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TGF-1 Plays an Important Role in the Mechanism of CD4⁺CD25⁺ Regulatory T Cell Activity in Both Humans and Mice

Kazuhiko Nakamura^*,, Atsushi Kitani^*, Ivan Fuss^*, Aasta Pedersen^*, Naohiko Harada, Hajime Nawata and Warren Strober^1,*

^* Mucosal Immunity Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan

	Abstract

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In previous studies, we have shown that murine CD4⁺CD25⁺ regulatory T cells produce high levels of TGF-1 in a cell surface and/or secreted form, and blockade of such TGF-1 by anti-TGF- curtails the ability of these cells to suppress CD25^- T cell proliferation and B cell Ig production in in vitro suppressor assays. In further support for the role of TGF-1 in suppression by CD4⁺CD25⁺ T cells, we show in this study that another TGF-1-blocking molecule, recombinant latency-associated peptide of TGF-1 (rLAP), also reverses suppression by mouse CD4⁺CD25⁺ T cells as well as their human counterparts, CD4⁺CD25^high T cells. In addition, we show that CD25^- T cells exposed to CD4⁺CD25⁺ T cells in vitro manifest activation of Smad-2 and induction of CD103, the latter a TGF--inducible surface integrin. In further studies, we show that while CD4⁺CD25⁺ T cells from TGF-1-deficient mice can suppress CD25^- T cell proliferation in vitro, these cells do not protect recipient mice from colitis in the SCID transfer model in vivo, and, in addition, CD4⁺LAP⁺, but not CD4⁺LAP^- T cells from normal mice protect recipient mice from colitis in this model. Together, these studies demonstrate that TGF-1 produced by CD4⁺CD25⁺ T cells is involved in the suppressor activity of these cells, particularly in their ability to regulate intestinal inflammation.

	Introduction

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There is accumulating evidence that the generation of regulatory T cells is necessary to maintain self-tolerance and prevent the onset of autoimmune disease. One such regulatory cell is a CD4⁺CD25⁺ T cell population that comprises 5–10% of the total peripheral CD4⁺ T cells in normal adult mice and expresses surface CD25 (IL-2R) before activation (1). In certain strains of mice, elimination of this CD4⁺CD25⁺ regulatory T cell population by early thymectomy results in various autoimmune diseases, which, in turn, are preventable by its restoration by adaptive transfer (2). Similarly, transfer of CD25⁺-depleted splenocytes into nude mice results in various autoimmune diseases that are prevented by cotransfer of CD4⁺CD25⁺ T cells (2). In more recent studies, CD4⁺CD25⁺ regulatory T cells very similar to the murine regulatory cell have also been shown to be present in humans (3, 4, 5, 6, 7).

In a series of in vitro studies, it has been shown that murine CD4⁺CD25⁺ T cells are relatively unresponsive to TCR-mediated stimulation and suppress T cell proliferation of cocultured CD4⁺CD25^- T cells (8, 9). In addition, it has been shown that such suppression acted via a cell-cell contact mechanism rather than through the secretion of suppressor cytokines such as TGF-1 or IL-10 (8, 9). However, in a previous report, we have shown that CD4⁺CD25⁺ T cells are in fact capable of producing TGF-1 either as a secreted or a cell surface protein, depending on the type and/or strength of TCR stimulation (10). Thus, when CD4⁺CD25⁺ T cells are stimulated with plate-bound anti-CD3 (under cross-linking conditions), they both secrete TGF-1 and also express cell surface-bound TGF-1; in contrast, when the same cells are stimulated by Ag plus APCs, they mainly express cell surface TGF-1 and secrete little, if any, of this cytokine. On this basis, it seems likely that under the stimulation conditions in which CD4⁺CD25⁺ T cell suppressor function is generally measured in vitro CD4⁺CD25⁺ T cells would express cell surface TGF-1, but not secrete TGF-1. Thus, we postulated that in vitro CD4⁺CD25⁺ T cell suppressor function is mediated by contact with cell surface TGF-1. In studies to support this concept, we showed that high concentrations of anti-TGF-1 inhibit the suppressor function of CD4⁺CD25⁺ T cells in in vitro coculture assays (10). In addition, other investigators reported that suppressor function of both mouse lymphocytes and human CD4⁺CD25⁺ thymocytes are at least partially reversed by neutralization of TGF- (11, 12).

Despite these findings, the involvement of TGF-1 in CD4⁺CD25⁺ T cell immunoregulatory function remains controversial because in previous studies neutralization of TGF- by anti-TGF- failed to reverse CD4⁺CD25⁺ suppressor function in vitro (8, 9). In addition, it has recently been shown that CD4⁺CD25⁺ T cells from normal mice could suppress the proliferation of cocultured CD4⁺CD25^- T cells from TGF-RII dominant-negative transgenic mice and Smad-3-deficient mice and, in addition, CD4⁺CD25⁺ T cells from TGF-1-deficient mice could suppress cocultured CD25^- T cells from normal mice, suggesting that TGF-1 is not necessary for suppressor function in in vitro coculture system (13). These contradictory studies did not, however, lead to an alternative suppressor mechanism, and led us to again examine the role of TGF-1 in CD4⁺CD25⁺ T cell regulatory function.

