The Journal of Immunology

Ets-1 Maintains IL-7 Expression in Peripheral T Cells

Roland Grenningloh,*,† Tzong-Shyuan Tai,* Nicole Frahm,†,‡,1 Tomoyuki C. Hongo,‡ Adam T. Chicoine,‡ Christian Brander,†,‡,x,{ Daniel E. Kaufmann,†,‡,‖ and I-Cheng Ho*,†

The expression of CD127, the IL-7–binding subunit of the IL-7 R, is tightly regulated during the development and activation of T cells and is reduced during chronic viral infection. However, the molecular mechanism regulating the dynamic expression of CD127 is still poorly understood. In this study, we report that the Ets-1 is required for maintaining the expression of CD127 in murine peripheral T cells. Ets-1 binds to and activates the CD127 promoter, and its absence leads to reduced CD127 expression, attenuated IL-7 signaling, and impaired IL-7–dependent homeostatic proliferation of T cells. The expression of CD127 and Ets-1 is strongly correlated in human T cells. Both CD127 and Ets-1 expression are decreased in CD8+ T cells during HIV infection. In addition, HIV-associated loss of CD127 is only observed in Ets-1low effector memory and central memory but not in Ets-1high naive CD8+ T cells. Taken together, our data identify Ets-1 as a critical regulator of CD127 expression in T cells. The Journal of Immunology, 2011, 186: 969–976.

nterleukin-7 signals are required for T cell development, GABPa or another Ets is responsible for maintaining maintaining the naive T cell pool, mounting proper primary CD127 expression in peripheral T cells is unknown. I responses, and inducing and maintaining CD4+ and CD8+ Ets-1 (E26 transformation-specific sequence) is the founding T cell memory (1–3). The IL-7R consists of the IL-7Ra–chain member of the Ets family of transcription factors and is expressed (CD127), which binds IL-7, and the common g-chain (CD132). at high levels in lymphoid cells (8, 11). Ets-1 has been shown to CD127 expression is dynamically regulated throughout T cell promote Th1 and inhibit Th17 differentiation (12, 13). Ets-1 is development, activation, and memory formation (2, 4). Many also recruited to the IL-5/IL-13/IL-4 and required for the extracellular stimuli can modulate the expression of CD127. For optimal expression of these cytokines (14). T cells express two example, TCR signals (5), IL-7 (6), and the HIV Tat protein (7) splice variants, the full-length Ets-1 p51 and the shorter Ets-1 p42 have been shown to inhibit the expression of CD127. However, that lacks exon VII (15, 16). The activity of Ets-1 is regulated by the mechanisms governing this dynamic regulation of CD127 are activating and inactivating phosphorylation events (11), but the poorly understood. The constitutive expression of CD127 is reg- role of these phosphorylation events in regulating the function of ulated by members of the Ets family of transcription factors in Th cells remains controversial (17). different lymphoid lineages. Ets are characterized by their In this study, we show that Ets-1 directly binds to and activates the DNA-binding Ets domain (8). The B cell-specific PU.1 is essential CD127 promoter. Ets-1–deficient (KO) T cells expressed reduced for the expression of CD127 in lin2 hematopoietic progenitors and levels of CD127 and displayed impaired IL-7–dependent survival pro-B cells (9), whereas GABPa is required for the expression of and homeostatic proliferation. Importantly, the level of CD127 in CD127 in early thymocytes (10). Both factors bind to a function- human T cells strongly correlates with that of Ets-1. Loss of CD127 ally critical Ets binding site in the CD127 promoter. But whether expression is a hallmark of CD8+ T cells during chronic viral in- fection (18, 19). The expression of Ets-1 is also reduced in CD8+ T cells of HIV-positive individuals mainly due to an expansion of *Division of Rheumatology, Immunology, and Allergy, Department of Medicine, † effector/effector memory cells. Notably, HIV-associated reduction in Brigham and Women’s Hospital, Boston, MA 02115; Harvard Medical School, low Boston, MA 02115; ‡Ragon Institute of MGH, MIT, and Harvard, Charlestown, CD127 occurs only in Ets-1 effector memory and central memory MA 02129; xCatalan Institution for Research and Advanced Studies, Barcelona, cells but not in Ets-1high naive cells. Thus, our data demonstrate that Spain; {AIDS Research Institute Irsicaixa-Catalan HIV Vaccine Program, Hospital ‖ Ets-1 is a critical regulator of CD127 in peripheral T cells. Germans Trias i Pujol, Badalona, Spain; and Infectious Disease Unit, Massachusetts General Hospital, Boston, MA 02114 1Current address: HIV Vaccine Trials Network, Fred Hutchinson Cancer Research Materials and Methods Center, Seattle, WA. Study subjects Received for publication June 25, 2010. Accepted for publication November 9, 2010. This work was supported in part by National Institutes of Health Grant AI081052 (to The study included 10 individuals chronically infected with HIV. Four of I.