Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2014 May 15.
Published in final edited form as: J Immunol. 2013 Apr 10;190(10):5057–5064. doi: 10.4049/jimmunol.1300065

GARP-TGFβ complexes negatively regulate Treg cell development and maintenance of peripheral CD4+ T cells in vivo

Angela X Zhou 1, Lina Kozhaya 1, Hodaka Fujii 4, Derya Unutmaz 1,2,3
PMCID: PMC3653571  NIHMSID: NIHMS456114  PMID: 23576681

Abstract

The role of surface bound TGFβ on regulatory T cells (Tregs) and the mechanisms mediating its functions are not well defined. We recently identified a cell surface molecule called GARP, which is expressed specifically on activated Tregs and was found to bind latent-TGFβ and mediate a portion of Treg suppressive activity in vitro. Here, we address the role of GARP in regulating Treg and conventional T cell development and immune suppression in vivo using a transgenic mouse expressing GARP on all T cells. We found that, despite forced expression of GARP on all T cells, stimulation through the T cell receptor (TCR) was required for efficient localization of GARP to the cell surface. In addition, IL-2 signals enhanced GARP cell surface expression specifically on Tregs. GARP-transgenic CD4+ T cells and Tregs, especially those expressing higher levels of GARP, were significantly reduced in the periphery. Mature Tregs, but not conventional CD4+ T cells, were also reduced in the thymus. CD4+ T cell reduction was more pronounced within the effector/memory subset, especially as the mouse aged. Additionally, GARP overexpressing CD4+ T cells stimulated through the TCR displayed reduced proliferative capacity, which was restored by inhibiting TGFβ signaling. Furthermore, inhibiting TGFβ signals greatly enhanced surface expression of GARP on Tregs and blocked the induction of FoxP3 in activated CD4+ T cells overexpressing GARP. These findings suggest a role for GARP in natural and induced Treg development through activation of bound latent TGFβ and signaling, which negatively regulates GARP expression on Tregs.

INTRODUCTION

Regulatory T cells (Tregs) are a crucial lymphocyte subset that suppress excessive immune activation and help maintain self-tolerance to prevent autoimmune diseases (1). Previously, we showed that Glycoprotein A Repetitions Predominant (GARP, or LRRC32) is specifically expressed on the surface of activated human Tregs and may play a role in Treg suppression (2, 3). Notably, GARP was found to bind to latent-TGFβ and is essential for anchoring TGFβ to the surface of Tregs (4, 5). GARP expression is also restricted to Tregs in mice and a recent study identified GARP as one of the differentially expressed genes in defective Tregs derived from NOD mice (6).

The expression of latent-TGFβ on the surface of Tregs, through its association with GARP, provides a conceptual framework to better understand the role of TGFβ in Treg development and function as a suppressive cytokine. TGFβ is a pleiotropic cytokine with essential roles in immune regulation (7, 8). While germline ablation of TGFβ1 is embryonically lethal, about a third of TGFβ-null mice on a mixed genetic background can survive up to 4 weeks before succumbing to severe multi-organ autoimmune disease, illustrating the importance of TGFβ in immune homeostasis (9). TGFβRII-conditional-knockout mice show similar pathology as TGFβ-null mice, with massive expansion of their T cells, which exhibit an activated phenotype (10, 11). A study of TGFβRI-conditional knockout mice also showed a block in the thymic development of FoxP3-expressing Tregs (12). In addition, TGFβ signals were shown to play an essential role in preventing autoimmunity and maintaining a healthy Treg population in the periphery as Treg numbers progressively decreased in mice that could not respond to TGFβ (11-15). Together with IL-2, TGFβ is the key cytokine in inducing the Treg master transcription factor FoxP3 in activated CD4+ T cells and in their conversion into suppressive cells, referred to as induced Tregs (iTregs) (16-20). However, it is not yet clear to what extent GARP associated with TGFβ on Tregs contribute to these important processes in regulating the immune system.

Critical for the understanding of TGFβ regulation is that TGFβ is secreted in a latent form where the active portion is noncovalently bound to the already cleaved portion of the TGFβ pro-protein called the latency-associated protein (LAP) (8). Upon activation through various mechanisms, the active TGFβ is released from LAP to bind to TGFβ receptors for signaling. The mechanisms of TGFβ activation are not entirely clear, but certain proteases, as well as physical interactions with proteins such as αVβ6 and αVβ8 integrins have been demonstrated to release active TGFβ (21-23). These αV-associated integrins are potentially also involved in activating GARP-associated TGFβ (24) and have been shown to be important for preventing autoimmunity (25-27). In this context, the relative contribution of cell-surface GARP-associated TGFβ on Tregs to its functional effects is not yet known.

To address the effects of GARP and GARP-bound TGFβ in immune regulation, we developed a transgenic mouse that expresses GARP on all mature T cell lineages and during thymic development. We found that TCR stimulation was required for efficient localization of GARP to the cell surface, even in transgenic T cells. Furthermore, expression of GARP specifically on Tregs was modulated negatively and positively by TGFβ and IL-2 signals, respectively. GARP-transgenic CD4+ T cells were progressively reduced in the periphery, especially within the memory subset, and displayed diminished proliferative capacity in vitro, which could be rescued to wild-type levels by inhibiting TGFβ signals. Within the thymus, conventional T cells were not affected by GARP transgene expression but Tregs were significantly reduced. Together, our findings suggest that GARP regulates both Treg development and CD4+ T cell activation in the periphery, through the activation of bound latent TGFβ.

MATERIALS AND METHODS

Transgenic Mice

Mouse GARP cDNA together with IRES-GFP was cleaved from the mGARP/pMXs-IG plasmid (3) and inserted into a human CD2 expression cassette (VA-hCD2) (28). After removal of vector sequences by digestion and agarose gel electrophoresis, the resultant transgene was injected into pronuclei of fertilized eggs from C56BL/6 mice as described previously (29). Mice were genotyped using tail biopsies by PCR with specific primers for GFP 5’- [5’-CGACGTAAACGGCCACAAGTTCAG-3’] and 3’- [5’-ATGCCGTTCTTCTGCTTGTC-3’]. C57BL/6 mice were purchased from Taconic Farms, Inc (Hudson, NY). All experiments were conducted in accordance with the approved protocols of the Institutional Animal Care and Use Committee.

qPCR

Total RNA was isolated and cDNA was synthesized as previously described (30). The cDNA was then used to perform qPCR using an Applied Biosystems 7300 apparatus (Foster City, CA). The following TaqMan primer and probe mixes were purchased from Applied Biosystems: GARP (Hs00194136_m1) and β–actin (Hs99999903_ml).

