The Transcription Factor PU.1 Regulates γδ T Cell Homeostasis

Background T cell development results in the generation of both mature αβ and γδ T cells. While αβ T cells predominate in secondary lymphoid organs, γδ T cells are more abundant in mucosal tissues. PU.1, an Ets family transcription factor, also identified as the spleen focus forming virus proviral integration site-1 (Sfpi1) is essential for early stages of T cell development, but is down regulated during the DN T-cell stage. Methodology/Principal Findings In this study, we show that in mice specifically lacking PU.1 in T cells using an lck-Cre transgene with a conditional Sfpi1 allele (Sfpi1 lck−/−) there are increased numbers of γδ T cells in spleen, thymus and in the intestine when compared to wild-type mice. The increase in γδ T cell numbers in PU.1-deficient mice is consistent in γδ T cell subsets identified by TCR variable regions. PU.1-deficient γδ T cells demonstrate greater proliferation in vivo and in vitro. Conclusions/Significance The increase of γδ T cell numbers in Lck-Cre deleter strains, where deletion occurs after PU.1 expression is diminished, as well as the observation that PU.1-deficient γδ T cells have greater proliferative responses than wild type cells, suggests that PU.1 effects are not developmental but rather at the level of homeostasis. Thus, our data shows that PU.1 has a negative influence on γδ T cell expansion.


Introduction
T cells are divided into two populations based on their surface expression of ab and cd T cell receptors (TCR). cd T cells function in immunosurveillance playing a significant role in innate immunity, autoimmunity and allergic responses [1]. cd T cells constitute only a small proportion (1-5%) of the lymphocytes that circulate in the blood and peripheral organs of most adult animals however; they are more widely distributed within epidermal and mucosal tissues, such as the skin, intestine and reproductive tract comprising up to 50% of T cells. Both ab and cd T cells arise from common multipotent DN precursors in the thymus that can be further separated into four DN subsets based on CD44 and CD25 expression [2,3]. Commitment to the T cell lineage is complete at the DN3 stage where the cells undergo extensive DNA rearrangements at the b, c and d TCR loci in order to express functional TCR chains and make a choice between two developmental programs, one generating ab T cell subsets and one generating the distinct characteristics and functions of cd T cells [4,5].
The molecular events involved in cd lineage commitment are poorly understood. Numerous signals impact on the cd T cell lineage including, TCR signal strength, notch signaling, IL-7R signaling and the presence of DP thymocytes [6][7][8][9][10]. Expression of the transcription factor Sox13 promotes cd T cell development while opposing ab T cell differentiation. Mice deficient in Sox13 exhibit impaired cd T cell development but not ab T cells [11]. The expression of c-Jun is not required for, and may antagonize, cd T cell development [12]. The role of other transcription factors in regulating cd T cell development and peripheral homeostasis has not been examined.
PU.1 is an Ets family transcription factor that is essential for lymphoid and myeloid development [13]. PU.1 mRNA is expressed in hematopoietic stem cells and in the earliest thymic precursors but is downregulated during the pro-T cell stage. Decreased expression of PU.1 is necessary for continued progress through T cell differentiation [14]. Our lab has previously shown that ab T cell development is normal in mice that conditionally lack PU.1 in T cells [15]. However the expression of PU.1 in T cells and subpopulations of Th2 cells contributes to heterogeneity in Th2 cytokine expression and TCR expression [16]. In this report we have shown that deletion of PU.1 results in increased numbers of cd T cells in various organs. Our results suggest that PU.1 has negative influence on cd T cell expansion in the periphery.