-C.H.), a Senior Research award from the Crohn’s and Colitis Foundation of America these subjects were treated with antiviral therapy, and six were untreated. (to I.-C.H.), National Institutes of Health Grant HL092565 (to D.E.K.), and a National All but one had plasma viral loads above the limit of detection of the Merit scholarship from the National Science Council, Taiwan (to T.-S.T.). quantitative assays used for clinical monitoring (.50 or .75 viral mRNA copies/ml). Median viral load was 21,200 viral mRNA copies/ml (range, Address correspondence and reprint requests to I-Cheng Ho, Smith Building, Room , 526, One Jimmy Fund Way, Boston, MA 02115. E-mail address: [email protected] 75 to 233,000). Additionally, 10 HIV-uninfected healthy volunteers were included as controls. Human subject protocols were approved by all par- The online version of this article contains supplemental material. ticipating hospitals and clinics, and all subjects provided written informed Abbreviations used in this article: ChIP, chromatin immunoprecipitation; CM, cen- consent prior to enrollment. tral memory; DN, double-negative; DP, double-positive; EM, effector memory; Ets-1, E26 transformation-specific sequence; HET, Ets-1 heterozygous (Ets-1+/2 ); KO, Ets- Mice 2/2 1 ; b2m, b2-microglobulin; SP, single-positive; WT, wild-type. Ets-1–deficient mice were described previously (20). Mice were back- Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 crossed to the C57BL/6 background for six generations and maintained www.jimmunol.org/cgi/doi/10.4049/jimmunol.1002099 970 Ets-1 CONTROLS IL7-R EXPRESSION by mating Ets-1+/2 male to Ets-1+/2 female mice. Ets-1+/+ (Ets-1 wild-type Luciferase assays and chromatin immunoprecipitation assay [WT]; designated WT), Ets-1+/2 (Ets-1 heterozygous; designated HET) and Ets-12/2 (KO) littermates were used throughout the studies. Congenic Luciferase assays were performed as described (17). The CD127 promoter CD45.1 C57BL/6 and CD45.1 Rag22/2 mice were purchased from Ta- construct pGL3-IL-7Rapr, the Ets-1 p51 expression vector pcDNA-Ets-1 conic (Hudson, NY). The animals were housed under specific pathogen- p51, and the Runx1 expression vector pcDNA-MycRunx1 have been de- free conditions, and all experiments were carried out in accordance with scribed elsewhere (12, 21, 22). Chromatin immunoprecipitation (ChIP) the institutional guidelines for animal care at the Dana-Farber Cancer In- assays were performed as described using in vitro differentiated Th1 cells stitute (Boston, MA). (12). Precipitated DNA fragments were amplified by quantitative PCR. The Abs used for immunoprecipitation were anti–Ets-1 C-20 and control rabbit IgG (both Santa Cruz Biotechnology). Specific binding was calcu- Cell lysates, Western blot, and Abs 2 lated as 2 (CtEts1 / CtCtrl). The following primer pairs were used: CD127 pro- Total cell lysates and nuclear or cytosolic extracts were adjusted for total moter Ets binding site, 59-ACCACAGACAGGGAACTATG-39 and 59-CAC- protein content and subjected to SDS-PAGE followed by Western blot. The ACTCTACCTCTCCGTTT-39;IFN-g promoter, 59-CTTTCAGAGAATCC- following Abs were used: anti-STAT5 and anti–phospho-STAT5 (Tyr694) CACAAG-39 and 59-TTAAGATGGTGACAGATAGGTG-39;TLR7,59-TC- (Cell Signaling Technology, Danvers, MA) and anti-Hsp90 (Santa Cruz TAGAGTCTTTGGGTTTCG-39 and 59-TTCTGTCAAATGCTTGTCTG-39. Biotechnology, Santa Cruz, CA). As secondary Abs, HRP-coupled goat- anti–rabbit IgG was used (Zymed Laboratories). Proteins were visualized Quantitative RNA analysis using an ECL kit (PerkinElmer). RNA isolation, reverse transcription, and real-time PCR were performed as described (13). The following primer pairs were used: mouse CD127, 59- Cell isolation and culture, reagents, flow cytometry GCGGACGATCACTCCTTCTG-39 and 59-AGCCCCACATATTTGAAA- TTCCA-39; human CD127, 59-CCCTCGTGGAGGTAAAGTGC-39 and Mouse T cells were isolated from lymph nodes and spleen using magnetic 