In the current series of studies, we showed first that in vitro suppressor function of both murine and human CD4⁺CD25⁺ regulatory T cells is inhibited by another TGF-1-blocking agent, recombinant latency-associated peptide of TGF-1 (rLAP),² in a dose-dependent fashion. We then showed that CD4⁺CD25⁺ T cells induce the expression of CD103 (_E integrin) and activated Smad-2 in cocultured CD25^- T cells, thus providing strong evidence that cell surface TGF-1 acts as a signaling molecule in the coculture situation. Moving then to in vivo studies, we showed that while CD4⁺CD25⁺ T cells from TGF-1-deficient mice do exhibit suppressor activity in vitro, such cells failed to prevent the development of colitis in the SCID transfer colitis model, indicating that cells from these mice do exhibit impaired suppressor function. In addition, these data show that CD4⁺CD25⁺ T cells exert suppressor activity in vivo by production of TGF-1 rather than by inducing other cells to do so. Finally, we showed that CD4⁺LAP⁺ T cells, but not CD4⁺LAP^- T cells, prevent colitis in the transfer model, showing that cell surface LAP could serve as a marker for regulatory T cells.

	Materials and Methods

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Mice

Specific pathogen-free, 8-wk-old female BALB/c mice were purchased from the National Cancer Institute (Frederick, MD). TGF-1-deficient mice (14) were a kind gift from J. Letterio (National Cancer Institute, National Institutes of Health, Bethesda, MD). Splenocytes were purified from TGF-1^+/+, TGF-1^+/-, and TGF-1^-/- mice at the age of 1–2 wk, as TGF-1^-/- mice die before age 3 wk due to systemic autoimmune inflammation. C57BL/6 Rag-2 knockout mice and C.B-17 SCID mice were purchased from Taconic Farms (Germantown, NY). Thy-1.1⁺ congenic BALB/c mice were a kind gift from J. Sprent (The Scripps Research Institute, La Jolla, CA). Animal use adhered to National Institutes of Health Animal Care Guidelines.

Reagents

Anti-mouse CD3 (145-2C11), anti-mouse CD28 (37.51), anti-mouse CTLA-4 (UC10-4F10-11), biotin-conjugated anti-mouse CD25 (7D4), FITC-conjugated anti-mouse CD4 (GK1.5 and RM4-5), PE-conjugated anti-mouse CD45RB (16A), biotin-conjugated anti-mouse IgG1, PE-conjugated streptavidin, CyChrome-conjugated streptavidin, FITC-conjugated anti-human CD4 (RPA-T4), PE-conjugated anti-human CD25 (M-A251), and 7-amino actinomycin D were purchased from BD PharMingen (San Diego, CA). Mouse anti-LAP (TGF-1) mAb (clone 27232.11, mouse IgG1) was a kind gift from R&D Systems (Minneapolis, MN). rLAP, anti-TGF- mAb (clone 1D11), biotin-conjugated goat anti-LAP Ab, and biotin-conjugated anti-LAP mAb (clone 27240) were purchased from R&D Systems. Streptavidin microbeads were purchased from Miltenyi Biotec (Auburn, CA). Anti-human CD3 mAb (OKT3) was purchased from Ortho Biotech (Raritan, NJ). Anti-phospho-Smad-2 (Ser⁴⁶⁵, Ser⁴⁶⁷) Ab was purchased from Upstate Biotechnology (Waltham, MA). Anti-Smad-2 mAb was purchased from Zymed Laboratories (South San Francisco, CA). Human rIL-2 was purchased from Life Technologies (Rockville, MD).

Cell purification

Mouse CD4⁺CD25⁺ and CD4⁺CD25^- T cells were purified, as described previously (10). Human CD4⁺CD25⁺ regulatory T cells were isolated from leukapheresis packs obtained from healthy donors after informed consent was given. In brief, PBMC isolated by Ficoll density-gradient centrifugation were passed over CD4⁺ T cell enrichment columns (R&D Systems) to obtain CD4⁺ T cells. The latter were then stained with FITC-conjugated anti-CD4 and PE-conjugated anti-CD25 and sorted by FACSVantage SE II (BD Biosciences, San Jose, CA) to obtain highly purified CD4⁺CD25^high and CD4⁺CD25^- T cell populations. The purity of the CD4⁺CD25^high and CD4⁺CD25^- T cells was >85 and >95%, respectively.