Human T cell purification

Peripheral blood mononuclear cells (PBMCs) from healthy individuals were prepared using Ficoll-plaque plus (GE Healthcare) from discarded buffy coats obtained anonymously from the New York Blood Center (New York, NY). CD4+ T cells were isolated using Dynal CD4 Positive Isolation kit (Invitrogen) directly from purified PBMCs and were >99% pure. The donor samples were not identifiable and did not involve any donor-specific information for data analysis and therefore consent forms were not required, according to guidelines of NYU School of Medicine Institutional Human Subjects Board.

T cell activation

Human and murine T cells were activated by anti-CD3− and anti-CD28 (αCD3/αCD28)–coated beads (1:4 bead/cell ratio; Invitrogen) and cultured in RPMI 1640 medium supplemented with 20 ng/ml IL-2 (R&D Systems, Minneapolis, MN), and 10% FBS (Fetal Bovine Serum) (Atlanta biologicals, Lawrenceville, GA). Alternatively, resting T cells were maintained with or without 10 U/ml recombinant human IL-2 (BD). Where indicated, murine T cells were activated with bone marrow-derived DCs (1:5 DC:CD4+ T cell ratio) and 10 μg/ml of anti-CD3 (clone 2c11). Bone marrow-derived DCs were generated as described by Inaba et al. (31). Briefly, cells were extracted from the femur of wild-type C57BL/6 mice and cultured in the presence of 20 ng/ml of GM-CSF for 7-10 days.

Antibodies and Reagents

Murine antibodies used for surface and intracellular staining include: TCRβ-PerCP/Cy5.5, CD4-PE/Cy7, CD8-Alexa700, CD11b-PE/Cy7, CD11c-PE, CD19-APC/Cy7, CD25-Pacific Blue, CD44-PE, CD62L-Alexa647, GARP-PE, LAP-APC, FoxP3-APC, FoxP3-Alexa488, Helios-Alexa488. Human antibodies include: CD25-Alexa700, CD45RO-Pacific Blue, GARP-PE, FoxP3-Alexa657, Helios-Alexa488. All antibodies were purchased from Biolegend. Recombinant TGFβ (R&D Systems; 2ng/ml) or TGF-β type I receptor inhibitor (SJN 2511 from Tocris Bioscience; 2.5μM) was added to cultures at the time of activation. In the experiments using PI-3K signaling inhibitors, cells were treated with PI-3K–specific inhibitor, LY294002 10ug/ml (EMD), or the Akt-specific inhibitor AKTi-1/2 5ug/ml (EMD) as previously described (32) and cultured in IL-2 supplemented media for 4 days.

Cell Sorting and Flow Cytometry

Cells from the spleen, thymus and mesenteric lymph nodes of mice were homogenized through a 70μm filter and were sorted on a FACS ARIA cell sorter (BD, San Jose, CA) based on expression of CD4, CD25, CD8, and/or GFP as indicated. Human Tregs were isolated from CD4+ T cells with FACSAria cell sorter (BD) into CD45RO+CD25+ cells. Cell surface staining was performed as previously described (33). Briefly, cells were stained with the relevant antibodies at 4°C for 30 min in PBS buffer containing 2% FBS and 0.1% sodium azide in the dark. Cells were washed twice then analyzed by flow cytometry using BD LSR-II (BD Biosciences, San Jose, CA). Live cells were gated based on Fixable Viability Dye eFluor780 or eFluor 450 (eBioscience, San Diego, CA). For intracellular staining, cells were fixed and permeabilized by commercially available FoxP3 intracellular staining kit (eBioscience, San Diego, CA) as per manufacturer’s protocol. After permeabilization and fixation, cells were washed with the permeabilization buffer (eBioscience, San Diego, CA) and incubated with the relevant antibodies at 4°C for 30 min. Cells were again washed twice with the permeabilization buffer before FACS analysis.

In Vitro Cell Proliferation and Suppression Assays

For in vitro proliferation and suppression assays, CD4+ CD25- T cells were labeled with CellTrace™ Violet (Invitrogen) following manufacturer’s instructions and activated by DCs and anti-CD3. Proliferation of labeled-cells was analyzed by BD LSR II on day 3-5 post-activation. For suppression assays, Tregs were added to conventional T cell cultures at a 1:1 ratio and suppressive ability was calculated based on reduction in proliferation of the conventional T cells as previously described (3).

Data Analysis

FACS data was analyzed using FlowJo (Tree Star, Ashland, OR) and statistical analysis was performed using Graphpad Prism software (Graphpad Inc., La Jolla, CA).

RESULTS

Characterization of GARP expression in GARP-transgenic mice

To create transgenic mice expressing T cell specific GARP, we used a human CD2 minilocus expression vector encoding murine GARP cDNA with IRES-GFP (Supplemental Fig. 1A). This vector has been described to give highly T cell-specific expression of the transgene (28). The mice were first screened for expression of the genomic GARP transgene using PCR and then for expression of GFP by FACS analysis of T cells in the spleen (Supplemental Fig. 1A).

Using quantitative RT-PCR, we found that GARP expression was ~30-60 fold higher in transgenic thymocyte populations as compared to wild-type mice (Fig. 1A). Highest expression of GARP was observed in the CD4 and CD8 single positive (SP) populations (Fig. 1A). Similarly, expression of GARP mRNA in CD4+ and CD8+ T cells from transgenic mouse spleens were also markedly increased (Fig. 1B). Interestingly, the level of GARP mRNA in Tregs from the transgenic mice was not significantly higher compared to wild-type animals, suggesting high levels of constitutive GARP expression in murine Tregs in vivo (Fig. 1C, 1D).

Figure 1. Characterization of GARP expression in GARP-transgenic mice.

Figure 1

Open in a new tab

(A) The expression of GARP mRNA in isolated T cell subsets from the thymus and (B) spleen, of either wild-type (WT) or transgenic (Tg) mice was determined by qRT-PCR. (C) GARP mRNA expression in isolated CD4+ CD25+ and CD25- subsets from the thymus and (D) spleen was determined by qRT-PCR. (E-H) Expression of GARP protein was determined by surface and intracellular (IC) staining of GARP after gating on the indicated T cell subsets. Representative FACS plot from (E) thymus and histogram plots from (G) spleen. The cumulative expressions from (F) thymus and (H) spleen are shown in bar graphs. Data are from at least three mice per group and were independently confirmed.

To determine the expression of GARP protein within the thymus of transgenic mice, we sorted T cells into CD4 single positive (SP), CD8 SP, CD4 and CD8 double positive (DP) and double negative (DN) subsets and probed for surface expression of GARP using a specific antibody. In contrast to the high levels of GARP mRNA, there was very low protein expression on the surface of any of these T cell subsets, despite expression of GFP from the transgene (Fig. 1E, 1F). However, when we performed an intracellular staining, we found that non-Tregs from the transgenic mice expressed significantly higher intracellular GARP compared to the wild-type controls (Fig. 1E and 1F).

