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. Author manuscript; available in PMC: 2012 Jan 15.
Published in final edited form as: J Immunol. 2010 Dec 10;186(2):969–976. doi: 10.4049/jimmunol.1002099

Ets-1 maintains IL-7 receptor expression in peripheral T cells

Roland Grenningloh *,†, Tzong-Shyuan Tai *, Nicole Frahm †,§, Tomoyuki C Hongo §, Adam T Chicoine §, Christian Brander †,§,||,#, Daniel E Kaufmann †,§,, I-Cheng Ho *,†
PMCID: PMC3074256  NIHMSID: NIHMS251999  PMID: 21148801

Abstract

The expression of CD127, the IL-7 binding subunit of the IL-7 receptor, 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. Here we report that the transcription factor 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 naïve CD8+ T cells. Taken together, our data identify Ets-1 as a critical regulator of CD127 expression in T cells.

Introduction

IL-7 signals are required for T cell development, maintaining the naïve T cell pool, mounting proper primary responses, and inducing and maintaining CD4+ and CD8+ T cell memory (1–3). The IL-7 receptor consists of the IL-7Rα chain (CD127), which binds IL-7, and the common gamma chain (γc, CD132). CD127 expression is dynamically regulated throughout T cell development, activation, and memory formation (2, 4). Many extracellular stimuli can modulate the expression of CD127. For example, TCR signals (5), IL-7 (6), and the HIV Tat protein (7) have been shown to inhibit the expression of CD127. However, the mechanisms governing this dynamic regulation of CD127 are poorly understood. The constitutive expression of CD127 is regulated by members of the Ets family of transcription factors in different lymphoid lineages. Ets proteins are characterized by their DNA-binding Ets domain (8). The B cell-specific PU.1 is essential for the expression of CD127 in lin− hematopoietic progenitors and pro-B cells (9), whereas GABPα is required for the expression of CD127 in early thymocytes (10). Both factors bind to a functionally critical Ets binding site in the CD127 promoter. But whether GABPα or another Ets protein is responsible for maintaining CD127 expression in peripheral T cells is unknown.

Ets-1 (E26 transformation-specific sequence) is the founding member of the Ets family of transcription factors and is expressed at high levels in lymphoid cells (8, 11). Ets-1 has been shown to promote Th1 and inhibit Th17 differentiation (12, 13). Ets-1 is also recruited to the IL-5/IL-13/IL-4 locus and required for the optimal expression of these cytokines (14). T cells express two splice variants, the full-length Ets-1 p51 and the shorter Ets-1 p42 that lacks ExonVII (15, 16). The activity of Ets-1 is regulated by activating and inactivating phosphorylation events (11), but the role of these phosphorylation events in regulating the function of Th cells remains controversial (17).

Here we show that Ets-1 directly binds to and activates the CD127 promoter. Ets-1-deficient (KO) T cells expressed reduced levels of CD127 and displayed impaired IL-7-dependent survival and homeostatic proliferation. Importantly, the level of CD127 in human T cells strongly correlates with that of Ets-1. Loss of CD127 expression is a hallmark of CD8+ T cells during chronic viral infection (18, 19). The expression of Ets-1 is also reduced in CD8+ T cells of HIV-positive individuals mainly due to an expansion of effector/effector memory cells. Interestingly, HIV-associated reduction in CD127 occurs only in Ets-1low effector memory and central memory cells but not in Ets-1high naïve cells. Thus, our data demonstrate that Ets-1 is a critical regulator of CD127 in peripheral T cells.

Material and methods

Study subjects

The study included 10 individuals chronically infected with HIV. Four of these subjects were treated with antiviral therapy (ARV) and six were untreated. All but one had plasma viral loads above the limit of detection of the quantitative assays used for clinical monitoring (>50 or >75 viral mRNA copies/mL). Median viral load was 21,200 viral mRNA copies/mL (range: <75 to 233,000). Additionally, ten HIV-uninfected healthy volunteers were included as controls. Human subject protocols were approved by all participating hospitals and clinics, and all subjects provided written informed consent prior to enrollment.

