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Conversion of Peripheral CD4+CD25− Naive T Cells to CD4+CD25+ Regulatory T Cells by TGF-β Induction of Transcription Factor Foxp3

Conversion of Peripheral CD4+CD25− Naive T Cells to CD4+CD25+ Regulatory T Cells by TGF-β... CD4 CD25 regulatory T cells (T ) are instrumental in the maintenance of immunological reg tolerance. One critical question is whether T can only be generated in the thymus or can reg differentiate from peripheral CD4 CD25 naive T cells. In this paper, we present novel evi- dence that conversion of naive peripheral CD4 CD25 T cells into anergic/suppressor cells /low that are CD25 , CD45RB and intracellular CTLA-4 can be achieved through costimu- lation with T cell receptors (TCRs) and transforming growth factor  (TGF-). Although transcription factor Foxp3 has been shown recently to be associated with the development of T , the physiological inducers for Foxp3 gene expression remain a mystery. TGF- induced reg Foxp3 gene expression in TCR-challenged CD4 CD25 naive T cells, which mediated their transition toward a regulatory T cell phenotype with potent immunosuppressive potential. These converted anergic/suppressor cells are not only unresponsive to TCR stimulation and produce neither T helper cell 1 nor T helper cell 2 cytokines but they also express TGF- and inhibit normal T cell proliferation in vitro. More importantly, in an ovalbumin peptide TCR transgenic adoptive transfer model, TGF-–converted transgenic CD4 CD25 suppressor cells proliferated in response to immunization and inhibited antigen-specific naive CD4 T cell expansion in vivo. Finally, in a murine asthma model, coadministration of these TGF-– induced suppressor T cells prevented house dust mite–induced allergic pathogenesis in lungs. Key words: anergy • IL-10 • OVA TCR transgenic • house dust mite • asthma Introduction CD4 CD25 regulatory T cells (T ) have emerged as a in the periphery (4, 5). One crucial question is whether reg unique population of suppressor T cells that maintain periph- T can be induced or converted from normal peripheral reg eral immune tolerance (1, 2). Although the immunoregula- CD4 T cells and if this occurs, which molecules and/or tory ability of T is no longer contested, where and how cytokines are responsible for the transition because acti- reg this population of CD4 T cells is generated and developed vated CD4 T cells expressing CD25 under neutral TCR still remains largely unknown. The major debate centers on stimulation conditions show no suppressive ability (1, 2). whether T are generated only in the thymus from a defined TGF- is a critical factor in regulation of T cell–mediated reg lineage and/or whether these cells represent a stage that immune responses and in the induction of immune toler- different types of CD4 T cells can acquire. Although most ance (for reviews see references 6, 7). When the TGF-1– analyses emphasize the thymus as the sole incubator for T mediated inhibitory pathway is abrogated specifically in T reg (3, 4), recent evidence suggests that T may also be induced cells by restrictive expression of dominant negative TGF- reg receptor II (8, 9), these mice develop unchecked T cell proliferation and inflammatory and autoimmune-like diseases, The online version of this article includes supplemental material. Address correspondence to WanJun Chen, Cellular Immunology Sec- tion, Oral Infection and Immunity Branch, National Institute of Dental Abbreviations used in this paper: 7-AAD, 7-amino-actinomycin D; CFSE, and Craniofacial Research, National Institutes of Health, Bethesda, carboxy-fluorescein diacetate succinimidyl ester; HDM, house dust mite; MD 20892. Phone: (301) 435-7168; Fax: (301) 402-1064; email: HPRT, hypoxanthineguanine phosphoribosyl transferase; PAS, periodic [email protected]; or Sharon M. Wahl. Phone: (301) 496-4178; acid schiff; P-Smad2/3, phosphorylated Smad2/3; T , CD4 CD25 reg email: [email protected] regulatory T cells. 1875 The Journal of Experimental Medicine • Volume 198, Number 12, December 15, 2003 1875–1886 http://www.jem.org/cgi/doi/10.1084/jem.20030152 The Journal of Experimental Medicine documenting a TGF-–dependent signal in T cell activa- synthesized and purified by reverse phase–HPLC (Synpep Cor- poration). The purity of the peptides is 98%. The following tion and tolerance in vivo. Although TGF- regulation of reagents were obtained from BD Biosciences: purified rat anti– immune responsiveness has been validated in vitro and in mouse mAbs to IL-2, IL-4, and IFN-, anti-CD28, FITC–anti- vivo, how TGF- accomplishes its suppressive role in T CD45RB, recombinant mouse IL-2, IL-4, and IFN-, PE- or cell activation remains unclear. A link between TGF- and purified anti-CD3 (145-2C11, NA/LE™), PE-anti–CTLA-4, induction of T has also been suggested in humans (10), reg hamster IgG isotypic control, FITC- or biotinylated antimurine but whether TGF- converts naive T cells to regulatory CD25 (Clone 7D4), FITC–rat IgM, PE- or purified anti-CD25 cells and, most importantly, the underlying molecular (Clone PC61, NA/LE™), and anti-FcRII/III. FITC or PE-anti– mechanisms await revelation. mouse CD4, anti-CD8, and the respective isotypic control In exciting new papers, Foxp3, which encodes a tran- mAbs and streptavidin-FITC, PE, or Tricolor were purchased scription factor that is genetically defective in an autoim- from Caltag. Recombinant human TGF-1, anti–TGF-1, 2,3 mAb, biotinylated chicken anti–TGF-1, and recombinant mu- mune syndrome in humans and mice (11, 12), has been rine IL-10 were purchased from R&D Systems. 7-amino-actino- shown to be not only specifically expressed in professional mycin D (7-AAD) was purchased from Calbiochem. T , but also required for their development (13–15). Nei- reg Cell Preparation. Spleens were gently minced in complete ther naive nor activated CD4 CD25 responder T cells DMEM containing 10% FBS (BioWhittaker), and CD4 T cells express Foxp3, distinguishing Foxp3 from other T associ- reg were purified using a mouse CD4 T cell column system (R&D ated molecules (CD25, CTLA-4, and GITR) that can be Systems; references 20, 21). T cell–depleted or whole spleen cells acquired in CD4 CD25 responder T cells once activated. (irradiated, 3,000 rad) of BALB/c or C57BL6 mice were used as Foxp3/Scurfin-deficient mice develop massive autoim- APCs as indicated. To isolate CD4 CD25 T cells, anti-CD25 mune and inflammatory disease, which shares many patho- antibody (PC61, 1 g/10 cells) was added into the antibody genetic features of mice deficient in CTLA-4 (16, 17) or cocktail and incubated with spleen cells before separation on the CD4 T cell column, to yield a purity of CD4 CD25 T cells TGF- (18, 19). Significantly, gene transfer of Foxp3 con- 90%. For T , the CD4 T cells were incubated with FITC– verts naive CD4 CD25 T cells toward a regulatory T cell reg anti-CD25 (1 g/10 cells; antibody depleted of sodium azide by phenotype similar to that of the professional T (13–15). reg dialysis in