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Originally published online as doi:10.1189/jlb.1006644 on May 2, 2007

Published online before print May 2, 2007
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(Journal of Leukocyte Biology. 2007;82:335-346.)
© 2007 by Society for Leukocyte Biology

TGF-β1 modulates Foxp3 expression and regulatory activity in distinct CD4+ T cell subsets

M. Pyzik and C. A. Piccirillo1

Department of Microbiology and Immunology and Strategic Training Centre in Infectious Diseases and Autoimmunity, McGill University, Montreal, Quebec, Canada

1 Correspondence: Department of Microbiology and Immunology, McGill University, 3775 University Street, Room 510, Lyman Duff Medical Building, Montreal, QC, Canada, H3A 2B4. E-mail: ciro.piccirillo{at}mcgill.ca


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ABSTRACT
 
Although forkhead box p3 (Foxp3) expression is restricted to naturally occurring CD4+ regulatory T cells (TREG), little is known about the various signals that regulate it in T cells. As TGF-β has been reported to modulate Foxp3 expression in T cells, we investigated its effects on the induction or maintenance of regulatory functions in different CD4+ T cell subsets. TGF-β1 priming was able to promote differentiation of TREG cells from nonregulatory CD4+CD25– T cells in a concentration-dependent manner through Foxp3 induction. As CD4+CD25– T cells remain a highly heterogeneous population with variable degrees of antigen experience, we then examined the effect of TGF-β1 on naive CD4+CD25–CD45RBHIGH T cells. Freshly isolated or TGF-β1-treated CD4+CD25–CD45RBHIGH T cells never displayed any regulatory functions or significant Foxp3 expression following TCR activation. In stark contrast, freshly isolated CD4+CD25–CD45RBLOW cells, albeit expressing low levels of Foxp3 mRNA and protein, were unable to suppress CD4+ effector T cell proliferation but acquired regulatory activity and de novo Foxp3 expression following TGF-β1 exposure. Furthermore, suppression was IL-10-dependent, as anti-IL-10 receptor antibody treatment abrogated this suppression completely, consistent with the ability of TGF-β1-treated CD4+CD25–CD45RBLOW to synthesize IL-10 upon restimulation in vitro. Last, we show that TGF-β1 treatment or blockade did not lead to enhanced expansion or function of naturally occurring CD4+CD25+ TREG cells, although it maintained Foxp3 mRNA and protein expression. Altogether, TGF-β1 promotes the induction of IL-10-secreting CD4+ TREG cells from CD4+CD25–CD45RBLOW precursors through de novo Foxp3 production and maintains natural TREG cell peripheral homeostasis by sustaining Foxp3 expression.

Key Words: CD4+CD25+ regulatory T cells • IL-10 • suppression • tolerance


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INTRODUCTION
 
Self/nonself discrimination is a key feature of immunological tolerance. Self-tolerance relies on various peripheral mechanisms, including expression of suppressor cytokines and action of various CD4+ regulatory T (TREG) cells, which act at multiple levels of an immune response to inhibit self-reactive T cells, having escaped central tolerance [1 ]. CD4+ TREG cells are potent modulators of T cell-mediated immune responses in vitro and in vivo and can be subdivided into two main groups: CD4+CD25+ naturally occurring TREG (nTREG) cells, which acquire their suppressive functions in thymus, and induced TREG (iTREG) cells, which acquire their functions in the periphery [2 ]. nTREG cells represent 5–10% of the peripheral CD4+ T cell pool in healthy rodents and humans, acquire their function during normal thymopoiesis, and prevent peripheral autoimmune responses. Functional abrogation of nTREG cells provokes multiorgan autoimmunity and also increases immunity to tumor, grafts, allergens, and microbial pathogens [1 ]. It has become evident that peripheral mechanisms of TREG cell generation exist as a result of decreasing thymic output of T lymphocytes with age yet equal levels of TREG cells among young and old individuals [3 ]. Indeed, induced CD4+CD25+ cells can be derived from CD4+CD25– T cell fractions and acquire or expand their suppressive activity as a consequence of in vivo or ex vivo activation of nTREG-depleted CD4+ T cells under unique stimulatory conditions in the periphery.

Forkhead box p3 (Foxp3) is essential for nTREG development and function, and its importance is best illustrated in Foxp3-deficient (–/–) mice and patients suffering from immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome, where mutations or deletions in the foxp3 gene halts the development of nTREG cells and consequentially, leads to a fatal autoimmune and inflammatory disease [4 5 6 7 8 ]. Furthermore, ectopic Foxp3 expression confers a suppressor phenotype in murine CD4+CD25– T cells similar to that of nTREG cells [9 ]. Although most studies support the notion that the thymus represents the major site of nTREG cell development, recent reports suggest that Foxp3+ T cell populations may also be induced in the periphery [10 11 12 13 ]. Although Foxp3 expression generally correlates with the suppressive activity of different iTREG cells, a number of exceptions have been documented [14 ]. Currently, the intrinsic and extrinsic factors, which induce and maintain Foxp3 expression/function and resulting suppressor activity in normal peripheral CD4+ T cells, remain poorly defined.

TGF-β1 is a potent, suppressive cytokine critically involved in the induction of tolerance and the regulation of immune responses [15 ]. This is best illustrated by the onset of a severe autoimmune-like syndrome in TGF-β1–/– mice, characterized by the spontaneous and progressive, multiorgan infiltration of mononuclear cells and pathogenic autoantibodies [8 ]. Furthermore, disruption of TGF-β1 signaling in T cells by deletion of the TGF-β Type II receptor or inactivation of the receptor-activated smad3 gene results in dysregulated T cell responses [16 17 18 ]. The mechanism through which TGF-β1 mediates its tolerogenic functions is not understood totally. The potential role of TGF-β1 in the development, differentiation, expansion, or suppressive mechanism of nTREG and iTREG cells remains of great importance. TGF-β1 has been shown recently to promote nTREG cell expansion as well as generation of Foxp3+ iTREG cells from CD4+CD25– T cells, although the underlying molecular mechanisms remain ill-defined.

