Late Chronic Stage of Pathogenic SIV Infection

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1 This information is current as of November 24, Loss of IL-17 Producing CD8 T Cells during Late Chronic Stage of Pathogenic SIV Infection Pragati Nigam, Suefen Kwa, Vijayakumar Velu and Rama Rao Amara J Immunol published online 10 December ol Supplementary Material Subscription Permissions Alerts 7.DC1 Why The JI? Submit online. Rapid Reviews! 30 days* from submission to initial decision No Triage! Every submission reviewed by practicing scientists Fast Publication! 4 weeks from acceptance to publication *average Information about subscribing to The Journal of Immunology is online at: Submit copyright permission requests at: Receive free -alerts when new articles cite this article. Sign up at: Downloaded from by guest on November 24, 2018 The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD All rights reserved. Print ISSN: Online ISSN:

2 Published December 10, 2010, doi: /jimmunol The Journal of Immunology Loss of IL-17 Producing CD8 T Cells during Late Chronic Stage of Pathogenic SIV Infection Pragati Nigam, Suefen Kwa, Vijayakumar Velu, and Rama Rao Amara Progressive disease caused by pathogenic SIV/HIV infections is marked by systemic hyperimmune activation, immune dysregulation, and profound depletion of CD4 + T cells in lymphoid and gastrointestinal mucosal tissues. IL-17 is important for protective immunity against extracellular bacterial infections at mucosa and for maintenance of mucosal barrier. Although IL-17 secreting CD4 (Th17) and CD8 (Tc17) T cells have been reported, very little is known about the latter subset for any infectious disease. In this study, we characterized the anatomical distribution, phenotype, and functional quality of Tc17 and Th17 cells in healthy (SIV2) and SIV+ rhesus macaques. In healthy macaques, Tc17 and Th17 cells were present in all lymphoid and gastrointestinal tissues studied with predominance in small intestine. About 50% of these cells coexpressed TNF-a and IL-2. Notably, 50% of Tc17 cells also expressed the co-inhibitory molecule CTLA-4, and only a minority (,20%) expressed granzyme B suggesting that these cells possess more of a regulatory than cytotoxic phenotype. After SIV infection, unlike Th17 cells, Tc17 cells were not depleted during the acute phase of infection. However, the frequency of Tc17 cells in SIV-infected macaques with AIDS was lower compared with that in healthy macaques demonstrating the loss of these cells during end-stage disease. Antiretroviral therapy partially restored the frequency of Tc17 and Th17 cells in the colorectal mucosa. Depletion of Tc17 cells was not observed in colorectal mucosa of chronically infected SIV+ sooty mangabeys. In conclusion, our results suggest a role for Tc17 cells in regulating disease progression during pathogenic SIV infection. The Journal of Immunology, 2011, 186: The gastrointestinal (GI) mucosa provides an interface between a sterile internal environment and a contaminated external environment. The GI tract becomes a key participant during HIV/SIV infections because it harbors 40% of all lymphocytes in the body (1, 2), and nearly 60 80% of the memory CD4 T cells in the GI tract are depleted within days after infection (3). This early loss of memory CD4 T cells compromises the ability of the host to generate secondary immune responses to pathogens and is thought to play a critical role in the progression to disease (3 5). In addition to the loss of memory CD4 T cells, HIV/SIV infections are also associated with impaired structure and function of gut mucosal tissue (6 10) culminating in the breakdown of the intestinal epithelial barrier (11, 12). Normal function of mucosal surfaces requires intact epithelium, with intact tight junctions. During HIV/SIV infections, there is downregulation of genes involved in intestinal epithelial cell growth and renewal, as well as increased expression of genes related to inflammation and immune activation (13 15). There is also an increase in proinflammatory cytokine secretion that could facilitate mucosal tissue damage Department of Microbiology and Immunology, Emory Vaccine Center, Yerkes National Primate Research Center and Emory University School of Medicine, Emory University, Atlanta, GA Received for publication August 18, Accepted for publication November 9, This work was supported by National Institutes of Health/National Institute of Allergy and Infectious Diseases Grants R01 AI057029, R01 AI074471, and R01 AI (to R.R.A.), Yerkes National Primate Research Center Base Grant P51 RR00165, and Emory Center for AIDS Research Grant P30 AI Address correspondence and reprint requests to Dr. Rama Rao Amara, Emory University, 954 Gatewood Road, Room 3024, Atlanta, GA address: ramara@ emory.edu The