We also noted that Tregs from wild-type mice as well as transgenics expressed considerable levels of GARP, especially intracellularly (Fig. 1E and 1F). A similar pattern of expression was observed in CD4+ and CD8+ T cells purified from the spleen and mesenteric lymph nodes (mLNs) (Fig. 1G, 1H, Supplemental Fig. 1B and 1C). We reasoned that GARP levels would be higher in cells expressing greater levels of GFP. As such, we isolated CD4+ T cells based on GFP intensity and compared the expression of GARP. We found that transgenic CD4+ T cells with low GFP levels had comparable GARP expression – either intracellular or surface – to wild-type CD4+ T cells, whereas GFP-high T cells expressed higher levels of intracellular GARP in non-Tregs (Supplemental Fig. 1B and 1C). GARP was also expressed on Tregs from both transgenic and wild-type mice at similar levels (Fig. 1G, H, Supplemental Fig. 1B and 1C).

Signaling requirements for GARP cell surface localization and binding to latent-TGFβ

To determine whether cell surface localization of murine GARP required signals from the TCR, we activated CD4+ and CD8+ T cells isolated from transgenic mouse spleens in vitro, and assessed surface and intracellular GARP expression after two days. Surface GARP expression was upregulated on Tregs after TCR stimulation (Fig. 2A), similar to previous reports in human cells (2, 3). In addition, GARP surface expression was dramatically increased in non-Treg CD4+ and CD8+ T cells from transgenic mice, which correlated with GFP expression from the transgene (Fig. 2A and Supplemental Fig. 2A). Intracellular GARP expression was comparable to surface expression after activation, suggesting that most preformed GARP protein had localized to the cell surface.

Figure 2. Signaling requirements for cell surface expression of GARP-TGFβ complex.

Figure 2

Open in a new tab

(A) Cell surface and IC expression of GARP was determined with GARP-PE antibodies in purified CD4+ and CD8+ T cells from the spleens of WT and Tg mice ex vivo (d0) and after 2 days of activation (d2) with anti-CD3/CD28 beads. (B) Presence of latent-TGFβ bound to GARP on CD4+ and CD8+ T cell was determined by staining with GARP-PE, LAP-APC antibodies extracellularly and FoxP3-Alexa488 antibodies intracellularly on day 2 post-activation. (C) Surface and intracellular GARP expression in CD4+ T cells isolated from murine spleens or (D) from blood of healthy human donors was determined at day 0 and after 1 (mouse) or 4 (human) days of culture in IL-2. Data are representative of at least three independent experiments.

Previous studies have shown that surface GARP can bind to latent-TGFβ and anchor the molecule to the cell surface (4, 5). Accordingly, we determined whether activated GARP-expressing transgenic T cells also bound to latent-TGFβ secreted from the T cells by staining cells with an anti-LAP antibody. We found that GARP and LAP were co-expressed on the surface of Tregs from wild-type mice as previously shown in human cells (4, 5) as well as on all transgenic T cells (Fig. 2B).

We and others have reported that GARP expression on human Tregs is dependent on activation of these cells through the TCR (2, 3, 5, 34). However, we observed that murine Tregs contained significant GARP in ex vivo analysis without any additional TCR stimulation (Fig. 1E-1H and Fig. 2C). We hypothesized that this disparity between mouse and human GARP expression was due to exposure of these cells to γc-cytokines such as IL-2 within the secondary lymphoid organs of mice where the cells were isolated from, which could provide signals for GARP localization to the surface; whereas, human T cells were isolated from the blood and thus were likely in a more quiescent state. Accordingly, a further increase in GARP expression was observed in murine Tregs after culture in IL-2 (Fig. 2C). This increase in GARP was limited to Tregs even in transgenic mice, suggesting that this regulation of GARP expression is Treg specific (Fig. 2D). We next determined whether IL-2 signals could also upregulate GARP on human Tregs. We purified resting human Tregs or conventional CD4+ T cells from blood and cultured in IL-2 alone for four days to determine GARP expression. Indeed, human Tregs also upregulated GARP expression considerably after culture with IL-2 (Fig. 2D). Further, upregulation of GARP via IL-2 signaling was dependent on downstream PI-3 kinase pathway (35), since inhibition of PI-3 kinase pathway signaling via specific inhibitors (Ly294002 or an AKT inhibitor) almost completely abolished IL-2-induced GARP expression (Supplemental Fig. 2B).

CD4+ T cells in GARP transgenic mice are reduced in the lymphoid organs

We next determined whether ectopic expression of GARP had an impact on proportions of immune cell populations within the lymphoid organs of transgenic mice. We found that in 4-8 week old mice, CD4+ T cells were significantly reduced both in proportion and absolute numbers, whereas the CD8+ subset was significantly increased (Fig 3A and 3B and Supplemental Fig. 3A). However, the proportions of total T cells, B cells and monocytes/dendritic cells were not different compared to wild-type littermates (Fig. 3A and Supplemental Fig. 3A). We then examined T cell subsets within the thymus of the GARP-transgenic animals and found that the proportions of single positive mature CD4+ and CD8+ T cells as well as double positive and double negative T cell subsets were comparable to wild-type controls (Fig. 3C). However, the total numbers of thymocytes were decreased about 2-fold in the thymi of transgenic mice compared to controls and this reduction remained constant in older mice (Fig. 3D). Morphologically, the thymi of transgenic mice were also noticeably reduced in size compared to wild-type.

Figure 3. Analysis of lymphocytes and myeloid cells in the lymphoid organs of GARP transgenic mice.

Figure 3

Open in a new tab

(A) Total numbers of splenocytes isolated from WT and Tg mice. (B) The distribution of cell types in the spleen was determined by flow cytometry analysis of total T cells (CD3), T cell subsets (CD4 and CD8), B cells (CD19) and monocytes (CD11b) and dendritic cells (CD11c). (C) Proportions of indicated T cell subsets within the thymus were determined based on CD4 and CD8 expression. (D) Total numbers of thymocytes in WT and Tg mice of the indicated ages. Data are from at least eight mice in each group. * = p<0.05, ** = p<0.01, *** = p<0.001

In older mice (~one year-old), we found even greater reduction of GARP-transgenic CD4+ T cells in the periphery compared to animals younger than 2 months (Fig. 4A). Since GFP expression correlated with GARP levels, we then compared proportions of CD4+ T cells that were GFP-high and GFP-low in young and old mice. The proportion of GFP-high CD4+ T cells in the periphery of older mice was significantly diminished compared to younger counterparts, whereas GFP-low CD4+ T cells and thymocytes were at comparable proportions (Fig. 4B). This loss of GFP expression was already apparent in memory CD4+ T cells in younger transgenic mice, as the ratio of GFP-high to GFP-low T cells was specifically reduced compared to the naïve cells. In older mice, there was a further decrease in this ratio in memory cells, whereas there was no significant change within the naïve T cell compartment in young versus old mice (Fig 4C). Together these findings suggest that T cells expressing higher levels of GARP are selectively reduced over time, and this correlates with increasing proportions of memory to naïve T cells as mice age.