Here, we evaluated the effect of TGF-β1 in inducing TREG cells from nonregulatory CD4+ T cell populations, as well as its contribution to the suppressive function and Foxp3 expression in the CD4+CD25+ nTREG population. We confirm that TGF-β1 is able to induce Foxp3 expression and suppressor function in nonregulatory CD4+CD25– T cell precursors in a dose-dependent manner and in the absence of APC costimulatory signals. However, TGF-β1 was unable to induce suppressive functions and Foxp3 expression in activated, naïve CD4+CD25–CD45RBHIGH T cells, and its effects were restricted solely to the antigen-experienced CD4+CD25–CD45RBLOW T cell subset. We also show that TGF-β1 priming was not required for the acquisition of nTREG cell proliferative and effector functions but could maintain Foxp3 expression in nTREG cells. Finally, the induction of TREG cells by TGF-β1 from antigen-experienced CD4+ T cells resulted in IL-10 expression, and anti-IL-10 receptor (IL-10R) treatment reversed the suppression mediated by iTREG cells in vitro. Thus, TGF-β1 sustains regulatory networks by positively modulating de novo Foxp3 expression and induces the post-thymic development of TREG cells.


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MATERIALS AND METHODS
 
Mice
Foxp3-GFP knock-in (kind gift from Alexander Rudensky, University of Washington, Seattle, WA, USA), C57BL/6, and OT-I mice (Taconic, Germantown, NY, USA) were bred and housed in a SPF animal facility at McGill University (Montreal, QC, Canada), and all animal studies were conducted according to institutional norms. Unless specified, mice used in all experiments were 6–10 weeks of age.

Reagents
Human recombinant (hr)IL-2 was a kind gift from the Surgery Branch [National Cancer Institute (NCI), National Institutes of Health (NIH), Bethesda, MD, USA]. hrTGF-β1 was a kind gift from John Letterio (NCI, NIH). Anti-TGF-β (1D11) and anti-CD3 {epsilon} (2C11) antibodies were produced in-house by supernatant purification on a protein G column. Anti-CD4, anti-FITC, and anti-PE magnetic beads were purchased from Miltenyi Biotec (Auburn, CA, USA). Anti-IL-10R antibody (1B1.3a) was a kind gift from Kevin Moore (DNAX, Palo Alto, CA, USA). Anti-mouse IL-4 (1B11) was purchased from BD PharMingen (San Diego, CA, USA). For cell culture, RPMI 1640 was supplemented with 10% heat-inactivated FCS, penicillin (100 U/ml), streptomycin (100 ug/ml), 2 mM L-glutamine, 10 mM Hepes, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate (Invitrogen Life Technologies, Carlsbad, CA, USA), and 50 µM 2-ME (Sigma-Aldrich, St. Louis, MO, USA).

Flow cytometric analysis
Single-cell suspensions prepared from lymph nodes (LN) of adult C57BL/6 or Foxp3-GFP knock-in reporter mice were stained and analyzed on FACSCalibur using CellQuest software (BD Biosciences, San Jose, CA, USA). For direct staining to determine the phenotype of lymphocyte populations, FcRs were blocked with anti-CD16/32 antibody (2.4G2, BD PharMingen) and then stained with the following conjugated antibodies: PE or allophycocyanin anti-CD25 (PC61, eBiosciences, San Diego, CA, USA), PE Cytocrome 7 anti-CD4 (L3T4), TriColor anti-CD4 (RM4-5, Caltag Laboratories, South San Francisco, CA, USA), PE or FITC anti-CD45RB (16A, eBiosciences), PE anti-CD69 (HI.2F3), PE anti-CD62 ligand (CD62L; MEL-14, BD PharMingen), as well as PE-IgG2a and PE-IgG1 isotype controls. For intranuclear Foxp3 staining, cells were stained with antibodies against surface antigens and then were fixed, permeabilized, and stained with PE anti-Foxp3 (PJK-16 s) according to the manufacturer’s protocol (eBiosciences). For intracellular IL-10 staining, cells were reactivated with plate-bound anti-CD3 (10 µg/ml) for 24 h. The cells were then collected, fixed, permeabilized, and stained with allophycocyanin anti-IL-10 (JES5-16E3, eBiosciences) using the BD Cytofix/Cytoperm kit, according to the manufacturer’s protocol (BD PharMingen).

Purification of T cell subsets
Different T cell subsets were isolated on a FACSAria (BD Biosciences) cell sorter as described previously [19 ]. Briefly, LN were collected from adult C57BL/6 or Foxp3-GFP knock-in reporter mice, and single-cell suspensions were prepared by macerating cells through a sterile wire mesh. The resulting cell suspension was stained with allophycocyanin anti-CD25 (10 µg/108 cells), PE anti-CD45RB (3.5 µg/108 cells), and PECy7 anti-CD4 (3 µg/108 cells) in PBS/2% FCS for 20 min at 4°C, washed, and resuspended in PBS/2% FCS. The purity of the final CD4+CD25–CD45RBLOW /HIGH preparation was typically >98%.

In vitro T cell priming assays
To generate iTREG cells in vitro, CD4+CD25– T cells (5x105) were cultured in 24-well, flat-bottom microtiter plates in cRPMI 1640 with plate-bound anti-CD3 (10 µg/ml) or soluble anti-CD3 (2 µg/ml) and an equal number of APC for 72 h in the presence or absence of titrated doses of hrTGF-β1 (0.1–10 ng/ml). Irradiated (3000 Rads), T cell-depleted spleen cells were used as APC and were prepared as described previously [19 ]. CD4+ or CD8+ responder T cells were prepared from LN of appropriate adult mice, as described previously [20 ]. To measure the effect of TGF-β1 on nTREG cells, CD4+CD25+ T cells (5x105) were cultured with 10 µg/ml plate-bound anti-CD3 for 72 h in the presence or absence of rhTGF-β1 (3 ng/ml) and IL-2 (5 ng/ml). In some instances, 10 µg/ml anti-TGF-β-neutralizing antibody was added to the culture.

In vitro proliferation/suppression assays
Proliferation assays were performed by stimulating responding T cells (2.5x104) in 96 flat-bottom microtiter plates in cRPMI 1640 with plate-bound (10 µg/ml) or with soluble anti-CD3 (2 µg/ml) and irradiated APC (1x105) for 72 h at 37°C in 5% CO2. For suppression assays, rhTGF-β1-treated or -untreated T cells (0.625–2.5x104) were cocultured with OT-I transgenic (Tg) CD8+ responder T cells (2.5x104) with soluble anti-CD3 (2 µg/ml) and APC (1x105) in 96-well plates for 72 h at 37°C/5% CO2. Cell cultures were pulsed with 0.5 µCi 3H-thymidine for the last 6–12 h to determine the extent of suppression. When specified, anti-IL-10R (3.5 µg/ml) or anti-IL-4 (10 µg/ml) was added to the cocultures.

