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. Author manuscript; available in PMC: 2013 May 1.
Published in final edited form as: J Immunol. 2012 Mar 28;188(9):4369–4375. doi: 10.4049/jimmunol.1102698

TGF-β converts apoptotic stimuli into the signal for Th9 differentiation

Mariko Takami *, Robert B Love †, Makio Iwashima *
PMCID: PMC3331903  NIHMSID: NIHMS361412  PMID: 22461692

Abstract

Naturally arising CD4+CD25+FoxP3+ regulatory T cells (nTregs) play an essential role in maintenance of immune homeostasis and peripheral tolerance. Previously, we reported that conventional CD4+ and CD8+ T cells undergo p53-induced CD28-dependent apoptosis (PICA) when stimulated with a combination of immobilized anti-CD3 and anti-CD28 antibodies while nTregs expand robustly under the same conditions, suggesting that there is a differential survival mechanism against PICA between conventional T cells and nTregs. Here, we demonstrate that TGF-β signaling is required for nTregs to survive PICA. Conversely, when an active form of exogenous TGF-β is present, conventional T cells become resistant to PICA and undergo robust expansion instead of apoptosis, with reduction of the pro-apoptotic protein Bim and FoxO3a. A substantial fraction of PICA-resisted T cells expressed IL-9 (TH9 cells). Moreover, the presence of IL-6 along with TGF-β led to generation of TH17 cells from conventional T cells. Together, the data demonstrate a novel role for TGF-β in the homeostasis of Tregs and effector T cell differentiation/ expansion.

Introduction

Naturally arising regulatory T cells (nTregs) develop in the thymus and are characterized by constitutive expression of CD25 and a transcription factor FoxP3 (1–3). FoxP3 plays critical roles in development and/or survival and functions of nTregs (2, 4–6) as depicted by severe autoimmune disorders caused by mutation in the foxp3 gene both in humans and mice (7–9). nTregs comprise up to 5–10% of the CD4+ T cell population in the periphery and relative increase/decrease of Tregs is often associated with immune regulation disorders (1). Thus, mechanisms of maintenance of the balance between nTregs and non-Tregs (conventional T cells) could play a significant role in the regulation of immunity against self- and non-self antigens.

We demonstrated previously that nTregs survive and expand when stimulated with immobilized anti-CD3 and anti-CD28 antibodies (by coating onto plastic plates) with the added presence of IL-2, while non-Treg T cells undergo apoptosis (10). Unlike classical AICD, this form of apoptosis was p53-dependent and requires engagement of CD28, and was hence named p53-induced CD28-dependent T cell apoptosis (PICA). Unlike conventional T cells, nTregs are resistant to PICA. When stimulated under the same conditions, Foxp3+ Tregs expanded more robustly than that seen with a more commonly used bead-based stimulation method and expanded over 7000 fold within 10 days. The data suggested that PICA might play a role in immune regulation by controlling the balance between nTregs and conventional T cells. The data also provided a potential explanation for previous observations on p53-deficient mice that exhibit earlier onset and exacerbated disease state in experimental autoimmune arthritis and other autoimmune disease models (11–13).

To determine the mechanism by which nTregs withstand PICA, we analyzed the role of transforming growth factor-β (TGF-β). TGF-β is a pleiotropic cytokine that is involved in various T cell responses including promotion of Foxp3+ iTreg induction and mediation of suppressive functions of Tregs, and is expressed by nTregs on the cell surface upon TCR activation (14–18). Here, we demonstrate that TGF-β signaling is required for survival of nTregs against PICA and TGF-β can render conventional T cells resistant to PICA without induction of Foxp3 expression. Strikingly, conventional T cells treated with TGF-β not only survived PICA, but differentiated to IL-9 producing T cells (TH9) and addition of exogenous IL-6 convert conventional T cells into IL-17 producing T cells (TH17). Together, the data show TGF-β as a key determinant of fate of T cells when they receive PICA-inducing stimuli.

Material and Method

Mice

C57BL/6 and CD4dnTgfbr2 mice were purchased from Jackson Laboratory (Bar Harbor, ME). All mice were maintained under specific pathogen-free condition. All procedures were approved and monitored by Institutional Animal Care and Use Committee of Loyola University Chicago.