PBS overnight) in 2% PBS-FBS for 30 min at 4 C, Although the identification of Foxp3 in T has greatly ex- reg washed, resuspended in X-Vivo 20 serum-free medium (Bio- panded our ability to decipher the development and func- Whittaker), and the T and CD4 CD25 T cells were purified reg tion of this unique population of regulatory T cells, how Plus using a FACStar ™ Cell Sorter (Becton Dickinson). The purity Foxp3 is controlled and by what physiological inducers re- of sorted cells was 95–99%. main mysteries to be solved. Cell Culture and T Cell Proliferation Assay. For normal In this paper, we present evidence that TGF- converts C57BL/6 CD4 T cell primary stimulation, purified CD4 , naive CD4 CD25 T cells into CD4 CD25 anergic/sup- CD4 CD25 , or T were stimulated with 0.5 g/ml anti-CD3 reg pressor T cells in the periphery. These suppressor T cells not in the presence of APCs in complete DMEM at 37 C and 5% CO for 7–10 d and CD4 T cells were isolated for further studies. In only exhibit unresponsiveness to TCR stimulation, but also some studies, 2 ng/ml TGF-1 was included in the cultures as in- suppress normal CD4 T cell activation and Th1 and Th2 dicated. In other experiments, CD4 CD25 or CD25 cells were cytokine production in vitro. Moreover, these suppressor T stimulated with 2 g/ml of platebound anti-CD3 and 2 g/ml cells also inhibit immune responses in vivo, as evident in soluble anti-CD28 in the absence or presence of 0.02, 0.2, 2, or 20 their striking prevention of allergic pathogenesis in a house ng/ml TGF-1, 1 ng/ml IL-10, or 100 U/ml IL-2 for 3 d. dust mite (HDM)–induced mouse asthma model and in their For DO11.10 TCR transgenic mice, spleen cells (2 significant suppression of antigen-specific CD4 T cell pro- cells/ml) were stimulated with 100 g/ml OVA in the absence liferation in an OVA peptide CD4 transgenic T cell adop- or presence of 2 ng/ml recombinant TGF-1 or 1 ng/ml murine tive transfer model. Significantly, we also show that TGF- IL-10 in complete DMEM for 7–10 d. Cells were harvested and conversion of naive CD4 CD25 T cells into CD4 CD25 extensively washed, and dead cells were eliminated by centrifuga- suppressor T cells involves induction of Foxp3 expression, tion over Ficoll-Paque (Amersham Biosciences). For secondary stimulation, CD4 cells purified from the primary cultures were the first identification of an inducer of this pathway, which restimulated with OVA or anti-CD3 in the presence of APCs as offers an opportunity to control tolerance. indicated. For activation with PMA, CD4 T cells were incu- bated with 5 ng/ml PMA and 250 ng/ml ionomycin. Cells were cultured at 37 C in 5% CO for 72 h and pulsed with 1 Ci Materials and Methods 3 [ H]thymidine for the last 6–16 h. Radioactivity incorporated was counted using a flatbed  counter (Wallac). In some experi- Mice. Normal BALB/c and DO11.10 transgenic mice ex- pressing a TCR with specificity for chicken OVA peptide 323- ments, 10 U/ml rIL-2 or 20 g/ml anti–TGF-1, 2,3 mAb and its isotypic antibody control were added at the beginning of the 339 were purchased from The Jackson Laboratory and provided by B. Kelsall (National Institute of Allergy and Infectious Dis- secondary culture. Cytokine Induction and Determination. For cytokine induc- eases, Bethesda, MD). C57BL/6 mice were obtained from The Jackson Laboratory and from in-house breeding at the National tion, cells were cultured with antigens or antibodies as indicated in complete DMEM for 24–96 h. Cell-free supernatants were Institute of Dental and Craniofacial Research. Antibodies and Reagents. Crystallized chicken OVA, hen egg collected for the determination of IL-2, IFN-, IL-4, and IL-13 production by ELISA using paired mAbs specific for the corre- lysozyme, PMA, and ionomycin were purchased from Sigma- Aldrich. OVA peptide 323-339 (ISQAVHAAHAEINEAGR) was sponding cytokines (BD Biosciences) or the respective ELISA kits TGF- Induces CD4 CD25 Regulatory T Cells through Induction of Foxp3 1876 (Biosource International and R&D Systems). A standard curve RT-PCR for Foxp3 Expression. RT-PCR was performed as was generated using known amounts of the respective purified described previously (13), except the number of cycles was 28 for recombinant murine cytokines. Foxp3. The primer sequences were as follows and synthesized by Coculture of CD4 T Cells. Freshly isolated C57BL/6 or Invitrogen: HPRT, 5 -GTTGGATACAGGCCAGACTTTG- BALB/c CD4 CD25 responder T cells were cultured in TTG-3 and 5 -GATTCAACTTGCTCTCATCTTAGGC-3 ; 96-well plates with anti-CD3, splenic APCs, and indicated and Foxp3, 5 -CAGCTGCCTACAGTGCCCCTAG-3 and 5 - CD4 CD25 cells for 72 h for proliferation assay. Transwell ex- CATTTGCCAGCAGTGGGTAG-3 . Normalized values for Foxp3 periments were performed as described previously (22). In brief, mRNA expression in each sample were calculated as the relative CD4 CD25 responder T cells were cultured in 24-well plates quantity of Foxp3 divided by the relative quantity of hypoxan- with APCs and 0.5 g/ml anti-CD3 in the presence or absence thineguanine phosphoribosyl transferase (HPRT). of TGF-–converted or control CD4 CD25 cells in the Trans- Statistical Analysis. Student’s t tests were used for the signifi- well™ (0.4 M pore size; Costar). For carboxy-fluorescein diac- cance of data comparison. etate succinimidyl ester (CFSE)–labeling assay, 10 cells/ml T Online Supplemental Material. Fig. S1 shows that TGF-– cells were incubated with 3 M CFSE in plain DMEM (without converted CD25 suppressor cells require cell contact to carry phenol red) at 37 C for 15 min. Cells were washed three times out their action, and that IL-10–induced CD25 T cells fail to and resuspended in complete DMEM for cell culture. suppress normal CD4 T cell proliferation in vitro. Fig. S2 shows Flow Cytometry Analysis. T cells were resuspended in PBS increased P-Smad2/3 expression in TGF-–converted CD25 containing 1% BSA (Irvine) and 0.1% sodium azide (Sigma- suppressor cells and that anti–TGF- reverses TGF-–induced Aldrich). For the staining of surface antigens, cells were incubated anergy of CD4 T cells. Fig. S3 shows that cotransfer of TGF-– with FITC-, PE-, or Tricolor-conjugated mAbs or their negative converted CD4 suppressor T cells blocked epithelial cell mucin control antibodies as indicated for 30 min on ice. For surface ac- production in airways in lungs induced by HDM as detected by tive TGF- staining, cells were stained with biotinylated chicken PAS staining. Online supplemental material is available at http:// anti–TGF-1 and FITC–anti-CD25 for 30 min, followed by PE- www.jem.org/cgi/content/full/jem.20030152/DC1. streptavidin incubation for an additional 30 min at 4 C. Intracel- lular staining of CTLA-4 was performed as described previously (23). Intracellular staining of phosphorylated Smad2/3 (P-Smad2/3) Results was performed as described in the figure legend for Fig. TCR and TGF- Costimulation Induces CD4 CD25 T S2 (available at http://www.jem.org/cgi/content/full/jem. 20030152/DC1. Cell Anergy, but Fails to