online version of this article contains supplemental material. Abbreviations used in this article: ART, antiretroviral therapy; DP, double-positive; GI, gastrointestinal; IEL, intraepithelial lymphocyte; LPL, lamina propria lymphocyte. Copyright Ó 2010 by The American Association of Immunologists, Inc /10/$16.00 (16 19). Using an in vitro system, a recent study demonstrated that exposure to HIV-1 directly impairs mucosal epithelial barrier integrity (20). This altered intestinal epithelial permeability is believed to permit bacterial translocation leading to hyperimmune activation, which has been shown to distinguish pathogenic and nonpathogenic SIV infections in nonhuman primates (21, 22). IL-17 producing CD4 T cells (Th17) are known to regulate permeability of the gut mucosa and microbial translocation. These cells can secrete two isoforms of IL-17, IL-17A and IL-17F, which are potent activators of neutrophilic inflammation at the gut mucosal tissue. In addition, Th17 cells produce IL-22, which plays a critical role in the maintenance of host defense and epithelialbarrier function. Recent studies have demonstrated that Th17 cells are depleted during HIV/SIV infections (4, 23 25) and suggested that the depletion of these cells may accelerate the progression to AIDS. However, very little is known about the influence of HIV/ SIV infections on the functional quality of Th17 cells during chronic infection. Furthermore, although these cells are depleted soon after SIV infection, it takes several months for development of simian AIDS suggesting the existence of compensatory immune mechanisms. It is increasingly becoming clear that a subset of CD8 T cells in humans and mice secrete IL-17 (Tc17), and these cells are yet to be characterized thoroughly (26 31). Importantly, very little is known about their anatomical distribution, phenotype, functional quality, and their role in host defense. A recent study characterized Tc17 cells in macaques and demonstrated that the frequency of these cells was higher in various tissues of pigtail macaques than that in rhesus macaques (32). However, very little is known about the modulation of these cells during chronic HIV/SIV infections (33), and no information is available on the status of these cells in the colorectal tissue, one of the major sites of HIV/SIV replication during chronic infection. In this study, we characterized the anatomical distribution, phenotype, and functional quality of Tc17 and Th17 cells in healthy rhesus macaques. Similar characterizations were also performed in SIV251-infected rhesus macaques

3 2 TC17 CELLS DURING SIV INFECTION during the acute and chronic phases of infection to understand the effect of pathogenic SIV infection on these cells. In addition, we sought to determine if the frequency of these cells could be restored after antiretroviral therapy (ART) in blood as well as colorectal mucosal tissue and if the depletion of IL-17 secreting T cells is specific to pathogenic infections. Materials and Methods Nonhuman primates and SIV infection Young adult rhesus macaques and sooty mangabeys from the Yerkes National Primate Research Center (Atlanta, GA) breeding colony were cared for under guidelines established by the Animal Welfare Act and the National Institutes of Health Guide for the Care and Use of Laboratory Animals using protocols approved by the Emory University (Atlanta, GA) Institutional Animal Care and Use Committee. Rhesus macaques were infected with SIVmac251 either intravenously or intrarectally at a dose of 100 or 1000 tissue culture infectious dose 50, respectively. Dr. Nancy Miller at the National Institutes of Health (Bethesda, MD) provided the challenge stock. Some of the SIV-infected rhesus macaques were treated with antiretroviral drugs PMPA (20 mg/kg), FTC (30 mg/kg), Kaletra (lopinavir, 12 mg/kg; ritonavir, 3 mg/kg), and AZT (5 mg/kg) at 8 wk after infection. Sooty mangabeys were housed in colonies of animals, and SIVsm is endemic in this population. None of the sooty mangabeys used in the study were experimentally infected. Cell isolation from blood, rectal biopsies, and tissues PBMCs were isolated from whole blood according to the standard Ficoll- Hypaque separation procedures as described previously (34). Lymphocytes from pinch biopsies from the rectum were obtained as described previously (35). Briefly, 10 to 20 pinch biopsies were collected in complete RPMI (RPMI 1640 supplemented with 10% FBS, 1% penicillin streptomycin, 1% HEPES buffer, and 0.1% gentamicin) and washed two times with icecold HBSS. Biopsies were digested with 150 U/ml collagenase IV (Worthington, Lakewood, NJ) and DNase I (Roche, Indianapolis, IN), passed through decreasing sizes of needles (16-, 18-, and 20-gauge, five to six times with each needle), and filtered through a 100-mm