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1 Plays an Important Role in the Mechanism of CD4+CD25+ Regulatory T Cell Activity in Both Humans and Mice
* 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
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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+CD25high 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. |
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) 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.
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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+CD25high and CD4+CD25- T cell populations. The purity of the CD4+CD25high 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+CD25high 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+CD25high 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 CD45RBhigh 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+CD45RBhigh and CD4+CD45RBlow T cells were purified by FACS sorting. Wild-type and TGF-1-deficient CD4+CD25+ T cells were purified, as described previously (10). CD4+CD45RBhigh T cells (3 x 105) were injected i.p. into Rag-2-deficient mice, accompanied, in some recipients, by CD4+CD45RBlow 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 CD45RBhigh 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+CD45RBhigh and CD4+CD45RBlow 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+CD45RBhigh T cells (3 x 105) were injected i.p. in C.B-17 SCID mice, accompanied in some recipients by CD4+CD45RBlow (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.
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Results |
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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).
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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+CD25high 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+CD25high and CD4+CD25- T cells. First, we isolated CD4+CD25high 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 CD25high 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.
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-dependent mechanism.
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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.
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1 signaling in CD25- target T cells, by measuring the appearance of activated Smad-2 in these cells following admixture with CD4+CD25+ suppressor T cells. In these studies, we mixed activated CD4+CD25+ or CD4+CD25- T cells with resting CD4+CD25- T cells (target cells) and then briefly centrifuged the cell mixture to promote cell-cell contact. We then reisolated the CD4+CD25- target cells and subjected lysates of these cells to Western blot analysis using anti-phospho-Smad-2 Ab. As shown in Fig. 5, CD4+CD25- target cells in contact with CD4+CD25+ T cells gave rise to a distinctly stronger phosphorylated Smad-2 signal than CD4+CD25- T cells in contact with CD4+CD25- T cells or CD4+CD25- T cells incubated alone. In contrast, total Smad-2 protein was similar in all cultures. Together, these studies show that TGF-1 on the surface of CD4+CD25+ T cells signals CD4+CD25- T cells: in one case, to induce expression of a TGF--dependent integrin, and, in another, to directly stimulate Smad-2.
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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.
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1-/- mice. In this case, as shown in Fig. 7A, Rag-2-deficient recipients of CD4+CD45RBhigh T cells derived from normal mice showed progressive weight loss that was inhibited by cotransfer of CD4+CD45RBlow T cells or CD4+CD25+ T cells from wild-type mice, whereas, in contrast, such weight loss was not inhibited by cotransfer of CD4+CD25+ T cells from TGF-1-/- mice. In addition, as shown in Figs. 7B and 8A, this pattern of weight loss corresponded to the macroscopic and microscopic changes present in the colons of the various groups. Finally, perhaps most importantly, these weight curves also corresponded with the pooled histological scores of mice in the various groups, which shows again that CD4+CD25+ T cells from TGF-1-/- did not protect Rag-2 knockout recipients of CD45RBhigh T cells from the development of colitis (Fig. 8B). It should be noted that these results cannot be explained by postulating that CD4+CD25+ T cells from TGF-1-/- have an intrinsic proinflammatory effect because the development of weight loss in recipients of these cells was limited to the colon and did not occur until after a long latent period, presumably due to the long time necessary for the development of colitis in the transfer model. It should also be noted that these results correlate with previous studies showing that anti-TGF- administration abrogates the protective effect of CD4+CD25+ T cells (19).
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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.
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1, and intracellular molecules such as FoxP3 (10, 18, 19, 20, 21, 22). However, the interrelation of these molecules or their final common pathway has yet to be defined. In a previous study, we showed that CD4+CD25+ T cells after stimulation express high levels of TGF-1 in a surface-bound and/or a secreted form, and addition of anti-TGF- to in vitro cocultures of CD4+CD25+ T cells and CD4+CD25- T cells, or CD4+CD25+, CD4+CD25- T cells, and B cells results in reversal of suppression of T cell proliferation and B cell Ig production, respectively (10). These findings have been subsequently corroborated by several other groups that have also found that addition of anti-TGF-1 or soluble TGF-R to in vitro cocultures reversed suppression of CD4+CD25+ T cells, at least in part (11, 12, 23). Nevertheless, the involvement of TGF-1 in the suppressor function of CD4+CD25+ T cells remains controversial, mainly because, as already mentioned, CD4+CD25+ T cells can still suppress CD25- T cells from mice in whom TGF-1 signaling is impaired and CD4+CD25+ T cells from TGF-1-deficient mice can still suppress CD4+CD25- T cells from normal mice in vitro (13). Therefore, in this study, we re-examined the involvement of TGF-1 in CD4+CD25+ T cell suppressor function and present new evidence buttressing the role of this molecule in the suppressor process.