Figure 4. Expression of GARP within transgenic CD4+ naïve and memory subsets in young and old mice.

Figure 4

Open in a new tab

(A) CD4+ T cells in the thymus (thy), spleen (spl) and mesenteric lymph nodes (ln) of young (≤2 months) or old (~one year) transgenic mice were determined as a proportion of total T cells by staining with CD3-PerCP/Cy5.5 and CD4-PE/Cy7 antibodies. The data for transgenic CD4+ T cells is graphed as percentage of CD4+ T cell proportions found in the age-matched wild-type controls. (B) After gating on total CD4+ T cells in isolated splenocytes, the percentage of cells that were GFP-high (GFPhi) or GFP-low (GFPlow) was determined by FACS. (C) CD4+ T cells isolated from spleens of transgenic mice were stained with CD4-PE/Cy7, CD62L-Alexa647 and CD44-PE antibodies to determine the ratio of GFPhi to GFPlow cells in naïve (CD62L+CD44-) or memory (CD62L-CD44+) CD4+ T cells. Data are from at least six mice each group. * = p<0.05, ** = p<0.01, *** = p<0.001

GARP-transgenic mice have reduced Tregs

To assess the impact of GARP overexpression on Tregs, we isolated CD4+ cells from transgenic mice and found a reduction in Treg proportions in both the thymus and in peripheral lymphoid organs (Fig. 5A). To determine whether this reduction was both in natural and induced Treg subsets, we further analyzed the proportion of Tregs either expressing both FoxP3 and Helios (nTregs) or FoxP3 alone (iTregs) (36). We found that the reduction was most significant in the FoxP3+ Helios+ subset of Tregs in the periphery (Fig 5B). In addition, this reduction in GARP-transgenic Tregs was restricted to cells expressing high levels of the transgene (Fig. 5C-5E). GFP-high FoxP3+ Helios+ and FoxP3+ Helios- Tregs were both reduced compared to GFP-low counterparts in both the periphery and the thymus, and the reduction in GFP-high nTreg subsets was more pronounced (Fig 5C-5E). These data suggest that increased levels of GARP within nTregs are detrimental for their survival or maintenance in the periphery but iTreg development was less affected.

Figure 5. Changes in Treg cell populations in GARP-transgenic mice.

Figure 5

Open in a new tab

(A) Proportions of Tregs in cells isolated from the thymus, spleen and mesenteric lymph nodes of WT and Tg mice was determined based on FoxP3 expression in CD3+CD4+ T cells. (B) Treg subsets from the thymus, spleen and mesenteric lymph nodes of WT and Tg mice were determined based on FoxP3 and Helios expression after gating on CD3+CD4+ T cells. (C-E) CD4+ T cells isolated from spleen, and mesenteric lymph nodes were FACS sorted into GFPhi and GFPlow subsets before intracellular staining for FoxP3 and Helios. Comparison of proportions of Treg subsets based on expression of Foxp3 and Helios in (C) spleen, (D) lymph nodes and (E) representative FACS plots of both. (F) Proportions of Tregs in thymus, spleen and lymph nodes of young and old mice determined using CD25 expression and graphed as percentage of age-matched WT mice. (G) Percentages of Tregs determined by CD25 expression that are GFP-high in thymus, spleen and lymph nodes of young and old mice. Data are from at least three mice in each group. * = p<0.05, ** = p<0.01, *** = p<0.001

We next determined the impact of age on Treg numbers in transgenic mice. In contrast to conventional CD4+ T cells, total Tregs in transgenic mice were increased in the periphery of older mice, and became comparable to wild-type controls (Fig. 5F). However, GFP-high Tregs in older mice were further reduced in the periphery (Fig. 5G). Despite the reduction in Treg numbers, transgenic Tregs retained full capacity to suppress T cell activation in vitro (Supplemental Fig. 3B and 3C).

GARP-overexpressing CD4+ T cells have lower proliferative capacity

Phenotypic changes in T cell populations in transgenic mice suggested that GARP overexpressing CD4+ cells have an impairment that manifests after cells are activated. Therefore, we assessed the activation and proliferation of transgenic CD4+ T cells that expressed high GFP. We found that GFP-high CD4+ T cells, stimulated through the TCR, displayed reduced proliferative capacity compared to GFP-low cells, the proliferation of which was comparable to wild-type CD4+ T cells (Fig. 6A). On the other hand, proliferation of CD8+ T cells from wild-type and transgenic mice were similar (Fig. 6A).

Figure 6. Activation of GARP-bound TGFβ and its effect on T cell proliferation in GARP-transgenic mice.

Figure 6

Open in a new tab

(A) Proliferation of CD4+ and CD8+ T cells from the GARP-tg mice. CD4+ and CD8+ T cells were purified from the spleen using FACS sorting and labeled with CellTrace™ Violet (VCT). The cells were then activated using bone marrow derived dendritic cells (BM-DCs) in the presence of anti-CD3 (clone 2C11). Proliferation was analyzed by flow cytometry on day 5 post-activation. (B) Model for GARP-bound TGFβ activity on CD4+CD25- T cells overexpressing GARP. Upon Treg activation, TGFβ is expressed on the cell surface bound to GARP and is subsequently activated and released locally where it can signal either in autocrine or paracrine fashion to proximal cells, thus inducing FoxP3 expression. This model predicts that blocking TGFβ receptor signals would inhibit expression of Foxp3. (C and D) GARP-Tg T cells can induce FoxP3 expression through TGFβ activation. CD4+CD25- T cells isolated from the spleen are activated using BM-DCs and anti-CD3 in the presence or absence of a TGFβ signaling inhibitor (SJN-2511) and then analyzed intracellularly for FoxP3 and Helios expression on day 5 post-activation. Data are presented as (C) representative FACS plot or (D) bar graph. (E) Blocking TGFβ signals to Tregs enhances GARP expression. CD4+CD25+ Tregs were isolated from the spleen using FACS sorting and were activated with anti-CD3/CD28 beads in the presence of either active TGFβ or an inhibitor of TGFβ signaling. The cells were then stained on the cell surface with GARP-PE and LAP-APC antibodies and intracellularly with FoxP3-Alexa488 antibodies and expression was assessed using FACS analysis. (F) Wild-type and transgenic CD4+ T cells were purified based on GFP expression through FACS sorting and then were tested for their ability to suppress the proliferation of wild-type CD4+ CD25- T cells as described in the methods section. (G) Proliferation of CD4+ CD25- T cells was determined as described above in the presence or absence of a TGFβ inhibitor. Data is representative of at least three separate experiments. * = p<0.05, ** = p<0.01, *** = p<0.001