RT-PCR
Evaluation of Foxp3 mRNA levels in TGF-β1-treated and -untreated CD4+ T cells was performed as described previously [9 ]. Briefly, total RNA was extracted from 0.5-1 x 106 cells using Trizol reagent (Invitrogen Life Technologies) and reverse-transcribed using the Superscript II RNase H RT (Invitrogen Life Technologies) and pd(N)6 random hexamers (0.05 µg/ul, GE Healthcare, UK). Foxp3 mRNA levels were quantified by RT-PCR using Chromo4 continuous fluorescence detector (Bio-Rad, Hercules, CA, US). Foxp3 primers and internal fluorescent TaqMan probes were used as described previously [9 ]. 18S rRNA primers and probe were purchased from Applied Biosystems (Foster City, CA, USA). A relative {Delta}{Delta} comparative threshold cycle method was used to quantify Foxp3 mRNA levels normalized to 18S rRNA. IL-10, TGF-β1, and G3PDH mRNA levels were quantified by semiquantitative PCR under the following conditions: 5 min at 95°C followed by 25 cycles at 1 min at 95°C, 1 min at 58°C, 1 min at 72°C, and 5 min final extension at 72°C. The primers for IL-4, IL-10, TGF-β1, and G3PDH were obtained from BD Biosciences. PCR products were analyzed on 2% agarose gels, and the intensity of each band was measured by the TotalLAB program.


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RESULTS
 
TGF-β1 induces CD4+ TREG cells from CD4+CD25– T cell precursors
Some recent reports have suggested that TGF-β1 can promote the development of TREG cells [10 11 12 ]. To assess its role in inducing TREG functions in nonregulatory CD4+ T cells, CD4+CD25– T cells were activated in the absence or presence of different concentrations of rhTGF-β1 for 3 days, after which, the suppressive activity was measured by coculturing treated cells with CD8+-responding T cells, soluble anti-CD3 (2 µg/ml), and irradiated, T cell-depleted splenocytes as source of APC. Although TGF-β1 displays potent, antiproliferative effects on activated T cells (Fig. 1A ), treatment with rhTGF-β1 induced regulatory functions in CD4+CD25– T cells. In vitro activation of CD4+CD25– T cells in the presence of high concentrations of TGF-β1 (10 ng/ml) induced cell dose-dependent suppression of proliferation, attaining 80% suppression at a 1:1 TREG:TEFF cell ratio (Fig. 1B , upper panels), in contrast to untreated CD4+CD25– T cells, which never inhibited the proliferation of CD8+ T cells. This regulatory function decreased concomitantly with lower TGF-β1-priming concentrations, such that 57% suppression (at a 1:1 TREG:TEFF cell ratio) was achieved at 0.1 ng/ml TGF-β1 pretreatment. It remained possible that TGF-β1 can mediate this effect indirectly via its action on APC or that APC could compete for the cytokine with responding T cells, thus leading to inferior induction of a regulatory phenotype. Therefore, we activated CD4+CD25– T cells under plate-bound anti-CD3 conditions (APC-free) in the presence of titrated doses of rhTGF-β1 (Fig. 1B , lower panels). Consistent with our previous results, the induction of TREG cells by TGF-β1 was dose-dependent, as the highest suppression levels were achieved with the highest TGF-β1 doses, and suppression decreased to 20% at a 1:1 TREG:TEFF cell ratio at lower TGF-β1 concentrations. It is intriguing that a higher induction of suppression was achieved in the presence of APC (57% vs. 20%) under low-dose TGF-β1 conditions. This data suggest that TGF-β1 can promote the development of TREG cells in the absence of APC-derived signals.


Figure 1
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  		Figure 1. TGF-β1 induces CD4+ TREG cells from CD4+CD25– T cell precursors. (A) Antiproliferative effect of TGF-β1 on freshly isolated CD4+CD25– T cells stimulated with plate-bound (5 µg/ml; right panel) or soluble anti-CD3 (2 µg/ml; left panel) with irradiated APC (2x104) in the absence or presence of increasing concentrations of rhTGF-β1 (0–10 ng/ml) for 3 days. (B) Three days postactivation, rhTGF-β1-treated or -untreated cells (1.25–5x104 cells) were cocultured with OT-I Tg CD8+ responder T cells (5x104), irradiated APC (2x105), and anti-CD3 (2 µg/ml) for 72 h. Cultures were pulsed with 0.5 µCi 3H-thymidine for the last 6–12 h of incubation. The data are shown as mean CPM ± SD (left panels) or percent suppression (right panels). The data are representative of three independent experiments. (C) TGF-β1 dose-dependent induction of Foxp3 mRNA levels and suppressor function at a 1:1 TREG:effector T (TEFF) cell ratio. Relative Foxp3 mRNA levels relative to 18S rRNA were assessed by quantitative RT-PCR (qRT-PCR) in TGF-β1-treated and -untreated CD4+CD25– T cells 3 days poststimulation. The results shown are representative of at least three separate experiments.

As Foxp3 has been associated with the induction of regulatory function in T cells, and TGF-β1 has been reported to induce its expression, we next compared the Foxp3 mRNA levels in TGF-β1-treated and -untreated CD4+CD25– T cells. qRT-PCR analysis revealed that TGF-β1 was able to induce Foxp3 gene expression in treated cells in a dose-dependent manner (Fig. 1C) . At low TGF-β1 doses (0.1 ng/ml), TGF-β1 induced a twofold increase in Foxp3 mRNA levels, and at higher doses, a 20-fold higher level of Foxp3 gene expression was observed compared with untreated T cells (Fig. 1C) . It is notable that the acquisition of suppressive activity in CD4+CD25– T cells by TGF-β1 pretreatment was concentration-dependent and correlated with greater synthesis of Foxp3 mRNA (Fig. 1C) . Collectively, these findings confirm the general observations from previous studies that TGF-β1 induction of suppressive activity in CD4+CD25– T cells is associated with Foxp3 up-regulation.

TGF-β1 induces CD4+ TREG cells only from CD4+CD25–CD45RBLOW T cells
Although TGF-β1 is able to induce regulatory activity in CD4+ T cell populations largely devoid of CD25+Foxp3+-expressing T cells, previous studies do not demonstrate directly that TGF-β1 can drive the differentiation of bona fide, naïve CD4+ T cells into a regulatory phenotype. As CD4+CD25– T cells remain a considerably heterogeneous population with variable degrees of antigen experience, we investigated the effect of TGF-β1 priming on a truly naïve subset within CD4+CD25– T cells, namely CD25–CD45RBHIGH T cells. The CD4+CD25–CD45RBHIGH T cell subset has been characterized previously as potently pathogenic as a result of its ability to transfer inflammatory bowel disease (IBD) in SCID recipient mice [21 ]. To this end, we FACS-sorted CD4+CD25–CD45RBHIGH T cells from LN of C57BL/6 mice, tested their suppressive activity directly ex vivo, or activated them in vitro with plate-bound anti-CD3 in the presence or absence of rhTGF-β1 (3 ng/ml) for 3 days and then assessed their suppressor activity (Fig. 2A ). Similar to freshly isolated CD25–CD45RBHIGH T cells, TGF-β1 treatment, strikingly, did not induce any regulatory functions in naïve CD4+CD25–CD45RBHIGH T cells, as treated cells never inhibited the proliferative capacity of responder T cells (Fig. 2C) .