Results
cd T cell numbers are expanded in the absence of PU.1 Previous work demonstrated that PU.1 functions in T cells by contributing to heterogeneity of TCR expression and Th2 cytokine production, and promoting the Th9 phenotype, while the development of ab T cells was not affected in the absence of PU.1 [15][16][17]. In the characterization of mice that conditionally lack PU.1 following Lck-Cre mediated deletion of Sfpi1, we examined the effects of PU.1-deficiency on other T cell subsets. Flow cytometry of splenocytes and thymocytes showed similar profiles of CD3+ abTCR and CD3+ NKT cells between wild-type and Sfpi1 lck2/2 mice (Fig. 1A). This is consistent with progressively diminished expression of Sfpi1 throughout the stages of DN T cell development in the thymus (Fig. 1B). However, Sfpi1 is expressed in peripheral CD4+ naïve ab T cells (Fig. 1C). We examined other T cell subsets and observed that splenic cd T cells had greater Sfpi1 expression than ab T cells (Fig. 1C). Sfpi1 expression was lower in cd T cells isolated from thymus, or intraepithelial lymphocytes, and was undetectable in cd T cells isolated from skin (Fig. 1C). These results are consistent with a previous report that detected PU.1 expression in cd T cells derived from fetal thymic organ culture [18].
Thus we next examined cd T cell in mice that lack expression of PU.1 in T cells. To determine if Lck-Cre deleted in cd T cells, genomic DNA was isolated from purified cd T cells from spleen and thymus and tested for deletion of the Sfpi1 allele. Although deletion was not complete as previously observed in doublepositive thymocytes [15], there was at least 50% deletion in cd T cell populations ( Fig. 2A). In contrast to the absence of an effect of PU.1 deletion on ab T cells, we observed a consistent increase in the numbers of cd T cells in a number of organs and tissues. Using flow cytometry to examine the percentages of cells positive for CD3 and cd TCR, we observed significantly increased percentages of cd T cells in spleen, thymus and among intra-epithelial lymphocytes (IEL) in the intestine, though not in the skin ( Fig. 2B and data not shown). Since PU.1-deficiency does not significantly affect the overall cell number in these organs, the increased percentages of cd T cells corresponds to increased numbers of cd T cells in these organs (Fig. 2B). We further examined the expression of other surface proteins on wild type and PU.1deficient splenic cd T cells. Greater than 90% of the cells were CD5-positive regardless of PU.1 expression, and the increase in cd T cells in the absence of PU.1 occurred similarly in CD24-positive and CD24-negative populations (data not shown). There were some differences in the expansion of cd T cell subsets that expressed CD62L and/or CD44, and though all populations demonstrated a trend towards increased numbers in the absence of PU.1, only the CD62Lhi, CD44hi population was significantly increased (Fig. 2C).

Sfpi1 lck2/2 mice contain enhanced numbers of cd T cell subsets
To determine if these expanded populations included all subsets of cd T cells identified by specific Vc and Vd TCRs, we examined the populations of cells that were positive for CD3 and Vc2 or Vd4. We observed significantly increased percentages of Vc2+ and Vd4+ T cells in all organs examined (Fig. 3). Concomitant with the increase in percentages, there was an increase in cell numbers of each of these cd T cell subsets (Fig. 3).

The absence of PU.1 results in enhanced cd T cells proliferation
We next examined whether PU.1-deficiency affected cd T cell function or gene expression. The expansion of cd T cells in the absence of PU.1 suggested that cell proliferation might be affected. To assess this in vivo we injected mice with BrdU to determine the percentage of cd T cells in cell cycle. We observed a significant increase in the percentage of BrdU-positive PU.1-deficient cd T cells, compared to wild type cells (Fig. 4A). To define proliferation in vitro, thymic cells were labeled with the fluorescent dye CFSE and activated with plate bound anti-CD3 and anti-CD28 for 72 h. Cell division was analyzed by flow cytometry. cd T cells from Sfpi1 lck2/2 mice had greater proliferation than cells from wild-type mice, determined by staining profile, mean fluorescence intensity of CFSE staining, and proliferative index ( Fig. 4B-C). In contrast, PU.1 had only modest effects on apoptosis (Fig. 4D). Using qPCR we tested the expression of transcription factors previously shown to affect cd T cell development, including Gata3, Sox13 and Jun [11,12,19]. We did not observe any significant differences in the expression of these transcription factors in cd T cells from wildtype and Sfpi1 lck2/2 mice (Fig. 4E). Previous reports have demonstrated that cd T cells can secrete IFNc and IL-17 in response to TCR stimulation [20][21][22] and the cytokine secretion pattern may vary with antigen exposure [23]. To test the cytokine production from PU.1-deficient cd T cells, we purified cd T cells from wild-type and Sfpi1 lck2/2 splenocytes using flow cytometry and stimulated them with anti-CD3. After 72 hours, supernatants were collected and tested for amounts of IFNc and IL-17 using ELISA. Amounts of IFNc produced were similar between wildtype and PU.1-deficient cd T cells, consistent with similar expression of Tbx21 and with similar percentages of cd T cells that were CD122-positive ( Fig. 4F and data not shown). There was a trend towards more IL-17 production from PU.1-deficient cd T cells than from wild type cells, though this was not statistically significant (Fig. 4G). Although there was no difference in the expression of Rorc between in wild-type and PU.1-deficient cd T cells, we did observe a significant increase in the numbers of cd T cells that were CCR6+, supporting a selective increase in the populations of some cytokine secreting cd T cells in the absence of PU.1 (Fig. 4H and data not shown).