59-CCTTCCCGATAGACGACACTC-39; human/mouse Ets-1 p51, 59-CT- cell sorting as described (12). For staining of human CD127, the Alexa 9 9 647-coupled Ab from eBioscience (San Diego, CA) was used; all other CCTATGACAGCTTCGACT-3 and 5 -ATCTCCTGTCCAGCTGATAA- 9 b b 9 9 Abs used for flow cytometry were purchased from BD Biosciences 3 ; human 2-microglobulin ( 2m), 5 -GTGGCCTTAGCTGTGCTCG-3 and 59-ACCTGAATGCTGGATAGCCTC-39; human Actin, 59-GTGACAG- (Franklin Lakes, NJ). Flow cytometry was performed on a FACSCanto CAGTCGGTTGGAG-39 and 59-AGGACTGGGCCATTCTCCTT-39;mouse (murine samples) or LSRII (human samples) and analyzed with FlowJo + Actin, 59-GGCTGTATTCCCCTCCATCG-39 and 59-CCAGTTGGTAAC- software. Isolation of CD8 T cell subsets from PBMCs of HIV-positive AATGCCATG-39. and HIV-negative subjects was performed on a FACSAria (BD Bio- sciences) equipped for biohazardous material. PBMCs were stained with lineage exclusion Abs (CD4, CD14, CD19, and CD56 conjugated to V450; Statistical analysis all BD Biosciences), anti-CD27 PE (BD), anti-CD45RA allophycocyanin Statistical analysis was done using Prism Software (GraphPad, La Jolla, (BD), and CD127-PerCP Cy 5.5 (eBioscience). To avoid prestimulation, CA). Paired or unpaired t tests were used as indicated in the figure legends + CD8 T cells were negatively selected (anti-CD3 or anti-CD8 Abs were of this article, and p , 0.05 was considered significant. not used) and CD8+ T cell subsets sorted based on the CD27/CD45RA phenotype. The FACSAria was operated at 70 lb/in.2 with a 70-mm nozzle. For all populations, at least 100,000 cells were collected directly into RLT Results lysis buffer and RNA extracted using the RNeasy Mini kit (Qiagen). Ets-1 is required for constitutive CD127 expression in murine peripheral T cells Bone marrow transduction PU.1 and GABPa have been shown to regulate the expression of Five days before bone marrow harvest, HET or KO donor mice were injected i.p. with 100 ml 50 mg/ml 5-fluorouracil (Sigma). Bone marrow CD127 in pro-B cells and early thymocytes, respectively (9, 10). cells were isolated, and SCA-1–positive cells were enriched using Because Ets-1 is expressed at high levels in thymocytes and ma- magnetic cell sorting. The cells were cultured in media containing 20 ng/ ture T cells, we hypothesized that Ets-1 might participate in reg- ml IL-3, 50 ng/ml stem cell factor, and 50 ng/ml IL-6 (all from ulating the expression of CD127. We first analyzed the expression PeproTech). Seventy-two hours and 96 h later, the cells were infected with a retrovirus expressing Ets-1 p51 or a control retrovirus (17). of CD127 at different stages of thymocyte development in KO Twenty-four hours after the last infection, bone marrow cells were in- mice. In agreement with a previous publication (23), we found that jected intravenously into irradiated (400 rad) Rag22/2 mice. KO thymi contained a higher percentage of double-negative (DN)

FIGURE 1. Ets-1 is required for CD127 expression in DN1 and mature thymic T cells. A, Expression of CD127 in immature thymocytes. Thymocytes from HET and KO mice were stained with Abs against CD4, CD8, CD25, and CD44. The gating used to identify DP cells and the DN1 through DN4 stages is shown. Histograms show expression of CD127 (black lines, anti-CD127; gray lines, iso- type control). B, Expression of CD127 on mature thymic T cells. Mature thymic T cells were identified as TCRbhi CD24lo and further separated into CD4+ or CD8+ T cells. Histograms show CD127 expression. C, Results from three HET and KO littermates were combined. *Sta- tistically significant differences (p , 0.05) in the level of CD127 expression (paired t tests). The Journal of Immunology 971 cells but fewer mature CD8 single-positive (SP) cells (Fig. 1A,1B) significantly reduced in naive and CM T cells from KO animals. than HET thymi. The expression of CD127 was reduced at the EM CD4+ and CD8+ T cells expressed a relatively low level of DN1 stage but normal in DN2 and DN3 cells in KO mice (Fig. CD127, and there was no difference between Ets-1 HET and KO 1A). CD127 is normally downregulated in the DN4 and double- EM T cells (Fig. 2B,2C). Thus, Ets-1 is required for constitutive positive (DP) stage and reappears in mature (TCRbhi CD24lo)SP expression of CD127 in DN1 and SP thymocytes, naive and CM cells. However, both KO CD4 and CD8 SP thymocytes displayed T cells, but not DN2 or DN3 thymocytes. The impaired expression significantly reduced CD127 expression (Fig. 1B,1C) compared of surface CD127 in naive Ets-1KO T cells correlated with a sig- with that of HET cells, which expressed a normal level of Ets-1 nificant reduction in the level of CD127 mRNA (Fig. 2D), sug- and, expectedly, CD127 (Supplemental Fig. 1). gesting that Ets-1 regulates the expression of CD127 at the tran- We then analyzed CD127 expression in peripheral T cells. Fig. scriptional level. 