Flow cytometry

Splenocytes from TGF-1^+/+, TGF-1^+/-, and TGF-1^-/- mice were stimulated with soluble anti-CD3 (10 µg/ml) and rIL-2 (50 U/ml) for 3 days. Cells were stained with anti-LAP mAb (clone 27232.11, mouse IgG1); washed, biotin-conjugated anti-mouse IgG1; and washed. Cells were then incubated with FITC-conjugated anti-CD4 and PE-conjugated streptavidin and washed. For exclusion of dead cells, cells were stained with 7-amino actinomycin D before analysis.

Purified human CD4⁺CD25^high and CD25^- T cells were stimulated with anti-CD3 (1 µg/ml), irradiated APC, and rIL-2 (50 U/ml) for 5 days. Cells were stained with biotin-conjugated goat anti-LAP Ab, washed, and stained with FITC-conjugated anti-CD4 and PE-streptavidin. Dead cells were excluded by staining with 7-amino actinomycin D.

The analysis was performed on FACScan (BD Biosciences) flow cytometer with CellQuest II software (BD Biosciences). In some experiments, 7-amino actinomycin D staining was necessary to exclude nonspecific high background on dead cells.

Cell culture

Cell cultures of mouse CD4⁺CD25⁺ and CD4⁺CD25^- T cells for in vitro suppression assays using [³H]thymidine incorporation readouts were performed, as described previously (10); for this purpose, indicated cell numbers of CD4⁺CD25⁺ and CD4⁺CD25^- T cells were placed in wells of 96-well plates. In some studies, rLAP was added to the cell cultures at various indicated concentrations.

For human CD4⁺ T cells, 2.5 x 10⁴ CD4⁺CD25^- T cells with or without the same number of CD4⁺CD25^high T cells were cultured in 96-well plates and stimulated with 1 µg/ml anti-CD3 (OKT3) and 5 x 10⁴ irradiated syngeneic PBMC for 4 days at 37°C and pulsed with 1 µCi of [³H]thymidine for the last 6 h of culture. RPMI 1640 supplemented with 3% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10 mM HEPES (pH 7.0) was used as culture medium for human cells.

FACS analysis of CD103 expression on CD4⁺CD25⁺ and CD4⁺CD25^- T cells

CD4⁺CD25⁺ and CD4⁺CD25^- T cells were isolated from Thy-1.1⁺ congenic BALB/c mice. In parallel, CD4⁺CD25⁺ T cells were purified from normal (Thy-1.2⁺) BALB/c mice. Thy-1.1⁺ CD25^- T cells were stimulated in the presence or absence of Thy-1.2⁺ CD25⁺ T cells with soluble anti-CD3 (10 µg/ml), irradiated APC, and rIL-2 (50 U/ml) for 5 days. For positive control, Thy-1.1⁺ CD25^- T cells were stimulated in the presence of rTGF-1 (5 ng/ml). In parallel, Thy-1.1⁺ CD25⁺ T cells were stimulated for comparison. After cultivation, cells were stained with FITC-conjugated anti-Thy-1.1, PE-conjugated anti-CD103, or isotype-matched control Ab, and CyChrome-conjugated anti-CD4. Expression of CD103 on Thy-1.1⁺ CD4⁺ cells was analyzed by FACScan (BD Biosciences) flow cytometer.

Detection of Smad-2 phosphorylation

To detect induction of Smad-2 phosphorylation in CD4⁺CD25^- T cells cocultured with CD4⁺CD25⁺ T cells, murine CD4⁺ T cells were purified by CD4 purification column (R&D Systems), and then separated into CD4⁺CD25⁺ and CD4⁺CD25^- T cells using PE-conjugated anti-CD25 and anti-PE microbeads (CD4⁺CD25⁺ regulatory T cell isolation kit; Miltenyi Biotec). CD4⁺CD25⁺ or CD4⁺CD25^- T cells were then stimulated with soluble anti-CD3 (10 µg/ml) plus irradiated adherent APCs (non-CD4 fraction) for 3 h. The nonadherent CD4⁺ T cells were then collected, washed three times, and mixed with freshly isolated resting CD4⁺CD25^- T cells. The cell mixtures thus obtained were added back to the washed original wells, briefly spun down to ensure cell-cell contact, and incubated at 37°C for 90 min. Negative controls consisted of CD4⁺CD25^- T cells incubated with irradiated APCs in the absence of CD4⁺CD25⁺ or CD4⁺CD25^- T cells. After incubation, CD4⁺CD25^- T cells were negatively repurified with biotin non-CD4 T cell Ab mixture, followed by the treatment of PE anti-mouse CD25, and solubilized in lysis buffer (1% Nonidet P-40, 150 mM NaCl, 20 mM Tris, pH 7.5, 2 mM EDTA, 10 mM NaF, and 1 mM Na₃VO₄ with protease inhibitors). Finally, a Western blot was performed, as described previously (10), using anti-phospho-Smad-2 Ab to detect phosphorylated Smad-2 and anti-Smad-2 mAb to detect total Smad-2 protein (on the stripped membrane).