filter. Cells were washed twice with RPMI and resuspended in complete RPMI for analysis. Small pieces from lymphoid tissues (axillary lymph node, colonic lymph node, and spleen) were processed in a Medimachine (Becton Dickinson) using 50-mm Medicons and filtered through 50-mm Filcons to obtain a homogenous suspension of cells that was resuspended in complete RPMI. Cells were pelleted and lysed with ACK lysing buffer for 7 min and then washed twice with RPMI and resuspended in complete RPMI for analysis. Small fractions of duodenum, jejunum, ileum, colon, and rectum were cut into small pieces (1 2 mm 2 ) and were then quickly washed with 1 mm DTT in HBSS. The sections were then placed in 2 mm EDTA solution with 2% FBS in HBSS for 20 min with rotation at 37 C. Intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) were processed separately and pooled at the end of cell isolation. Cells that were released into EDTA solution were considered as IELs and were pelleted and resuspended in complete RPMI and placed on ice. The remainder of tissues was then processed as the pinch biopsies from rectum, and cells isolated from the tissues (LPLs) were collected in complete RPMI. IELs and LPLs were then pooled for further analysis. Intracellular cytokine staining analysis Intracellular cytokine production was assessed as previously described with few modifications (36). Briefly, 2 million cells were stimulated in 200 ml RPMI with 10% FBS in a 5-ml polypropylene tube. PMA and ionomycin were used at 25 ng/ml and 0.5 mg/ml, respectively. Cells were incubated at 37 C in the presence of 5% CO 2 for 6 h. Brefeldin A (10 mg/ml) was added after 2 h of incubation. At the end of stimulation, cells were washed once with PBS containing 2% FBS, stained with Live/Dead marker (Invitrogen), followed by surface staining for 30 min at 4 C with anti-human CD4 (clone L200; BD Pharmingen, San Diego, CA), anti-human CD3 (clone SP34-2; BD Pharmingen), and anti-human CD8 (clone SK1; BD Biosciences, San Jose, CA). Cells were then fixed with BD FACS Lyse solution (BD Pharmingen) for 10 min and permeabilized with BD Perm 2 solution (BD Pharmingen) for 10 min. Cells were washed with PBS containing 2% FBS and then incubated for 30 min at 4 C with anti-human IFN-g Ab (clone B27; BD Pharmingen), IL-17 (clone ebio64dec17; ebioscience, San Diego, CA), IL-2 (clone MQ1-167H12; BD Pharmingen), and TNF-a (clone MAb11; ebioscience). Cells were then washed twice with 2% FBS in PBS and resuspended in 1% formalin in PBS. Approximately 500,000 lymphocytes were acquired on the LSRII (BD Immunocytometry Systems) and analyzed using FlowJo software (Tree Star, San Carlos, CA). Dead cells were excluded from analysis. Lymphocytes were identified based on their scatter pattern, and CD3 + CD8 2 CD4 + cells were considered as CD4 T cells, and CD3 + CD8 + CD4 2 cells were considered as CD8 T cells. These CD4 or CD8 T cells were then gated for cytokine-positive cells. For phenotypic analysis, PMA/ionomycin-stimulated cells were stained with anti-human CD4 (clone L200; BD Pharmingen), anti-human CD3 (clone SP34-2; BD Pharmingen), and anti-human CD8 (clone SK1; BD Biosciences) and costained for surface expression of CTLA-4 (clone BNI3; BD Pharmingen), b7 (clone FIB504; BD Pharmingen), CCR6 (clone 11A9; BD Biosciences), and intracellular granzyme B (clone GB11; BD Biosciences), anti-human IFN-g Ab (clone B27; BD Pharmingen), and IL-17 (clone ebio64dec17; ebioscience). Boolean combination gating was performed on cytokine-positive T cells to calculate the frequencies of coexpression profiles corresponding with the four different combinations of cytokines (TNF-a + IL-2 +, TNF-a + or IL-2 +, and TNF-a 2 IL-2 2 ) by Th1, Tc1, Th17, Tc17, CD4 double-positive (DP) and CD8 DP T cells by using the FlowJo software. After subtracting the background values, the proportions of the four different subsets were expressed as percentages of total cytokine-positive cells. The results for each subset were plotted. Responses corresponding with more than 0.05% of total response were considered for analysis. Quantitation of SIV RNA plasma load The SIV copy number was determined using quantitative real-time PCR as previously described (36). All specimens were extracted and amplified in duplicate, and the mean results are reported. Statistical analysis The Student t test was used to compare the frequency of T cell subsets in blood and colorectal tissue and between different time points after SIV infection. The Wilcoxon rank-sum test was used to compare the frequency of T cell subsets in the different tissues of SIV2 and SIV+ rhesus macaques and for phenotypic analysis. The Wilcoxon signed-rank test was used for analysis of the effect of ART on the frequency of T cell subsets. A p value,0.05 was considered statistically