2A showsthegatingstrategyusedtoidentifynaive(CD44lo CD62Lhi), effector memory (EM; CD44hiCD62Llo), and central Ets-1 binds to and activates the CD127 promoter memory (CM; CD44hiCD62Lhi) T cells. As described by others, The CD127 promoter contains a conserved Ets binding site that is we observed an increased percentage of CD4+ and CD8+ EM critical for activation by PU.1 or GABPa (Fig. 3A) (10, 22). We cells in KO mice (23) (Fig. 2A). The expression of CD127 was therefore speculated that Ets-1 might bind to this site in mature

FIGURE 3. Ets-1 binds to the CD127 promoter and drives CD127 ex- pression. A, Schematic diagram of the CD127 promoter. The locations of the conserved Ets and Runx1 binding are marked. Arrows indicate the location of primers used for the detection of Ets-1 binding by ChIP. TSS, transcriptional start site. B, Binding of Ets-1 to the CD127 and IFN-g promoters and to the TLR7 in CD4+ T cells. Binding of Ets-1 to the indicated was determined by ChIP, and binding strength was cal- culated as enrichment over control IgG. Mean values and error bars from three independent experiments are shown. C, Activation of the CD127 promoter by Ets-1, Runx1, and PU-1 in the human embryonic kidney cell line 293T. 293T cells were transfected with expression plasmids encoding the indicated transcription factors and a reporter construct containing the minimal CD127 promoter shown in A. After 24 h, luminescence was measured. Luminescence was normalized to empty expression vector FIGURE 2. Reduced CD127 expression in peripheral Ets-1–deficient (pcDNA). Assays were done in triplicate, and means and SEs are shown. T cells. A, Splenocytes and lymph node cells from HET and KO mice were Representative results from one of three independent experiments are 2 stained with Abs against CD3 and CD8 to identify CD4+ (CD3+CD8 ) and shown. D and E, Retroviral expression of Ets-1 p51 restores CD127 ex- CD8+ (CD3+CD8+) T cells, which were further separated into naive pression in peripheral naive T cells. Bone marrow cells from HET or KO (CD62LhiCD44lo), EM (CD62LloCD44hi), and CM (CD62LhiCD44hi) mice were isolated, enriched for SCA-1–positive cells, and infected with cells. B, Each population of cells was further stained with anti-CD127 the indicated retroviruses. The cells were injected into Rag22/2 mice to (black lines) or an isotype control (gray lines). Histograms from one allow reconstitution of the lymphoid system, and CD127 expression on representative experiment are shown. C, Combined data from five HET and naive (CD44lo) CD4+ or CD8+ T cells was analyzed 8 wk later. D, His- KO littermate pairs. *p , 0.05 (paired t test). D, RNA was harvested from tograms from CD127 staining from one representative mouse are shown. sorted naive CD4+ and CD8+ T cells, and the level of CD127 transcripts Black lines, CD127; gray lines, isotype control. E, Combined data from was measured using quantitative PCR. The data shown are the level of three individual mice. An unpaired t test was used to identify significantly expression relative to Actin. different CD127 expression: *p , 0.05. 972 Ets-1 CONTROLS IL7-R EXPRESSION

T cells and directly regulate the transcription of CD127. By ChIP The infected bone marrow cells were then transferred to Rag2 assay, we found that Ets-1 indeed bound to the endogenous CD127 knockout (Rag22/2) mice. Peripheral T cells were harvested from promoter with an affinity even higher than its binding to the IFN-g the host animals 8 wk later and analyzed. As shown in Fig. 3D and promoter, a known target of Ets-1 (Fig. 3B) (12). In contrast, there 3E, retrovirally expressed Ets-1 efficiently restored CD127 levels was no specific binding of Ets-1 to the TLR7 gene, which served in both peripheral naive CD4+ and CD8+ T cells. as a negative control. In addition, Ets-1 activated a reporter con- struct containing the 200-bp minimal promoter of mouse CD127, including the Ets site (22) (Fig. 3C). The p42 isoform of Ets-1, which lacks part of the inhibitory domain, was slightly