Transfer of wild-type and TGF-1-deficient CD4⁺CD25⁺ T cells with CD45RB^high T cells into Rag-2 knockout recipients

Splenocytes from C57BL/6 mice were stained with FITC-conjugated anti-mouse CD4 (RM4-5) and PE-conjugated anti-mouse CD45RB, after which CD4⁺CD45RB^high and CD4⁺CD45RB^low T cells were purified by FACS sorting. Wild-type and TGF-1-deficient CD4⁺CD25⁺ T cells were purified, as described previously (10). CD4⁺CD45RB^high T cells (3 x 10⁵) were injected i.p. into Rag-2-deficient mice, accompanied, in some recipients, by CD4⁺CD45RB^low T cells (1.5 x 10⁵) from normal C57BL/6 or CD4⁺CD25⁺ T cells (1.2 x 10⁵) from wild-type or TGF-1-deficient mice. After transfer, recipient mice were weighted weekly until they were euthanized for histopathologic analysis.

Transfer of LAP⁺ and LAP^-CD4⁺ T cells with CD45RB^high T cells into C.B-17 SCID recipients

Splenocytes from BALB/c mice were stained with FITC-conjugated anti-mouse CD4 and PE-conjugated anti-mouse CD45RB, after which CD4⁺CD45RB^high and CD4⁺CD45RB^low T cells were purified by FACS sorting. In parallel, CD4⁺ T cells stimulated with plate-bound anti-CD3 (10 µg/ml), soluble anti-CD28 (2 µg/ml), and rIL-2 (50 U/ml) for 3 days were collected and stained with FITC-conjugated anti-mouse CD4 (RM4-5) and anti-LAP mAb (27232.11), biotin-conjugated anti-mouse IgG1, and PE-conjugated streptavidin. CD4⁺LAP⁺ and CD4⁺LAP^- T cells were purified by FACS sorting. Dead cells were excluded by 7-amino actinomycin D staining. CD4⁺CD45RB^high T cells (3 x 10⁵) were injected i.p. in C.B-17 SCID mice, accompanied in some recipients by CD4⁺CD45RB^low (1.5 x 10⁵), CD4⁺LAP⁺ (1 x 10⁵), or CD4⁺LAP^- (1 x 10⁵) T cells. After transfer, recipient mice were weighted weekly until they were euthanized for histopathologic analysis.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
rLAP inhibits suppressor function of murine CD4⁺CD25⁺ regulatory T cells

To explore further the role of TGF- in the suppressor function of murine CD4⁺CD25⁺ T cells in vitro, we conducted suppressor arrays in the presence and absence of rLAP, a molecule that could block TGF- activity by capturing active TGF- or by blocking the association of latent TGF- with an activating molecule, such as thrombospondin-1 and _v6 integrin (15, 16, 17). Accordingly, we set up cultures in which CD4⁺CD25^- T cells were stimulated with soluble anti-CD3 and APC in the presence or absence of CD4⁺CD25⁺ T cells, as well as with various amounts of rLAP. As shown in Fig. 1, in the absence of rLAP, CD4⁺CD25^- T cells proliferated well in response to stimulation, while CD4⁺CD25⁺ T cells proliferated poorly. In addition, coculture of CD4⁺CD25^- T cells with CD4⁺CD25⁺ T cells resulted in profound suppression of CD4⁺CD25^- T cell proliferation. However, in the presence of rLAP, proliferation of CD4⁺CD25^- T cells was further increased and, more importantly, suppression by CD4⁺CD25⁺ T cells was significantly reversed in a dose-dependent fashion. Of note, reversal of suppression was obtained with much lower concentrations of rLAP than reversal by anti-TGF- (10). These results thus lend additional credence to the idea that such reversal involves a TGF-1-mediated mechanism. The fact that CD4⁺ CD25^- T cell proliferation increases in the presence of rLAP may be due to the fact that, as shown previously, CD4⁺CD25^- T cells do produce some TGF- when stimulated with a variety of conditions (10) or are being suppressed by TGF- known to be present in the FCS in the culture medium. Such augmentation of proliferation was previously seen when anti-TGF- was added to the cultures (10). Finally, it should be noted that while rLAP caused substantial inhibition of suppression by CD4⁺CD25⁺ T cells, such inhibition was not complete. Several possible explanations for this can be put forward, but we think the most cogent is that CD4⁺CD25⁺ T cells inhibit CD25^- T cells via a second mechanism as well under certain stimulation conditions (see discussion below).

View larger version (11K): [in this window] [in a new window]   FIGURE 1. rLAP reverses suppressor function of murine CD4+CD25+ T cells. Mouse CD4+CD25- T cells (4 x 104/well) were stimulated with 10 µg/ml soluble anti-CD3 and irradiated APC in the presence or absence of CD4+CD25+ T cells (3 x 104/well). Various concentrations of rLAP were added to the cell culture. •, CD4+CD25- T cell; , CD4+CD25- and CD4+CD25+ T cell; , CD4+CD25+ T cell. Representative results of three independent experiments are shown.