Flow cytometry

Fluorochrome-conjugated antibodies specific for Foxp3 (FJK-16s) and IL-17A (ebio17B7) were from eBioscience (San Diego, CA). Anti-CD4 (GK1.5) and anti-IL-9 (RM9A4) were from BioLegend (San Diego, CA). Annexin V, 7- aminoactinomycin D (7AAD), anti-CD25 (7D4), anti-Fas (Jo2) and anti-FasL (MFL3) were from BD Biosciences (San Jose, CA). Cell surface staining was performed on ice (30 min. unless stated otherwise) with appropriately conditioned antibodies. For Foxp3 staining, cells were fixed and permeabilized using eBioscience FOXP3 Staining Buffer Set as described by the manufacturer’s protocol. For intracellular cytokine staining, cells were harvested then restimulated with 50ng/ml phorbol 12-myristate 13-acetate (PMA) and 1µM ionomycin in the presence of monencin for 4 hours. Cells were then fixed and permialized for staining with anti-IL-17 or anti-IL-9 antibodies. Data was collected by a FACS Canto flow cytometer (BD Biosciences) or an Accuri’s C6 flow cytometer (Accuri Cytometers, Ann Arbor, MI) and analyzed using FlowJo software (TreeStar, Ashland, OR).

Cell preparation

Splenic CD4+ T cells were purified by depletion of non-CD4+ T cells by the panning method. Briefly, cells were labeled with anti-CD8 (3–155) antibody, washed and then allowed to adhere to plate-bound goat anti-mouse immunoglobulin. After 30min, non-adherent cells were collected. This crude fraction of CD4+ T cells were then labeled with fluorochrome-conjugated anti-CD4 and anti-CD25 antibodies and sorted into CD4+CD25− cells /CD4+CD25+ cells fractions by a FACS ARIA cell sorter (BD Biosciences). Sorted cells were rested overnight at 4 °C before then used for each experiment.

For plate bound anti-CD3/anti-CD28 antibodies stimulation, sorted CD4+CD25− or CD4+CD25+ (1.5×105) T cells were placed into 5 ml culture medium in 60 mm dishes that had been pre-coated overnight at room temperature with 2 ml of anti-CD3 (2C11, Biolegend) and anti-CD28 (37–51, Biolegend) antibodies (5µg/ml each) in 0.1M Borate buffer pH 8.5. The culture medium was RPMI 1640 medium supplemented with 10% FCS (Atlanta Biologicals), β-mercaptoethanol (50µM), glutamine, sodium pyruvate (1mM), and non-essential amino acids (Invitrogen Life Technologies, Grand Island, NY) in the presence of recombinant IL-2 (10ng/ml). To block TGF-β signaling, 5µg/ml anti-TGF-β1, 2, 3 antibody (1D11, R&D Systems, Minneapolis, MN ) or 10µM SB431542 (Sigma-Aldrich, St. Louis, MO) were added into culture medium. Recombinant human TGF-β (2.5ng/ml) (R&D Systems) was used for an active form of TGF-β. To block IL-4 signaling, 10% 11B11 hybridoma culture supernatant which contains anti-IL-4 was added into culture medium.

Western Blot

Cells were directly lysed in SDS sample buffer (2% SDS, 125mM DTT, 10% glycerol, 62.5mM Tris-HCl, pH 6.8). Cell lysates were boiled for 10min, then equal amount (based on cell count) were loaded onto SDS PAGE gels (8–15%). After gel electrophoresis, separated proteins were blotted onto PVDF membranes. The membranes were probed with following antibodies. Anti-phospho-Akt (Ser473), phospho-Erk1/2, phospho-FoxO1 (Ser256), FoxO1, phospho-FoxO3a (Ser253), FoxO3a, and Bim were from Cell Signaling Technology (Denvers, MA). Anti-Akt antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Erk1/2 antibody was from Millipore (Billerica, MA). Anti-β-actin was from Sigma-Aldrich. The membranes were further probed with anti-rabbit, anti-goat or anti-mouse HRP conjugated antibodies (Cell Signaling Technology). Signals were detected by the ECL system (GE Healthcare, Piscataway, NJ). Band intensity of scanned data from films was quantified using ImageJ software (National Institutes of Health).

Statistical analysis

Statistical significance was determined by 2-tailed Student T tests.