significant. Statistical analyses were performed using the S-PLUS 7.0 software program. Results Tc17 and Th17 cells are predominant in the small intestines of normal macaques We measured the frequencies of IL-17 and IFN-g secreting CD4 and CD8 T cells in various lymphoid and intestinal tissues of normal (SIV2) rhesus macaques to understand the relative levels and distribution of these cells (Fig. 1). We stimulated cells isolated from respective compartments with PMA/ionomycin and quantified the levels of IFN-g producers (hereafter called Th1 or Tc1 for IFN-g secreting CD4 or CD8 T cells, respectively), IL-17 producers (hereafter called Th17 or Tc17 for IL-17 secreting CD4 or CD8 T cells, respectively), and IFN-g plus IL-17 double producers (hereafter called either CD4 DP or CD8 DP T cells) using an intracellular cytokine assay and multicolor flow cytometry (Fig. 1A, 1B). All three subsets were present in all the tissues studied with IFN-g producers being the most dominant subset and IFN-g plus IL-17 double producers being the least dominant subset. The mean frequency of Th17 cells in different compartments ranged from as low as 1.7% to as high as 13.9% (Fig. 1B). These were the lowest in spleen and highest in duodenum. In general, these were predominant in the small intestine. Similarly, the mean frequency of Tc17 cells in different compartments ranged from as low as 0.7% to as high as 12.6% and were predominant in the small intestine with the highest levels in duodenum (Fig. 1B). As expected, high frequencies of Th1 and Tc1 cells were detected in every compartment studied, with mean frequencies of % of respective total CD4 or CD8 T cells. CD4 DP and CD8 DP subsets were also detected in every compartment, albeit at much lower levels and with predominance at the intestinal mucosa.

4 The Journal of Immunology 3 FIGURE 1. Distribution of T cell subsets in normal rhesus macaques. A, Gating pattern for Th1, Tc1, Th17, Tc17, CD4 DP, and CD8 DP cells in peripheral blood and jejunum. T cells were gated on lymphocyte gates, followed by exclusion of dead cells and gating on CD3 + T cells. CD3 + CD4 + CD8 2 T cells were identified as CD4 + T cells, and CD3 + CD4 2 CD8 + cells were identified as CD8 + T cells. IL-17 secreting CD4 + or CD8 + T cells were identified as Th17 or Tc17 cells, respectively. IFN-g secreting CD4 + or CD8 + T cells were identified as Th1 or Tc1 cells, respectively. IL-17 + IFN-g + CD4 + or CD8 + T cells were identified as CD4 DP or CD8 DP cells, respectively. B, Summary of the frequency of Th1, Tc1, Th17, Tc17, CD4 DP, and CD8 DP cells and the ratio between Th1/Th17 and Tc1/Tc17 in various tissues. Error bars represent means 6 SEM; n = 5orn = 6.*p, 0.05 (significantly higher or lower compared with blood). ALN, axillary lymph node; CLN, colonic lymph node. To understand the relative proportions of Th1 over Th17 cells in different compartments, we calculated the ratio between these cell subsets in each compartment (Fig. 1B). We observed that for the majority of compartments studied, there was a predominance of Th1 over Th17 T cells. In blood, there were 4 Th1 cells for every Th17 cell, whereas in spleen, there were 12 Th1 cells for every Th17 cell. However, in both the duodenum and jejunum, there were only 1 2 Th1 cells for every Th17 cell. Similarly, for CD8 T cells, we observed predominance of Tc1 over Tc17; however, the ratios were 10-fold higher than the ratios of Th1 and Th17 cells. Consistent with the predominance of Tc17 cells in the small intestine, the ratios of Tc1/Tc17 cells were the lowest (ranging from 9.4 to 11) in this compartment (Fig. 1B). Proportion of polyfunctional cells is greater for Tc17 cells compared with that for Tc1 cells We next characterized Th17 and Tc17 cells for their ability to coproduce TNF-a and IL-2 (polyfunctionality) and compared them with Th1 and Tc1 cells, respectively (Fig. 2). It is important to study these cytokines because TNF-a has been shown to influence the permeability of intestinal epithelial barrier by acting on the tight junctions and by synergizing with IL-17 in inducing chemokine production by epithelial cells (37, 38), and IL-2 is a cytokine that drives proliferation and effector T cell differentiation. It is well known that Th1 and Tc1 cells coproduce TNF-a and IL-2; however, it is not known whether Th17 and Tc17 cells can coproduce these cytokines. A significant proportion of Tc17 and Th17 cells in the blood (Fig. 2) and gut mucosal tissue (Supplemental Fig. 1) was polyfunctional. Based on the coexpression of TNF-a and IL-2, we categorized each of the IFN-g producers, IL-17 producers, or DP cells into four subsets consisting of TNF-a + IL-2 + cells, TNF-a + or IL-2 + cells, and TNF-a 2 IL-2 2 cells (Fig. 2B). About 50% of Tc17 cells in the blood of normal macaques coproduced