Mice

Ets-1 deficient mice were described previously (20). Mice were backcrossed to the C57BL/6 background for six generations and maintained by mating Ets-1−/− male to Ets-1+/− female mice. Ets-1+/− (designated “HET”) and Ets-1−/− (designated “KO”) littermates were used throughout the studies. Congenic CD45.1 C57BL/6 and CD45.1 Rag2−/− mice were purchased from Taconic (Hudson, NY). The animals were housed under specific pathogen free conditions, and all experiments were carried out in accordance with the institutional guidelines for animal care at the Dana-Farber Cancer Institute.

Cell lysates, Western Blot, and Antibodies

Total cell lysates, nuclear or cytosolic extracts were adjusted for total protein content and subjected to SDS-PAGE followed by Western Blot. The following antibodies were used: anti-STAT5 and anti-Phospho-STAT5 (Tyr694) (Cell Signaling Technology, Danvers, MA), and anti-Hsp90 (both Santa Cruz Biotechnology, Santa Cruz, CA). As secondary antibodies, HRP-coupled goat-anti-rabbit IgG was used (Zymed Laboratories). Proteins were visualized using an ECL kit (Perkin Elmer).

Cell isolation and culture, reagents, flow cytometry

Mouse T cells were isolated from lymph nodes and spleen using magnetic cell sorting as described (12). For staining of human CD127 the Alexa647-coupled antibody from eBioscience was used (San Diego, CA); all other antibodies used for flow cytometry were purchased from BD Biosciences (Franklin Lakes, NJ). Flow cytometry was performed on a FACS Canto (murine samples) or LSRII (human samples) and analyzed with FlowJo software. Isolation of CD8+ T cell subsets from PBMC of HIV-positive and HIV-negative subjects was performed on a FACS Aria (BD Biosciences) equipped for biohazardous material. PBMC were stained with lineage exclusion antibodies (CD4, CD14, CD19 and CD56 conjugated to V450, all BD Biosciences), anti-CD27 PE (BD), anti-CD45RA APC (BD) and CD127-PerCP Cy 5.5 (eBioscience). In order to avoid pre-stimulation, CD8+ T cells were negatively selected (anti-CD3 or anti-CD8 antibodies were not used) and CD8+ T cell subsets sorted based on the CD27/CD45RA phenotype as outlined in Figure S4. The FACS Aria was operated at at 70 pounds per square inch with a 70 μm nozzle. For all populations, at least 100,000 cells were collected directly into RLT lysis buffer and RNA extracted using the RNeasy Mini kit (Qiagen).

Bone marrow transduction

Five days before bone marrow harvest, HET or KO donor mice were injected intraperitoneally with 100 μl of 50 mg/ml 5-Fluorouracil (Sigma). Bone marrow cells were isolated, and SCA-1 positive cells were enriched using magnetic cell sorting. The cells were cultured in media containing 20 ng/ml IL-3, 50 ng/ml SCF, and 50 ng/ml IL-6 (all from Peprotech). 72 hours and 96 hours later the cells were infected with a retrovirus expressing Ets-1 p51 or a control retrovirus (17). 24 hours after the last infection, bone marrow cells were injected intravenously into irradiated (400 rad) Rag2−/− mice.

Luciferase assays and chromatin immunoprecipitation (ChIP) assay

Luciferase assays were performed as described (17). The CD127 promoter construct pGL3-IL-7Rαpr, the Ets-1 p51 expression vector pcDNA-Ets-1 p51, and the Runx1 expression vector pcDNA-MycRunx1 have been described elsewhere (12, 21, 22). ChIP assays were performed as described using in vitro differentiated Th1 cells (12). Precipitated DNA fragments were amplified by quantitative PCR. The antibodies used for immunoprecipitation were anti-Ets1 C-20 and control rabbit IgG (both Santa Cruz Biotechnology). Specific binding was calculated as 2^-(CtEts1/CtCtrl). The following primer pairs were used: CD127 promoter Ets binding site, 5′-ACCACAGACAGGGAACTATG-3′ and 5′-CACACTCTACCTCTCCGTTT-3′, IFN-γ promoter, 5′-CTTTCAGAGAATCCCACAAG-3′ and 5′-TTAAGATGGTGACAGATAGGTG-3′, TLR7, 5′-TCTAGAGTCTTTGGGTTTCG-3′ and 5′-TTCTGTCAAATGCTTGTCTG-3′.