Results

Exogenous TGF-β renders CD4+CD25− T cells resistant to PICA

Previous reports showed that TGF-β is involved both in apoptosis (19–24) as well as in cell survival (25–27). Since TGF-β is differently expressed by nTregs and other T cells (28), we hypothesized that resistance to PICA by Tregs may be mediated in part by TGF-β. Our hypothesis predicted that inhibition of the TGF-β signaling pathway will abrogate PICA resistance by Tregs while addition of exogenous TGF-β will increase the frequency of live cells that survive PICA. Thus, we cultured purified CD4+CD25− T cells under PICA-inducing conditions in the presence or absence of exogenous TGF-β. After 3 days of culturing, we harvested cells and assessed their survival. As observed previously, cells that were stimulated by plate-bound anti-CD3/anti-CD28 antibodies underwent apoptotic cell death detected by an increase of Annexin V+ cells (Fig. 1A). When exogenous TGF-β was added to the culture, the frequency of apoptotic/dead cells decreased substantially. This change with TGF-β was due to expansion of the number of live cells and not due to a decrease of Annexin V+ cell numbers (Fig.1B). When CD4+CD25− T cells were stimulated with plate-bound anti-CD3/anti-CD28 antibodies, the final live cell number after 3 days was about the same as the starting sample (1.2 fold increase). In contrast, the Annexin V− cell number increased by 2.8 fold when CD4+CD25− cells were stimulated with plate-bound anti-CD3/anti-CD28 antibodies in the presence of TGF-β. These data show that TGF-β renders CD4+ CD25− T cells resistant to PICA and allows them to expand.

Fig. 1. Effect of TGF-β on PICA by CD4+CD25− T cells.

Fig. 1

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(A, B) CD4+CD25− T cells isolated from the spleen of C57BL/6 mice were stimulated with plate-bound anti-CD3/anti-CD28 antibodies in the presence or absence of recombinant TGF-β in media supplemented with IL-2. (A) Cells were harvested at day 3, stained with Annexin V, and analyzed by flow cytometry. (B) Total number of live cells was determined when cells were harvested at day 3 by Trypan Blue uptake. (C) CD4+CD25− T cells isolated from the spleen of C57BL/6 were stimulated with either plate-bound anti-CD3/anti-CD28 antibodies or plate-bound anti-CD3 and soluble anti-CD28 antibodies in the presence or absence of TGF-β in media supplemented with IL-2. Cells harvested at day 3 were stained for CD4 and Foxp3, and analyzed by flow cytometry. The data are representative of three independent experiments. ** p<0.01.

It is well established that TGF-β can induce differentiation of naïve CD4+ T cells into Foxp3+ inducible Tregs (iTregs)(16, 17). Thus, the survival of CD4+CD25− T cells observed with exogenous TGF-β may have been due to conversion of CD4+CD25− T cells to Foxp3+ iTregs. To test this possibility, we stimulated sorted CD4+CD25− T cells with plate-bound anti-CD3 plus either soluble or plate-bound anti-CD28 antibodies with the culture medium conditioned for induction of iTregs (including IL-2)(29). After 3 days of stimulation, expression of Foxp3 by expanded cells was examined by flow cytometry. When stimulated by plate-bound anti-CD3 and soluble anti-CD28 antibodies in the presence of TGF-β, a significant proportion of cells (37.3%) expressed Foxp3 (Fig. 1C). In contrast, only 5.3% of cells expanded with both the anti-CD3 and anti-CD28 antibodies being plate-bound expressed Foxp3. The level of Foxp3+ cells from plate-bound anti-CD28-stimulated cells was comparable to those stimulated without TGF-β. Together, the data show that resistance of CD4+CD25− T cells against PICA by TGF-β is due to anti-apoptotic responses of CD4+CD25− T cells and is not caused by enhanced induction of iTregs.

TGF-β receptor signaling is required for survival of CD4+CD25+ Tregs against PICA

The data presented above showed TGF-β may also play a role in survival of nTregs against PICA since CD4+CD25+ nTregs but not other T cell populations express TGF-β on their cell surface (14). Thus, we determined if TGF-β receptor signaling is required for survival of nTregs. To inhibit TGF-β receptor signaling in nTregs, we used a TGF-β super-family type I receptor kinase inhibitor (SB431542) or TGF-β neutralizing antibody (anti-TGF-β-1,2,3 antibody). Purified CD4+CD25+ nTregs were stimulated by plate bound anti-CD3/anti-CD28 antibodies in the presence of SB431542 or anti-TGF-β neutralizing antibody. After three days of stimulation, cells were harvested and analyzed by flow cytometry. CD4+CD25+ nTregs expanded ~2 fold compared to the starting cell number. When SB431542 was added, cell growth was substantially blocked and the cell number decreased approximately 5 fold. Similarly, when CD4+CD25+ Tregs were treated with anti-TGF-β antibody, live cell number decreased substantially compared to the starting number (Fig. 2A). Flow cytometric analysis showed that this decrease in cell numbers corresponds to an increase in Annexin V+ apoptotic/dead cell frequency (Fig. 2B).