Figure 2
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  		Figure 2. TGF-β1 promotes the development of CD4+ TREG cells uniquely from CD4+CD25–CD45LOW T cells. (A) CD4+CD25–CD45RBLOW, CD4+CD25–CD45RBHIGH, and CD4+CD25+ populations were FACS-sorted from LN of C57BL/6 mice according to the illustrated gates. (B) Flow cytometric analysis of CD69, CD62L, and Foxp3 expression in CD45RBLOW and CD45RBHIGH subsets within CD4+CD25– T cells. The regulatory function of CD4+CD25–CD45RBHIGH (C) and CD4+CD25–CD45RBLOW (D) T cells, relative to CD4+CD25+ nTREG cells, was measured directly ex vivo (left panel) or after in vitro rhTGF-β1 priming (middle and right panels). In brief, specific cell subsets (5x105) were activated with plate-bound anti-CD3 (10 µg/ml) in the presence or absence of rhTGF-β1 (3 ng/ml) for 3 days. TGF-β1-treated or -untreated cells were then cocultured at different ratios (6.25x103:2.5x104) with OT-I Tg CD8+ responder T cells (2.5x104) and stimulated with anti-CD3 (2 µg/ml) in the presence of APC (1x105). Cultures were pulsed with 3H-thymidine during the last 8 h and harvested at 72 h. Results of the suppression assay are shown as the mean CPM ± SD (left and middle panels) or percent suppression (right panels). The results shown are representative of at least three separate experiments.

As CD4+CD25– T cells contain 30–40% of antigen-experienced CD4+CD25–CD45RBLOW T cells in adult mice [22 ], we postulated that this subset might represent the cellular target of TGF-β1 in our system. We first confirmed the antigen experience of this population by examining the frequency of activated/memory cells. Although the CD69 expression profile was identical in CD25–CD45RBHIGH and CD25–CD45RBLOW T cells, CD4+CD25–CD45RBLOW T cells contained ~40% of CD62LLOW cells (Fig. 2B) . To examine CD4+CD25–CD45RBLOW T cells functionally, we FACS-sorted CD25–CD45RBLOW T cells from LN of C57BL/6 mice, tested their suppressive activity directly ex vivo, or activated them in vitro as described in Figure 1 and then assessed their in vitro suppressor activity relative to CD4+CD25+ nTREG cells (Fig. 2D) . Freshly isolated CD4+CD25–CD45RBLOW T cells did not suppress proliferation of CD8+ T cells compared with nTREG cells, which readily resulted in more than 85% suppression at a 1:1 TREG:TEFF cell ratio (Fig. 2C) . It is most important that TGF-β1-pretreated CD25–CD45RBLOW cells were able to suppress proliferation of OT-I cells up to 75% at a 1:1 TREG:TEFF cell ratio, in contrast to cells cultured in the absence of TGF-β1, as proliferation levels were equivalent or higher than OT-I cells alone. These results clearly identify the antigen-experienced CD25–CD45RBLOW T cell subset as a cellular target for the regulatory inducing functions of TGF-β1.

TGF-β1 induces Foxp3 mRNA levels in CD4+CD25–CD45RBLOW T cells
TGF-β1 is characterized by its ability to inhibit the proliferative capacity of responding T cells. To this end, the proliferation of freshly isolated CD25–CD45RBLOW and CD25–CD45RBHIGH T cells was assessed following anti-CD3 stimulation in the absence or presence of rhTGF-β1. Our results indicate that untreated, CD25–CD45RBLOW cells were hypoproliferative compared with CD25–CD45RBHIGH cells (Fig. 3A ) but upon TGF-β1 priming, the proliferation of both cell subsets was decreased substantially, and CD25–CD45RBHIGH T cells showed a greater susceptibility to the growth inhibitory effects of TGF-β1 than CD25–CD45RBLOW cells (60% vs. 44%, respectively, P<0.02). In summary, our results show that TGF-β1 can selectively induce the expression of Foxp3 in antigen-experienced CD25–CD45RBLOW T cells and not in naive CD4+ T cells.


Figure 3
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  		Figure 3. Preferential TGF-β1 effects on proliferation and induction of Foxp3 mRNA levels in CD4+CD25–CD45RBLOW T cells. (A) CD4+CD25–CD45RBLOW or CD4+CD25–CD45RBHIGH T cell subsets (5x105) were activated with plate-bound anti-CD3 (10 µg/ml) in the absence or presence of rhTGF-β1 (3 ng/ml) for 3 days. Cultures were pulsed with 3H-thymidine during the last 8 h and harvested at 72 h. Results of the proliferation assay are shown as the mean CPM ± SD of triplicate cultures (#, P<0.02; *, P<0.002). (B) Total RNA was isolated from resting or Day 3, TGF-β1-stimulated cells (0.5–1x106) and reverse-transcribed, and relative Foxp3 mRNA levels, normalized to 18S rRNA, were assessed by qRT-PCR. All data are shown as mean of duplicates and are representative of at least three separate experiments (#, P<0.002; *, P<0.003). The results shown are representative of at least three separate experiments.

We next examined Foxp3 mRNA levels in freshly isolated and TGF-β1-primed CD4+CD25–CD45RBLOW and CD4+CD25–CD45RBHIGH T cell subsets relative to CD4+CD25+ nTREG cells. It is interesting that CD4+CD25–CD45RBLOW T cells expressed up to a tenfold increase in Foxp3 mRNA levels than CD4+CD25–CD45RBHIGH T cells directly ex vivo, although at significantly lower levels than CD4+CD25+ nTREG cells (Fig. 3B) . This observation is consistent with 5–10% of CD4+CD25–CD45RBLOW T cells expressing Foxp3 directly ex vivo in contrast to naïve T cells (Fig. 2B) , albeit not at the same frequency as nTREG cells (data not shown). After 3 days of TGF-β1 priming, no detectable increase in Foxp3 could be observed in naïve T cells, a finding consistent with the apparent absence in regulatory function in these cells following TGF-β1 treatment. In stark contrast, TGF-β1 pretreatment induced approximately a threefold increase in Foxp3 mRNA levels in CD4+CD25–CD45RBLOW cells compared with untreated cells and represented a 30-fold increase over CD4+CD25–CD45RBHIGH-treated T cells (P<0.002, Fig. 3B ). These levels represented almost 50% of the Foxp3 levels detected in resting CD4+CD25+ nTREG (P<0.003, Fig. 3B ).