Discussion
The development of cd T cells is still not completely understood. Development is regulated by extracellular signals and the expression of cell-intrinsic factors that regulate development and expansion. In this report we identify PU.1 as a transcription factor that regulates numbers of cd T cells in secondary lymphoid organs and at mucosal sites. This represents a distinct function of PU.1 between ab and cd T cells, since ab T cell development and numbers in various lymphoid organs is unaltered by PU.1-deficiency. Thus, this study has identified a novel function of PU.1 and contributes further to our understanding of transcription factor control of cd T cell homeostasis.
PU.1 is expressed in cd T cells from several organs. Splenic cd T cells have higher expression of Sfpi1 than naïve CD4+ T cells, although Sfpi1 is expressed in lower amounts by thymic and intraepithelial intestinal cd T cells, and not expressed in dermal cd T cells. Since dermal cd T cells have a fetal thymic origin [1], it is possible that there may be a difference in PU.1 expression between cd T cells that derive during embryonic development and mature mice. PU.1 expression was observed in cd T cells derived from fetal thymic organ culture suggesting that a fetal thymus does not result in the absence of PU.1 expression in cd T cells [18]. Further studies will be required to determine precisely when Sfpi1 is regulated during cd T cell development.
The point at which PU.1-deficiency affects cd T cell homeostasis is difficult to define. Although cd T cell commitment during DN T cell development may occur at several stages [24] deletion by Lck-Cre, which occurs after Sfpi1 expression is diminished between DN1 and DN2 and extinguished by DN3, is unlikely to affect cd T cell development. In support of this idea, we did not see alterations in the expression of transcription factors that mediate ab/cd T cell lineage decisions. Moreover, it is likely that the phenotype observed is cell-intrinsic, and not due to the reported effects of ab T cells on cd T cell development [10] because deletion of Sfpi1 by CD4-Cre, where Sfpi1 is deleted in all ab T cells did not effect cd T cell numbers in spleen or thymus (data not shown). We did observe that proliferation of thymic cd T cells was increased in vivo and in vitro in the absence of PU.1. However, this does not distinguish between proliferation of cd T cells during or after development, and it is still possible that PU.1 functions at both stages. The precise functions PU.1 regulates will require a greater understanding of cd T cell homeostasis.
We demonstrate that PU.1-deficiency increases in vivo proliferation of cd T cells, and in vitro TCR-induced proliferation of cd T cells. In several cell types PU.1 can function as a tumor suppressor Figure 2. Increased cd T cells in Sfpi1 lck2/2 mice. (A) cd TCR+ thymocytes and cdTCR+ splenocytes were sorted from wild-type and Sfpi1 lck2/2 mice and genomic DNA was analyzed for the presence of the wild-type (WT; +), floxed (fl), or deleted (D) allele. (B) Flow cytometric analysis of cd T cells in spleen, thymus and intestine (intra-epithelial lymphocytes) from wild-type and Sfpi1 lck2/2 mice using antibodies specific for CD3 and pan-cd TCR. Numbers in dot plots represent the mean 6 SEM of 10-12 mice. The absolute number of cd T cells was calculated by multiplying the total cell number recovered from each organ by the percentage of cd T cells. Results are an average of 10-12 mice. (C) Flow cytometric analysis of cd T cells in spleen from wild-type and Sfpi1 lck2/2 mice using antibodies specific for pan-cd TCR, CD62L and CD44. Numbers are cd T cells in each subpopulation and are averages 6 SEM of three mice. *Significantly different from WT, p,0.05 determined by Student's t test. doi:10.1371/journal.pone.0022189.g002 [25,26]. The development of myeloid leukemia in mice that have decreased expression of PU.1 is dependent on Jun [27]. Thus while Jun is not required for cd T cell development [12], and we did not observe differences in the expression of Jun, it is possible that in the absence of PU.1, Jun may help to promote expansion of T cells. In various cell types PU.1 has also been shown to induce expression of TRAIL and Ink4b, which respectively induce apoptosis and inhibit cell proliferation [28,29]. It may ultimately be a combination of effects on multiple genes that allows PU.1 to regulate cd T cell expansion.
In the initial descriptions of PU.1, it was thought to be largely restricted to myeloid lineages [30]. During T cell development PU.1 expression is decreased between DN1 and DN2, and is extinguished by DN3. However, PU.1 is expressed in naïve CD4+ T cells, and we have shown that it regulates the expression of TCR in T cells, decreasing the threshold of activation [15]. We have also demonstrated that PU.1 limits the expression of Th2 cytokines and contributes to Th2 heterogeneity [15,16]. Importantly, PU.1 promotes the development of Th9 cells [17]. In contrast to these effects, we did not observe significant effects of PU.1-deficiency on  cytokine production from cd T cells. Thus, PU.1 appears to have distinct effects in various types of T cells, negatively regulating cd T cell numbers, negatively regulating Th2 cytokine production and TCR expression, while promoting IL-9 expression [15][16][17].
In this report we describe the effects of PU.1-deficiency on the cd T cell population. The transcription factor network that promotes the development and homeostasis of cd T cells is not well-defined. Our demonstration that deletion of PU.1 results in expansion of cd T cells, an effect that is restricted to this subset, adds to our understanding of the regulation of cd T cells. Further work will help to define how this factor limits cd T cell expansion.