more ef- ficient than the full-length p51 isoform. The degree of activation was comparable with that induced by PU.1, which we used as a positive control (22). The CD127 promoter contains a conserved Runx site next to the Ets site (22) (Fig. 3A). Runx1 is required for CD127 expression in CD4+ T cells (24) and can physically in- teract with Ets-1 (25). Although Runx1 also transactivated the CD127 promoter, coexpression of Runx1 and Ets-1 only led to additive, but not synergistic, activation. Next, we isolated bone marrow cells from HET or KO mice and transduced the cells in vitro with a control retrovirus expressing only GFP (GFP-RV) or a virus expressing GFP along with Ets-1 p51 (RV-Ets1 p51). We have previously shown that such retroviral transduction can re- store the expression of Ets-1 to a near physiological level (17).

FIGURE 5. Impaired expansion of Ets-1–deficient T cells in a lympho- FIGURE 4. Impaired STAT5 phosphorylation and survival of Ets-1– penic host. A, A mixture of naive, CFSE-labeled WT (CD45.22), and KO deficient (KO) T cells in response to IL-7. A, Reduced STAT5 phosphor- (CD45.2+) CD4+ T cells (at an ∼1:1 ratio) was injected intravenously into ylation in naive KO CD4+ T cells in response to IL-7. Naive CD4+ T cells Rag22/2 mice (1 million cells/mouse). At day 7 and 29, inguinal, axillary, were sorted based on CD44loCD62Lhi expression and either left untreated and mandibular lymph nodes were isolated. Donor T cells (CD4+TCRb+) (Media) or stimulated with 10 ng/ml IL-7 for 20 min. Total STAT5 levels were analyzed for the expression of CD44 and content of CFSE (left and phosphorylation of Tyr694 (P-STAT5) were examined by Western panels). WT and KO donor cells were distinguished according to the ex- analysis. Hsp90 expression was determined to ensure equal loading. B, pression of CD45.2 (middle panels). The CFSE2 and CFSE+ populations Naive CD4+ T cells from congenic CD45.1 C57BL/6 (WT) or KO mice represent cells undergoing endogenous and homeostatic proliferation, re- were sorted based on CD45RBhi expression. The cells were mixed at a 1:1 spectively. The percentage of CFSE2 and CFSE+ WT or KO T cells from ratio (left panel), labeled with CFSE, and CD127 expression was analyzed one representative mouse is shown in the middle panels. The means and (black, CD127; gray, isotype control). C, Decreased survival of KO CD4+ SEs from three mice are shown in the right panels. B and C, The indicated T cells in response to IL-7. The WT (CD45.22) and KO (CD45.2+) cell populations were further analyzed for the expression of CD127 on day 7 mixture from B was cultured in media alone or in media containing 10 ng/ and day 29. Data from one representative mouse are shown in B. Com- ml IL-7. After 72 h, the cells were stained for CD45.2, and the percentage bined mean values and SD from three mice are shown in C. An unpaired t of live cells was determined by flow cytometry based on their position in test was used to detect significant differences between WT and KO: *p , the forward/sideward scatter (left panels). The WT and KO cells within the 0.05. D, The cell numbers for each indicated donor population recovered at live populations were further separated according to the expression of different time points after transfer were calculated by normalizing the CD45.2 (right panels). D, The percentage of WT and KO live cells within percentage of T cells against the percentage of host NK cells. Mean values the total population was calculated from C and is shown. and SEs from three individual mice are shown. The Journal of Immunology 973

Impaired IL-7 signaling and survival of KO T cells It has been shown that naive T cells transferred into lymphopenic Binding of IL-7 to CD127 leads to phosphorylation of STAT5 and is hosts will undergo both endogenous and homeostatic proliferation. required for survival and homeostatic proliferation of peripheral Endogenous proliferation occurs rapidly and is dependent on TCR T cells. To examine whether the impaired CD127 expression had signals and possibly IL-6. In contrast, homeostatic proliferation has any impact on these IL-7–induced downstream events in KO slower kinetics and requires IL-7 (26, 27). Accordingly, reduced T cells, we first tested the ability of KO T cells to phosphorylate CD127 expression should impair homeostatic, but not endoge- STAT5 in response to IL-7. We sorted naive CD4+ T cells from nous, proliferation of KO T