Recently, it was reported that human CD4⁺CD25⁺ T cells, comprising 5–15% of CD4⁺ peripheral blood T cells, contain a subpopulation with suppressor function similar to that exhibited by murine CD4⁺CD25⁺ T cells (3, 4, 5, 6). However, further work reported previously and corroborated in this study indicated that only CD4⁺CD25^high T cells, comprising 1–2% of circulating CD4⁺ T cells, possess suppressor capability (7). In view of these findings, we investigated expression of cell surface TGF-1 on CD4⁺CD25^high and CD4⁺CD25^- T cells. First, we isolated CD4⁺CD25^high and CD4⁺CD25^- T cells from human peripheral blood by flow cytometric sorting (Fig. 2A). We then stimulated the cells with soluble anti-CD3, APC, and rIL-2, and after 5 days stained the cells thus activated with polyclonal goat anti-LAP Ab. As shown in Fig. 2B, the expression level of cell surface LAP was high on CD25^high T cells after stimulation, whereas only a small amount of LAP was found on CD25^- T cells. Similar results were obtained with another biotin-conjugated mAb against LAP (clone 27240) (data not shown). However, using an Ab against active TGF-1, we could detect only marginal amount of TGF-1 on the cell surface; this contrasts with results obtained in mouse cells.

View larger version (22K): [in this window] [in a new window]   FIGURE 2. Purification of human CD4+CD25high T cells and expression of LAP on activated CD4+CD25high T cells. A, Purified human CD4+ T cells were stained with FITC-conjugated anti-CD4 and PE-conjugated anti-CD25. CD4+CD25high and CD4+CD25- T cells were then purified by FACS sorting. B, Purified CD4+CD25high and CD4+CD25- T cells were stimulated with soluble anti-CD3 (1 µg/ml), irradiated APC, and rIL-2 (50 U/ml) for 5 days. Cells were then stained with FITC-conjugated anti-CD4, biotin-conjugated anti-LAP, and PE-conjugated streptavidin. Expression of LAP on CD4+ gate is shown. Results are representative of six independent experiments.

View larger version (11K): [in this window] [in a new window]   FIGURE 3. rLAP reverses suppressor function of human CD4+CD25high T cells. Human CD4+CD25- T cells were stimulated with 1 µg/ml soluble anti-CD3 and irradiated APC in the presence or absence of CD4+CD25high T cells. Various concentrations of rLAP were added to the cell culture. •, CD4+CD25- T cell; , CD4+CD25- and CD4+CD25+ T cell. Results are representative of three independent experiments.

CD4⁺CD25⁺ regulatory T cells induce TGF-R signaling in the target cells

To provide further evidence that cell surface TGF- on the surface of CD4⁺CD25⁺ T cells is involved in the suppression mediated by these cells, we sought evidence that such TGF- signals the CD25^- target cells. In an initial series of experiments along these lines, we took advantage of the fact that CD103 expression can be induced by TGF-1, and thus, expression of this integrin on CD4⁺CD25^- target cells should increase during coculture with CD4⁺CD25⁺ T cells in the presence of cell surface-bound TGF-1. Accordingly, we cocultured Thy-1.2⁺ CD4⁺CD25⁺ T cells with Thy-1.1⁺ CD4⁺CD25^- target cells and stimulated with anti-CD3, APC, and rIL-2. We then stained the cells and discriminated suppressors and targets on the basis of Thy-1.1 expression. As shown in Fig. 4, cultured Thy-1.1⁺ CD4⁺CD25^- T cells contained very few CD103⁺ cells (1.1%), whereas the same cells cultured in the presence of CD4⁺CD25⁺ T cells contain as many CD103⁺ cells as the same cells cultured with rTGF-1. As reported previously (18), CD4⁺CD25⁺ T cells contained a substantial subpopulation of CD103⁺ cells, possibly due to autoactivation.

View larger version (35K): [in this window] [in a new window]   FIGURE 4. CD4+CD25+ T cells induce CD103 expression on CD4+CD25- T cells. Thy-1.1+CD4+CD25+ T cells (left panels), Thy-1.1+CD4+CD25- T cells in the absence (second left panels) and the presence (second right panels) of rTGF-1 (5 ng/ml), and Thy-1.1+CD4+CD25- in the presence of Thy-1.2+CD4+CD25+ T cells (right panels) were stimulated with soluble anti-CD3 (10 µg/ml), irradiated APC, and IL-2 (50 U/ml) for 5 days. Cells were stained with FITC-conjugated anti-Thy-1.1, PE-conjugated anti-CD103 or isotype control, and CyChrome-conjugated anti-CD4. Top panels, The expression of Thy-1.1 and CD4. Staining with anti-CD103 (bottom panels) and isotype control Ab (middle panels) on Thy-1.1+CD4+ gate is shown. These data are representative of three independent experiments.