Quantitative RNA Analysis

RNA isolation, reverse transcription, and real-time PCR were performed as described (13). The following primer pairs were used: mouse (ms)CD127 5′-GCGGACGATCACTCCTTCTG-3′ and 5′-AGCCCCACATATTTGAAATTCCA-3′, human (hu)CD127 5′-CCCTCGTGGAGGTAAAGTGC-3′ and 5′-CCTTCCCGATAGACGACACTC-3′, hu/msEts1 p51 5′-CTCCTATGACAGCTTCGACT-3′ and 5′-ATCTCCTGTCCAGCTGATAA-3′, huβ2m 5′-GTGGCCTTAGCTGTGCTCG-3′ and 5′-ACCTGAATGCTGGATAGCCTC-3′, huActin 5′-GTGACAGCAGTCGGTTGGAG-3′ and 5′-AGGACTGGGCCATTCTCCTT-3′, msActin 5′-GGCTGTATTCCCCTCCATCG-3′ and 5′-CCAGTTGGTAACAATGCCATG-3′

Statistical analysis

Statistical analysis was done using Prism Software (GraphPad, La Jolla, CA). Paired or unpaired t tests were used as indicated in the Figure legends. p < 0.05 was considered significant.

Results

Ets-1 is required for constitutive CD127 expression in murine peripheral T cells

PU.1 and GABPα have been shown to regulate the expression of CD127 in pro-B cells and early thymocytes, respectively (9, 10). Since Ets-1 is expressed at high levels in thymocytes and mature T cells, we hypothesized that Ets-1 might participate in regulating the expression of CD127. We first analyzed the expression of CD127 at different stages of thymocyte development in KO mice. In agreement with a previous publication (23), we found that KO thymi contained a higher percentage of DN cells but fewer mature CD8 SP cells (Figures 1a and 1b) than HET thymuses. The expression of CD127 was reduced at the DN1 stage but normal in DN2 and DN3 cells in KO mice (Figure 1a). CD127 is normally downregulated in the DN4 and DP stage and reappears in mature (TCRβhi CD24lo) SP cells. However, both KO CD4 and CD8 SP thymocytes displayed significantly reduced CD127 expression (Figure 1b and 1c) compared to HET cells, which expressed a normal level of Ets-1 and, expectedly, CD127 (Supplemental Figure 1).

Figure 1. Ets-1 is required for CD127 expression in DN1 and mature thymic T cells.

Figure 1

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(a) Expression of CD127 in immature thymocytes. Thymocytes from Ets-1HET and Ets-1KO mice were stained with antibodies 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, grey lines: isotype control). (b) Expression of CD127 on mature thymic T cells. Mature thymic T cells were identified as TCRβhiCD24lo and further separated into CD4+ or CD8+ T cells. Histograms show CD127 expression. (c). Results from three HET and KO littermates were combined. Paired t tests were performed and statistically significant differences in the level of CD127 expression were denoted with *.

We then analyzed CD127 expression in peripheral T cells. Figure 2a shows the gating strategy used to identify naïve (CD44lo CD62Lhi), effector memory (EM, CD44hi CD62Llo), and central memory (CM, CD44hi CD62Lhi) T cells. As described by others, we observed an increased percentage of CD4+ and CD8+ EM cells in KO mice (23) (Figure 2a). The expression of CD127 was significantly reduced in naïve and CM T cells from KO animals. EM CD4+ and CD8+ T cells expressed a relatively low level of CD127, and there was no difference between Ets-1 HET and KO EM T cells (Figure 2b and 2c). Thus, Ets-1 is required for constitutive expression of CD127 in DN1 and SP thymocytes, naïve and CM T cells but not DN2 or DN3 thymocytes. The impaired expression of surface CD127 in naïve Ets-1KO T cells correlated with a significant reduction in the level of CD127 mRNA (Figure 2d), suggesting that Ets-1 regulates the expression of CD127 at the transcriptional level.

Figure 2. Reduced CD127 expression in peripheral Ets-1 deficient T cells.