TNF-a and IL-2 demonstrating that a significant proportion of these cells is polyfunctional (Fig. 2B). Notably, the proportion of these cells was significantly greater in the Tc17 subset than that in the Tc1 subset (p, ). A similar trend was also observed in duodenum, where 60% of Tc17 cells and 40% of Tc1 cells coproduced TNF-a and IL-2 (Supplemental Fig. 1). Similarly, the proportion of polyfunctional cells was significantly greater in the CD8 DP subset than that in the Tc1 subset (p, 0.05). Similar to Tc17 cells, 45% of Th17 cells also coproduced TNF-a and IL-2 (Fig. 2C). However, the proportion of these cells in the Th17 cells was not significantly different from that of Th1 cells. Notably, nearly 60% of the CD4 DP subset consisted of polyfunctional cells, and this percentage was significantly greater than the polyfunctionality exhibited by Th17 and Th1 subsets (p, 0.001). Tc17 cells have more regulatory and less cytotoxic phenotype than Tc1 cells We next characterized the Th17 and Tc17 cells for expression of b7 and CCR6 (gut-homing potential), CTLA-4 (co-inhibitory receptor), and granzyme B (cytotoxic potential) (Fig. 3). Consistent

5 4 TC17 CELLS DURING SIV INFECTION FIGURE 2. Polyfunctionality of T cell subsets. A, Representative flow plots depicting TNF-a or IL-2 secretion by Th17, Th1, CD4 DP, Tc17, Tc1, or CD8 DP cells. B, Cytokine coexpression profiles by Tc17, Tc1, and CD8 DP cells. C, Cytokine coexpression profiles by Th17, Th1, and CD4 DP cells. Error bars represent means 6 SEM; n = 5. *Significantly higher than the respective Tc1 subset (p, 0.05); **significantly higher than respective Th17 or Th1 subset (p, 0.01). with their predominance in the small intestine, 25 65% of Tc17 and Th17 cells expressed gut-homing markers b7 and CCR6. Importantly, both Tc17 and Th17 cells expressed higher levels of these gut-homing markers than those of Tc1 and Th1 cells, respectively. a4b7 but not aeb7 promotes migration to gut. However, a previous study showed that the majority of b7 + T cells in the blood are a4b7 + (39), and thus the anti-b7 Ab that we used here should primarily mark a4b7 + cells. Notably, a significant proportion of Tc17 and Th17 cells exhibited an inhibitory phenotype and did not possess cytolytic potential. CTLA-4 is an inhibitory receptor that is constitutively expressed on regulatory T cells and on some activated CD4 T cells. It is known to deliver negative signals that inhibit expansion of T cells (40). Only a small fraction of CD8 T cells typically express CTLA-4. Of interest, 90% of Th17 and 50% of Tc17 cells expressed CTLA-4 postactivation. These may not represent regulatory T cells as none of these cells costained for FOXP3 (data not shown). Importantly, these levels were much higher compared with those of CTLA-4 expression on Th1 and Tc1 cells. Only 50% of the Th1 cells and 6% of Tc1 cells expressed CTLA-4. A small fraction of Tc17 cells expressed granzyme B suggesting that only a minority of these cells may have killing potential. Only 10% of Tc17 cells were positive for intracellular granzyme B, and this was 6-fold lower than granzyme B expression by Tc1 cells. We could not measure expression of perforin, another molecule required for killing potential, because perforin is rapidly released from the cells after stimulation and thus is hard to stain for on stimulated cells. The CD4 DP and CD8 DP T cells also expressed high levels of b7, CCR6, and CTLA-4. In general, these were higher than those of the respective Th1 and Tc1 cells and similar to those of the respective Th17 and Tc17 cells. Similarly, the expression of granzyme B by CD8 DP T cells was lower than that of Tc1 cells and similar to that of Tc17 cells. These results suggest that DP T cells share more of a Th17 and Tc17 lineage rather than Th1 and Tc1 lineage. Tc17 cells but not Tc1 cells are depleted during the end-stage SIV infection IL-17 has been shown to be important in controlling extracellular bacteria at mucosal surfaces and for enterocyte homeostasis. Consistently, our results demonstrate that Tc17 and Th17 cells are predominant in the small intestine (Fig. 1B), one of the primary sites of viral replication. So, it is important to study the dynamics of Tc17 and Th17 cells during the course of SIV infection to better understand the relative roles of these cells. We quantified the frequencies of Tc17, Tc1, and CD8 DP cells in the blood (Fig. 4A) and colorectal mucosa (Fig. 4B) of 12 rhesus macaques prior to SIV infection and at 2 and 16 wk postinfection. The viral load in these macaques at 16 wk postinfection ranged from to