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 CD45RBhigh 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 CD45RBhigh T cells and protective CD45RBlow 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-LAPhigh 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.
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1 knockout mice. In addition, we acknowledge Akiyo Kondo for her technical assistance and Sara Kaul for editorial assistance. |
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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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 R Duchmann and M Zeitz T regulatory cell suppression of colitis: the role of TGF-{beta} Gut, May 1, 2006; 55(5): 604 - 606. [Full Text] [PDF]
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Z.-Z. Yang, A. J. Novak, M. J. Stenson, T. E. Witzig, and S. M. Ansell Intratumoral CD4+CD25+ regulatory T-cell-mediated suppression of infiltrating CD4+ T cells in B-cell non-Hodgkin lymphoma Blood, May 1, 2006; 107(9): 3639 - 3646. [Abstract] [Full Text] [PDF]
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X. Zhang, J. Reddy, H. Ochi, D. Frenkel, V. K. Kuchroo, and H. L. Weiner Recovery from experimental allergic encephalomyelitis is TGF-{beta} dependent and associated with increases in CD4+LAP+ and CD4+CD25+ T cells Int. Immunol., April 1, 2006; 18(4): 495 - 503. [Abstract] [Full Text] [PDF]
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M. S. Longhi, M. J. Hussain, R. R. Mitry, S. K. Arora, G. Mieli-Vergani, D. Vergani, and Y. Ma Functional Study of CD4+CD25+ Regulatory T Cells in Health and Autoimmune Hepatitis J. Immunol., April 1, 2006; 176(7): 4484 - 4491. [Abstract] [Full Text] [PDF]
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C. Chen, W.-h. Lee, L. Zhong, and C.-P. Liu Regulatory T Cells Can Mediate Their Function through the Stimulation of APCs to Produce Immunosuppressive Nitric Oxide J. Immunol., March 15, 2006; 176(6): 3449 - 3460. [Abstract] [Full Text] [PDF]
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S. Suvas, A. K. Azkur, and B. T. Rouse Qa-1b and CD94-NKG2a Interaction Regulate Cytolytic Activity of Herpes Simplex Virus-Specific Memory CD8+ T Cells in the Latently Infected Trigeminal Ganglia J. Immunol., February 1, 2006; 176(3): 1703 - 1711. [Abstract] [Full Text] [PDF]
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M.-A. Alyanakian, F. Grela, A. Aumeunier, C. Chiavaroli, C. Gouarin, E. Bardel, G. Normier, L. Chatenoud, N. Thieblemont, and J.-F. Bach Transforming Growth Factor-{beta} and Natural Killer T-Cells Are Involved in the Protective Effect of a Bacterial Extract on Type 1 Diabetes Diabetes, January 1, 2006; 55(1): 179 - 185. [Abstract] [Full Text] [PDF]
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U. C. Rogner, F. Lepault, M.-C. Gagnerault, D. Vallois, J. Morin, P. Avner, and C. Boitard The Diabetes Type 1 Locus Idd6 Modulates Activity of CD4+CD25+ Regulatory T-Cells Diabetes, January 1, 2006; 55(1): 186 - 192. [Abstract] [Full Text] [PDF]
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S. Sugita, T. F. Ng, P. J. Lucas, R. E. Gress, and J. W. Streilein B7+ Iris Pigment Epithelium Induce CD8+ T Regulatory Cells; Both Suppress CTLA-4+ T Cells J. Immunol., January 1, 2006; 176(1): 118 - 127. [Abstract] [Full Text] [PDF]
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N. N. Choileain, M. MacConmara, Y. Zang, T. J. Murphy, J. A. Mannick, and J. A. Lederer Enhanced Regulatory T Cell Activity Is an Element of the Host Response to Injury J. Immunol., January 1, 2006; 176(1): 225 - 236. [Abstract] [Full Text] [PDF]
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I. P. Lewkowich, N. S. Herman, K. W. Schleifer, M. P. Dance, B. L. Chen, K. M. Dienger, A. A. Sproles, J. S. Shah, J. Kohl, Y. Belkaid, et al. CD4+CD25+ T cells protect against experimentally induced asthma and alter pulmonary dendritic cell phenotype and function J. Exp. Med., December 5, 2005; 202(11): 1549 - 1561. [Abstract] [Full Text] [PDF]