Figure 2

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(a) Splenocytes and lymph node cells from HET and KO mice were stained with antibodies against CD3 and CD8 to identify CD4+ (CD3+CD8−) and CD8+ (CD3+CD8+) T cells, which were further separated into naïve (CD62LhiCD44lo), effector memory (EM, CD62LloCD44hi), and central memory (CM, CD62LhiCD44hi) cells. (b) Each population of cells was further stained with anti-CD127 (black lines) or an isotype control (grey). Histograms from one representative experiment are shown. (c) Combined data from five HET and KO littermate pairs. * indicates significantly different expression (paired t test). (d) RNA was harvested from sorted naïve CD4+ and CD8+ T cells and the level of CD127 transcripts was measured using quantitative PCR. The data shown are the level of expression relative to actin.

Ets-1 binds to and activates the CD127 promoter

The CD127 promoter contains a conserved Ets binding site that is critical for activation by PU.1 or GABPα (Figure 3a) (10, 22). We therefore speculated that Ets-1 might bind to this site in mature T cells and directly regulate the transcription of CD127. By chromatin immunoprecipitation assay (ChIP) we found that Ets-1 indeed bound to the endogenous CD127 promoter with an affinity even higher than its binding to the IFN-γ promoter, a known target of Ets-1 (Figure 3b) (12). In contrast, there was no specific binding of Ets-1 to the TLR7 gene, which served as a negative control. In addition, Ets-1 activated a reporter construct containing the 200 bp minimal promoter of mouse CD127, including the Ets site (22) (Figure 3c). The p42 isoform of Ets-1, which lacks part of the inhibitory domain, was slightly more efficient than the full-length p51 isoform. The degree of activation was comparable to 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) (Figure 3a). Runx1 is required for CD127 expression in CD4+ T cells (24) and can physically interact with Ets-1 (25). While Runx1 also transactivated the CD127 promoter, co-expression 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 restore the expression of Ets-1 to a near physiological level (17). The infected bone marrow cells were then transferred to Rag2KO mice. Peripheral T cells were harvested from the host animals eight weeks later and analyzed. As shown in Figure 3d and 3e, retrovirally expressed Ets-1 efficiently restored CD127 levels in both peripheral naïve CD4+ and CD8+ T cells.

Figure 3. Ets-1 binds to the CD127 promoter and drives CD127 expression.

Figure 3

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(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 stands for transcriptional start site. (b) Binding of Ets-1 to the CD127 and IFN-γ promoters and to the TLR7 gene in CD4+ T cells. Binding of Ets-1 to the indicated genes was determined by ChIP, and binding strength was calculated 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 hours, luminescence was measured. Luminescence was normalized to empty expression vector (pcDNA). Assays were done in triplicates, and means and standard errors are shown. Representative results from one of three independent experiments are shown. (d) and (e) Retroviral expression of Ets-1 p51 restores CD127 expression in peripheral naïve T cells. Bone marrow cells from HET or KO mice were isolated, enriched for Sca-1 positive cells, and infected with the indicated retroviruses. The cells were injected into Rag2−/− mice to allow reconstitution of the lymphoid system, and CD127 expression on naive (CD44lo) CD4+ or CD8+ T cells was analyzed 8 weeks later. (d) Histograms from CD127 staining from one representative mouse are shown. Black: CD127, grey: isotype control. (e) Combined data from three individual mice. An unpaired t test was used to identify significantly different CD127 expression (*: p<0.05).

Impaired IL-7 signaling and survival of Ets-1KO T cells

Binding of IL-7 to CD127 leads to phosphorylation of STAT5 and is required for survival and homeostatic proliferation of peripheral T cells. To examine whether the impaired CD127 expression had any impact on these IL-7-induced downstream events in Ets-1KO T cells, we first tested the ability of KO T cells to phosphorylate STAT5 in response to IL-7. We sorted naïve CD4+ T cells from HET or KO mice, stimulated the cells with 10 ng/ml IL-7 for 20 minutes, and analyzed the total protein levels for STAT5 as well as phosphorylation of STAT5 Tyr694 by Western Blot. Unexpectedly, we found that KO T cells expressed a higher than normal level of total STAT5. While treatment with IL-7 readily induced Tyr694 phosphorylation in HET naïve CD4+ T cells, very little phospho-STAT5 was detected in KO T cells despite the increase in the level of total STAT5 (Figure 4a). We have shown earlier that Ets-1KO Th cells, once the expression of CD25 is induced, are fully capable of phosphorylating STAT5 in response to IL-2 (13), indicating that the downstream machinery for phosphorylating STAT5 is intact in these cells.