6 The Journal of Immunology 5 FIGURE 3. Characterization of T cell subsets. A, Representative flow plots for expression of CTLA-4, b7, CCR6, and granzyme B by Th1, Th17, Tc1, Tc17, CD4 DP, and CD8 DP T cells. Black dots represent respective cytokine-positive cells, and gray contours represent total CD4 or CD8 T cells. B, Summary of expression levels of CTLA-4, b7, CCR6, and granzyme B. Black bars represent Th1 or Tc1 cells, open bars represent Th17 or Tc17 cells, and gray bars represent CD4 DP or CD8 T cells. Error bars represent means 6 SEM; n = 4. The Wilcoxon rank-sum test was used for statistical analysis of phenotypic data. *p, 0.05; **p, 0.01; ***p, with geometric mean of copies/ml plasma. The frequencies of these cells were also quantified in a group of macaques that were chronically infected with SIV for wk and were euthanized due to simian AIDS (Fig. 4C). The viral load in these macaques at euthanasia ranged from to with geometric mean of copies/ml plasma. We calculated the ratio of Tc1 over Tc17 cells to understand the relative proportions of these cell subsets at different time points and in various tissues studied (Fig. 4). In blood, a transient reduction in the frequencies of Tc17 cells was observed at 2 wk after SIV infection that recovered by 16 wk (Fig. 4A). However, the frequency of these cells in animals with AIDS was significantly lower compared with that of normal (SIV2) animals demonstrating a loss of these cells during the end stage of disease. A similar trend was also observed for the frequency of CD8 DP cells in intestinal and lymphoid tissues after SIV infection (Fig. 4C). In contrast, the frequencies of Tc1 cells showed a trend toward transient increase at 2 wk after SIV infection and were similar to preinfection levels at 16 wk postinfection and at end-stage disease. These results demonstrate a preferential loss of Tc17 T cells during end-stage AIDS. This preferential loss resulted in an altered balance between Tc1 and Tc17 cells. This loss was not observed for Tc1 cells. Prior to infection, there were 31 Tc1 cells for every Tc17 cell in colon, whereas at end-stage infection, there were 435 Tc1 cells for every Tc17 cell. The difference in ratio was even greater in the liver, with 376 Tc1 cells for every Tc17 cell prior to infection and 3126 Tc1 cells for every Tc17 cell at end-stage disease. We also performed temporal evaluation of Th17, Th1, and CD4 DP T cells after infection and at end-stage disease (Supplemental Fig. 2). For these analyses, we calculated the absolute number rather than percentages because CD4 T cells are infected and killed by the virus. Consistent with previous reports (4, 24, 25, 39), we observed a profound depletion of Th17 cells in blood (Supplemental Fig. 2A) and lymphoid and intestinal tissues (Supplemental Fig. 2B) as early as 2 wk postinfection that was sustained until the end stage of disease. A similar pattern was also observed for CD4 DP cells. The ratios of Th1/Th17 cells also indicated a preferential depletion of Th17 cells after infection. ART preferentially restores Th1 over Th17 cells We next studied the influence of ART on restoration of Tc17 and Th17 cells both in blood and colorectal mucosa in a cohort of eight rhesus macaques that received therapy at 18 wk after SIV infection for a period of 8 wk (Fig. 5). This treatment resulted in a 2- to 3- fold log reduction in the viral load (data not shown). Eight weeks after initiation of ART, blood and colorectal tissue were sampled from the macaques to assess levels of Tc17 and Th17 cells. In colorectal tissue, the frequency of Tc17 increased by 1.5- to 2-fold in 5 of 8 animals (Fig. 5A). Similarly, the frequency of Tc1 cells increased by 1.3- to 3-fold in 7 of 8 animals (Fig. 5A). However, the ratio of Tc1/Tc17 cells did not change significantly demonstrating similar effects of ART on both of these subsets. In blood, the frequency of Tc17 or Tc17 cells did not change significantly after ART (Fig. 5A). A different pattern was observed for Th17 and Th1 cells. The absolute number of Th17 and Th1 cells increased in blood, but in colorectal mucosa significant increases were observed only for the Th1 subset. The increases were higher for Th1 than Th17 resulting in a higher ratio of Th1/Th17 cells in both compartments. No significant changes were observed for CD8 DP or CD4 DP T cells. These results demonstrate that ART increases the frequency of Tc17 and Tc1 cells proportionately and the number of Th17 and Th1 cells disproportionately in the colorectal mucosa. We also tested the potential of Th17 and Tc17 cells to coproduce TNF-a or IL-2, and both subsets of cells were capable of secreting these cytokines at levels similar to those prior to infection (data not shown). Tc17 and Th17 cells are not depleted in SIV-infected sooty mangabeys Previous studies demonstrated a preferential depletion of Th17 cells in pathogenic (rhesus macaques) but not in nonpathogenic (sooty mangabeys) SIV infections. However, no information is available on the status of Tc17 cells in SIV-infected sooty mangabeys. We determined the frequency of Tc17 and Th17 cells in the blood and colorectal tissue of SIV2 and SIV+ sooty mangabeys to determine the effect of SIV infection on these cells (Fig. 6). The frequency of Tc17 cells and the absolute number of Th17 cells in the blood and colorectal tissue of SIV2 and SIV+ sooty man-