Based on these findings, we hypothesized that reduced proliferation of GFP-high CD4+ transgenic T cells was due to higher amounts of cell-bound TGFβ, which was locally activated. To test this hypothesis, we determined the activity of cell-surface TGFβ through its ability to induce FoxP3 expression in autocrine or paracrine signaling (Fig 6B). We found significantly higher FoxP3 expression in GFP-high cells compared to GFP-low and wild-type controls, which was abolished by a TGFβ signaling inhibitor (Fig. 6C, 6D). Interestingly, blocking TGFβ signals did not affect FoxP3 expression in the Treg population (Fig. 6E). However, we noted that inhibiting TGFβ signals on activated Tregs significantly upregulated GARP expression, but not surface bound latent-TGFβ, whereas there was slightly lower expression of GARP in some mice after treatment with active TGFβ (Fig. 6E and Supplemental Fig. 4A). These findings were recapitulated in human Tregs, which also showed a significant reduction in GARP expression upon culture with exogenous active TGFβ (Supplemental Fig. 4B and 4C).

Given the role of TGFβ as a mechanism of Treg suppression, we tested whether the overexpression of GARP on non-Tregs endowed them with the ability to suppress proliferation of target T cells in vitro. Indeed, GFP-high cells displayed higher suppressive ability compared to both GFP-low and wild-type controls (Fig. 6F). These findings mirror results from previous studies in human T cells (3), and suggests that the increased activation of TGFβ can inhibit proliferation. Based on these data, we postulated that activation of GARP-bound TGFβ could account for the diminished in vitro proliferative capacity observed in GFP-high transgenic CD4+ T cells. In agreement with this, we found that blocking TGFβ signals could rescue proliferation of GFP-high CD4+ T cells to wild-type levels (Fig. 6G).

DISCUSSION

In this study we show that ectopic and constitutive GARP expression on all T cell subsets in a transgenic mouse model results in diminished Treg development in the thymus and reduced differentiation of CD4+ T cells into memory subsets. The proliferative capacity of GARP-transgenic CD4+ T cells was also reduced and could potentially account for their reduction in vivo. We mechanistically mapped this defect to enhanced TGFβ signaling – potentially due to higher GARP expression. In addition, our findings revealed further complexity in the regulation of both GARP mRNA and cell surface localization in response to cytokine or TCR signaling.

Our result that GARP potentiates TGFβ signaling is supported by another report, where active simian TGFβ1 was overexpressed in murine T cells (37). The study found a similar progressive reduction in CD4+ T cells in the spleen, with no effects on CD8+ T cells, and decreased thymocyte numbers (37). The reduction of CD4+ T cells in the periphery of our transgenic mice is likely accounted for by the decrease in proliferative capacity of naïve GARP+ CD4+ T cells, as demonstrated in vitro (Fig. 6A). Our finding that we could rescue the GARP transgenic CD4+ T cell proliferation defects by blocking TGFβ signals supports known anti-proliferative effects of this cytokine (7, 38). In fact, previous studies have shown that blocking TGFβ signals in mice exhibit the converse effect – an increased expansion of T cells with higher activated/memory phenotype (10, 11).

A key difference between the above study by Schramm et al. and ours is that constitutive secretion of active TGFβ led to increased Treg numbers (37), whereas in our model we observed somewhat an opposite effect. This could be due to the nature of GARP-bound latent TGFβ, which requires further activation to be released and act locally, while secreted active TGFβ is less restricted in its activity. It is conceivable that factors that can activate GARP-bound latent TGFβ are present sub-locally or contextually in the thymus and have more precise control on TGFβ effects in Treg cell development. This spatial and temporal presentation of TGFβ signals through GARP could potentially account for the differences in generation or expansion of Tregs in the thymus. Alternatively, the over-abundance of GARP on all T cells in transgenic mice may act as a sink that sequesters TGFβ and neutralizes signals before they act on thymic Tregs. Indeed, in a study where TGFβ signals were blocked, the negative selection of thymocytes was increased and the number of Tregs was diminished (39).

In the periphery of GARP transgenic mice there appears to be a compensatory increase of Tregs that lack transgene expression, especially as the mice aged, possibly due to position-effect variegation of the transgene. We did not observe a significant change in suppressive ability displayed by transgenic Tregs. Although there is an overall decrease in thymic Treg development, the finding that Tregs increase in the periphery suggests homeostatic proliferation and expansion of these cells in the periphery, which has been shown to occur in a lymphopenic environment (40). Alternatively, GARP-bound surface TGFβ in the periphery may be inducing Treg differentiation from conventional T cells, accounting for similar proportions of FoxP3+ Helios- Tregs in transgenic and wild-type mice. Indeed, we found that GARP-overexpressing CD4+ T cells from transgenic mice had enhanced induction of FoxP3 in vitro, resembling iTregs induced by TGFβ. Previous studies have observed enhanced FoxP3 expression in human T cells overexpressing GARP as well (3, 34). Recent studies have shown that DCs, especially those expressing αVβ8 integrins, are important players in peripheral Treg induction through integrin-mediated activation of TGFβ (25, 26, 41, 42). GARP has also been shown to interact with these integrins to activate cell-bound latent-TGFβ (24). Our findings suggest a possible role for GARP in the differentiation of Tregs in the periphery and in the ability of Tregs to confer “infectious tolerance” through interactions with DCs that can activate cell-bound TGFβ as had been suggested (43).

One of the novel findings in this study was that TGFβ bound to GARP, when activated, could negatively regulate the expression of GARP. We showed that TGFβ signals downregulate GARP expression in human Tregs after TCR stimulation, whereas inhibiting TGFβ signals upregulates GARP expression in both human and mouse (Fig. 6E and Supplemental Fig. 4A and B). However, we did not observe a significant reduction in GARP expression in murine Tregs with TGFβ treatment after TCR-stimulation, possibly because they are either less sensitive to the concentrations of TGFβ used or autocrine TGFβ signaling is already at saturating levels. Regulation of GARP expression by TGFβ may be an additional mechanism for spatial and temporal regulation of TGFβ signaling from Tregs, along with the requirement of TGFβ to be activated when bound to GARP. This tight mechanism of regulation may allow Tregs to sense TGFβ concentrations in the environment and induce or inhibit expression of GARP, as necessary, to fine tune TGFβ signals.

Our study also revealed that GARP is not only dependent on TCR-signals (2, 3, 5) but that IL-2 alone could induce its expression on resting mouse and human T cells without concurrent TGFβ secretion (data not shown). This raises the possibility that Tregs can express empty GARP molecules on the cell surface in conditions of inflammation and immune activation to bind environmental latent-TGFβ secreted by other cells. Depending on the surrounding cell populations and signals, GARP could act either to enhance activation of TGFβ, perhaps to limit inflammation, or to sequester the cytokine on its cell surface. It will be important in future studies to determine how bystander or non-antigen signals from the microenvironment impact GARP-bound TGFβ expression and function on Tregs.