View larger version (34K): [in this window] [in a new window]   FIGURE 5. CD4+CD25+ T cells induce Smad-2 phosphorylation in CD4+CD25- T cells. Purified CD4+CD25+ and CD4+CD25- T cells were stimulated with soluble anti-CD3 (10 µg/ml) in the presence of irradiated APC. Then freshly isolated CD4+CD25- T cells were mixed with or without activated CD4+CD25+ T cells, CD4+CD25- T cells, or rTGF-1, and briefly spun down and incubated for 90 min. Then CD4+CD25- T cells were repurified by magnetic beads and lysed. Phosphorylated Smad-2 (top panel) and total Smad-2 protein (bottom panel) were detected by Western blot. CD4+CD25- T cells were mixed with CD4+CD25+ T cells and APC (lane 1); mixed with CD4+CD25- T cells and APC (lane 2); incubated with APC (lane 3); and incubated with rTGF-1 (5 ng/ml) and APC (lane 4).

TGF-1 produced from CD4⁺CD25⁺ T cells is important in suppression of intestinal inflammation in vivo

The above results strongly support the idea that TGF-1 is involved in CD4⁺CD25⁺ T cell-mediated suppression of T cell proliferation in vitro. However, the opposite conclusion was drawn from studies in which CD4⁺CD25⁺ T cells from TGF-1^-/- mice were shown to mediate suppression in vitro (13). To further explore these results, we assessed the suppressor function of CD4⁺CD25⁺ T cells from TGF-1^-/- mice both in vitro or in vivo. In initial in vitro studies shown in Fig. 6, we found that TGF-1-deficient CD4⁺CD25⁺ T cells from TGF-1^-/- could indeed suppress T cell proliferation in vitro to the same changes as CD4⁺CD25⁺ T cells from wild-type mice.

View larger version (10K): [in this window] [in a new window]   FIGURE 6. TGF-1-deficient CD4+CD25+ T cells mediate suppression in vitro. CD4+CD25- T cells (4 x 104/well) from wild-type mice were stimulated with 10 µg/ml of soluble anti-CD3 and irradiated APC in the presence or absence of CD4+CD25+ T cells (4 x 104/well) from wild-type or TGF-1-null mice.

View larger version (25K): [in this window] [in a new window]   FIGURE 7. In vivo suppressor function of CD4+CD25+ T cells from TGF-1-deficient mice in the SCID transfer model of colitis. Weight curves and gross appearance of colons. A, Rag-2-deficient mice were reconstituted with CD4+CD45RBhigh T cells (), CD4+CD45RBhigh and CD4+CD45RBlow T cells (), CD4+CD45RBhigh and wild-type CD4+CD25+ T cells (), or CD4+CD45RBhigh and TGF-1-deficient CD4+CD25+ T cells (). The change in weight was monitored weekly after the transfer. B, The gross appearance of colons in each group. Recipients of a, CD4+CD45RBhigh T cells; b, CD4+CD45RBhigh and CD4+CD45RBlow T cells; c, CD4+CD45RBhigh and wild-type CD4+CD25+ T cells; and d, CD4+CD45RBhigh and TGF-1-deficient CD4+CD25+ T cells.

View larger version (58K): [in this window] [in a new window]   FIGURE 8. In vivo suppressor function of CD4+CD25+ T cells from TGF-1-deficient mice in SCID transfer model of colitis. Microscopic appearance and colonic score. A, Representative microscopic appearance of colon of the Rag-2-deficient recipients reconstituted with CD4+CD45RBhigh T cells, CD4+CD45RBhigh and CD4+CD45RBlow T cells, CD4+CD45RBhigh and wild-type CD4+CD25+ T cells, or CD4+CD45RBhigh and TGF-1-deficient CD4+CD25+ T cells. B, The score of colonic inflammation in each group is shown. Similar results were obtained in two independent experiments.

In a final series of studies, we examined the capacity of TGF-1-bearing T cells from normal mice to mediate regulatory function in vivo, using a SCID transfer model of colitis. In initial studies, we determined the percentage of spleen CD4⁺ T cells expressing LAP on the cell surface after activation. As shown in Fig. 9A, 7.5% of activated CD4⁺ T cells obtained from wild-type mice were cell surface LAP positive after staining with anti-LAP mAb. To confirm the specificity of the staining, we stained CD4⁺ T cells obtained from TGF-1^-/- mice, and, as expected, they were negative for LAP expression. In addition, 3.5% of CD4⁺ T cells from TGF-1^+/- heterozygous mice expressed cell surface LAP, approximately half as many cells as in wild-type mice.