7 6 TC17 CELLS DURING SIV INFECTION FIGURE 4. Kinetics of CD8 + T cell subsets after pathogenic SIV infection. A and B, Kinetics of the frequencies of Tc17, Tc1, and CD8 DP cells and ratios of Tc1/Tc17 cells in (A) blood and (B) colorectal mucosa. C, Frequencies of Tc17, Tc1, and CD8 DP cells and ratios of Tc1/Tc17 cells in different tissues (upper row: duodenum, jejunum, ileum, colon, and rectum; lower row: PBMCs, axillary lymph node, colonic lymph node, spleen, and liver). Error bars represent means 6 SEM. Twelve rhesus macaques were followed after SIV infection. Rhesus macaques euthanized for tissue analysis: n = 5orn = 6 for normal uninfected macaques and n = 4 for macaques with clinical AIDS. Time to develop AIDS was wk postinfection. The Wilcoxon rank-sum test was used to compare the frequency of T cell subsets in the different tissues of SIV2 and SIV+ rhesus macaques. *p, 0.05 (significantly higher or lower compared with SIV2 animals). ALN, axillary lymph node; CLN, colonic lymph node. gabeys were not significantly different demonstrating that these cells are not depleted after SIV infection. Furthermore, the frequencies of Tc17 and Th17 cells coproducing TNF-a and IL-2 were also not significantly different (data not shown). Discussion Chronic HIV/SIV infections are associated with impaired gut permeability and microbial translocation. IL-17 plays a critical role in regulating the permeability of gut epithelium and control of microbial and fungal infections. Previous studies have shown a rapid and preferential depletion of IL-17 producing CD4 T cells (Th17) at the gut mucosa after pathogenic SIV infection of rhesus macaques and suggested a role for these cells in faster disease progression. However, it is increasingly becoming clear that IL-17 can also be produced by a subset of CD8 T cells (Tc17), and it is important to study the status of these cells during chronic HIV/ SIV infection because these cells most likely are not killed by the virus. Our study, which for the first time to our knowledge characterizes the anatomical distribution, phenotype, and functional quality of Tc17 cells in normal and SIV-infected macaques, demonstrates that in contrast to Th17 cells, the magnitude and functional quality of Tc17 cells is not significantly altered during the acute phase of infection. Importantly, our results demonstrate that Tc17 cells indeed are depleted during end-stage disease in multiple tissues. In addition, they demonstrate that Tc17 cells are not depleted in the blood and colorectal tissue of nonpathogenic SIV infection of sooty mangabeys. Although we show that Tc17 cells are not detectable at end-stage disease in the tissues we studied, our results do not discriminate between depletion and exhaustion. It is possible that by using other surface markers for identification of Tc17 cells, we may discover that these cells are still present but unable to secrete IL-17. The lack of depletion/exhaustion of Tc17 cells during the acute and early chronic phases of pathogenic SIV infection suggests that these cells may compensate for the loss of Th17 cells and may play a role in controlling disease progression by possibly maintaining partial functionality of the epithelial barrier. We quantified the frequency of Tc17 cells in the blood of a small group of normal and ART naive, chronically infected HIV+ individuals (infected for y with no signs of AIDS) in an attempt to study the status of these cells in HIV infection (data not shown). Tc17 cells could be detected in both groups of individuals but were similar in frequency suggesting the lack of depletion of these cells in blood. However, based on our macaque studies, it is

8 The Journal of Immunology 7 FIGURE 5. Effect of ART on T cell subsets. A, Frequencies of Tc17, Tc1, and CD8 DP cells before and after ART in colorectal mucosa and PBMCs. Right panel summarizes the ratios of Tc1/Tc17 cells before and after ART. B, Number of Th17, Th1, and CD4 DP cells per 100,000 lymphocytes before and after ART in colorectal mucosa and PBMCs. Right panel summarizes the ratios of Th1/Th17 cells before and after ART. ART was initiated 18 wk after SIV infection. Samples were collected 16 wk after SIV infection and 8 wk after initiation of ART. Data from colorectal mucosa and peripheral blood are shown. Each symbol represents an individual macaque. The Wilcoxon signed-rank test was used for analysis of the effect of ART on the frequency of T cell