cells. We therefore transferred a + HET or KO mice, stimulated the cells with 10 ng/ml IL-7 for 20 mixture of CFSE-labeled naive WT and KO CD4 T cells into 2/2 min, and analyzed the total protein levels for STAT5 as well as Rag2 knockout (Rag2 ) mice and analyzed cell numbers and phosphorylation of STAT5 Tyr694 by Western blot. Unexpectedly, CFSE dilution after 7 and 29 d. As described by others (26), by we found that KO T cells expressed a higher than normal level of day 7 the transferred T cells had split into two populations un- total STAT5. Whereas treatment with IL-7 readily induced Tyr694 dergoing endogenous (fast) and homeostatic (slow) proliferation phosphorylation in HET naive CD4+ T cells, very little phospho- and expressing high and low CD44 levels, respectively (Fig. 5A, STAT5 was detected in KO T cells despite the increase in the level left upper panel). The population dividing faster had completed at of total STAT5 (Fig. 4A). We have shown earlier that KO Th cells, least seven divisions and become CFSE-negative (CFSE2). It was once the expression of CD25 is induced, are fully capable of made up of KO and WT cells at a near 1:1 ratio (Fig. 5A, middle phosphorylating STAT5 in response to IL-2 (13), indicating that upper and right upper panels). Thus, the endogenous proliferation the downstream machinery for phosphorylating STAT5 is intact in of KO T cells was undisturbed. In contrast, whereas WT cells these cells. undergoing homeostatic proliferation had divided up to two times We then examined whether the impaired IL-7 signaling would and remained CFSE+, their KO counterparts had not divided at all, affect IL-7–dependent survival of KO T cells. To this aim, we set resulting in a substantial reduction in the ratio between KO and up cocultures of sorted naive CD4+ T cells from KO mice or WT populations (Fig. 5A, middle upper and right upper panels). congenic WT C57BL/6 mice expressing CD45.1. Naive T cells Thus, IL-7–dependent homeostatic proliferation is impaired in were sorted based on high CD45RB expression. WT and KO cells the absence of Ets-1. This defect in IL-7–dependent homeostatic were mixed and labeled with CFSE. Fig. 4B shows the expression proliferation may in part contribute to the reduction in the number of CD127 on WT and KO cells in the starting coculture. The cells of naive peripheral T cells in KO mice (Fig. 2A, Supplemental were then either cultured in media alone or in media containing 10 Fig. 2). ng/ml IL-7 for 72 h. As shown in Fig. 4C, only ∼4% of the We also found that those T cells that had undergone endogenous unstimulated cells fell in the live gate after 72 h of culture, and the proliferation expressed a high level of CD127 compared with those ratio between KO and WT cells remained largely unchanged. undergoing homeostatic proliferation (Fig. 5B,5C, day 7). This More live cells (∼30%) were detected in the culture containing upregulation of CD127 was independent of Ets-1 as both WT and IL-7. But the ratio between KO and WT cells within the live KO populations expressed comparable levels of CD127. However, population was significantly reduced. Thus, whereas IL-7 sub- Ets-1 was required for maintaining the high level of CD127 in stantially increased the percentage of live HET CD4+ T cells, it those cells at later time points. When examined at 29 d after only had a minimal effect on KO CD4+ T cells (Fig. 4D). The transfer, the WT cells that had become CFSE2 still expressed change in the ratio was not due to a difference in proliferation a high level of CD127 and the population continued to expand between WT and KO T cells because the content of CFSE (Fig. 5D). In contrast, the KO CFSE2 cells could not maintain the remained unaltered even in the presence of IL-7 (Fig. 4C, right high level of CD127 (Figure 5B,5C), and their number did not panels). further increase (Fig. 5D), resulting in a significant reduction in