Expand Professional T . Although reg Adoptive Transfer Experiments. Freshly isolated CD4 KJ1- TGF- suppression of normal CD4 T cell activation has 26 transgenic T cells were injected i.p. into unirradiated synge- been validated, it remains unclear whether TGF- action is neic BALB/c recipients. Some mice were cotransferred i.p. with through induction of CD4 CD25 T cell anergy or by pro- control, TGF-–, or IL-10–pretreated naive CD4 KJ1-26 T moting growth of existing T . First, we studied whether reg cells. Animals were immunized s.c. with 100 g of P323-339 TCR and TGF- costimulation could expand a population emulsified in IFA (Difco). Cells from draining lymph nodes (in- of existing T in vitro. As expected, freshly isolated T reg reg guinal) were harvested at indicated time points and stained ex were unresponsive to anti-CD3 stimulation, but could be vivo with Tricolor–anti-CD4, PE–anti-CD25, and FITC–KJ1- coerced to proliferate upon stimulation with anti-CD3 in 26 mAbs to determine the expansion of CD4 KJ1-26 T cells in the presence of a high dose of exogenous IL-2 (100 U/ml; vivo. For in vitro restimulation assay, draining lymph node cells were cultured with OVA peptide 323-339 for 3 d to determine references 1, 2; unpublished data). However, after primary T cell proliferation or for 6 h in the presence of GolgiPlug™ culture with anti-CD3 only or with anti-CD3 and TGF- (BD Biosciences) to determine intracellular cytokines as de- in the presence of APCs for 7 d, most of the T died. There reg scribed previously (24). was no increase in the number of T in TGF-–treated cul- reg HDM-induced Allergic Pathogenesis. Allergen-induced asthma tures, but the surviving anti-CD3 plus TGF-–treated T , reg was induced as described previously (25) with some modifica- similar to only anti-CD3–treated cells, remained unrespon- tions. In brief, 6–8-wk-old C57BL/6 mice were immunized by sive to anti-CD3 restimulation (Fig. 1 A), indicating that ex- i.p. injection of 10 g HDM antigen (Greer Laboratories) in 0.1 ogenous TGF- in vitro in the absence of other factors, such ml PBS or PBS alone (unpublished data) at days 1 and 7, followed as IL-2, was insufficient to expand the original T . reg by intratracheal challenge with 100 g HDM antigen in 40 l Alternatively, to determine whether TGF- could aner- PBS or an equivalent volume of PBS as a control at days 14 and gize CD4 CD25 naive T cells, purified CD4 CD25 T 21, respectively. 4 d after the last challenge, mice were killed, and tissues were harvested for immunohistopathologic analysis or in cells from spleens of normal C57BL/6 (Fig. 1) or BALB/c vitro cultures. The mucin expression in the airways was deter- (not depicted) mice were cultured with anti-CD3 and APCs mined with periodic acid schiff (PAS) staining (25). Where indi- in the presence and absence of TGF- for 1 wk, harvested, cated, TGF-–anergized or control CD4 T cells were injected washed, and restimulated with anti-CD3 and APCs. Consis- i.v. into the mice on day 1 and again on day 14. tent with previous papers (1, 2), control CD4 T cells, pre- Western Blot Analysis. Western blot analysis was performed cultured with anti-CD3 and APCs (neutral stimulation as described previously (24) with an antibody to P-Smad2/3 (rab- condition), proliferated vigorously in response to TCR re- bit polyclonal IgG, 1:200) or to Smad2/3 (goat polyclonal IgG, stimulation (Fig. 1 A). In contrast, anti-CD3 plus TGF-– 1:200; Santa Cruz Biotechnology, Inc.) followed by horseradish pretreated cells were unresponsive to TCR restimulation peroxidase–conjugated goat anti–rabbit IgG or donkey anti–goat (Fig. 1 A) without increased apoptosis (not depicted), consis- IgG (Santa Cruz Biotechnology, Inc.) as recommended by the tent with TGF- induction of CD4 CD25 T cell anergy. manufacturer. Chen et al. 1877 Figure 1. Costimulation of TCR and TGF- induces CD4 CD25 T cell anergy, but fails to expand existing T . (A) reg C57BL/6 CD25 naive cells or T (5 reg 10 ) were cultured (primary) with anti-CD3 and APCs (2 10 ) in the absence (CD3 med) and presence (CD3  TGF-) of 2 ng/ml TGF- for 7 d. 3 10 harvested viable CD4 responder T cells or 5 T were restimulated with anti-CD3 and reg APCs for 72 h to monitor their prolifera- tion. The data are representative of three separate experiments. (B–F) TGF- induces OVA TCR transgenic CD4 T cells (KJ1- 26 ) anergy. 2 10 cells/ml spleen cells were cultured with OVA in the presence or absence of TGF- for 7–10 d (primary). Viable CD4 T cells were purified and re- stimulated with 100 g/ml OVA, 100 g/ml hen egg lysozyme (HEL), 1 g/ml anti- CD3 mAb, or 10 U/ml IL-2 as indicated in the presence of BALB/c APCs or with PMA and ionomycin (secondary stimulation). The values are expressed as mean SD of triplicate wells for H incorporation (CPM, 10 T cells) (B and C) or of duplicate wells of the ELISA (D–F, 2 10 T cells). (B) TGF- induces transgenic CD4 TCR-specific anergy. (C) Inclusion of exogenous IL-2 in primary cultures blocks TGF-–induced CD4 T cell anergy. (D–F) TGF- induces both Th1 and Th2 cell anergy. Cytokine levels of IL-2 (D), IFN- (E), and IL-4 (F) in secondary culture supernatants (after 24–48 h) were determined by ELISA. The data shown were repeated from two to six times with similar results. Extending this study to CD4 T stimulation with a spe- of T cell proliferation (Fig. 1 B) and Th1 and Th2 cytokine cific peptide antigen in TCR transgenic mice, similar results production in TGF-–anergized T cells (Fig. 1, D–F). To were obtained. Using TCR transgenic mice DO11.10 in study IL-2 effects on the induction phase of the TGF-– which the majority of the peripheral CD4 T cells express induced anergy, IL-2 was included in the primary culture clonotypic TCR (KJ1-26 , V 8.2) recognizing the OVA with OVA plus TGF- for 7 d, and cells were washed and peptide 323-339, of TGF- inhibited OVA-specific activa- restimulated with OVA or anti-CD3 in the absence of IL-2. tion of these TCR transgenic T cells in primary culture in a Under these conditions, TGF-–induced suppression of dose-dependent manner (unpublished data). When TGF- TCR-specific T cell proliferation was abrogated (Fig. 1 C), was included in primary spleen cell cultures with OVA for 1 documenting that IL-2 not only reverses TGF-–induced wk and washed, and the purified transgenic CD4 KJ1-26 anergy, but also abolishes the development of TGF-– T cells were rechallenged to monitor their secondary re- induced CD4 T cell anergy. Thus, in two discrete sys- sponses, the TGF-–pretreated CD4 T cells exhibited tems, TGF- was uniquely able to induce CD4 CD25 profound antigen-specific unresponsiveness to OVA or cells into an anergic state. anti-CD3 in the presence of syngeneic (BALB/c) APCs TCR and TGF- Converts CD4 CD25 T Cells to (Fig. 1 B), in contrast to the vigorous proliferation of con- CD4 CD25 Suppressor T Cells In Vitro. We studied trol cells (Fig. 1 B, OVA  Medium). However, these whether the TGF-–anergized CD4 T cells were func- TGF-–treated transgenic CD4 T cells proliferated