TGF-β1 induces de novo Foxp3 expression and regulatory function in CD4+Foxp3–CD45RBLOW T cell precursors
As CD4+CD25–CD45RBLOW T cells contain a small (5–10%), endogenous Foxp3+ subset, it remained possible that the TGF-β1 inhibitory functions were induced through preferential expansion/differentiation of these cells, in contrast to de novo Foxp3 expression in this subset [23 ]. To discriminate the effects of TGF-β1 on Foxp3+ and Foxp3– cells within the CD4+CD25–CD45RBLOW T cell subset, CD4+CD45RBLOWFoxp3+, CD4+CD45RBLOWFoxp3–, and CD4+CD45RBHIGHFoxp3– T cells were isolated from Foxp3-GFP knock-in reporter mice, as described (Fig. 4A ). First, the proliferation of freshly isolated CD25–CD45RBLOWFoxp3– and CD25–CD45RBHIGH Foxp3– T cells was assessed following anti-CD3 stimulation in the absence or presence of rhTGF-β1. The CD45RBLOW subset depleted of Foxp3+ cells was consistently hypoproliferative compared with Foxp3–CD45RBHIGH cells (data not shown), but upon TGF-β1 treatment, the proliferation of both cell types was decreased substantially, and Foxp3–CD45RBHIGH T cells showed a greater susceptibility to the growth-inhibitory effects of TGF-β1 than Foxp3–CD45RBLOW cells (59% vs. 44%, respectively, P<0.03). Moreover, Foxp3–CD45RBLOW cells, pretreated for 3 days with TGF-β1, were able to suppress proliferation of OT-I cells up to 55% at a 1:1 TREG:TEFF cell ratio contrary to Foxp3–CD45RBHIGH-treated cells (Fig. 4B) , demonstrating that regulatory functions are induced in populations initially lacking nTREG cells but not from truly naïve T cells. To determine whether TGF-β1 induces Foxp3 protein expression, rhTGF-β1-treated and -untreated cells were examined for Foxp3/GFP levels by FACS. Our results show that TGF-β1 induces de novo expression of Foxp3 only in the activated CD45RBLOW population (2±1% vs. 23±5% of rhTGF-β1-treated cells, P<0.03; Fig. 4C ). Thus, we show that TGF-β1 priming selectively induces de novo Foxp3 protein expression and regulatory functions in CD4+CD25–CD45RBLOW T cells devoid of any Foxp3+ T cells.


Figure 4
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  		Figure 4. TGF-β1 induces de novo Foxp3 expression and regulatory function in CD4+Foxp3–CD45RBLOW T cell precursors. (A) CD4+Foxp3–CD45RBLOW, CD4+Foxp3–CD45RBHIGH, and CD4+Foxp3+ populations were FACS-sorted from LN of Foxp3/GFP knock-in mice. Gates used are illustrated in the FACS profile shown. Histograms represent a Foxp3/GFP profile at Day 0 of the three sorted populations. (B) CD4+Foxp3–CD45RBLOW or CD4+Foxp3–CD45RBHIGH T cells (5x105) were activated with plate-bound anti-CD3 (10 µg/ml) and rhTGF-β1 (3 ng/ml) for 3 days. Treated or untreated cells (1.25–5x104 cells) were then cocultured with OT-I Tg CD8+ responder T cells (5x104), irradiated APC (2x105), and anti-CD3 (2 µg/ml). Cultures were pulsed with 3H-thymidine during the last 8 h and harvested at 72 h. Results of the proliferation assay are shown as percent suppression ± SD at a 1:1 ratio. (C) Flow cytometric analysis of Foxp3/GFP expression in initially Foxp3–CD45RBLOW and Foxp3–CD45RBHIGH subsets, treated or not with rhTGF-β1, as described above. Left panel represents mean percent Foxp3/GFP+ cells ± percent SD; right panel, a FACS histogram of Foxp3/GFP induction in cell subsets treated with rhTGF-β1. The cells were gated on live events based on forward- and side-scatter and represented ~80% of total acquired cells, independent of cell subset or condition tested. All data are representative of at least three separate experiments.

TGF-β1 maintains Foxp3 expression in CD4+CD25+ nTREG cells without potentiating their expansion or function
Recent reports concluded that a latent, membrane-bound form of TGF-β1 on CD4+CD25+ nTREG cells was responsible for their suppressive functions in vitro and in vivo, albeit never formally demonstrating a direct effect of TREG cell-derived TGF-β1 on responder T cells [24 ]. Furthermore, these studies did not examine the possibility that cell surface TGF-β1, from autocrine or paracrine sources, mediates its effects by actively signaling in CD4+CD25+ TREG cells themselves, possibly maintaining their differentiation, expansion, or suppressive function, as suggested by recent reports [25 ]. To this end, we purified CD4+CD25+ T cells, activated them in vitro, and assessed their proliferation and functional activity, following TGF-β1 neutralization or exogenous priming. TGF-β1 treatment did not promote the expansion of CD4+CD25+ T cells in vitro (Fig. 5A ). In addition, neutralization of TGF-β1 or preconditioning with rhTGF-β1 in vitro failed to abrogate or promote TREG function, respectively, suggesting that autocrine or paracrine sources of TGF-β1 do not modulate the proliferation or suppressor phase of nTREG activity (Fig. 5B) . Addition of greater concentrations of antibody (50 ug/ml isotype control or anti-TGF-β1) was never able to abrogate suppression in vitro (data not shown; ref. [14 ]). However, when nTREG cells were incubated with rhTGF-β1 for 3 days in the presence of IL-2 (5 ng/ml), an important increase in Foxp3 mRNA levels was detected (P<0.003, Fig. 5C ). To investigate the effect of TGF-β1 directly on Foxp3 protein expression in nTREG cells, CD4+Foxp3+ cells were sorted from the peripheral LN of Foxp3-GFP knock-in mice and activated with plate-bound anti-CD3 in the presence or absence of rhTGF-β1. Thereafter, Foxp3 levels, as determined by GFP expression, were measured by FACS 24 h poststimulation. We show that in the presence of rhTGF-β1, no significant change in Foxp3 expression was observed, and all cells remained Foxp3+ (Fig. 5D) . It is interesting that 12–17% of Foxp3+ cells lost Foxp3 expression in the absence of exogenous rhTGF-β1 stimulation. The viability of TGF-β1-treated CD4+CD25+ TREG cells was no different than untreated cells, as determined by FACS, and represented 85–90% of total acquired cells, independent of cell subset or condition tested. Thus, this reduction in Foxp3 is not a result of a selective loss in cell viability but more so, a result of the loss of exogenous TGF-β1-triggering in nTREG cells. Our results show that TGF-β1 does not affect the expansion or function of CD4+CD25+ nTREG cells but that TGF-β1 maintains Foxp3 expression in nTREG cells.