FIGURE 6. Ets-1 and CD127 expression is correlated in human T cells. Total (A, n = 10 individuals) and indicated subsets of peripheral CD8+ T cells (C, n = 6 individuals) were purified from peripheral blood of healthy donors according to the gating criteria shown in B. Each population of cells was further stained with anti-CD127 (black lines) or an isotype control (gray lines). Histograms from one representative experiment are shown in B. The transcript level of CD127 and Ets-1 p51 was quantified with real-time PCR and normalized against the level of Actin. The correlation between CD127 and Ets-1 was tested with a Spearman non-parametric test. The r2 for naive cells is 0.7927. 974 Ets-1 CONTROLS IL7-R EXPRESSION the ratio between CFSE2 KO and CFSE2 WT populations (Fig. 5A, lower panels). At this late time point, the WT CFSE+ cells started to merge with the CFSE2 cells (Fig. 5A, lower panels), and their numbers increased slightly despite a reduction in per- centage (Fig. 5D). Instead, very few KO CFSE+ cells were de- tected, and these cells still expressed low levels of CD127 and stayed segregated from the CFSE2 population. The transcript level of Ets-1 correlates with the level of CD127 in human T cells To determine whether the level of Ets-1 correlates with the ex- pression of CD127 in human T cells, we isolated peripheral blood CD8+ T cells from healthy donors and analyzed their expression of CD127 and Ets-1. We found a strong linear correlation between the transcript level of CD127 and Ets-1 (Fig. 6A). Using live cell sorting of primary cell subsets, we further fractionated CD8+ T cells into naive (CD27+CD45RA+), effector (CD272CD45RA+), EM (CD272CD45RA2), and CM (CD27+CD45RA2) populations (Fig. 6B). As expected, naive T cells expressed high levels of CD127 (Fig. 6B,6C). There was a strong correlation between Ets- 1 and CD127 mRNA levels in this subset (Fig. 6C). Effector cells lost both Ets-1 and CD127 expression (Fig. 6C) but partially regained CD127 when they became EM cells. Notably, CM cells fully regained CD127 expression despite a low level of Ets-1. These data suggest that Ets-1 also participates in maintaining the CD127 level in human naive T cells and that additional transcription factors are required for the reexpression of CD127 in human CM cells.

Loss of CD127 expression during HIV infection occurs only in Ets-1low T cells FIGURE 7. Ets-1 and CD127 expression is correlated in human T cells. + Loss of CD127 expression in CD8 T cells is a hallmark of HIV A, CD127 expression (black) on CD8 T cells from an HIV-negative and an infection (18, 19, 28). We wondered whether the HIV-associated HIV-positive donor. The gray lines are histograms of staining with an loss of CD127 expression correlated with downregulation of Ets-1. isotype control Ab. B and C, Total CD8+ T cells from the 10 HIV-negative Therefore, we also analyzed peripheral blood CD8+ T cells from (shown in Fig. 3A) and 10 HIV-positive donors were purified by MACS. HIV-positive donors. As expected, the surface level of CD127 was All of the samples with the exception of two HIV-negative ones were significantly lower in CD8+ T cells of HIV-positive donors than analyzed for surface expression of CD127 (B). All samples were also those of healthy controls (Fig. 7A,7B). The reduction in the level analyzed for transcript levels of CD127, Ets-1 p51, and b2m with real-time of surface CD127 was also reflected in the level of CD127 tran- PCR. The levels were normalized against that of Actin and are shown in corresponding panels (C). Significant differences between HIV-negative script (Fig. 7C, left panel), suggesting that the downregulation of and HIV-positive donors were detected using an unpaired t test: *p , 0.05. CD127 is mediated mainly by a transcriptional mechanism rather D, Indicated subsets of CD8+ T cells were sorted from six HIV-negative than a posttranscriptional mechanism. Notably, the transcript level and six HIV-positive donors described in B. The transcript levels of CD127 of Ets-1 p51 was also significantly reduced in HIV-infected sub- and Ets-1 were quantified and are shown in corresponding panels. *p , jects compared with HIV-negative individuals (Fig. 7C, middle 0.05; **p , 0.01. E, The percentage of each subset within total CD8+ panel). In contrast, the transcript level of b2m was comparable T cells is shown. between healthy and HIV-infected individuals (Fig. 7C, right panel). To examine further whether downregulation of Ets-1 may of HIV-positive individuals contained more effector and EM cells contribute to the loss of CD127 expression during HIV infection, than that of healthy donors (Fig. 7E). Thus, expansion of Ets-1low we also fractionated CD8+ T cells of HIV-positive donors into effector and EM cells at the expense of Ets-1high naive cells naive, effector, EM, and CM populations and measured the tran- contributes to the reduction in the level of Ets-1 in bulk CD8+ script levels of Ets-1 and CD127 in each