nor- tionally active and able to suppress responder T cell prolif- mally to signals from PMA and ionomycin stimulation (Fig. eration. Freshly isolated CD4 CD25 naive T cells were 1 B). Both OVA-specific production of Th1 (Fig. 1, D and cultured with anti-CD3 and APCs in the absence and E, IL-2 and IFN-) and Th2 (Fig. 1 F, IL-4) cytokines were presence of TGF-. After 1 wk, viable CD4 T cells blunted in TGF-–pretreated transgenic CD4 T cells. were stained with FITC–anti-CD25 and sorted with flow This antigen-specific T cell unresponsiveness was not due to cytometry into four populations (Fig. 2 A): Control cell death because TGF-–treated transgenic T cells mani- CD4 CD25 (control 25 ) and CD4 CD25 (control fested similar numbers of apoptotic cells compared with 25 ), and TGF-–treated CD4 CD25 (TGF- 25 ) and control as determined by DNA dye 7-AAD (20, 21) after CD4 CD25 (TGF- 25 ). The individual populations 16-h restimulation with OVA and APCs (7-AAD OVA  were restimulated with anti-CD3 and APCs (Fig. 2 B). Medium [13%] vs. OVA  TGF- [17%]). These data sug- Both control 25 ( H uptake mean CPM 35,661) and gest that TGF-1 induces both antigen-specific Th1 and control 25 (mean CPM 65,057) cells proliferated vig- Th2 cell anergy in transgenic CD4 T cells. orously (Fig. 2 B). In contrast, TGF- 25 exhibited re- Because exogenous IL-2 reportedly blocks T cell anergy duced TCR-triggered proliferation (mean CPM 14,684) (26), we included IL-2 in secondary restimulation cultures as did TGF- 25 cells (Fig. 2 B, mean CPM 2,471). and found that it reversed the antigen-specific suppression Most importantly, when these individual populations were TGF- Induces CD4 CD25 Regulatory T Cells through Induction of Foxp3 1878 contact because no inhibition occurred when TGF-–con- verted suppressor T cells were separated from the responder cells in Transwell™ plates (Fig. S1, E and F). If TGF- was replaced with IL-10, another potent immunoregulatory cy- tokine, in parallel cultures of naive CD4 CD25 T cells for 7 d, the resultant CD4 CD25 T cells (IL-10 25 ) ex- hibited no suppression to normal CD4 T cell proliferation in vitro (Fig. S1, G and H). The data support a role for TGF- in the conversion of CD4 CD25 naive/re- sponder into CD4 CD25 anergic/suppressor T cells. Phenotype of TGF-–induced Anergic/Suppressor CD4 T Cells. We determined whether TGF-–induced anergic/ suppressor T cells exhibited a phenotype comparable to /low that of T (e.g., CD25 , CD45RB ) and intracellular reg CTLA-4 (1, 23). OVA TCR transgenic CD4 T cells were stimulated with OVA in the presence or absence of TGF-. After 1 wk, TGF-–anergized viable CD4 trans- genic T cells were found to be smaller in size (Fig. 3 A), to express CD25 (Fig. 3 B), and to show reduced CD45RB /low (Fig. 3 C, CD45RB ). Next, we focused on the expression of CTLA-4 associ- ated with T (23, 27, 28). After primary culture for 7 d, reg viable transgenic CD4 T cells were isolated and main- tained in complete DMEM for up to 56 h without addition of any growth factors or cytokines to rest the T cells com- pletely. The CD4 T cells were doubly stained with anti– CTLA-4 antibody together with KJ1-26, and both surface and intracellular CTLA-4 levels were examined. Although Figure 2. TGF- converts naive CD4 CD25 T cells to CD4 almost all surviving control CD4 transgenic T cells lost CD25 anergic/suppressor T cells. (A) Schematic for the experi- ment. Freshly isolated B6 CD4 CD25 T cells were stimulated with their intracellular (8%) and surface (1.5%) CTLA-4 as typi- anti-CD3 and APCs in the absence and presence of TGF- for 1 wk. cal resting T cells (Fig. 3 D), TGF-–anergized CD4 Viable CD4 T cells were stained with FITC–anti-CD25 mAb, and four KJ1-26 T cells retained intracellular CTLA-4 (56%), al- populations of cells (control 25 , control 25 , TGF- 25 , and TGF- Plus beit not on the surface (2.2%; Fig. 3 D and not depicted). 25 ) were sorted by FACStar ™ Cell Sorter. (B) The individual popu- 4 5 lations (5 10 ) were restimulated with anti-CD3 and APCs (2 10 ) Similar results were obtained when normal B6 and BALB/c to monitor their proliferative response. The data are shown as mean of CD4 T cells were cultured with anti-CD3 plus TGF- 3 4 duplicate wells of H incorporation (CPM). (C) 1.5 10 freshly iso- (unpublished data). Thus, TGF-–induced anergic/sup- lated CD4 CD25 responder T cells were stimulated with anti-CD3 pressor CD4 T cells exhibit a similar phenotype to T . and APCs in the absence (naive CD25 alone) or presence of the four reg individual populations of cells (5 10 ) to examine their suppressive TGF-–induced Anergic/Suppressor CD4 T Cells Express ability for naive responder T cell activation. The data are representative Cell Membrane–bound Active TGF-. Previous papers have of three experiments. shown that the T express cell membrane–bound TGF-, reg which is involved in T cell contact–dependent immuno- reg suppression (references 29, 30; unpublished data). We ex- cocultured with freshly isolated CD4 CD25 naive re- amined whether TGF-–induced anergic/suppressor T sponder T cells, only the TGF- 25 cells inhibited the cells express increased TGF-. Naive C57BL/6 CD4 anti-CD3–induced responder T cell proliferation (Fig. 2 C; CD25 T cells were stimulated with anti-CD3 and APCs Fig. S1 H, available at http://www.jem.org/cgi/content/ in the presence or absence of TGF- for 3 d, washed full/jem.20030152/DC1). Neither control 25 nor control extensively, and stained doubly with antibody to active 25 had any suppressive action (Fig. 2 C; Fig. S1 H). Inter- TGF- and anti-CD25. As expected, most of the CD4 T estingly, TGF-–treated cells that remained 25 cells also cells (90%) expressed CD25 after TCR stimulation for 3 d. lacked suppressive action to normal T cell proliferation, de- The striking finding was that the majority of TGF-– spite their anergy to TCR stimulation (Fig. 2 B). Similar treated CD25 T cells (50–80%) exhibited cell surface ac- results were obtained in normal BALB/C mice (unpub- tive TGF-, whereas only a few CD25 T cells (3–5%) in lished data). When CD4 CD25 responder T cells were control cultures (anti-CD3 alone) were positive for surface labeled with CFSE and cocultured with TGF- 25 or TGF-, determined by flow cytometry (Fig. 3 E) and im- control 25 T cells, only TGF- 25 , but not control 25 munofluorescence microscopy (Fig. 3 F). To establish a link T cells, blocked CFSE-labeled reduction as a marker of between their increased surface TGF- and functional an- proliferation in CD25 responder cells (Fig. S1, A–D). The ergy to TCR stimulation, anti-CD3 restimulation showed immunosuppressive action in vitro was dependent on cell increased phosphorylation of Smad2/3, which are key me- Chen et al. 1879 Figure 3. Phenotype of TGF- –induced anergic/suppressor T cells. (A–D) DO11.10 TCR transgenic spleen cells were stim- ulated with OVA in the absence (OVA  Med) and presence of TGF- (OVA  TGF-) for 7 d. CD4 T cells were purified and stained with PE–anti-CD4 and FITC–anti-CD25, or