The regulation of TGFβ signals through the expression of GARP on Tregs has implications for their function in physiological or pathologic conditions such as cancer and fibrosis where TGFβ plays significant roles. For example, in several cancers, TGFβ has well-characterized positive and negative effects on tumor development (44). In addition, Tregs have been found to suppress tumor immunity (45-48) at least partly through TGFβ signals (49, 50), which also contributes to Treg generation (51, 52). This is especially relevant since Tregs have been reported to be elevated in patients with a variety of cancers (53), with one recent study showed that elevated cell subsets also express GARP (54), and Treg depletion has been shown to have beneficial effects in some cancer therapies (55). In light of our findings, it is conceivable that modulating GARP expression could also be a mechanism to control Treg-mediated effects in these malignant processes. Further work is necessary to determine the contribution of GARP-bound TGFβ on Tregs in cancer and other immunological diseases.

The role of TGFβ in the maintenance of immune homeostasis and Treg development and function has been studied extensively. However, the regulation of TGFβ in the immune system remains unclear. In this study, we have demonstrated a role for Treg cell surface GARP in regulating the activity of TGF β in vivo, which has effects on Treg development both in the thymus and the periphery as well as on conventional T cell populations in the secondary lymphoid organs. Our findings provide a mechanistic link between GARP expression and regulation of cell-bound TGFβ signals in vivo. In addition, these findings provide a framework to dissect how TGFβ expressed on the Treg cell surface, fine tunes immune homeostasis and impacts the pathogenesis of diseases such as cancer.

Supplementary Material

1

Acknowledgments

We thank Francie Mercer, Alka Khaitan and Stephen Rawlings for critical reading and suggestions, the Transgenic Core Facility of New York University Cancer Institute for generation of transgenic mice, Akemi Hoshino and Shifra Liba Klein for technical assistance.

This work was supported by Grant-in-Aid for Scientific Research (C) (#23590569) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Osaka Clinical Immunology Foundation, Takeda Science Foundation, Senshin Medical Research Foundation, Japan Rheumatism Foundation, Itoh Chubei Foundation to H.F., New York University School of Medicine Development Funds and National Institutes of Health (NIH) grant R01AI065303 to D.U and NIH training grant 5T32AI007647 to AZ.