View larger version (23K): [in this window] [in a new window]   FIGURE 9. Purification of LAP+ and LAP- cells from activated mouse CD4+ T cells. A, Expression of cell surface LAP on wild-type, but not on TGF-1-deficient CD4+ T cells. Total splenocytes purified from TGF-1+/+, TGF-1+/-, and TGF-1-/- mice were stimulated with soluble anti-CD3 (10 µg/ml) and rIL-2 (50 U/ml) for 3 days. Cells were stained with FITC-conjugated anti-CD4 and anti-LAP mAb (clone 27232.11, mouse IgG1), biotin-conjugated anti-mouse IgG1, and PE-conjugated streptavidin. Cells were stained with 7-amino actinomycin D before analysis for dead cell exclusion. CD4+ cells were gated, and the expression of LAP was shown. Results shown are representative of three independent experiments. B, LAP+ and LAP- CD4+ T cells were purified by FACS sorting.

View larger version (25K): [in this window] [in a new window]   FIGURE 10. In vivo suppressor function of LAP+ and LAP- T cells in the SCID transfer colitis. Weight curves and gross appearance of colons. A, C.B-17 SCID mice were reconstituted with CD4+CD45RBhigh T cells (), CD4+CD45RBhigh and CD4+CD45RBlow T cells (), CD4+CD45RBhigh and CD4+LAP+ T cells (), or CD4+CD45RBhigh and CD4+LAP- T cells (). The change in weight was monitored weekly after the transfer. B, The gross appearance of colons in each group. Recipients of a, CD4+CD45RBhigh T cells; b, CD4+CD45RBhigh and CD4+CD45RBlow T cells; c, CD4+CD45RBhigh and CD4+LAP+ T cells; and d, CD4+CD45RBhigh and CD4+LAP- T cells.

View larger version (53K): [in this window] [in a new window]   FIGURE 11. In vivo suppressor function of LAP+ and LAP- T cells in SCID transfer colitis. Microscopic appearance and colonic score. A, Representative microscopic appearance of colon of the C.B-17 SCID mice reconstituted with CD4+CD45RBhigh T cells, CD4+CD45RBhigh and CD4+CD45RBlow T cells, CD4+CD45RBhigh andCD4+LAP+ T cells, or CD4+CD45RBhigh and CD4+LAP- T cells. B, The score of colonic inflammation in each group is shown. Similar results were obtained in another independent experiment.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Recognizing that the results of studies with anti-TGF- have been variable in their ability to demonstrate inhibition of CD4⁺CD25⁺ T cell-mediated suppression, we sought another inhibiting agent. We ultimately chose to evaluate rLAP as a possible inhibitor because this molecule is as TGF-1 specific as anti-TGF-1 and binds to TGF-1 with at least as high an affinity as anti-TGF-1 (24). This substance could conceivably block TGF-1 suppressor activity by either of two mechanisms: 1) by combining with newly released active TGF-1 and converting it back into a latent form; and 2) by competitive inhibition of latent TGF-1 (LAP) for potential binding sites at which conversion of latent TGF-1 to an active form normally occurs. Indeed, we found that rLAP inhibited CD4⁺CD25⁺ T cell-mediated suppression in the in vitro assay in a dose-dependent fashion and was able to do so in assays of either murine or human cells. These results provide independent confirmation that TGF-1 is at least partially involved in the in vitro suppressor activity of CD4⁺CD25⁺ T cells. In retrospect, we would suggest that the failure of some investigators to obtain inhibition of suppression with anti-TGF-1 can be attributed to either the use of too low a concentration of Ab or an Ab that does not block the ability of latent TGF-1 to bind to sites where conversion to an active form must occur or alternatively to block the ability of TGF-1 to bind to the TGF-R after conversion to an active form. In addition, we have noted that blockade by anti-TGF- varies with the degree of suppression and that at higher levels of suppression one observes less blockade. This may relate to the fact that at higher levels of suppression there is sufficient redundancy in the system to preclude effective blockade at reasonable Ab doses. Thus, the study of inhibition by anti-TGF- should be done at a variety of E:T cell ratios. Finally, because TGF-1-mediated suppression would necessarily involve both an activation step (resulting in dissociation from LAP) and a receptor interaction step and because these steps may be interrelated, it may be difficult to inhibit with Ab, unless one uses a great excess of Ab. LAP would be less subject to this difficulty because it can act before activation of TGF-1.

In a second approach to investigating whether TGF-1 is acting as a suppressor factor in CD4⁺CD25⁺ T cell-mediated suppression, we sought evidence that TGF-1 could signal CD25^- T cells via TGF-R. We showed that this was indeed true in that CD25^- target cells in the in vitro assay when mixed with CD4⁺CD25⁺ T cells (but not with CD4⁺CD25^- T cells) undergo Smad-2 phosphorylation. Such activation indicates that the target cells have been signaled via TGF-R. TGF-1 signaling of target cells was also demonstrated somewhat more indirectly by the fact that CD25^- target cells cocultured with CD4⁺CD25⁺ T cells were induced to express CD103 (_E integrin), an integrin previously shown to be regulated by TGF-1 (25). This finding has added weight in the light of the recent finding that stimulated CD4⁺ T cells in which TGF-1 signaling is blocked by overexpression of Smad-7 do not express _E7, indicating that TGF-1 is necessary for _E7 expression, at least in vitro (26). Finally, it is interesting that a significant fraction of CD4⁺CD25⁺ T cells expresses CD103, presumably because they are autostimulated by TGF-1.