FITC– anti-CD45RB. CD4 T cells (98% KJ1-26 ) were gated, and histogram profiles of cell size on FSC (A) and CD25 (B) are displayed. Profile of dual CD4 and CD45RB expression (C) CD4 T cells purified at day 7 after the primary cultures were rested in complete DMEM for 56 h. Viable CD4 T cells were stained with FITC-anti–KJ1-26 and intracellular PE-anti– CTLA-4. (D) TGF-–induced anergic/suppressor T cells express membrane-bound TGF- (E and F). B6 CD4 CD25 T cells were cultured with anti-CD3 and APCs in the absence (panels a and b, CD3  Med) or pres- ence of TGF- (panels c and d, CD3  TGF-) for 3 d. After extensive washes, cells were stained with FITC–anti-CD25 and biotinylated chicken anti– TGF-1, followed by streptavi- din-PE. Cells were analyzed on flow cytometry and under im- munofluorescence microscopy. diators of TGF- signaling (6, 7) and which is indicative of CD25 naive/responder T cells, switching them toward a TGF- signal transduction, by Western blot analysis (Fig. regulatory phenotype. S2 A) or intracellular immunofluorescence staining (Fig. S2 Freshly purified (99%) T (CD25 ), but not CD4 reg B). Furthermore, when anti–TGF- neutralizing monoclo- CD25 (CD25 ) naive responder T cells, in normal nal antibody was included in secondary restimulation cul- C57BL/6 mice express Foxp3 (Fig. 4 A; references 13–15). tures of these anergic T cells, anti–TGF-, but not isotypic When naive responder T cells were stimulated with anti- control antibodies, reversed their proliferation (Fig. S2 C), CD3 and anti-CD28, the Foxp3 gene was not up-regulated as well as IL-2, IFN-, and IL-4 production (not depicted). (Fig. 4 B), consistent with previous findings (13–15). Signif- Anti–TGF- had no apparent effects on control CD4 T icantly, stimulation of naive responder T cells with anti- cells (Fig. S2 C, OVA  Med), consistent with their lack of CD3 and anti-CD28 (Fig. 4 B) or with anti-CD3 and APCs surface TGF- (Fig. 3, E and F). Thus, TGF-–induced (Fig. 4 C) in the presence of active TGF- dramatically in- anergic/suppressor CD4 T cells expressed increased TGF-, duced Foxp3 expression, which was dependent on the levels which likely mediates their own anergic state, as well as of TGF- (Fig. 4 D), whereas TGF- treatment alone their suppression of normal T cell activation. failed to elicit Foxp3 (Fig. 4 B). Similar results were ob- TGF- Induces Foxp3 Expression in CD4 CD25 Naive/ served in BALB/c mice (unpublished data), indicating a Responder T Cells. Although TGF- converts peripheral generality of the effect. By comparison, stimulation of naive CD4 CD25 T cells into anergic/suppressor T cells, Foxp3-expressing T with anti-CD3 and anti-CD28 and a reg the molecular mechanisms by which TGF- drives this high dose of IL-2 (100 U/ml), a regimen for their prolifera- transition are unknown. Transcription factor Foxp3 is a re- tion (1), did not increase constitutive Foxp3 (Fig. 4 E; refer- cently described gene associated with the development of ence 13), nor did the addition of TGF- (Fig. 4 E). Separa- regulatory T cells, and retroviral gene transfer of Foxp3 into tion of TGF- and anti-CD3–cultured CD4 CD25 cells naive responder T cells converts them toward a regulatory (day 7) into CD25 suppressor and CD25 nonsuppressor T cell phenotype (13–15). The physiological inducers for cells by FACS (Fig. 2 A and Fig. 4 F) revealed that the Foxp3 gene expression remain a mystery, but we reasoned TGF-–converted CD25 subset had much higher levels of whether TGF- might trigger Foxp3 expression in CD4 Foxp3 than did the CD25 subpopulation (Fig. 4 G). As ex- TGF- Induces CD4 CD25 Regulatory T Cells through Induction of Foxp3 1880 Figure 4. TGF- and TCR costimulation induces Foxp3 expression in CD4 CD25 naive responder T cells. (A) B6 spleen cells were sorted into CD4 CD25 (CD25 ) and T reg (CD25 ) populations. cDNA from each population was sub- jected to nonsaturating PCR using Foxp3 or HPRT-specific primers, and data are presented as Foxp3/HPRT ratio. (B) CD25 cells were cultured with medium or 2 ng/ml TGF-1 (24 h) or stimulated with platebound anti- CD3 and soluble anti-CD28 in the absence or presence of TGF- 1 (72 h) and assessed for the expression of Foxp3 by RT-PCR. (C) CD25 cells were activated with soluble anti-CD3 and APCs with or without TGF- for 3 d and assessed for Foxp3 expression. (D) Dose depen- dence of TGF- and failure of IL-10 on Foxp3 induction in CD25 naive T cells. CD25 cells were cultured as in B in the presence of indicated concentra- tions of TGF- or recombinant murine IL-10. (E) Both TGF- and IL-10 failed to further enhance Foxp3 expression in T . Freshly isolated T were activated with platebound anti-CD3, soluble anti-CD28, and IL-2 reg reg (100 U/ml) with or without TGF- or IL-10, and Foxp3 expression was assessed by RT-PCR. (F and G) Flow cytometry analysis (F) and Foxp3 expression (G) of CD25 or CD25 T cells sorted from TGF-– and anti-CD3–costimulated naive CD25 T cells (day 7). (H) No Foxp3 expression in both CD25 and CD25 subsets purified from control (anti-CD3 only) stimulated CD25 naive cells (day 7). The data in the figure are representative of at least three experiments. pected, both CD25 and CD25 subsets from control cul- levels of CD25 (70%) and preserved their specific unre- tures had undetectable Foxp3 (Fig. 4 H). On the other sponsiveness to OVA peptide 323-339 restimulation in hand, IL-10 failed to induce Foxp3 expression in naive vitro (unpublished data). CD4 KJ1-26 T cells recovered CD4 CD25 cells (Fig. 4 D) or to further enhance Foxp3 from mice receiving Control Cell had much lower levels in T (Fig. 4 E). Thus, costimulation of Foxp3-negative of CD25 (28%) and proliferated vigorously to OVA pep- reg CD4 CD25 naive responder T cells with TCR engage- tide 323-339 (unpublished data). When cotransferred with ment in the presence of TGF- induces de novo expression naive/responder CD4 transgenic T cells, the TGF- of Foxp3, concomitant with conversion to a population of Cell, but not Control Cell, blocked antigen-specific ex- CD4 CD25 Foxp3 T cells phenotypically and function- pansion of naive CD4 KJ1-26 T cells in draining lymph ally indistinguishable from professional T . nodes (Fig. 5, A–C and E). Importantly, the recovered reg TGF-–induced CD4 Transgenic Anergic/Suppressor T CD4 KJ1-26 T cells from the mice injected with naive Cells Suppress OVA-specific T Cell Proliferation In Vivo. CD4 KJ1-26 cells plus TGF- Cell also expressed much Extending these studies in vivo, first we used an adoptive higher levels of CD25 (Fig. 5 c) compared with that in transfer model (31, 32) in which transgenic CD4 KJ1- mice injected with naive CD4 T cells alone (Fig. 5 a) or 26 T cells were transferred into syngeneic BALB/c mice plus Control Cell (Fig. 5 b). Interestingly, in contrast to followed by OVA peptide immunization. The prolifera- their nonsuppressive features in vitro, IL-10–induced tion of the transgenic CD4 T cells in host mice was CD4 transgenic T cells (IL-10 Cell) suppressed antigen- monitored using clonotypic antibody KJ1-26 (Fig. 5). In driven CD4 KJ1-26 T cell proliferation in vivo (Fig. 5, contrast to their anergic state in vitro, the TGF-–con- D and E). However, the recovered CD4 KJ1-26 T cells verted CD4 CD25 transgenic suppressor T cells (TGF- from mice receiving IL-10 cells expressed reduced CD25 Cell) expanded at similar levels as control CD4 trans- (Fig. 5 d) compared with those from mice receiving TGF- genic T cells (Control Cell) in vivo after OVA peptide Cell (Fig. 5 c). 