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M. L. Fields, B. D. Hondowicz, M. H. Metzgar, S. A. Nish, G. N. Wharton, C. C. Picca, A. J. Caton, and J. Erikson CD4+CD25+ Regulatory T Cells Inhibit the Maturation but Not the Initiation of an Autoantibody Response J. Immunol., October 1, 2005; 175(7): 4255 - 4264. [Abstract] [Full Text] [PDF]
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X. Huang, J. Zhu, and Y. Yang Protection against Autoimmunity in Nonlymphopenic Hosts by CD4+CD25+ Regulatory T Cells Is Antigen-Specific and Requires IL-10 and TGF-{beta} J. Immunol., October 1, 2005; 175(7): 4283 - 4291. [Abstract] [Full Text] [PDF]
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 S. P Cobbold T cell tolerance induced by therapeutic antibodies Phil Trans R Soc B, September 29, 2005; 360(1461): 1695 - 1705. [Abstract] [Full Text] [PDF]
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T. Banovic, K. P. A. MacDonald, E. S. Morris, V. Rowe, R. Kuns, A. Don, J. Kelly, S. Ledbetter, A. D. Clouston, and G. R. Hill TGF-{beta} in allogeneic stem cell transplantation: friend or foe? Blood, September 15, 2005; 106(6): 2206 - 2214. [Abstract] [Full Text] [PDF]
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H. H. Birdsall, W. J. Porter, J. Trial, and R. D. Rossen Monocytes Stimulated by 110-kDa Fibronectin Fragments Suppress Proliferation of Anti-CD3-Activated T Cells J. Immunol., September 1, 2005; 175(5): 3347 - 3353. [Abstract] [Full Text] [PDF]
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 A. Asgharpour, C. Gilchrist, D. Baba, S. Hamano, and E. Houpt Resistance to Intestinal Entamoeba histolytica Infection Is Conferred by Innate Immunity and Gr-1+ Cells Infect. Immun., August 1, 2005; 73(8): 4522 - 4529. [Abstract] [Full Text] [PDF]
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V. Gillan and E. Devaney Regulatory T Cells Modulate Th2 Responses Induced by Brugia pahangi Third-Stage Larvae Infect. Immun., July 1, 2005; 73(7): 4034 - 4042. [Abstract] [Full Text] [PDF]
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 L. Liu, Z. Xu, R. C. Fuhlbrigge, V. Pena-Cruz, J. Lieberman, and T. S. Kupper Vaccinia Virus Induces Strong Immunoregulatory Cytokine Production in Healthy Human Epidermal Keratinocytes: a Novel Strategy for Immune Evasion J. Virol., June 15, 2005; 79(12): 7363 - 7370. [Abstract] [Full Text] [PDF]
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S. Sharma, S.-C. Yang, L. Zhu, K. Reckamp, B. Gardner, F. Baratelli, M. Huang, R. K. Batra, and S. M. Dubinett Tumor Cyclooxygenase-2/Prostaglandin E2-Dependent Promotion of FOXP3 Expression and CD4+CD25+ T Regulatory Cell Activities in Lung Cancer Cancer Res., June 15, 2005; 65(12): 5211 - 5220. [Abstract] [Full Text] [PDF]
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T. L. Denning, G. Kim, and M. Kronenberg Cutting Edge: CD4+CD25+ Regulatory T Cells Impaired for Intestinal Homing Can Prevent Colitis J. Immunol., June 15, 2005; 174(12): 7487 - 7491. [Abstract] [Full Text] [PDF]
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L. Myers, M. Croft, B. S. Kwon, R. S. Mittler, and A. T. Vella Peptide-Specific CD8 T Regulatory Cells Use IFN-{gamma} to Elaborate TGF-{beta}-Based Suppression J. Immunol., June 15, 2005; 174(12): 7625 - 7632. [Abstract] [Full Text] [PDF]
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T. M. Brusko, C. H. Wasserfall, M. J. Clare-Salzler, D. A. Schatz, and M. A. Atkinson Functional Defects and the Influence of Age on the Frequency of CD4+CD25+ T-Cells in Type 1 Diabetes Diabetes, May 1, 2005; 54(5): 1407 - 1414. [Abstract] [Full Text] [PDF]