Abbreviations used

GARP

Glycoprotein A Repetitions Predominant

LAP

Latency Associated Protein

FOXP3

Forkhead Box P3

References

  • 1.Sakaguchi S, Yamaguchi T, Nomura T, Ono M. Regulatory T cells and immune tolerance. Cell. 2008;133:775–787. doi: 10.1016/j.cell.2008.05.009. [DOI] [PubMed] [Google Scholar]
  • 2.Wang R, Kozhaya L, Mercer F, Khaitan A, Fujii H, Unutmaz D. Expression of GARP selectively identifies activated human FOXP3+ regulatory T cells. Proc Natl Acad Sci U S A. 2009;106:13439–13444. doi: 10.1073/pnas.0901965106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Wang R, Wan Q, Kozhaya L, Fujii H, Unutmaz D. Identification of a regulatory T cell specific cell surface molecule that mediates suppressive signals and induces Foxp3 expression. PLoS One. 2008;3:e2705. doi: 10.1371/journal.pone.0002705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Tran DQ, Andersson J, Wang R, Ramsey H, Unutmaz D, Shevach EM. GARP (LRRC32) is essential for the surface expression of latent TGF-beta on platelets and activated FOXP3+ regulatory T cells. Proc Natl Acad Sci U S A. 2009;106:13445–13450. doi: 10.1073/pnas.0901944106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Stockis J, Colau D, Coulie PG, Lucas S. Membrane protein GARP is a receptor for latent TGF-beta on the surface of activated human Treg. Eur J Immunol. 2009;39:3315–3322. doi: 10.1002/eji.200939684. [DOI] [PubMed] [Google Scholar]
  • 6.D’Alise AM, Ergun A, Hill JA, Mathis D, Benoist C. A cluster of coregulated genes determines TGF-beta-induced regulatory T-cell (Treg) dysfunction in NOD mice. Proc Natl Acad Sci U S A. 2011;108:8737–8742. doi: 10.1073/pnas.1105364108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Gorelik L, Flavell RA. Transforming growth factor-beta in T-cell biology. Nat Rev Immunol. 2002;2:46–53. doi: 10.1038/nri704. [DOI] [PubMed] [Google Scholar]
  • 8.Li MO, Wan YY, Sanjabi S, Robertson AK, Flavell RA. Transforming growth factor-beta regulation of immune responses. Annu Rev Immunol. 2006;24:99–146. doi: 10.1146/annurev.immunol.24.021605.090737. [DOI] [PubMed] [Google Scholar]
  • 9.Shull MM, Ormsby I, Kier AB, Pawlowski S, Diebold RJ, Yin M, Allen R, Sidman C, Proetzel G, Calvin D, et al. Targeted disruption of the mouse transforming growth factor-beta 1 gene results in multifocal inflammatory disease. Nature. 1992;359:693–699. doi: 10.1038/359693a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Marie JC, Liggitt D, Rudensky AY. Cellular mechanisms of fatal early-onset autoimmunity in mice with the T cell-specific targeting of transforming growth factor-beta receptor. Immunity. 2006;25:441–454. doi: 10.1016/j.immuni.2006.07.012. [DOI] [PubMed] [Google Scholar]
  • 11.Li MO, Sanjabi S, Flavell RA. Transforming growth factor-beta controls development, homeostasis, and tolerance of T cells by regulatory T cell-dependent and - independent mechanisms. Immunity. 2006;25:455–471. doi: 10.1016/j.immuni.2006.07.011. [DOI] [PubMed] [Google Scholar]
  • 12.Marie JC, Letterio JJ, Gavin M, Rudensky AY. TGF-beta1 maintains suppressor function and Foxp3 expression in CD4+CD25+ regulatory T cells. J Exp Med. 2005;201:1061–1067. doi: 10.1084/jem.20042276. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Fahlen L, Read S, Gorelik L, Hurst SD, Coffman RL, Flavell RA, Powrie F. T cells that cannot respond to TGF-beta escape control by CD4(+)CD25(+) regulatory T cells. J Exp Med. 2005;201:737–746. doi: 10.1084/jem.20040685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Gorelik L, Flavell RA. Abrogation of TGFbeta signaling in T cells leads to spontaneous T cell differentiation and autoimmune disease. Immunity. 2000;12:171–181. doi: 10.1016/s1074-7613(00)80170-3. [DOI] [PubMed] [Google Scholar]
  • 15.Lucas PJ, Kim SJ, Melby SJ, Gress RE. Disruption of T cell homeostasis in mice expressing a T cell-specific dominant negative transforming growth factor beta II receptor. J Exp Med. 2000;191:1187–1196. doi: 10.1084/jem.191.7.1187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Chen W, Jin W, Hardegen N, Lei KJ, Li L, Marinos N, McGrady G, Wahl SM. Conversion of peripheral CD4+CD25- naive T cells to CD4+CD25+ regulatory T cells by TGF-beta induction of transcription factor Foxp3. J Exp Med. 2003;198:1875–1886. doi: 10.1084/jem.20030152. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Fantini MC, Becker C, Monteleone G, Pallone F, Galle PR, Neurath MF. Cutting edge: TGF-beta induces a regulatory phenotype in CD4+CD25- T cells through Foxp3 induction and down-regulation of Smad7. J Immunol. 2004;172:5149–5153. doi: 10.4049/jimmunol.172.9.5149. [DOI] [PubMed] [Google Scholar]
  • 18.Fu S, Zhang N, Yopp AC, Chen D, Mao M, Zhang H, Ding Y, Bromberg JS. TGF-beta induces Foxp3 + T-regulatory cells from CD4 + CD25 - precursors. Am J Transplant. 2004;4:1614–1627. doi: 10.1111/j.1600-6143.2004.00566.x. [DOI] [PubMed] [Google Scholar]
  • 19.Zheng SG, Wang JH, Gray JD, Soucier H, Horwitz DA. Natural and induced CD4+CD25+ cells educate CD4+CD25- cells to develop suppressive activity: the role of IL-2, TGF-beta, and IL-10. J Immunol. 2004;172:5213–5221. doi: 10.4049/jimmunol.172.9.5213. [DOI] [PubMed] [Google Scholar]
  • 20.Chen Q, Kim YC, Laurence A, Punkosdy GA, Shevach EM. IL-2 controls the stability of Foxp3 expression in TGF-beta-induced Foxp3+ T cells in vivo. J Immunol. 2011;186:6329–6337. doi: 10.4049/jimmunol.1100061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Taylor AW. Review of the activation of TGF-beta in immunity. J Leukoc Biol. 2009;85:29–33. doi: 10.1189/jlb.0708415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Munger JS, Huang X, Kawakatsu H, Griffiths MJ, Dalton SL, Wu J, Pittet JF, Kaminski N, Garat C, Matthay MA, Rifkin DB, Sheppard D. The integrin alpha v beta 6 binds and activates latent TGF beta 1: a mechanism for regulating pulmonary inflammation and fibrosis. Cell. 1999;96:319–328. doi: 10.1016/s0092-8674(00)80545-0. [DOI] [PubMed] [Google Scholar]
  • 23.Mu D, Cambier S, Fjellbirkeland L, Baron JL, Munger JS, Kawakatsu H, Sheppard D, Broaddus VC, Nishimura SL. The integrin alpha(v)beta8 mediates epithelial homeostasis through MT1-MMP-dependent activation of TGF-beta1. The Journal of cell biology. 2002;157:493–507. doi: 10.1083/jcb.200109100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Wang R, Zhu J, Dong X, Shi M, Lu C, Springer TA. GARP regulates the bioavailability and activation of TGFbeta. Mol Biol Cell. 2012;23:1129–1139. doi: 10.1091/mbc.E11-12-1018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Travis MA, Reizis B, Melton AC, Masteller E, Tang Q, Proctor JM, Wang Y, Bernstein X, Huang X, Reichardt LF, Bluestone JA, Sheppard D. Loss of integrin alpha(v)beta8 on dendritic cells causes autoimmunity and colitis in mice. Nature. 2007;449:361–365. doi: 10.1038/nature06110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Lacy-Hulbert A, Smith AM, Tissire H, Barry M, Crowley D, Bronson RT, Roes JT, Savill JS, Hynes RO. Ulcerative colitis and autoimmunity induced by loss of myeloid alphav integrins. Proc Natl Acad Sci U S A. 2007;104:15823–15828. doi: 10.1073/pnas.0707421104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Yang Z, Mu Z, Dabovic B, Jurukovski V, Yu D, Sung J, Xiong X, Munger JS. Absence of integrin-mediated TGFbeta1 activation in vivo recapitulates the phenotype of TGFbeta1-null mice. The Journal of cell biology. 2007;176:787–793. doi: 10.1083/jcb.200611044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Zhumabekov T, Corbella P, Tolaini M, Kioussis D. Improved version of a human CD2 minigene based vector for T cell-specific expression in transgenic mice. J Immunol Methods. 1995;185:133–140. doi: 10.1016/0022-1759(95)00124-s. [DOI] [PubMed] [Google Scholar]