Ethics Statement
Mice were maintained in pathogen-free conditions and all studies were approved by the Indiana University School of Medicine Animal Care and Use committee.

Mice
Wild-type C57BL/6 female mice were purchased from Harlan Bioscience. Mice with conditional deletion of the PU.1 gene (Sfpi1 lck2/2 ) on the C57BL/6 background were previously described [31] and mated to mice carrying a Cre transgene under control of an Lck promoter (B6(CBA)-Tg(Lck-cre)I540Jxm/J). The examination of allele deletion was performed as previously described [31]. Mice were used at age 6-8 weeks.

Cell Isolation and Flow cytometry
Splenocytes and thymocytes of both wild-type and Sfpi1 lck2/2 were harvested and single cell suspensions were obtained. Viable cells were counted and determined by trypan blue exclusion. IELs were isolated by incubation of cleaned intenstine followed by Ficoll gradient centrifugation as described previously [32]. Splenocytes, thymocytes and IELs were then preincubated with Fc-block (2.4G2, BD pharmingenTM) for 10 minutes, followed by incubation with anti-CD3 PE, anti-cdTCR FITC, anti-Vc2dTCR FITC, anti-Vd4TCR FITC or antibodies to other surface proteins for 30 minutes at 4uC. Stained cells were analyzed with the BD FACSCalibur flow cytometer. All antibodies were purchased from BD Pharmingen unless otherwise stated.

Cell proliferation and apoptosis assay
For BrdU studies, mice were administered 2 mg of BrdU intra peritoneally 24 h prior to analysis. To detect incorporation of BrdU, cells were stained with mAb specific to BrdU using a FITC-BrdU flow kit (BD Biosciences) following manufacturer's instructions.
For CFSE labeling, cd T cells were isolated from both wild-type and Sfpi1 lck2/2 by staining splenocytes and thymocytes with PE anti-cdTCR before sorting with FACSAria (Becton Dickinson). Sorted cd T cells from spleen of wild-type and Sfpi1 lck2/2 mice were washed in PBS, resuspended at a concentration of 10 6 cells/ ml and incubated with 5 mM of CFSE (Invitrogen) at 37uC for 10 minutes. Cells were then washed with cold culture media supplemented with 10% FBS. CFSE labeled cells were cultured for 3 days in anti-CD3 coated plates and soluble anti-CD28 (anti-CD3 4 mg/ml). The cell division status of cells was determined by measuring CFSE fluorescence after 3 days. Apoptosis was measured by annexin V staining according to the manufacturer's protocol (BD Pharmingen). Briefly, cd T cells were washed with PBS supplemented with 10% FBS and then resuspended at a concentration of 10 6 cells/ml in binding buffer. Cells were then stained with PE-Annexin V and incubated for 15 minutes. After incubation cells were resuspended in 400 ml of binding buffer and analyzed by flow cytometry within 1 h.

Cytokine secretion assays
The purified cd T cells were stimulated in vitro at 1610 6 cells/ well in duplicate with 2 mg/mL plate-coated anti-CD3 antibody (BD Biosciences, San Jose, CA) for 72 hours. Supernatants were collected and assayed for the presence of cytokines IFN-c and IL-17 by ELISA.

Genomic DNA extraction and polymerase chain reaction analysis
The DNA was extracted from cd T cells sorted from spleen or thymus using DNeasy Blood and Tissue kit from Qiagen. PCR was performed using primer/probe sets as described previously [31]. RNA was extracted from sorted cd T cells from the indicated organs using Trizol. Quantitative real-time polymerase chain reaction was performed using Taqman Fast Universal PCR Master Mix and the plate was run on a 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA). RNA was normalized to expression levels of b2-microglobulin and relative expression was calculated using the -DDCt method.