Figure 4. Impaired STAT5 phosphorylation and survival of Ets-1 deficient T cells in response to IL-7.

Figure 4

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(a) Reduced STAT5 phosphorylation in naïve KO CD4+ T cells in response to IL-7. Naïve CD4+ T cells were sorted based on CD44loCD62Lhi expression, and either left untreated (Media) or stimulated with 10 ng/ml IL-7 for 20 minutes. Total STAT5 levels and phosphorylation of Tyr694 (P-STAT5) were examined by Western analysis. Hsp90 expression was determined to ensure equal loading. (b) Naïve CD4+ T cells from congenic CD45.1 C57BL/6 (WT) or Ets-1 KO mice were sorted based on CD45RBhi expression. The cells were mixed at a 1:1 ratio (left panel), labeled with CFSE, and CD127 expression was analyzed (black: CD127, grey: isotype control). (c) Decreased survival of KO CD4+ T cells in response to IL-7. The WT (CD45.2−) and KO (CD45.2+) cell mixture from b) was cultured in media alone or in media containing 10 ng/ml IL-7. After 72 hours, the cells were stained for CD45.2, and the percentage of live cells was determined by flow cytometry based on their position in the forward/sideward scatter (left panels). The WT and KO cells within the live populations were further separated according to the expression of CD45.2 (right panels). (d) The percentage of WT and KO live cells within the total population was calculated from (c) and shown.

We then examined whether the impaired IL-7 signaling would affect IL-7-dependent survival of Ets-1KO T cells. To this aim, we set up co-cultures of sorted naïve CD4+ T cells from Ets-1KO or congenic wild type mice expressing CD45.1 (WT). Naïve T cells were sorted based on high CD45RB expression. WT and KO cells were mixed and labeled with CFSE. Figure 4b shows the expression of CD127 on WT and KO cells in the starting co-culture. The cells were then either cultured in media alone or in media containing 10 ng/ml IL-7 for 72 hours. As shown in Figure 4c, only about 4% of the unstimulated cells fell in the live gate after 72 hours of culture, and the ratio between KO and WT cells remained largely unchanged. More live cells (approximately 30 %) were detected in the culture containing IL-7. But the ratio between KO and WT cells within the live population was significantly reduced. Thus, while IL-7 substantially increased the percentage of live HET CD4+ T cells, it only had a minimal effect on KO CD4+ T cells (Figure 4d). The change in the ratio was not due to a difference in proliferation between WT and KO T cells because the content of CFSE remained unaltered even in the presence of IL-7 (Figure 4c, right panels).

It has been shown that naïve T cells transferred into lymphopenic hosts will undergo both endogenous and homeostatic proliferation. Endogenous proliferation occurs rapidly and is dependent on TCR signals and possibly IL-6. In contrast, homeostatic proliferation has slower kinetics and requires IL-7 (26, 27). Accordingly, reduced CD127 expression should impair homeostatic, but not endogenous, proliferation of Ets-1KO T cells. We therefore transferred a mixture of CFSE-labeled naïve WT and KO CD4+ T cells into Rag2KO mice and analyzed cell numbers and CFSE dilution after 7 and 29 days. As described by others (26), by day 7 the transferred T cells had split into two populations undergoing endogenous (fast) and homeostatic (slow) proliferation and expressing high and low CD44 levels, respectively (Figure 5a, left upper panel). The population dividing faster had completed at least 7 divisions and become CFSE-negative (CFSE−). It was made up of KO and WT cells at a near 1:1 ratio (Figure 5a, middle upper and right upper panels). Thus, the endogenous proliferation of Ets-1KO T cells was undisturbed. In contrast, while WT cells undergoing homeostatic proliferation had divided up to two times and remained CFSE+, their Ets-1KO counterparts had not divided at all, resulting in a substantial reduction in the ratio between KO and WT populations (Figure 5a, middle upper and right upper panels). Thus, IL-7-dependent homeostatic proliferation is impaired in the absence of Ets-1. This defect in IL-7 dependent homeostatic proliferation may in part contribute to the reduction in the number of naïve peripheral T cells in KO mice (Figure 2a and Supplemental Figure 2).