Fig. 2. Effect of TGF-β signaling blockade on PICA of CD4+CD25+ Tregs.

Fig. 2

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(A, B) CD4+CD25+ Tregs isolated from the spleen of C57BL/6 mice were stimulated with plate-bound anti-CD3/anti-CD28 antibodies in the presence or absence of SB431542 (TGF-β super-family type I receptor kinase inhibitor) or anti-TGF-β antibody in media supplemented with IL-2. (A) Cells were harvested at day 3 and total number of live cells was determined. (B) At day 3, cells were stained with anti-Annexin V antibody and analyzed by flow cytometry. (C, D) CD4+CD25+ Tregs isolated from the spleen of dnTgfbr2 mice or wild type littermate were stimulated with plate-bound anti-CD3/anti-CD28 antibodies. (C) Cells were harvested at day 3 and total number of live cells was determined. (D) At day 3, cells were stained for Annexin V and analyzed by flow cytometry. The data are representative of three independent experiments. ** p<0.01.

To further confirm these results, we examined PICA resistance by CD4+CD25+ Tregs isolated from transgenic mice expressing a dominant-negative form of TGF-β receptor type II under the control of mouse CD4 promoter (CD4dnTgfbr2). These mice have a normal level of Foxp3+CD4+CD25+ nTregs (~8 weeks old) although TGF-β receptor signaling is substantially blocked in T cells (30). We isolated splenic CD4+CD25+ nTregs from CD4dnTgfbr2 mice or their wild type littermate control mice and stimulated them with plate bound anti-CD3/anti-CD28 antibodies in the presence of IL-2. After 3 days of culture, we harvested cells and assessed their survival (Fig. 2C). While the cell number of wild type littermate nTregs increased from day 0, numbers of CD4dnTgfbr2 nTregs were less than 10% of the control and decreased compared to the starting sample cell number. As observed with the chemical inhibitor and blocking antibody, the frequency of AnnexinV+ cells was about 2 fold higher in CD4dnTgfbr2 T cell culture compared to that of the littermate control (Fig. 2D). Together, the data show that TGF-β is required for survival of nTregs against PICA. Since we did not add exogenous TGF-β to the culture, the data strongly suggest that CD4+CD25+ Tregs provide TGF-β in an autocrine manner to maintain nTregs resistance against PICA.

TGF-β signaling reduces expression of Bim by activated CD4+CD25− T cells and CD4+CD25− Tregs

Previously, we demonstrated that PICA requires expression of Bim and Fas/FasL, which are known molecules for apoptosis by T cells (10). Since TGF-β rescued CD4+CD25− T cells from PICA, we determined if addition of exogenous TGF-β reduces expression of Bim and/or Fas ligand by CD4+CD25− T cells when stimulated by plate-bound anti-CD3/anti-CD28 antibodies (Fig. 3A). Unstimulated CD4+CD25− T cells expressed two forms (L and EL isoforms) of Bim at a low level. When stimulated with anti-CD3/anti-CD28 antibodies, CD4+CD25− T cells expressed both forms of Bim at a level clearly higher than that seen in unstimulated T cells. Stimulated but also TGF-β treated T cells, on the other hand, showed a markedly reduced level of Bim protein expression, even lower than that in unstimulated T cells (EL isoform). In contrast to Bim expression, TGF-β treatment caused a mild reduction in expression of FasL by CD4+CD25− T cells (Fig. 3B) while expression of Fas did not differ between TGF-β treated or untreated samples (Supplemental Fig. 1A). Together, the data clearly show that TGF-β suppresses expression of molecules required for apoptosis, particularly Bim, by CD4+CD25− cells stimulated by PICA-inducing conditions.

Fig. 3. Effect of TGF-β on Bim expression by CD4+CD25− T cells and CD4+CD25+ Tregs.