Figure 5
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  		Figure 5. TGF-β1 maintains Foxp3 expression in CD4+CD25+ nTREG cells without potentiating their expansion or function. (A) CD4+CD25+ nTREG cells (5x105) were activated with plate-bound anti-CD3 (10 µg/ml) in the absence or presence of rhTGF-β1 (3 ng/ml) and IL-2 (5 µg/ml), and proliferation was assessed on Day 3 (P<0.002). (B) CD4+CD25+ TREG cells (5x105) were activated with plate-bound anti-CD3 (10 µg/ml) and IL-2 (5 µg/ml), alone or with rhTGF-β1 (3 ng/ml) or anti-TGF-β-blocking antibodies (10 µg/ml) for 3 days. Treated or untreated cells (1.25–5x104 cells) were then cocultured with OT-I Tg CD8+ responder T cells (5x104), irradiated APC (2x105), and anti-CD3 (2 µg/ml). Cultures were pulsed with 3H-thymidine during the last 8 h and harvested at 72 h. Results of the proliferation assay are shown as the mean CPM ± SD of triplicate cultures. (C) Total RNA was isolated from resting or Day 3, TGF-β1-stimulated CD4+CD25+ nTREG cells (0.5–1x106), and relative Foxp3 mRNA levels were measured by qRT-PCR (P<0.003). (D) FACS analysis of GFP expression of freshly isolated CD4+Foxp3+ T cells (black lines; 5x105) activated with plate-bound anti-CD3 (10 µg/ml) in the absence or presence of rhTGF-β1 (3 ng/ml) for 24 h. All data are representative of at least three separate experiments.

TGF-β1-induced TREG cell effector function is IL-10-dependent
In recent years, a variety of iTREG cells, capable of controlling T cell responses in vitro and in vivo, has been described, and their regulatory functions have largely been attributed to the production of certain signature suppressor cytokines after antigen priming, such as TGF-β1 or IL-10 [14 , 26 ]. To determine whether the in vitro-suppressive function of TGF-β1-iTREG cells is cytokine-dependent, we measured IL-10, TGF-β1, and IL-4 mRNA levels by RT-PCR in resting or PMA/ionomycin-restimulated CD25–CD45RBHIGH and CD25–CD45RBLOW or restimulated Foxp3–CD45RBLOW and Foxp3–CD45RBHIGH T cells, initially activated in the presence or absence of TGF-β1, and TGF-β1 gene expression was not detected in CD25–CD45RBHIGH or CD25–CD45RBLOW T cells, irrespective of stimulation (data not shown). Moreover, negligible amounts of IL-4 mRNA were detected in both populations tested, irrespective of TGF-β1 treatment (Fig. 6B and data not shown). It is interesting that although low levels of IL-10 mRNA were detected in resting, TGF-β1-treated CD25–CD45RBLOW cells, significantly higher levels of IL-10 mRNA were detected in reactivated, TGF-β1-treated CD25–CD45RBLOW cells (Fig. 6A) . CD25–CD45RBHIGH or Foxp3–CD45RBHIGH T cell subsets did not express IL-10 in any condition tested (Fig. 6A and data not shown). Similarly, comparable IL-10 mRNA expression levels were observed in reactivated, TGF-β1-treated Foxp3–CD45RBLOW (Fig. 6B) but not in Foxp3–CD45RBHIGH T cell subsets (data not shown). We then tested the capacity of TGF-β1-treated CD4+Foxp3–CD45RBLOW cells to produce IL-10. To this end, treated or untreated CD4+Foxp3–CD45RBLOW and CD4+Foxp3–CD45RBHIGH subsets were restimulated with plate-bound anti-CD3 (10 µg/ml) and stained for intracellular cytokine IL-10. We show that IL-10 was readily detected only in TGF-β1-treated CD4+Foxp3–CD45RBLOW T cells post-restimulation, and 40% of induced Foxp3+ was able to produce it, compared with less than 6% in TGF-β1-untreated cells or less than 3% in CD4+Foxp3–CD45RBHIGH cells (Fig. 6C and 6D) . These results strongly suggest that TGF-β1 selectively induces Foxp3 and IL-10 expression only in CD4+Foxp3–CD45RBLOW cells and that TCR triggering is required for IL-10 production in TGF-β1 iTREG cells.


Figure 6
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  		Figure 6. TGF-β1-induced TREG cells express and secrete IL-10. (A) Day 3, rhTGF-β1-treated or -untreated CD4+CD25–CD45RBLOW T cells were reactivated or not with PMA (10 ng/ml) and ionomycin (250 ng/ml) for 6 h, and analysis of IL-10, relative to G3PDH gene expression, was preformed by RT-PCR on total RNA. Data are presented as a IL-10:G3PDH ratio relative to the IL-10:G3PDH ratio of CD4+CD25–CD45RBHIGH-untreated cells (set as 1). (B) CD4+Foxp3–CD45RBLOW T cell (5x105) subsets were activated with plate-bound anti-CD3 (10 µg/ml) and rhTGF-β1 (3 ng/ml) for 3 days and reactivated with PMA (10 ng/ml) and ionomycin (250 ng/ml) for 6 h. Analysis of IL-10 and IL-4 mRNA, relative to G3PDH gene expression, was preformed by RT-PCR on total RNA. Data are presented as a IL-10:G3PDH or IL-4:G3PDH ratio, relative to untreated CD4+Foxp3–CD45RBHIGH cells (set as 1). The data are representative of three experiments. (C) CD4+Foxp3–CD45RBLOW or CD4+Foxp3–CD45RBHIGH T cells (5x105) were treated as above for 3 days, reactivated with plate-bound anti-CD3 (10 µg/ml) for 24 h, and analyzed for Foxp3 expression (GFP) and intracellular IL-10 production by FACS. The cells were gated on live events based on forward- and side-scatter and represented ~85% of total acquired cells, independent of cell subset or condition tested. (D) FACS analysis of intracellular IL-10 production in TGF-β1-treated T cell subsets described above (black lines). Filled histograms represent isotype controls. The results shown are representative of at least three separate experiments.