Figure 5. Impaired expansion of Ets-1 deficient T cells in a lymphopenic host.

Figure 5

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(a) A mixture of naïve, CFSE labeled WT (CD45.2−) and KO (CD45.2+) CD4+ T cells (at an approx 1:1 ratio) was injected intravenously into Rag2−/− mice (one million cells/mouse). At day 7 and 29, inguinal, axillary, and mandibular lymph nodes were isolated. Donor T cells (CD4+TCRβ+) were analyzed for the expression of CD44 and content of CFSE (left panels). WT and KO donor cells were distinguished according to the expression of CD45.2 (middle panels). The CFSE− and CFSE+ populations represent cells undergoing endogenous and homeostatic proliferation, respectively. The percentage of CFSE− and CFSE+ WT or KO T cells from one representative mouse is shown in the middle panels. The means and standard errors from three mice are shown in the right panels. (b) and (c) The indicated populations were further analyzed for the expression of CD127 on day 7 and day 29. Data from one representative mouse are shown in (b). Combined mean values and standard deviation from 3 mice are shown in c). An unpaired t test was used to detect significant differences between WT and KO (*: p<0.05). (d) The cell numbers for each indicated donor population recovered at different time points after transfer were calculated by normalizing the percentage of T cells against the percentage of host NK cells. Mean values and standard errors from three individual mice are shown.

We also found that those T cells that had undergone endogenous proliferation expressed a high level of CD127 compared to those undergoing homeostatic proliferation (Figure 5b and 5c, day 7). This upregulation of CD127 was independent of Ets-1 since both WT and KO populations expressed comparable levels of CD127. However, Ets-1 was required for maintaining the high level of CD127 in those cells at later time points. When examined at 29 days after transfer, the WT cells that had become CFSE− still expressed a high level of CD127 and the population continued to expand (Figure 5d). In contrast, the KO CFSE− cells could not maintain the high level of CD127 (Figure 5b and 5c) and their number did not further increase (Figure 5d), resulting in a significant reduction in the ratio between CFSE− KO and CFSE− WT populations (Figure 5a, lower panels). At this late time point, the WT CFSE+ cells started to merge with the CFSE− cells (Figure 5a, lower panels) and their numbers increased slightly despite a reduction in percentage (Figure 5d). Instead, very few KO CFSE+ cells were detected, and these cells still expressed low levels of CD127 and stayed segregated from the CFSE− 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 expression 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 (Figure 6a). Using live cell sorting of primary cell subsets, we further fractionated CD8+ T cells into naïve (CD27+CD45RA+), effector (CD27−CD45RA+), effector memory (CD27−CD45RA−, EM), and central memory (CD27+CD45RA−, CM) populations (Figure 6b). As expected, naive T cells expressed high levels of CD127 (Figure 6b and 6c). There was a strong correlation between Ets-1 and CD127 mRNA levels in this subset (Figure 6c). Effector cells lost both Ets-1 and CD127 expression (Figure 6c) but partially regained CD127 when they became EM cells. Interestingly, CM cells fully regained CD127 expression despite a low level of Ets-1. These data suggest that Ets-1 also participates in maintaining CD127 level in human naïve T cells and that additional transcription factors are required for the re-expression of CD127 in human CM cells.

Figure 6. Ets-1 and CD127 expression is correlated in human T cells.

Figure 6

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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 (grey). 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 naïve cells is 0.7927.

Loss of CD127 expression during HIV infection occurs only in Ets-1low T cells

Loss of CD127 expression in CD8+ T cells is a hallmark of HIV infection (18, 19, 28). We wondered whether the HIV-associated loss of CD127 expression correlated with down-regulation of Ets-1. Therefore, we also analyzed peripheral blood CD8+ T cells from HIV-positive donors. As expected, the surface level of CD127 was significantly lower in CD8+ T cells of HIV-positive donors than those of healthy controls (Figures 7a and 7b). The reduction in the level of surface CD127 was also reflected in the level of CD127 transcript (Figure 7c left panel), suggesting that the down-regulation of CD127 is mediated mainly by a transcriptional mechanism rather than a post-transcriptional mechanism. Interestingly, the transcript level of Ets-1 p51 was also significantly reduced in HIV-infected subjects compared to HIV-negative individuals (Figure 7c middle panel). In contrast, the transcript level of β2m was comparable between healthy and HIV-infected individuals (Figure 7c right panel).