323-339 immunization (Fig. 5 E and not depicted; with- When draining lymph node cells were restimulated in out immunization, recovered CD4 KJ1-26 T cells were vitro with OVA peptide 323-339, the antigen-specific T 0.1 10 in draining lymph nodes in all groups). Signif- cell proliferation from mice cotransferred with TGF- icantly, the recovered CD4 KJ1-26 T cells from drain- Cell was significantly inhibited compared with that of ing lymph nodes receiving TGF- Cell expressed high mice receiving naive CD4 KJ1-26 T cells alone (Fig. 5 Chen et al. Figure 5. TGF-–converted CD4 sup- pressor T cells inhibited antigen-specific expansion of transgenic CD4 T cells in vivo. Freshly isolated CD4 KJ1-26 T cells (DO11.10 CD4 cells) or with equal number of OVA only (Control cell), plus 2 ng/ml TGF- (TGF- cell) or plus 1 ng/ml IL-10– converted (IL-10 cell) CD25 KJ1-26 T cells were injected i.p into normal BALB/c mice. Some mice were injected with 2.5 10 of Control cell, TGF- cell, or IL-10 cell alone. Mice were immunized with Peptide 323-339 emulsified with IFA on day 2. On day 11, draining lymph nodes (inguinal) were harvested, and cells were stained with Tricolor–anti-CD4, FITC– KJ1-26, and PE–anti-CD25 and analyzed on FACSCalibur™. (A–D) Live cells were gated, and the profiles between CD4 versus KJ1-26 are displayed. The numbers repre- sent CD4 KJ1-26 cell frequency within lymph node cells. The numbers in the paren- theses are injected cells. Each group con- tained two mice and the lymph node cells were pooled before staining. (a–d) CD4 KJ1- 26 T cells in corresponding A–D were gated, and CD25 frequency is shown. (E) Total CD4 KJ1-26 T cells in draining lymph nodes per mouse (percentage of CD4 KJ1-26 the number of total lymph node cells/2). (F) 2 10 lymph node cells labeled with 3 M CFSE were cultured with 1 g/ml Peptide 323-339 in 24-well plates for 72 h. Cells were stained with Tricolor–anti-CD4 and PE–KJ1-26 and analyzed on FACSCalibur™. CD4 KJ1- hi 26 cells were gated, and the CFSE cells are shown. The marker was set according to CFSE fluorescence of the live CD4 KJ1- 26 T cells in parallel cultures with medium only. (G) 2 10 lymph node cells were cultured with Peptide 323-339 in the presence of GolgiPlug™ for 5–6 h. Cells were stained with Tricolor–anti-CD4 and FITC– KJ1-26 antibodies before being fixed and intracellularly stained for IL-4 or IFN- cytokines with respective antibodies (PE-anti–IL-4 or PE-anti–IFN-, 0.5 g/10 cells). 40,000–80,000 cells were acquired on FACSCalibur™, and percentages of KJ1-26 IL-4 or IFN- cells within CD4 T cells are shown. The experiments were repeated twice with similar results. F). The antigen-specific IL-4 and IFN- production in sence or presence of TGF- for 3–4 d, washed extensively, recovered CD4 KJ1-26 T cells was also inhibited in and rested in complete medium containing IL-2 for an ad- mice cotransferred with TGF- suppressor cells (Fig. 5 ditional 3 d. Viable cells were harvested and delivered in- G). The recovered CD4 KJ1-26 T cells from mice travenously into mice immunized and challenged with cotransferred with IL-10 Cell exhibited less inhibition of HDM as depicted in Fig. 6 A. After HDM intraperitoneal OVA-specific T cell proliferation and cytokine produc- immunization and intratracheal challenge, massive inflam- tion (Fig. 5, F and G). Clearly, TGF-–converted CD25 matory cell infiltrates were evident with mucus obstruction T cells have regulatory ability for antigen-induced naive of the airway in the lung (Fig. 6 C), compared with the CD4 T cell activation in vivo, consistent with the func- lung tissues in naive or PBS-injected mice (Fig. 6 B and tional features of T (31, 32), but distinct from IL-10– not depicted). Administration of control CD4 T cells reg induced CD25 T cells. (Control Cell) pretreated with anti-CD3 alone failed to re- TGF-–induced CD4 Anergic/Suppressor T Cells Prevent duce the inflammatory cell infiltration significantly, al- HDM-induced Allergic Pathogenesis In Vivo. The immuno- though mucus was less obvious in the airways (Fig. 6 D). In regulatory effect of TGF-–induced anergic/suppressor contrast, administration of TGF-–converted CD4 sup- CD4 T cells in vivo was further assessed in an antigen- pressor T cells (TGF- cell) dramatically reduced HDM- induced allergic model. C57BL/6 CD4 CD25 naive T induced inflammatory cell infiltration and preserved the cells were cultured with anti-CD3 and APCs in the ab- integrity of airway structures (Fig. 6 E). Cotransfer of TGF- Induces CD4 CD25 Regulatory T Cells through Induction of Foxp3 Figure 6. TGF-–converted-CD4 sup- pressor T cells prevent HDM-induced allergic pathogenesis in vivo. B6 CD4 CD25 T cells were cultured with anti-CD3 and APCs in the absence (Control cell) and presence of TGF- (TGF- cell) for 3–5 d, extensively washed and rested in DMEM complete medium containing 10 U/ml of IL-2 for an additional 3 d. Viable cells were harvested, washed, and resuspended in PBS for injection. For induction of allergic immune responses in lungs, B6 mice were injected with HDM allergen as depicted in schematic plan (A). 4 d after the last HDM intratracheal challenge, mice were killed, and lungs were immediately fixed with 10% PBS-Formalin for immunohistological staining (H&E). One representative lung from each group (three to five mice) is shown. The magnification of the images is 20. (B) Naive lung. (C) HDM-injected lung. (D) HDM-injected lung coinjected 6 5 with Control cell (10 on day 1 and 5 on day 14, i.v.). (E) HDM-injected lungs on day 1 coinjected with TGF- cell (10 and 5 10 on day 14, i.v.). (inset) Boxed images are mucin staining in airways by PAS (40 ). The red staining represents mucin positive cells. (F–H) Spleen cells were harvested and pooled from three to five mice per group. 