Fig. 3

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(A, B) CD4+CD25− T cells isolated from the spleen of C57BL/6 mice were stimulated with plate-bound anti-CD3/anti-CD28 antibodies in the presence or absence of exogenous TGF-β in media supplemented with IL-2. (A) Cells were harvested at day 3. Cells were directly lysed into SDS sample buffer and subjected for Western blot analysis using anti- Bim and anti-β actin (for loading control) antibodies. EL and L forms are indicated. The relative expression level of Bim was determined by setting the band intensity of Day 0 sample as 1 using Image J software. Loading amount of each sample was normalized using the levels of β-actin expression. (B) Cells harvested at day 3 were stained for FasL expression and analyzed by flow cytometry. (C, D) CD4+CD25+ Tregs isolated from the spleen of C57BL/6 mice were stimulated with plate-bound anti-CD3/anti-CD28 antibodies in the presence or absence of SB431542 (TGF-β super-family type I receptor kinase inhibitor) in media supplemented with IL-2 for 2 days. (C) Cells were harvested and were directly lysed in SDS sample buffer and subjected for Western blot analysis using anti-Bim or anti-β-actin (for loading control) antibodies. Band intensity was quantified as in A. (D) Cells were stained for FasL expression and analyzed by flow cytometry. The data are representative of two independent experiments.

We next determined if TGF-β signaling is required for nTregs resistance against PICA for the same reason as conventional T cells. If TGF-β receptor signaling in nTregs acts to keep Tregs resistant to PICA, it was predicted that nTregs treated with TGF-β signaling inhibitor would express higher levels of Bim. To test this, CD4+CD25+ Tregs were purified and stimulated with plate-bound anti-CD3/anti-CD28 antibodies for 2 days with or without SB431542. Cells were harvested and tested for the expression of Bim, Fas and FasL (cell surface) (Fig. 3C, D and Supplemental Fig. 1B). Stimulated Tregs expressed a lower level of Bim protein to unstimulated cells and showed a stark contrast to Bim expression by CD4+CD25− T cells as we reported previously (10). In contrast, Tregs that were stimulated in the presence of TGF-β signaling inhibitor showed a substantial upregulation of both isoforms of Bim expression (Fig. 3C). EL form is considered to play a major role in apoptosis by inducing release of apoptotic proteins Bax and Bak (31). Unlike Bim, Fas and FasL expression by stimulated CD4+CD25+ nTregs did not change with TGF-β treatment (Fig. 3D and Supplemental Fig. 1B). Taken together with the data from studies with CD4+CD25− T cells, the data demonstrate that TGF-β suppresses Bim protein expression under PICA inducing conditions and blocks apoptosis.

TGF-β promotes differentiation of TH9 cells under PICA-inducing condition

TGF-β is not only involved in iTreg differentiation but also for other helper T cell subset differentiations, such as TH9 or TH17 (32, 33). Since TGF-β rescued CD4+CD25− T cells from PICA without inducing Foxp3+ Tregs, we determined whether cells survived PICA in the presence of TGF-β differentiated into other effector T cell subsets. To address this question, we stimulated purified CD4+CD25− T cells with plate-bound anti-CD3 plus either soluble or plate-bound anti-CD28 antibodies in the presence or absence of TGF-β. After 3 days of stimulation, cells expressing IL-9 or IL-17 were assessed by intracellular cytokine staining. CD4+CD25− T cells stimulated by plate-bound anti-CD3 plus anti-CD28 without TGF-β did not express IL-9, but a significant portion of the cells stimulated by the same manner in the presence of TGF-β expressed IL-9 (14%) (Fig. 4A). Culture supernatant from cells stimulated with plate-bound antibodies and TGF-β showed a substantial increase in IL-9 compared to the samples from cells stimulated without TGF-β (Fig. 4B). Actual cell number producing IL-9 also increased significantly with TGF-β (Fig. 4C), showing that TGF-β induced differentiation and/or expansion of a group of CD4+CD25− T cells into TH9 cells under PICA-inducing conditions. In contrast, CD4+CD25− T cells stimulated by plate-bound anti-CD3 plus soluble anti-CD28 express a significantly lower level of IL-9 with TGF-β (Fig. 4B). No increase in TH17 cells was observed under either of these conditions (Fig. 4A, C).

Fig. 4. Effect of TGF-β and IL-6 on PICA by CD4+CD25− T cells.