To further assess the involvement of IL-10 in the suppression function of TGF-β1 iTREG cells, we cocultured TGF-β1-pretreated CD25–CD45RBLOW or CD25–CD45RBHIGH cells with responder CD8+ T cells in the presence or absence of a neutralizing, anti-IL-10R-blocking antibody. As discussed, TGF-β1-treated or -untreated CD25–CD45RBHIGH T cells never displayed any in vitro regulatory function under any conditions tested (Fig. 7A , upper panels). In contrast, addition of an anti-IL-10R-blocking antibody to TGF-β1-treated CD25–CD45RBLOW T cells almost completely abrogated suppression, reducing suppression levels from 73% (isotype control) to 17% (anti-IL-10R antibody) at a 1:1 TREG:TEFF cell ratio (Fig. 7A , lower panels). Similar results were obtained with Foxp3–CD45RBLOW T cells activated in the presence of TGF-β1 (Fig. 7B) . To definitively demonstrate the absence of IL-4 function in the suppressive mechanism of TGF-β1 iTREG cells, the suppressive capacity of TGF-β1-treated or -untreated Foxp3–CD45RBLOW and Foxp3–CD45RBHIGH cells was evaluated in the presence or absence of neutralizing anti-IL-4 antibody. Despite the presence of IL-4-blocking antibody, the suppressive activity of TGF-β1-treated Foxp3–CD45RBLOW remained unchanged and was similar to isotype control, demonstrating that TGF-β1 iTREG cells mediate their effect in an IL-4-independent manner (Fig. 7B) . These results clearly demonstrate that TGF-β1 iTREG cells produce IL-10 and that their in vitro-suppressive function is IL-10-dependent, consistent with the requirement of reactivation of these cells to reveal their regulatory potential.


Figure 7
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  		Figure 7. TGF-β1-induced TREG cell effector function is IL-10-dependent. (A) Three days of TGF-β1 treatment, CD4+CD25–CD45RBLOW and CD4+CD25–CD45RBHIGH cells were cocultured at different ratios (6.25x103:2.5x104) with OT-I Tg CD8+ T cells (2.5x104) and stimulated with anti-CD3 (2 µg/ml) in the presence of irradiated APC (1x105) and in the presence or absence of anti-IL-10R or IgG isotype control (3.5 µg/ml). (B) CD4+Foxp3–CD45RBLOW or CD4+Foxp3–CD45RBHIGH T cells (5x105) were activated with plate-bound anti-CD3 (10 µg/ml) and rhTGF-β1 (3 ng/ml) for 3 days. Suppressive properties were assessed as described above at a 1:1 TREG:TEFF cell ratio in the presence or absence of anti-IL-10R (3.5 µg/ml), anti-IL-4 (10 µg/ml), or IgG isotype control (3.5 µg/ml). Results of the proliferation assays are shown as the mean percent suppression ± SD of triplicate cultures. The data are representative of three experiments.


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DISCUSSION
 
The present study evaluated the ability of TGF-β1 to induce, maintain, or enhance suppressive functions in different CD4+ T cell subsets. We show that TCR engagement in conjunction with TGF-β1 stimulation can up-regulate Foxp3 expression and drive the differentiation of TREG cells from peripheral CD4+CD25– T cell progenitors. It is striking that we demonstrate that bona fide, naïve, CD25–CD45RBHIGH T cells, known to be potently proinflammatory in vitro and in vivo [21 ], did not acquire regulatory functions or up-regulate Foxp3 upon TGF-β1 treatment. The effect of TGF-β1 on the fate of T cell activation is determined, not only by an essential TCR trigger but also on the TGF-β1 dose, as the suppressor potency and de novo Foxp3 gene expression increase with the priming dose. We also show that this ability to promote de novo Foxp3 expression and TREG cell function by TGF-β1 is restricted to antigen-experienced Foxp3–CD45RBLOW T cells and not naïve, Foxp3–CD45RBHIGH responder T cells. In a similar manner, TGF-β1 maintains Foxp3 expression in CD4+CD25+ nTREG cells, however, without potentiating their expansion or function. Last, we show that TGF-β1 drives the expression and secretion of IL-10 upon TCR reactivation in primed T cells, and in vitro blockade of IL-10 abrogated suppression.

A number of studies show that TGF-β1 is a natural inducer of Foxp3 expression and regulatory activity in nTREG cell-depleted CD4+ T cells, namely CD4+CD25– T cells [10 11 12 ]. Although these TGF-β1 effects have been substantiated in in vitro and in vivo models, the authors from these reports fail to demonstrate whether these effects can be achieved in truly naïve CD4+ T cell populations. Another fundamental difference between these reports and the present study may depend on the time necessary to develop regulatory functions, with naïve T cells requiring prolonged stimulation contrary to memory T cells. Recently, human nTREG cells have been shown to derive from rapid turnover of memory populations in vivo, supporting the claim that the antigen-experienced fraction serves as the possible reservoir, which is more prone to develop regulatory functions under specific stimulatory conditions [27 ]. The generation of iTREG cells from naïve T cells isolated from antigen-specific TCR Tg mice, which do not possess nTREG cells, on a recombination-activating gene-deficient background has been described; however, it remains unclear whether TGF-β1 played any role in this process [13 , 28 , 29 ]. Other signals, such as vitamin D3 and dexamethasone, have been demonstrated to induce IL-10-producing, Foxp3– iTREG cells, suggesting that the induction of Foxp3 is a not a prerequisite for TREG development [14 ].