Figure 7. Ets-1 and CD127 expression is correlate in human T cells.

Figure 7

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(a) CD127 expression (black) on CD8 T cells from an HIV− and an HIV+ donor. The grey lines are histograms of staining with an isotype control antibody. (b and c) Total CD8+ T cells from the 10 HIV− (shown in Figure 3a) and 10 HIV+ donors were purified by MACS. All of the samples with the exception of two HIV− ones were analyzed for surface expression of CD127 (c). All samples were also analyzed for transcript levels of CD127, Ets-1 p51, and beta2-microglobulin (β2m) with real-time PCR. The levels were normalized against that of Actin and shown in corresponding panels (c). Significant differences between HIV− and HIV+ donors were detected using an unpaired t test (* p<0.05, ** p<0.01, *** p<0.001). (d) Indicated subsets of CD8+ T cells were sorted from six HIV− and six HIV+ donors described in b. The transcript levels of CD127 and Ets-1 were quantified and shown in corresponding panel. (e) The percentage of each subset within total CD8+ T cells was shown.

To further examine whether down-regulation of Ets-1 may contribute to the loss of CD127 expression during HIV infection, we also fractionated CD8+ T cells of HIV-positive donors into naïve, effector, EM, and CM populations and measured the transcript levels of Ets-1 and CD127 in each subset. We found that HIV-associated loss of CD127 was observed only in EM and CM cells (Figure 7d left panel), which expressed a low level of Ets-1 (Figure 7d right panel). In contrast, no reduction in CD127 expression was detected in naïve T cells, which expressed the highest level of Ets-1. We did not detect any difference in the level of CD127 between healthy and HIV-positive effector cells probably because these cells already expressed a nearly undetectable level of CD127. However, there was no difference in the transcript level of Ets-1 in any of the subsets between healthy and HIV-positive individuals (Figure 7d right panel), indicating that down-regulation of Ets-1 alone is insufficient to induce the loss of CD127 during HIV infection. We also found that peripheral blood of HIV-positive individuals contained more effector and EM cells than that of healthy donors (Figure 7e). Thus, expansion of Ets-1low effector and EM cells in the expense of Ets-1high naïve cells contributes to the reduction in the level of Ets-1 in bulk CD8+ T cells of HIV-positive individuals.

Discussion

The expression of CD127 is tightly regulated and varies swiftly in response to environmental cues. But very little is known regarding the molecular mechanism controlling the dynamic expression of CD127. The present report fills some of the gaps in our knowledge of CD127 regulation and a) identifies Ets-1 as a critical regulator for CD127 expression in mature T cells and b) provides evidence suggesting that down-regulation of Ets-1 may contribute to the loss of CD127 expression in CD8+ T cells during HIV infection.

Our data expand on the role of Ets members in regulating the expression of CD127 in lymphoid cells. GABPα has been shown to control the expression of CD127 in early thymocytes (DN1 and DN2 cells) (10). Deficiency of Ets-1 also affects CD127 expression in DN1 but not DN2 thymocytes. In addition, Ets-1KO naïve and central memory T cells also express a low level of CD127, suggesting that Ets-1 is the dominant Ets factor that controls CD127 expression in peripheral T cells. However, since Ets-1KO peripheral T cells are not completely negative for CD127, other Ets factors such as Ets-2, ELF, ELK, or GABPα may compensate for the loss of Ets-1. It is noteworthy that a recent publication proposes a role for GABPα in regulating CD127 in peripheral T cells (29). We did, however, not detect any significant change in the level of GABPα in Ets-1KO T cells (data not shown). The role of other Ets factors in maintaining the constitutive expression of CD127 in T cells remains to be determined.