Fig. 4

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CD4+CD25− T cells isolated from the spleen of C57BL/6 mice were stimulated with either plate-bound anti-CD3/anti-CD28 antibodies or plate-bound anti-CD3 and soluble anti-CD28 antibodies in the presence or absence of TGF-β, IL-6 or anti-IL-4 antibody in media supplemented with IL-2. (A) Cells harvested at day 3 were re-stimulated with PMA+ Ionomycin for 4 hours in the presence of monensin and stained for IL-9 and IL-17 expression, then analyzed by flow cytometry. (B) Culture supernatant was collected at day 3. IL-4, IL-17 and IL-9 productions were determined by ELISA. (C) Total number of IL-17+ cells and IL-9+ cells were determined based on the data obtained in (A). The data are representative of three independent experiments. ** p<0.01

IL-4 plays a pivotal role in generation of TH9 cells (34). Indeed, addition of anti-IL-4 antibody abrogated induction of TH9 cells by TGF-β and plate-bound anti-CD3/anti-CD28 antibodies (Fig. 4A). Whereas IL-4 producing cells were not detectable by cytokine staining after three days of stimulation (data not shown), culture supernatants from cells stimulated with plate-bound anti-CD3/anti-CD28 antibodies contained a clearly detectable level of IL-4 either in the presence or absence of TGF-β (Fig. 4B). TGF-β abrogated IL-4 production from cells stimulated with plate-bound anti-CD3 and soluble anti-CD28 while no decrease of IL-4 was observed for cells stimulated with plate-bound anti-CD3/anti-CD28 antibodies (Fig. 4B).

T cells from BALB/c mice showed the same responses when stimulated by plate-bound anti-CD3 and anti-CD28 antibodies (Supplemental Fig.2A). TGF-β rescued CD4+CD25− T cells from PICA and induced TH9 differentiation. A difference was found when T cells were stimulated by soluble anti-CD28 antibody. Unlike T cells from C57.BL/6 mice, a substantial amount of BALB/c mouse T cells developed into TH9 cells after simulation by soluble anti-CD28 antibodies in the presence of TGF-β. This is likely due to a high level of IL-4 production with soluble anti-CD28 antibody stimulation (Supplemental Fig. 2B). While IL-4 expression by C57.BL/6 T cells was abrogated by TGF-β when anti-CD28 antibody was provided in a soluble form (Fig.3B), TGF-β increased IL-4 production by BALB/c T cells stimulated under the same conditions. The data are in agreement with those observed with C57.BL/6 mouse T cells and show the significance of IL-4 in TH9 generation by plate-bound anti-CD3/anti-CD28 antibodies plus TGF-β. Together, the data suggest that T cells stimulated with plate-bound anti-CD3/anti-CD28 antibodies differentiate into TH9 in part due to the presence of autocrine IL-4.

In contrast to the effect on IL-4, TGF-β suppressed production of IFN-γ regardless of how anti-CD28 antibodies were provided (supplemental Fig.3). No differentiation of IFN-γ+ cells were observed from cells resisted PICA by TGF-β addition. TGF-β also suppressed expression of IFN-γ by BALB/c T cells (Supplemental Fig. 2A,B).

IL-6 plays a critical role in regulating the balance between TH17 and Tregs and induces TH17 along with TGF-β (35). Since T cells do not produce IL-6, we tested if exogenous IL-6 changes the fate of CD4+CD25− T cells under PICA-inducing conditions. When CD4+CD25− T cells were stimulated in the presence of TGF-β and IL-6, the frequency of IL-17+ cells showed a modest increase over the TGF-β only control groups (Fig. 4A). The increase was higher for the plate-bound anti-CD28 antibody stimulation than soluble anti-CD28 stimulation (3.7% over 1.4%). In addition, we observed a significant increase in the amount of IL-17 detected in the culture supernatant for cells stimulated with plate-bound anti-CD28 antibody than with soluble anti-CD28 controls (Fig. 4B). Addition of IL-6 increased the total cell number and IL-17+ cells (Fig. 4C, Supplemental Fig. 4). Therefore, a marked increase in IL-17 production by plate-bound anti-CD28 antibody simulated T cells may be due to an increase in total live cell numbers in the presence of exogenous IL-6. Together, the data show that TGF-β promotes differentiation and/or expansion of TH17 cells in the presence of IL-6 when T cells are stimulated by plate-bound anti-CD3 and anti-CD28 antibodies. IL-6 also increased the secreted IL-9 by T cells stimulated with plate bound anti-CD28 in the presence of TGF-β although IL-9+ cells were at the level comparable to cells stimulated without TGF-β suggesting an increase in the level of IL-9 production per individual cell (Fig 4B, C).

