differentiation from CD252 TN precursors did not examine TNregs, and they may have used culture growth that selected for outgrowth of CCR6+ cells (41, 42). Furthermore, the presence of CCR6+ naive cells, which are poised to differentiate into Th17 cells in cord blood, supports the notion that a subset of Th17 lineage cells is developmentally regulated. Indeed, similar to bona fide Tregs, some Th17-like cells have been shown to emerge directly from the thymus and to be activated by self-antigen in the periphery (44). Although we were constrained by limited amounts of CCR6+ cells in umbilical cord blood samples and were not able to further subdivide them into CD25+CCR6+ and CD252CCR6+ cells, a portion of these cells reproducibly differentiated into FOXP3+IL-17+ cells. However, it is not clear why there are more CCR6+ naive precursors of Th17

and IL-17+ Tregs in adult blood. It is conceivable that this is due to preferential homeostatic expansion of CCR6+ T cells owing to the environmental cytokine milieu. It is also possible that TN or TNreg cells can be conditioned toward a lineage commitment program, for example based on the cytokine milieu, thus expressing CCR6 in the periphery. A major difficulty in defining Treg subsets, especially in humans, has been the expression of FOXP3 in non-Tregs and the similarity of other Treg markers (such as CD25) to the phenotype of activated T cells (19, 33). However, single-cell cloning of FOXP3+IL-17+ cells shows that ex vivo IL-17+ Tregs are indeed suppressive (6, 8, 45). Furthermore, suppression assays performed by Valmori et al. (31) demonstrated the suppressive activity of polarized TNreg cultures. Consistent with these findings, polarized CCR6+ TNregs displayed potent suppressive activity, and the IL-17+FOXP3+ cells therein were strikingly different from FOXP3+IL-17+ cells from TN cultures with regard to expression of the Treg markers (Fig. 4A, 4B). Additionally, these markers exclude cells that produce IFN-g (data not shown), which is associated with another described subset of Th17 cells (24). Future studies assessing the methylation pattern at the Tregspecific demethylation region will be useful in assessing the stability of CCR6+ TNregs and IL-17+ Tregs. Both Treg and Th17 cells are crucial for maintaining mucosal barrier integrity and a balanced immune response in the lamina propria (24, 40). Quantitative assessments of IL-17+ Tregs in gut tissues are difficult to make in humans, and limited data in healthy donors are available. However, several studies now demonstrate an accumulation of IL-17+ Tregs in the gut during inflammatory states (7–9), when their function may be more pertinent. One possible role for IL-17+ Tregs could be both suppressing the formation of professional memory responses by T cells while also securing barrier integrity and ensuring a basal level of microbial control via secretion of IL-17 (40, 46). For example, IL-17+ Tregs could prevent

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FIGURE 4. Characterization of in vitro–derived IL17+ Tregs. (A) Expanded and polarized CCR6+ subsets from Fig. 3 were subjected to an in vitro assay for suppression of T cell proliferation. Ex vivo mature Tregs were used as a positive control. Target fresh TN cells were labeled with a cell trace and stimulated with DCs and anti-CD3 Ab, in the presence or absence of candidate “suppressor” cells at various suppressor/target ratios noted. After 5 d, dilution of cell trace in the target cells was assessed by flow cytometry. (B) Expanded TNregs or TN cells were stimulated with PMA and ionomoycin and stained for surface markers, followed by intracellular staining for FOXP3 and IL-17. IL-17+ subsets were gated based on their FOXP3 expression, and the FOXP3+ versus FOXP32 portions of each are compared with histogram overlaps. One donor representative of three is shown. (C) Ex vivo–sorted CD4+ T cells were cultured overnight in IL-2–containing media and then stimulated, stained, and subjected to the same analysis as in (B).

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adaptive immune responses against commensal bacteria while using IL-17 to keep the growth of commensals to an acceptable level and to repair epithelial damage caused by pathogens. Indeed, host tolerance of pathogens through employment of basic repair mechanisms may allow the immune response to proceed in a less vigorous manner, thus minimizing immunopathology (47). Conversely, in specific contexts, IL-17+ Tregs may also contribute to mucosal disease, as they were recently implicated in development of colon cancer (7, 9) and possibly inflammatory bowel disease (8). In the setting of HIV infection, we found that IL-17+ Tregs in HIV+ individuals on active antiretroviral therapy with suppressed viremia were significantly reduced. Interestingly, in these HIV+ subjects the levels of FOXP3+HELIOS2 cells were also greatly decreased as a proportion of total FOXP3+ cells, and this correlated with a decreased proportion of IL-17+ Tregs in the Th17 subset. In contrast, FOXP3+HELIOS+ Tregs were overall higher in these HIV+ subjects compared with healthy controls. As such,

these findings reveal an immunological hole within the IL-17– secreting and Treg compartments in vivo, despite effective antiretroviral therapy. These results also support our in vitro evidence that the HELIOS2IL-17+ Tregs represent a separate compartment within Treg or Th17 subsets. It is unclear why these cells are preferentially reduced in the blood during HIV infection and what biological outcome this may have in HIV+ individuals. Given that the gut mucosal barrier is likely to be breached during HIV infection (39, 40) and that there is a propensity of IL-17–secreting cells to migrate to the gut mucosa, it is conceivable that IL-17+ Tregs are preferentially targeted by the virus and, at least in some individuals, do not recover. Thus, it is tempting to speculate that disruption of IL-17+ Tregs in certain HIV+ subjects contributes to disruption of mucosal barriers and may have unknown long-term adverse consequences. In future studies, it may be important to assess the consequences of IL-17+ Treg deficiency in the context of HIV infection in a humanized mouse models. Ultimately, our

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FIGURE 5. The IL-17+ Treg compartment is perturbed in HIV+ individuals. PBMCs from HIV+ subjects on antiretroviral therapy or HIV2 controls were stained for proteins, including CD4, FOXP3, HELIOS, and IL-17. (A and B) The percentage of memory CD4+ T cells that are (A) FOXP3+HELIOS+ or (B) FOXP3+HELIOS2 in controls or HIV+ subjects. (C and D) PBMCs were cultured overnight in IL-7, stimulated with PMA and ionomycin in the presence of GolgiStop, and then stained for surface markers and intracellular proteins. The proportion within total CCR6+ memory cells of (C) total IL-17+IFN-g2 cells or (D) FOXP3+IL-17+IFN-g2 cells is shown. (E and F) Correlation between the proportion of FOXP3+HELIOS2 T cells in the total FOXP3+ compartment shown on the x-axis and the proportion of IL-17+ Tregs within the total IL-17+ compartment on the y-axis among (E) the controls and (F) HIV-infected cohort. *p , 0.05, **p , 0.005.

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DIFFERENTIATION OF IL-17+ Tregs FROM CCR6+ NAIVE T CELLS

findings that IL-17+ Tregs differentiate from a discreet subset of precursor TNreg cells may enable better manipulation of these cells as a therapeutic approach during HIV infection and other disease states where they are perturbed.

Acknowledgments We thank Drs. Mark Sundrud, Jan Vilcek, Angela Zhou, and Stephen Rawlings for critical reading and suggestions on the manuscript. Dr. Nathaniel Landau and Nicolin Bloch provided useful discussions and suggestions.

Disclosures The authors have no financial conflicts of interest.

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Differentiation of IL-17-producing effector and regulatory human T cells from lineage-committed naive precursors.

A subset of human regulatory T cells (Tregs) secretes IL-17 and thus resembles Th17 effector cells. How IL-17(+) Tregs differentiate from naive precur...
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