  • 29.Suzuki M, Abe K, Yoshinaga K, Obinata M, Furusawa M. Specific arrest of spermatogenesis caused by apoptotic cell death in transgenic mice. Genes Cells. 1996;1:1077–1086. doi: 10.1046/j.1365-2443.1996.d01-228.x. [DOI] [PubMed] [Google Scholar]
  • 30.Oswald-Richter K, Grill SM, Shariat N, Leelawong M, Sundrud MS, Haas DW, Unutmaz D. HIV infection of naturally occurring and genetically reprogrammed human regulatory T-cells. PLoS biology. 2004;2:E198. doi: 10.1371/journal.pbio.0020198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Inaba K, Inaba M, Romani N, Aya H, Deguchi M, Ikehara S, Muramatsu S, Steinman RM. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J Exp Med. 1992;176:1693–1702. doi: 10.1084/jem.176.6.1693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Wan Q, Kozhaya L, ElHed A, Ramesh R, Carlson TJ, Djuretic IM, Sundrud MS, Unutmaz D. Cytokine signals through PI-3 kinase pathway modulate Th17 cytokine production by CCR6+ human memory T cells. J Exp Med. 2011;208:1875–1887. doi: 10.1084/jem.20102516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Oswald-Richter K, Grill SM, Leelawong M, Tseng M, Kalams SA, Hulgan T, Haas DW, Unutmaz D. Identification of a CCR5-expressing T cell subset that is resistant to R5-tropic HIV infection. PLoS Pathog. 2007;3:e58. doi: 10.1371/journal.ppat.0030058. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Probst-Kepper M, Geffers R, Kroger A, Viegas N, Erck C, Hecht HJ, Lunsdorf H, Roubin R, Moharregh-Khiabani D, Wagner K, Ocklenburg F, Jeron A, Garritsen H, Arstila TP, Kekalainen E, Balling R, Hauser H, Buer J, Weiss S. GARP: a key receptor controlling FOXP3 in human regulatory T cells. Journal of cellular and molecular medicine. 2009;13:3343–3357. doi: 10.1111/j.1582-4934.2009.00782.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Rochman Y, Spolski R, Leonard WJ. New insights into the regulation of T cells by gamma(c) family cytokines. Nat Rev Immunol. 2009;9:480–490. doi: 10.1038/nri2580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Thornton AM, Korty PE, Tran DQ, Wohlfert EA, Murray PE, Belkaid Y, Shevach EM. Expression of Helios, an Ikaros transcription factor family member, differentiates thymic-derived from peripherally induced Foxp3+ T regulatory cells. J Immunol. 2010;184:3433–3441. doi: 10.4049/jimmunol.0904028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Schramm C, Huber S, Protschka M, Czochra P, Burg J, Schmitt E, Lohse AW, Galle PR, Blessing M. TGFbeta regulates the CD4+CD25+ T-cell pool and the expression of Foxp3 in vivo. Int Immunol. 2004;16:1241–1249. doi: 10.1093/intimm/dxh126. [DOI] [PubMed] [Google Scholar]
  • 38.Kehrl JH, Wakefield LM, Roberts AB, Jakowlew S, Alvarez-Mon M, Derynck R, Sporn MB, Fauci AS. Production of transforming growth factor beta by human T lymphocytes and its potential role in the regulation of T cell growth. J Exp Med. 1986;163:1037–1050. doi: 10.1084/jem.163.5.1037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Ouyang W, Beckett O, Ma Q, Li MO. Transforming growth factor-beta signaling curbs thymic negative selection promoting regulatory T cell development. Immunity. 2010;32:642–653. doi: 10.1016/j.immuni.2010.04.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Gavin MA, Clarke SR, Negrou E, Gallegos A, Rudensky A. Homeostasis and anergy of CD4(+)CD25(+) suppressor T cells in vivo. Nat Immunol. 2002;3:33–41. doi: 10.1038/ni743. [DOI] [PubMed] [Google Scholar]
  • 41.Coombes JL, Siddiqui KR, Arancibia-Carcamo CV, Hall J, Sun CM, Belkaid Y, Powrie F. A functionally specialized population of mucosal CD103+ DCs induces Foxp3+ regulatory T cells via a TGF-beta and retinoic acid-dependent mechanism. J Exp Med. 2007;204:1757–1764. doi: 10.1084/jem.20070590. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Sun CM, Hall JA, Blank RB, Bouladoux N, Oukka M, Mora JR, Belkaid Y. Small intestine lamina propria dendritic cells promote de novo generation of Foxp3 T reg cells via retinoic acid. J Exp Med. 2007;204:1775–1785. doi: 10.1084/jem.20070602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Gravano DM, Vignali DA. The battle against immunopathology: infectious tolerance mediated by regulatory T cells. Cellular and molecular life sciences : CMLS. 2012;69:1997–2008. doi: 10.1007/s00018-011-0907-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Ikushima H, Miyazono K. TGFbeta signalling: a complex web in cancer progression. Nature reviews. Cancer. 2010;10:415–424. doi: 10.1038/nrc2853. [DOI] [PubMed] [Google Scholar]
  • 45.Curiel TJ, Coukos G, Zou L, Alvarez X, Cheng P, Mottram P, Evdemon-Hogan M, Conejo-Garcia JR, Zhang L, Burow M, Zhu Y, Wei S, Kryczek I, Daniel B, Gordon A, Myers L, Lackner A, Disis ML, Knutson KL, Chen L, Zou W. Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat Med. 2004;10:942–949. doi: 10.1038/nm1093. [DOI] [PubMed] [Google Scholar]
  • 46.Turk MJ, Guevara-Patino JA, Rizzuto GA, Engelhorn ME, Sakaguchi S, Houghton AN. Concomitant tumor immunity to a poorly immunogenic melanoma is prevented by regulatory T cells. J Exp Med. 2004;200:771–782. doi: 10.1084/jem.20041130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Antony PA, Piccirillo CA, Akpinarli A, Finkelstein SE, Speiss PJ, Surman DR, Palmer DC, Chan CC, Klebanoff CA, Overwijk WW, Rosenberg SA, Restifo NP. CD8+ T cell immunity against a tumor/self-antigen is augmented by CD4+ T helper cells and hindered by naturally occurring T regulatory cells. J Immunol. 2005;174:2591–2601. doi: 10.4049/jimmunol.174.5.2591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Woo EY, Chu CS, Goletz TJ, Schlienger K, Yeh H, Coukos G, Rubin SC, Kaiser LR, June CH. Regulatory CD4(+)CD25(+) T cells in tumors from patients with early-stage non-small cell lung cancer and late-stage ovarian cancer. Cancer research. 2001;61:4766–4772. [PubMed] [Google Scholar]
  • 49.Chen ML, Pittet MJ, Gorelik L, Flavell RA, Weissleder R, von Boehmer H, Khazaie K. Regulatory T cells suppress tumor-specific CD8 T cell cytotoxicity through TGF-beta signals in vivo. Proc Natl Acad Sci U S A. 2005;102:419–424. doi: 10.1073/pnas.0408197102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Thomas DA, Massague J. TGF-beta directly targets cytotoxic T cell functions during tumor evasion of immune surveillance. Cancer cell. 2005;8:369–380. doi: 10.1016/j.ccr.2005.10.012. [DOI] [PubMed] [Google Scholar]
  • 51.Li X, Ye F, Chen H, Lu W, Wan X, Xie X. Human ovarian carcinoma cells generate CD4(+)CD25(+) regulatory T cells from peripheral CD4(+)CD25(-) T cells through secreting TGF-beta. Cancer letters. 2007;253:144–153. doi: 10.1016/j.canlet.2007.01.024. [DOI] [PubMed] [Google Scholar]
  • 52.Liu VC, Wong LY, Jang T, Shah AH, Park I, Yang X, Zhang Q, Lonning S, Teicher BA, Lee C. Tumor evasion of the immune system by converting CD4+CD25- T cells into CD4+CD25+ T regulatory cells: role of tumor-derived TGF-beta. J Immunol. 2007;178:2883–2892. doi: 10.4049/jimmunol.178.5.2883. [DOI] [PubMed] [Google Scholar]
  • 53.Zou W. Regulatory T cells, tumour immunity and immunotherapy. Nat Rev Immunol. 2006;6:295–307. doi: 10.1038/nri1806. [DOI] [PubMed] [Google Scholar]
  • 54.Schuler PJ, Schilling B, Harasymczuk M, Hoffmann TK, Johnson J, Lang S, Whiteside TL. Phenotypic and functional characteristics of CD4+ CD39+ FOXP3+ and CD4+ CD39+ FOXP3neg T-cell subsets in cancer patients. Eur J Immunol. 2012;42:1876–1885. doi: 10.1002/eji.201142347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Zou W. Immunosuppressive networks in the tumour environment and their therapeutic relevance. Nature reviews. Cancer. 2005;5:263–274. doi: 10.1038/nrc1586. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS456114-supplement-1.pdf (1.2MB, pdf)

RESOURCES