Normal, healthy mice harbor a significant frequency of naturally activated T cells, often found within the CD4+CD45RBLOW pool, which may represent physiological autoreactivity, as they are also found in germ-free or antigen-free mice. Several lines of evidence suggest that this pool may represent a reservoir from which TREG cells originate in the periphery [21 ]. Many models show that CD4+CD45RBLOW cells limit the pathogenicity of the counterpart CD4+CD45RBHIGH, naive T cells, as the former include nTREG cells [1 , 30 ]. Other studies show that nTREG are not contained exclusively within CD25-expressing CD4+ T cells, as nTREG markers such as CD103, glucocorticoid-induced TNF receptor, CTLA-4, and Foxp3 are also found in the CD45RBLOWCD25– subpopulation and are absent in the CD25–CD45RBHIGH subset [1 , 31 ]. Zelenay et al. [32 ] has shown that freshly isolated CD4+CD45RBLOW CD25–Foxp3+ T cells, albeit not directly suppressive ex vivo, constitute a reservoir of committed TREG cells, which regain CD25 expression upon homeostatic expansion. Furthermore, Fontenot et al. [23 ] demonstrated clearly, by means of mice containing a GFP-Foxp3 knock-in allele, that CD4+Foxp3+CD25– (largely CD45RBLOW) and CD4+Foxp3+CD25+ possess an identical, suppressive function in vitro and display different gene expression signatures as CD4+Foxp3–CD25+ T cells (largely CD45RBHIGH). Thus, although the thymic origin of Foxp3+CD4+ nTREG cells is unquestioned, our results also point to another nonmutually exclusive possibility, whereby TGF-β1 may actually serve as TREG-differentiating factor for distinct lineage(s) of TREG-like cells found within this subset of CD4+Foxp3–CD25–. In this manner, extrinsic signals such as TGF-β1 could possibly favor the terminal differentiation of pre-existing, non-CD25+ thymically derived, TREG-like cells in the periphery and contribute to the peripheral development of those cells.

Our results illustrate that TGF-β1-induced CD4+Foxp3+ T cells, generated from CD25–CD45RBLOW precursor T cells, have increased secretion of IL-10 and little or no TGF-β1 or IL-4 production upon TCR re-engagement, and the suppressor activity is abrogated with IL-10 neutralization in vitro but not IL-4. Chen et al. [10 ] has demonstrated that IL-10 fails to induce Foxp3 in CD4+CD25– T cells in vitro, indicating that IL-10 is not involved directly in the induction of the TREG phenotype. The major inhibitory role of IL-10 is to act on APC function to decrease costimulation and secretion of proinflammatory cytokines [33 , 34 ]. As the action of IL-10 on activated T cells causes re-expression of TGF-β receptor II (TGF-βRII) and in turn, promotes TGF-β1 signaling, it is possible that IL-10 potentiates TGF-β1 effects [35 ]. TGF-β1-induced Foxp3 can down-regulate Smad7 expression in CD4+ T cells by a direct effect on smad7 promoter activity, thereby down-modulating the key, negative pathway in TGF-β1 signaling [11 ]. Thus, iTREG cells could themselves be more refractive to TGF-β1-inhibitory effects and simultaneously increase the susceptibility of other T cells to this cytokine by secretion of IL-10.

TGF-β1 has also been shown to promote the development, expansion, or effector function of Foxp3+CD4+ nTREG cells, and the apparent expression of a latent, membrane-bound form of TGF-β1 on nTREG cells isolated from inflamed tissues has been at the root of this premise [24 , 36 , 37 ]. CD4+CD25+ T cells from mice expressing a dominant-negative form of TGF-βRII failed to expand in vivo and to suppress dextran sulfate sodium-induced colitis [38 ]. We recently showed that nTREG cells from wild-type or neonatal TGF-β1–/– mice are potently suppressive in vitro and can equally suppress the incidence and severity of T cell-induced IBD [39 ]. In this report, we show that exposure of nTREG cells to exogenous TGF-β1 in vitro did not induce the growth of nTREG cells in the presence or absence of IL-2, an established critical signal for the induction of nTREG cell function in vitro and in vivo. Our results also show that the suppressive activity was not affected when freshly isolated CD4+CD25+ nTREG cells were activated in the presence or absence of exogenous TGF-β1 or anti-TGF-β1-blocking antibody, suggesting that autocrine or paracrine sources of TGF-β1 do not modulate nTREG cell effector function in vitro. This is consistent with the observation that TGF-β-resistant Smad3–/– nTREG cells are equivalent to wild-type nTREG cells in their capacity to suppress IBD [39 ]. However, our results show that Foxp3 gene and protein expression in nTREG cells are increased modestly but significantly or maintained following TGF-β1 priming, indicating that TGF-β1 may be a stabilizer of Foxp3 expression and ultimately, sustaining nTREG peripheral homeostasis. However, this observation suggests that in the absence of TGF-β1, Foxp3 mRNA levels are not maintained and result in a temporal decline in Foxp3 activity in wild-type nTREG cells. Thus, TGF-β1 would not necessarily be inducing Foxp3 transcription in nTREG cells but rather, maintaining Foxp3 mRNA expression levels. This observation is consistent with a recently published study by Marie et al. [25 ], showing that antibody-mediated TGF-β1 neutralization or TGF-β1 gene deletion in mice results in a substantial decline of Foxp3 levels in peripheral nTREG cells, suggesting that TGF-β1 signaling in nTREG cells may promote Foxp3 expression. Thus, although TGF-β1 is dispensable for mediating nTREG activity, long-term TGF-β1 signaling may be required to sustain regulatory networks by promoting the development of Foxp3+ TREG cells from antigen-experienced CD4+ T cells and to bolster Foxp3 expression in CD4+ nTREG cells.

In summary, the present study provides evidence that TGF-β1 plays an essential role in the generation of IL-10-producing, CD4+ TREG cells in the periphery via the induction of Foxp3 in a subset of antigen-experienced CD4+ T cells. In addition, TGF-β1 stabilizes Foxp3 expression in nTREG cells without affecting their expansion or effector function. Our results suggest that TGF-β1 sustain regulatory networks by positively modulating de novo Foxp3 expression and inducing the post-thymic development of TREG cells and maintaining nTREG cell peripheral homeostasis by bolstering Foxp3 expression. The concept that TREG cells can be generated in vitro from a large pool of antigen-experienced CD4+ T cells via TGF-β1 leads to the possibility that novel, therapeutic strategies may be developed to potentiate the development or function of these cells in the periphery for the treatment of autoimmune and other chronic inflammatory diseases. Alternatively, manipulation of the TGF-β1/Foxp3 axis may abrogate dominant, tolerogenic mechanisms and consequentially, promote immunity to pathogens and cancers.


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ACKNOWLEDGEMENTS
 
We acknowledge the financial support of the Canadian Institutes for Health Research (CIHR; MOP 67211), Canadian Foundation for Innovation (CFI), and Canadian Diabetes Association (CDA). M. P. is a recipient of the Masters Canadian Graduate Scholarship from CIHR. C. A. P. is the recipient of the Canada Research Chair in Regulatory Lymphocytes of the Immune System. We are grateful for the McGill Flow Cytometry Facilities for their effort in cell sorting. We also thank the excellent technical assistance provided by the animal care staff at the McGill Animal Center. We show our appreciation to Dr. Silvia M. Vidal for her assistance in completing this manuscript.

Received October 25, 2006; revised March 13, 2007; accepted March 30, 2007.


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