In addition to the Ets family of transcription factors, other transcription factors are also important for the expression of CD127. It was recently reported that Foxo1-deficient T cells also displayed a profound defect in the expression of CD127, did not respond to IL-7, and had impaired homeostatic expansion (30, 31). Foxo1 binds to the promoter of CD127 in a region upstream of the putative Ets-1 binding site and is required for the cytokine withdraw-mediated induction of CD127. Given our findings, it would be very interesting to examine whether there is any functional interaction between Ets-1 and Foxo1. Our data also for the first time demonstrate that the transcriptional regulation of CD127 varies among different peripheral T cells subsets. Human CM T cells still express a high level of CD127 despite a “sub-naïve” level of Ets-1 and the expression of CD127 in murine EM cells is not affected by the absence of Ets-1. Examining the differential expression of the aforementioned transcription factors in each of the subsets will yield important insights into the molecular mechanism regulating CD127 expression in T cells.

Ets-1KO T cells expanded poorly in lymphopenic hosts. Previous studies have demonstrated that IL-7 is required for homeostatic proliferation, whereas endogenous proliferation depends on TCR-mediated signals and IL-6 (27). We indeed found that homeostatic proliferation but not endogenous proliferation of Ets-1KO T cells was impaired at day 7. Interestingly, Ets-1KO T cells that had undergone endogenous proliferation could not maintain the expression of CD127 and failed to expand beyond day 7. This observation suggests that the survival or continuous proliferation of these cells may also become dependent on IL-7 after the initial expansion through IL-7-independent endogenous proliferation. CD127 has recently been shown to be critical for the development of Th cells-induced colitis in lymphopenic mice (32). We have previously shown that Ets-1KO Th cells are unable to cause colitis in SCID mice (12). The molecular mechanism mediating this phenomenon is still unclear. It is very likely that the impaired CD127 expression contributes to the incapacity of Ets-1KO Th cells to induce colitis. This scenario is now being examined.

IL-7 signals were recently shown to induce Runx3 expression and promote CD8 lineage choice in post-selected thymocytes (33). Deficiency of Ets-1 leads to abnormal thymic development characterized by a reduced number of mature CD8SP thymocytes and the presence of TCRhi DP cells (34). We have shown that this phenotype is caused by impaired Runx3 expression, resulting in incomplete silencing of CD4 gene in CD8 SP thymcoytes. Although Ets-1 can be recruited to the runx3 gene and can potentially transactivate Runx3 promoter, our data strongly suggest that Ets-1 can also indirectly regulate the level of Runx3 through controlling CD127 expression and IL-7 signaling.

Our data also provide insights into the molecular mechanism mediating the loss of CD127 expression during HIV infection. Both HIV-derived Tat protein and abnormally high levels of serum IL-7 detected in HIV-positive individuals have been shown to cause down-regulation of surface CD127 even in uninfected T cells. We found that HIV-associated loss of CD127 was observed only in EM and CM, but not naïve, CD8+ T cells. As naive T cells are also susceptible to the influence of Tat and IL-7, additional mechanisms or signals are required for shutting down the expression of CD127 in EM and CM cells during HIV infection. It is intriguing to notice that both EM and CM, but not naïve, cells expressed a low level of Ets-1. This observation raises the possibility that down-regulation of Ets-1 is a pre-requisite, but insufficient, for the loss of CD127 expression. It will be of great interest to examine whether forced expression of Ets-1 will render T cells resistant to HIV-associated loss of CD127 expression and how Ets-1 is down-regulated in antigen-experienced T cells.

Supplementary Material

Supplemental Figures

Acknowledgments

We would like to thank Dr. Koichi Ikuta for generously providing the CD127 promoter reporter, and Dr. Mark Brockman for providing the human β-Actin primers. We would also like to thank Dr. Sung-Yun Pai for critical review of the manuscript.

This work was supported in part by a NIH grant AI081052 (ICH), a Senior Research Award (ICH) from Crohn’s and Colitis Foundation of America, a NIH grant HL092565 (DEK), and a National Merit Scholarship (TST) from National Science Council, Taiwan.

Abbreviations

Ets-1KO

Ets-1-deficient

Ets-1HET

Ets-1 heterozygous

DN

double negative

SP

single positive

EM

effector memory

CM

central memory

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