subset. We found that T cells of HIV-positive individuals. HIV-associated loss of CD127 was observed only in EM and CM cells (Fig. 7D, left panel), which expressed a low level of Ets-1 Discussion (Fig. 7D, right panel). In contrast, no reduction in CD127 ex- The expression of CD127 is tightly regulated and varies swiftly in pression was detected in naive T cells, which expressed the response to environmental cues. But very little is known regarding highest level of Ets-1. We did not detect any difference in the level the molecular mechanism controlling the dynamic expression of of CD127 between healthy and HIV-positive effector cells prob- CD127. The current report fills some of the gaps in our knowledge ably because these cells already expressed a nearly undetectable of CD127 regulation and a) identifies Ets-1 as a critical regulator level of CD127. However, there was no difference in the transcript for CD127 expression in mature T cells and b) provides evidence level of Ets-1 in any of the subsets between healthy and HIV- suggesting that downregulation of Ets-1 may contribute to the loss positive individuals (Fig. 7D, right panel), indicating that down- of CD127 expression in CD8+ T cells during HIV infection. regulation of Ets-1 alone is insufficient to induce the loss of Our data expand on the role of Ets members in regulating the CD127 during HIV infection. We also found that peripheral blood expression of CD127 in lymphoid cells. GABPa has been shown to The Journal of Immunology 975 control the expression of CD127 in early thymocytes (DN1 and HIV-derived Tat protein and abnormally high levels of serum IL-7 DN2 cells) (10). Deficiency of Ets-1 also affects CD127 expres- detected in HIV-positive individuals have been shown to cause sion in DN1 but not DN2 thymocytes. In addition, KO naive and downregulation of surface CD127 even in uninfected T cells. We central memory T cells also express a low level of CD127, sug- found that HIV-associated loss of CD127 was observed only in EM gesting that Ets-1 is the dominant Ets factor that controls CD127 and CM, but not naive, CD8+ T cells. As naive T cells are also expression in peripheral T cells. However, because KO peripheral susceptible to the influence of Tat and IL-7, additional mecha- T cells are not completely negative for CD127, other Ets factors nisms or signals are required for shutting down the expression of such as Ets-2, ELF, ELK, or GABPa may compensate for the loss CD127 in EM and CM cells during HIV infection. It is intriguing of Ets-1. It is noteworthy that a recent publication proposes a role to notice that both EM and CM, but not naive, cells expressed for GABPa in regulating CD127 in peripheral T cells (29). We a low level of Ets-1. This observation raises the possibility that did, however, not detect any significant change in the level of downregulation of Ets-1 is a prerequisite, but insufficient, for the GABPa in KO T cells (data not shown). The role of other Ets loss of CD127 expression. It will be of great interest to examine factors in maintaining the constitutive expression of CD127 in whether forced expression of Ets-1 will render T cells resistant to T cells remains to be determined. HIV-associated loss of CD127 expression and how Ets-1 is In addition to the Ets family of transcription factors, other downregulated in Ag-experienced T cells. transcription factors are also important for the expression of CD127. It was recently reported that Foxo1-deficient T cells also Acknowledgments displayed a profound defect in the expression of CD127, did not We thank Dr. Koichi Ikuta for generously providing the CD127 promoter respond to IL-7, and had impaired homeostatic expansion (30, 31). reporter and Dr. Mark Brockman for providing the human b-Actin primers. Foxo1 binds to the promoter of CD127 in a region upstream of the We would also like to thank Dr. Sung-Yun Pai for critical review of the putative Ets-1 binding site and is required for the cytokine manuscript. withdraw-mediated induction of CD127. Given our findings, it would be very interesting to examine whether there is any func- Disclosures tional interaction between Ets-1 and Foxo1. To our knowledge, The authors have no financial conflicts of interest. our data also demonstrate for the first time that the transcriptional regulation of CD127 varies among different peripheral T cells subsets. 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