Signaling differences between plate-bound and soluble anti-CD28 antibody stimulation in T cells treated with TGF-β

Our data presented here demonstrate fundamental differences in T cell activation when CD28 is engaged by the plate-bound or soluble form of anti-CD28. To determine the underling mechanism that controls apoptosis or cell survival/differentiation, signaling processes involved in Bim expression were compared between soluble anti-CD28 and plate-bound anti-CD28 antibody stimulated T cells. CD4+CD25− T cells were purified from total splenocytes and stimulated with plate-bound anti-CD3 plus soluble- or plate-bound anti-CD28 antibodies in the presence or absence of TGF-β. After one day of stimulation, total cell lysates were prepared and analyzed by Western blot (Fig. 5).

Fig. 5. TGF-β effect on the FoxO3a-related signaling processes in plate-bound and soluble anti-CD28 antibody stimulated T cells.

Fig. 5

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Splenic CD4+CD25− T cells from C57BL/6 mice were stimulated by ant-CD3 antibodies (plate-bound) either with plate-bound anti-CD28 (plate) or soluble anti-CD28 antibodies (soluble) in the presence (+) or absence (−) of TGF-β. Cells were harvested after 1 day and were directly lysed by SDS sample buffer and subjected for Western blot analysis using anti-phospho-Akt (p-Akt), anti-Akt, anti-phospho-FoxO3a (p-FoxO3a), anti-FoxO3a, anti-phospho-Erk1/2 (p-Erk1/2), anti-Erk1/2, anti-phospho-FoxO1 (p-FoxO1), anti-FoxO1, and anti-β-actin (for loading control) antibodies. Relative intensity of each band (shown below each lane) was determined against day 0 data after standardization for the loading amount using β-actin as the control.

We determined if plate-bound and soluble anti-CD28 antibody stimulation differs in inducing the signaling process of the Akt/FoxO3a axis since previous studies on cytokine deprivation-induced apoptosis of T cells showed that FoxO3a, a Forkhead transcription family member, induced expression of Bim while Akt suppressed Bim expression via inhibitory phosphorylation of FoxO3a (36, 37). Expression of FoxO3a showed a substantial increase in plate-bound antibody stimulated T cells over unstimulated or soluble anti-CD28 stimulated samples. Addition of TGF-β, which renders T cells resistant to PICA, caused a marked decrease of FoxO3a expression by plate-bound anti-CD28 antibody stimulated samples while no obvious change was observed for soluble anti-CD28 stimulated T cells. Inhibitory phosphorylation of FoxO3a at Ser 253 was not substantially changed by TGF-β either in plate-bound or soluble anti-CD28 antibody stimulated samples.

Expression of Akt, a negative regulator of FoxO3a, increased after soluble- and plate-bound anti-CD28 antibody stimulation. TGF-β did not cause significant changes in Akt protein levels. However, TGF-β upregulated the level of activating phosphorylation of Akt at residue 473 only in plate-bound anit-CD28 stimulated samples, suggesting that TGF-β inhibited FoxO3a expression in part by activation of Akt.

FoxO1 is another Forkhead transcription factor that is regulated by Akt (38). Expression and phosphorylation of FoxO1 was markedly induced by TGF-β in cells stimulated by plate-bound and soluble anti-CD28 antibodies to a comparable extent. Therefore, FoxO1 expression is a downstream target of TGF-β but not linked to plate-bound anti-CD28 antibody stimulation or PICA. The data suggest that expression of FoxO3a is one of the unique downstream signaling events that differs between plate-bound and soluble anti-CD28 antibody stimulation and is potentially involved in PICA. ERK1/2 is known to down regulate FoxO3a via MDM-mediated degradation (39). A mild increase in the level of activated ERK was observed in plate-bound anti-CD28 antibody stimulated samples compared to unstimulated T cells or T cells stimulated by soluble anti-CD28 antibodies. However, TGF-β did not enhance ERK activation or expression. Thus, ERK activity did not correlate with the level of FoxO3a expression. Together, the data show a correlative link between PICA and expression of FoxO3a, which is negatively regulated by TGF-β under PICA-inducing conditions.

Discussion

In this study, we demonstrated that TGF-β signaling renders CD4+CD25− T cells resistant to PICA and is required for survival and expansion by nTregs ex vivo when stimulated by plate-bound anti-CD3/anti-CD28 antibodies. TGF-β rendered CD4+CD25− T cells resistant to PICA and differentiated them to TH9 or TH17 cells, depending on the presence of IL-4 and IL-6, respectively. These data suggest that TGF-β signaling plays another role in controlling numbers of conventional and regulatory CD4+ T cells during antigen stimulation.