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T. M. Brusko, C. H. Wasserfall, A. Agarwal, M. H. Kapturczak, and M. A. Atkinson An Integral Role for Heme Oxygenase-1 and Carbon Monoxide in Maintaining Peripheral Tolerance by CD4+CD25+ Regulatory T Cells J. Immunol., May 1, 2005; 174(9): 5181 - 5186. [Abstract] [Full Text] [PDF]
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S. M. Pop, C. P. Wong, D. A. Culton, S. H. Clarke, and R. Tisch Single cell analysis shows decreasing FoxP3 and TGF{beta}1 coexpressing CD4+CD25+ regulatory T cells during autoimmune diabetes J. Exp. Med., April 18, 2005; 201(8): 1333 - 1346. [Abstract] [Full Text] [PDF]
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I. Lee, L. Wang, A. D. Wells, M. E. Dorf, E. Ozkaynak, and W. W. Hancock Recruitment of Foxp3+ T regulatory cells mediating allograft tolerance depends on the CCR4 chemokine receptor J. Exp. Med., April 4, 2005; 201(7): 1037 - 1044. [Abstract] [Full Text] [PDF]
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D. W. Smith and C. Nagler-Anderson Preventing Intolerance: The Induction of Nonresponsiveness to Dietary and Microbial Antigens in the Intestinal Mucosa J. Immunol., April 1, 2005; 174(7): 3851 - 3857. [Abstract] [Full Text] [PDF]
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M. Kleinewietfeld, F. Puentes, G. Borsellino, L. Battistini, O. Rotzschke, and K. Falk CCR6 expression defines regulatory effector/memory-like cells within the CD25+CD4+ T-cell subset Blood, April 1, 2005; 105(7): 2877 - 2886. [Abstract] [Full Text] [PDF]
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C. Di Giacinto, M. Marinaro, M. Sanchez, W. Strober, and M. Boirivant Probiotics Ameliorate Recurrent Th1-Mediated Murine Colitis by Inducing IL-10 and IL-10-Dependent TGF-{beta}-Bearing Regulatory Cells J. Immunol., March 15, 2005; 174(6): 3237 - 3246. [Abstract] [Full Text] [PDF]
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H.-K. Kang, M. A. Michaels, B. R. Berner, and S. K. Datta Very Low-Dose Tolerance with Nucleosomal Peptides Controls Lupus and Induces Potent Regulatory T Cell Subsets J. Immunol., March 15, 2005; 174(6): 3247 - 3255. [Abstract] [Full Text] [PDF]
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D. C. Gondek, L.-F. Lu, S. A. Quezada, S. Sakaguchi, and R. J. Noelle Cutting Edge: Contact-Mediated Suppression by CD4+CD25+ Regulatory Cells Involves a Granzyme B-Dependent, Perforin-Independent Mechanism J. Immunol., February 15, 2005; 174(4): 1783 - 1786. [Abstract] [Full Text] [PDF]
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C Veltkamp, R B Sartor, T Giese, F Autschbach, I Kaden, R Veltkamp, D Rost, B Kallinowski, and W Stremmel Regulatory CD4+CD25+ cells reverse imbalances in the T cell pool of bone marrow transplanted TG{varepsilon}26 mice leading to the prevention of colitis Gut, February 1, 2005; 54(2): 207 - 214. [Abstract] [Full Text] [PDF]
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P. E. Rao, A. L. Petrone, and P. D. Ponath Differentiation and Expansion of T Cells with Regulatory Function from Human Peripheral Lymphocytes by Stimulation in the Presence of TGF-{beta} J. Immunol., February 1, 2005; 174(3): 1446 - 1455. [Abstract] [Full Text] [PDF]
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W. R. Godfrey, D. J. Spoden, Y. G. Ge, S. R. Baker, B. Liu, B. L. Levine, C. H. June, B. R. Blazar, and S. B. Porter Cord blood CD4+CD25+-derived T regulatory cell lines express FoxP3 protein and manifest potent suppressor function Blood, January 15, 2005; 105(2): 750 - 758. [Abstract] [Full Text] [PDF]
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, CD4+CD25- and CD4+CD25+ T cell; 
, CD4+CD25+ T cell. Representative results of three independent experiments are shown.
), CD4+CD45RBhigh and CD4+CD45RBlow T cells (
), CD4+CD45RBhigh and wild-type CD4+CD25+ T cells (
), or CD4+CD45RBhigh and TGF-
). 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-