A finding that is seemingly at odds with the TGF-1 blocking and signaling studies reported in this work concerns the fact that, as reported previously, CD4⁺CD25⁺ T cells from TGF-1-deficient mice were able to mediate suppression of CD4⁺CD25^- T cells in the in vitro assay system. There are a number of possible explanations of these discordant results, but the one that appears most likely to us is that in the absence of TGF-1, CD4⁺CD25⁺ T cells express and/or up-regulate alternative suppressor mechanisms that ordinarily play a secondary role in the mechanism of suppression. This possibility would accord with the fact mentioned above that anti-TGF- blockade is more effective at lower levels of suppression (when secondary mechanisms may be less evident) and the finding of Chen and Wahl (23) that CD4⁺CD25⁺ T cells from TGF-1-deficient mice suppressed less well at the usual ratio of regulatory cells to target cells, but could exert suppression at high ratios. This explanation notwithstanding, further work will be necessary to resolve this discrepancy. Finally, the finding that CD4⁺CD25⁺ T cells are capable of suppressing CD25^- T cells from mice with TGF-1 signaling defects (13) is not persuasive to us because these cells had leaky defects. Thus, in our hands, cells from either Smad3-deficient mice or mice bearing a dominant-negative TGF-R were still subject to inhibition of proliferation by the addition of rTGF- to the cultures (data not shown). Thus, these cells do not allow a rigorous test of TGF--mediated suppressor function.

Additional support for the involvement of TGF-1 in CD4⁺CD25⁺ T cell regulatory function came from in vivo studies in which we showed that while CD4⁺CD25⁺ T cells from TGF-1-deficient mice manifest suppressor function in the in vitro suppressor assay, they cannot prevent colitis mediated by CD45RB^high T cells transferred to Rag-2-deficient recipients, whereas as shown previously and again in this study, such cells from wild-type mice can prevent colitis. We believe this is a more robust test of the role of TGF-1 in the CD4⁺CD25⁺ T cell regulatory cell function of cells from TGF-1-deficient mice than the in vitro study, because in this case the regulatory cells are presumably acting via surface expression or secretion of TGF-1, which cannot be compensated by another suppressor mechanism.

Previous studies have shown that anti-TGF- administration to SCID or Rag-2-deficient mice transferred CD45RB^high T cells and protective CD45RB^low abrogates the protective effect and that CD4⁺CD25⁺ T cells are the subpopulation that mediate protection (19, 27). These studies left unanswered the question of whether the CD4⁺CD25⁺ T cells were producing the TGF-1 themselves or inducing other cells to produce this cytokine. This question is answered by the present findings showing that the TGF-1 must in fact come from the CD4⁺CD25⁺ T cells themselves.

In related studies, we showed that CD4⁺LAP⁺ T cells, but not CD4⁺LAP^- T cells, from normal mice prevent colitis in the transfer model. This again provides yet additional evidence for the link between cell surface TGF-1 expression and regulatory function. Recently, Oida et al. (28) reported that CD25^-LAP^high cells also prevent colitis in the transfer model and such prevention is also TGF- dependent. This cell population may represent a population in a different state of activation or differentiation than the cells investigated in this study, but nevertheless, the results are consistent with our finding that LAP⁺ cells (cells bearing cell surface TGF-1) are suppressor cells whether or not they express CD25⁺.

Taken together, these studies show that TGF-1 produced by CD4⁺CD25⁺ regulatory T cells are an important mechanism by which these cells exert suppression in vitro and more, particularly, in vivo. However, they leave open the possibility that other suppressor mechanisms may also exist and may come to the fore under some circumstances. Finally, studies presented in this work and previously strongly suggest that the presence of LAP on the cell surface is an excellent marker of regulatory cells, perhaps better than CD25 itself.

	Acknowledgments

 
We thank Calvin Eigsti for technical assistance with FACS sorting. We also thank Dr. Mizuko Mamura for valuable help in providing TGF-1 knockout mice. In addition, we acknowledge Akiyo Kondo for her technical assistance and Sara Kaul for editorial assistance.

	Footnotes

 
¹ Address correspondence and reprint requests to Dr. Warren Strober, Mucosal Immunity Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 10, Room 11N238, 10 Center Drive, Bethesda, MD 20892. E-mail address: wstrober{at}niaid.nih.gov

² Abbreviation used in this paper: LAP, latency-associated peptide of TGF-.

Received for publication July 10, 2003. Accepted for publication November 4, 2003.

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