subsets. *p, 0.05; ***p, important to study the frequency of these cells at the end stage of disease both in blood and gut to see whether these cells will be depleted in HIV-infected humans. Phenotypic characterization of Tc17 and Th17 cells revealed that these cells express high levels of the co-inhibitory marker CTLA-4, a finding that has not been appreciated before. This is particularly interesting for Tc17 cells because CD8 T cells do not normally express high levels of CTLA-4 as has been observed for Tc1 cells in our study. CTLA-4 binds to CD80 and CD86, which are normally expressed on APCs, such as dendritic cells, and diminishes their T cell activation potential. The role of CTLA-4 expressed on Th17 and Tc17 cells is not clear but perhaps could be important to regulate the excessive Th1 and Tc1 response (hyperimmune activation) in the gut. It is well established that hyperimmune activation plays a critical role in faster disease progression during pathogenic SIV infection, and thus it is possible that loss of Th17 and Tc17 cells contributes to enhanced hyperimmune activation during pathogenic SIV infection. It will be important to study whether Tc17 cells can modulate Tc1 responses. Unexpectedly, we found that Tc17 cells are similar to Th17 cells for many immune parameters that we studied. Both subsets were predominant in the small intestine, consisted of high proportions of cells coproducing TNF-a and IL-2, and expressed markers associated with gut-homing potential. Furthermore, Tc17 cells, despite being CD8 +, expressed very little granzyme B. This suggests that the function of Tc17 cells could be more helper rather than cytotoxic. If this is true, the specific role of Tc17 cells is not clear. This requires FIGURE 6. Comparison of T cell subsets in sooty mangabeys. Number of Th17, Th1, and CD4 DP cells per 100,000 lymphocytes and frequencies of Tc17, Tc1, and CD8 DP cells in uninfected and chronically infected sooty mangabeys (n = 8 per group).

9 8 TC17 CELLS DURING SIV INFECTION further characterization of these cells with respect to production of other cytokines, such as IL-22, cytotoxicity, suppressive potential, and Ag specificity. Furthermore, it is possible that their anatomical location within the mucosal tissue may be different. For example, CD8 T cells are enriched in the intraepithelial compartment and CD4 T cells are enriched in the lamina propria, and correspondingly, the Tc17 and Th17 cells also may be enriched in specific gastrointestinal compartments. However, in our studies, we did not analyze IELs and LPLs separately, thus is difficult to address this question. ART therapy increased the levels of Tc17 T cells in colorectal mucosa of six of eight macaques and increased levels of Th17 T cells in colorectal mucosa of half of the macaques undergoing antiretroviral treatment. This, along with low viral burden, may contribute to the partial restoration of GI mucosa and immune function, as well as the alleviation of symptoms such as diarrhea caused by GI infections. However, ART preferentially restored Th1 cells over Th17 cells resulting in an altered balance between the two subsets promoting a more proinflammatory response. The reasons for this preferential restoration of Th1 cells are not clear. Perhaps longer duration of ART could further restore Th17 cells and reestablish the relative proportion of these cells to what was seen prior to infection. IL-21 is secreted by Th17 cells and drives IL-17 secretion in an autocrine manner. Also, the levels of IL-21 have been shown to be decreased in HIV-infected individuals, and ART is only partially capable of restoring production of this cytokine (41). So, it is possible that combining ART with IL-21 therapy may restore Th17 and thus enhance the function of gut mucosa. The reasons for depletion of Tc17 cells during end-stage disease in pathogenic infections remain unclear. It is possible that the depletion of these cells is due to loss of CD4 help combined with higher activation levels; however, further experiments are needed for confirmation. Based on our results, we speculate that there is greater permeability of gut mucosal epithelial barrier and infiltration of microbes during late chronic phase than during the acute phase of infection. In conclusion, our results suggest a role for Tc17 cells in regulating disease progression during pathogenic SIV infection and that therapeutic approaches that enhance the magnitude and function of these cells may have benefit in disease outcome. Acknowledgments We thank H. Drake-Perrow for outstanding administrative support, Lakshmi Chennareddi for statistical analysis, and the Yerkes Division of Research Resources for the consistent excellence of veterinary care and pathology support. 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