Our data show that TGF-β reduced expression of Bim and FoxO3a. Recent reports showed that TGF-β regulates expression of Bim in non-lymphoid cells and mitogen- and stress-activated protein kinase-1 (MSK-1) played a critical role in the anti-apoptotic function of TGF-β (40, 41). Currently, it is not known if MSK1 plays any role in T cell activation or death but investigations to determine the role, if any, of MSK1 in PICA are ongoing. It should also be noted that reduction of FoxO3a expression by TGF-β in T cells has not been reported. The data presented here is correlative evidence, and whether or not the reduction of FoxO3a by TGF-β plays a functional role in PICA is currently under investigation.

Though the underlying mechanism is not clear, the data also demonstrate that induction of FoxO3a by anti-CD28 antibody immobilized on the plastic surface, but not by soluble anti-CD28 antibody. This FoxO3a expression was reduced by TGF-β. A recent report showed that TGF-β causes inactivation of FoxO3a and reduction of Bim expression in a PI3K dependent manner in mesangial cells (42). In this study, it was shown that TGF-β caused activation of Akt and inactivating phosphorylation/degrdation of FoxO3a. Our data also show that addition of TGF-β causes reduction of FoxO3a and a mild but reproducible increase in Akt phosphorylation, suggesting that reduction of FoxO3a by TGF-β is mediated by activation of the PI3K/Akt pathway. Although CD28 engagement is known to provoke PI3K signaling and its downstream process involving Akt and mTOR (43), multiple residues in the cytoplasmic region of CD28 are known to play functional roles other than activation of PI3K (44, 45). Therefore, it is possible that differential signaling is provided to the PI3K/Akt signaling pathway from CD28 when stimulation is provided by plate-bound or by soluble anti-CD28 antibody.

TGF-β promotes nTreg cell survival during negative selection where Bim plays a critical role (46). Though thymic negative selection does not require p53, the data suggest that TGF-β signaling can be anti-apoptotic under certain conditions in connection to Bim expression. Though PICA is an ex vivo event established by use of anti-receptor antibodies, our previous work showed that PICA can be induced by extended stimulation from allogeneic dendritic cells in vitro (10). Therefore, it will be interesting to see if TGF-β rescues conventional T cells from PICA in vivo. PICA may be utilized by chronically infecting agents and/or tumor cells that establish their survival by expansion of nTregs. Conversely, since TGF-β is critical for the survival of nTregs against PICA, inhibition of TGF-β signaling could lead to loss of nTregs and abrogation of suppression and/or tolerance.

Complex and intricate regulation of Bim by TGF-β potentially reflects what has been reported on the role of miR-25 (47). In both CD4+CD25− and CD4+CD25+ T cells, Bim protein level is regulated negatively by TGF-β. Notably, recent reports showed that miR-25, which regulates Bim protein synthesis and promotes anti-apoptotic responses, was much reduced in Tregs from patients with multiple sclerosis (48, 49). Loss of this miRNA could lead to an increase in Bim protein expression by Tregs and their death, hence less effective maintenance of self-tolerance. We are currently investigating the potential role of this miR-25 in Bim expression in Tregs under PICA-inducing conditions.

Data presented here also showed that TGF-β promotes differentiation of CD4+CD25− T cells that receive PICA-inducing stimuli. Currently, the molecular mechanism underlying this phenomenon is unknown. TGF-β may be simply providing signaling required for survival of T cells and IL-4 provides differentiation signaling for TH9. Similarly, TGF-β might allow T cells to survive PICA so that exogenous IL-6 can induce differentiation of surviving cells into TH17 cells. Alternatively, TGF-β is also providing signaling required for initiation/establishment of differentiation. In either case, the plasticity of T cell differentiation provided by TGF-β and PICA-inducing stimulation could play significant roles in determining the outcomes of in vivo immune responses. Further studies are needed to elucidate the potential roles of PICA and TGF-β under physiological and pathological conditions.

Supplementary Material

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Acknowledgement

The authors thank Drs. Phong Le, Pandelakis Koni, Kathleen Jaeger, and Shauna Marvin for critical reading of the manuscript, Dr. Yoichi Seki for suggestions, and Patricia Simms (Loyola FACS core facility) for cell sorting.

This work was supported by NIH R01 AI055022 (MI) and Van Kampen cardiovascular research fund (RBL).

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Supplementary Materials

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NIHMS361412-supplement-1.doc (60KB, doc)
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NIHMS361412-supplement-3.tif (15.3MB, tif)
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