4 10 spleen cells were cultured with 100 g/ml HDM (white bars) or 0.5 g/ml anti-CD3 (black bars) for 24–96 h, the cell-free supernatants were collected to measure IFN- (F, 48 h), IL-4 (G, 24 h), and IL-13 (H, 96 h), respec- tively, by ELISA. IL-4 levels are shown as OD (OD value of test supernatants  OD value of culture medium; OD 0.107). n.d., not done. Discussion TGF-–converted CD4 suppressor T cells blocked epi- thelial cell mucin production in airways induced by HDM Of many unresolved questions regarding T , where and reg as detected by PAS staining, whereas control CD4 T cells how they are generated remains significant (2, 33). In this re- did not (Fig. 6, B–E; Fig. S3, available at http://www. port, we have provided evidence that T can be induced/ reg jem.org/cgi/content/full/jem.20030152/DC1). Spleen T converted from peripheral CD4 CD25 naive responder T cells from mice treated with HDM and TGF-–induced cells through costimulation of TCR and TGF- signaling. anergic/suppressor CD4 T cells produced much lower Several important conclusions can be drawn from the current levels of HDM-specific, as well as anti-CD3 driven, Th1 work. First, TGF- treatment in the presence of TCR stim- (IFN-) and Th2 (IL-4 and IL-13) cytokines compared ulation converts naive CD4 CD25 responder T cells to an- with mice receiving HDM alone (Fig. 6, F–H). In contrast, ergized CD4 CD25 T cells, but fails to promote growth of spleen T cells from mice treated with HDM and control existing T , at least in vitro. The effect of TGF- on the fate reg CD4 T cells produced higher levels of IFN- (Fig. 6 F), of T cell activation is determined not only by TGF- con- although these cells produced reduced levels of IL-4 and centration, but also by the level of IL-2. Importantly, exoge- IL-13 (Fig. 6, G and H). Thus, TGF-–induced anergic/ nous IL-2 not only reverses unresponsiveness to TCR restim- suppressor CD4 T cells dampen inflammation and associ- ulation of TGF-–anergized CD4 T cells but also ated mucin production in allergic immune responses in antagonizes the induction of CD4 T cell anergy by TGF-. vivo. Second, TGF-1–converted anergic CD4 T cells are Chen et al. 1883 suppressor cells. Phenotypically, TGF-–converted aner- cells under conditions involving either anti-CD3 and anti- gic/suppressor T cells retain surface CD25 and exhibit CD28 costimulation in the absence of APCs or with anti- /low CD45RB , similar to professional T (2, 34). Another CD3 in the presence of APCs, suggesting the dispensability reg key finding is that TGF-–anergized CD4 CD25 T cells of APCs. The Foxp3 induction is dependent on the levels express intracellular CTLA-4 as do T (23, 27, 28). TGF- of TGF-, suggesting the causal influence of TGF-. Sec- reg regulation of intracellular CTLA-4 in CD4 T cells does ond, in the absence of TCR stimulation, TGF- itself fails not appear to involve induction of CTLA-4 synthesis, but to induce or enhance Foxp3 expression, indicating the ne- rather prevention of its degradation (unpublished data). cessity of TCR engagement. Moreover, when CD25 and Functionally, when cocultured with normal CD4 T cells, CD25 subsets were isolated from TGF-– and TCR- TGF-1–anergized T cells inhibit proliferation and cyto- costimulated cultures, CD25 T cells exhibited much higher kine production of the target T cells. This suppressive ac- levels of Foxp3 than the CD25 subset, supporting the no- tivity is mainly attributed to the CD25 subpopulation of tion that Foxp3 may be involved in T immunoregulation reg anergized cells, because TGF-–anergized cells lacking (38). Lastly, TGF- had no further enhancement on exist- CD25, at least in vitro, have no obvious suppressive ability. ing Foxp3 gene in professional T , consistent with the ev- reg Neither CD25 nor CD25 T cells from control cultures idence that Foxp3 expression is stable in T (13). The fail- reg (without TGF-) have suppressive activity, consistent with ure of IL-10 to induce Foxp3 in CD25 naive T cells previous observations (1, 2, 34, 35). The level of immuno- indicates that IL-10 is not a physiological inducer for Foxp3 suppression is dependent on the cell number and reversible expression. Although mechanistically elusive, the observa- by exogenous IL-2. Consistent with T , TGF-–con- tion that IL-10–induced CD25 T cells lack characteristic reg verted CD25 suppressor T cells also require cell contact to suppression in vitro, but are functional in vivo suggests that carry out their suppression, which can be mediated by cell these cells are distinct from TGF-–converted CD25 reg- membrane–bound TGF- (29, 30). ulatory cells. The data are consistent with the notion that The data that TGF-–converted CD4 CD25 T cells IL-10 is more likely a switch for CD4 T cells into Trl cells express cell surface TGF- in active form provide further (39). These discriminating results not only provide a mo- evidence that the professional T can be converted by lecular mechanism for the TGF-–driven transition of na- reg TGF- and TCR costimulation from peripheral naive ive T cells toward regulatory T cells, but also unveil the CD4 T cells. Although not unanimous, increasing evi- first physiological inducer for Foxp3 expression in CD4 dence has demonstrated that T produce/express higher naive/responder T cells, which may enable manipulation of reg levels of TGF-, which plays a significant role in immune this specific population of regulatory cells. regulation in vitro and in vivo (27, 29, 30, 36, 37). That More importantly, these TGF-1–converted CD4 TGF-–converted CD25 suppressor T cells express cell CD25 suppressor cells also inhibit T cell responses surface TGF- not only supports the concept that these when transferred in vivo. Evidence from the OVA trans- suppressor cells possess the same phenotype as the profes- genic adoptive transfer model document that TGF-–con- sional T but also offers an explanation for how profes- verted CD25 suppressor cells resemble T . First, TGF- reg reg sional T are developed. Although how these TGF- sup- –converted CD25 suppressor T cells proliferate in vivo reg pressor T cells acquire surface TGF- remains elusive, it is upon specific antigen immunization, yet preserve their an- conceivable that TGF- enhances its own production as tigen specific anergy when recultured in vitro. Second, evident by exogenous TGF- up-regulation of its own transgenic CD4 KJ1-26 T cells recovered from mice re- mRNA expression in CD4 T cells (reference 7; unpub- ceiving TGF-–converted suppressor cells expressed ele- lished data) and/or TGF- binds onto its cognate receptors vated CD25. Significantly, TGF-–converted KJ1-26 on these suppressor T cells, because TCR stimulation en- CD25 suppressor cells inhibit antigen-driven CD4 T cell hances TGF- receptor expression in CD4 T cells (un- growth in vivo. Again, the recovered CD4 KJ1-26 cells published data). The cell membrane–bound TGF- is from the suppressed draining lymph nodes show high levels likely involved in unresponsiveness to TCR engagement of CD25 and fail to respond to specific antigen stimulation. and suppression of normal T cells by these TGF- anergic/ All these phenotypic and functional features of TGF-– suppressor T cells because restimulation of the suppressor converted suppressor T cells are consistent with those of cells drives a higher level of phosphorylation of Smad2/3, professional T (31, 32). Moreover, TGF-–converted reg and anti–TGF- antibody reverses their anergy and abro- suppressor T cells may carry out their suppression in vivo gates their suppressive action. in a nonspecific manner because TGF-–converted The strongest evidence that TGF- converts CD4 BALB/c CD25 T cells harvested from the primary cul- CD25 naive T cells to CD25 regulatory cells tures exhibit the inhibition of antigen-driven expansion of comes from our surprising findings that TGF- and TCR the transferred CD4 KJ1-26 T cells (unpublished data). costimulation induces transcription factor Foxp3 gene ex- In HDM-induced allergic immune responses in lungs, pression in CD4 CD25 T cells. The observation that TGF-–converted suppressor T cells ameliorate allergen- TGF- induces Foxp3 expression in CD4 CD25 naive T driven inflammatory cell infiltration and mucin production cells has led to several unprecedented conclusions. First, in airways. Anergic/suppressor T cells appear to inhibit TGF- can induce Foxp3 expression in CD4 CD25 T both Th1 and Th2 responses, whereas administration of TGF- Induces CD4 CD25 Regulatory T Cells through Induction of Foxp3 control CD4 cells enhanced Th1 IFN-, which might 7. Chen, W., and S.M. Wahl. 2002. TGF-beta: receptors, sig- naling pathways and autoimmunity. Curr. Dir. Autoimmun. contribute to the inhibition of Th2 cytokines, but failed to 5:62–91. significantly limit the inflammatory response in the lungs, 8. Gorelik, L., and R.A. Flavell. 2000. Abrogation of TGFbeta indicating the complexity of the Th1 effector T cell role in signaling in T cells leads to spontaneous T cell differentiation allergic lung inflammation (40). Interestingly, we also ob- and autoimmune disease. Immunity. 12:171–181. served an increase in TGF- production in the TGF- 9. Lucas, P.J., S.J. Kim, S.J. Melby, and R.E. Gress. 2000. 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To respond or not to in CD4()CD25() regulatory T cell-mediated immuno- respond: T cells in allergic asthma. Nat. Rev. Immunol. 3:405– suppression. Cytokine Growth Factor Rev. 14:85–89. 412. 31. Walker, L.S., A. Chodos, M. Eggena, H. Dooms, and A.K. TGF- Induces CD4 CD25 Regulatory T Cells through Induction of Foxp3 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Experimental Medicine Pubmed Central

Conversion of Peripheral CD4+CD25− Naive T Cells to CD4+CD25+ Regulatory T Cells by TGF-β Induction of Transcription Factor Foxp3

Chen, WanJun; Jin, Wenwen; Hardegen, Neil; Lei, Ke-jian; Li, Li; Marinos, Nancy; McGrady, George; Wahl, Sharon M.
The Journal of Experimental Medicine , Volume 198 (12) – Dec 15, 2003
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Copyright © 2003, The Rockefeller University Press
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0022-1007
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1540-9538
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10.1084/jem.20030152
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Abstract

CD4 CD25 regulatory T cells (T ) are instrumental in the maintenance of immunological reg tolerance. One critical question is whether T can only be generated in the thymus or can reg differentiate from peripheral CD4 CD25 naive T cells. In this paper, we present novel evi- dence that conversion of naive peripheral CD4 CD25 T cells into anergic/suppressor cells /low that are CD25 , CD45RB and intracellular CTLA-4 can be achieved through costimu- lation with T cell receptors (TCRs) and transforming growth factor  (TGF-). Although transcription factor Foxp3 has been shown recently to be associated with the development of T , the physiological inducers for Foxp3 gene expression remain a mystery. TGF- induced reg Foxp3 gene expression in TCR-challenged CD4 CD25 naive T cells, which mediated their transition toward a regulatory T cell phenotype with potent immunosuppressive potential. These converted anergic/suppressor cells are not only unresponsive to TCR stimulation and produce neither T helper cell 1 nor T helper cell 2 cytokines but they also express TGF- and inhibit normal T cell proliferation in vitro. More importantly, in an ovalbumin peptide TCR transgenic adoptive transfer model, TGF-–converted transgenic CD4 CD25 suppressor cells proliferated in response to immunization and inhibited antigen-specific naive CD4 T cell expansion in vivo. Finally, in a murine asthma model, coadministration of these TGF-– induced suppressor T cells prevented house dust mite–induced allergic pathogenesis in lungs. Key words: anergy • IL-10 • OVA TCR transgenic • house dust mite • asthma Introduction CD4 CD25 regulatory T cells (T ) have emerged as a in the periphery (4, 5). One crucial question is whether reg unique population of suppressor T cells that maintain periph- T can be induced or converted from normal peripheral reg eral immune tolerance (1, 2). Although the immunoregula- CD4 T cells and if this occurs, which molecules and/or tory ability of T is no longer contested, where and how cytokines are responsible for the transition because acti- reg this population of CD4 T cells is generated and developed vated CD4 T cells expressing CD25 under neutral TCR still remains largely unknown. The major debate centers on stimulation conditions show no suppressive ability (1, 2). whether T are generated only in the thymus from a defined TGF- is a critical factor in regulation of T cell–mediated reg lineage and/or whether these cells represent a stage that immune responses and in the induction of immune toler- different types of CD4 T cells can acquire. Although most ance (for reviews see references 6, 7). When the TGF-1– analyses emphasize the thymus as the sole incubator for T mediated inhibitory pathway is abrogated specifically in T reg (3, 4), recent evidence suggests that T may also be induced cells by restrictive expression of dominant negative TGF- reg receptor II (8, 9), these mice develop unchecked T cell proliferation and inflammatory and autoimmune-like diseases, The online version of this article includes supplemental material. Address correspondence to WanJun Chen, Cellular Immunology Sec- tion, Oral Infection and Immunity Branch, National Institute of Dental Abbreviations used in this paper: 7-AAD, 7-amino-actinomycin D; CFSE, and Craniofacial Research, National Institutes of Health, Bethesda, carboxy-fluorescein diacetate succinimidyl ester; HDM, house dust mite; MD 20892. Phone: (301) 435-7168; Fax: (301) 402-1064; email: HPRT, hypoxanthineguanine phosphoribosyl transferase; PAS, periodic [email protected]; or Sharon M. Wahl. Phone: (301) 496-4178; acid schiff; P-Smad2/3, phosphorylated Smad2/3; T , CD4 CD25 reg email: [email protected] regulatory T cells. 1875 The Journal of Experimental Medicine • Volume 198, Number 12, December 15, 2003 1875–1886 http://www.jem.org/cgi/doi/10.1084/jem.20030152 The Journal of Experimental Medicine documenting a TGF-–dependent signal in T cell activa- synthesized and purified by reverse phase–HPLC (Synpep Cor- poration). The purity of the peptides is 98%. The following tion and tolerance in vivo. Although TGF- regulation of reagents were obtained from BD Biosciences: purified rat anti– immune responsiveness has been validated in vitro and in mouse mAbs to IL-2, IL-4, and IFN-, anti-CD28, FITC–anti- vivo, how TGF- accomplishes its suppressive role in T CD45RB, recombinant mouse IL-2, IL-4, and IFN-, PE- or cell activation remains unclear. A link between TGF- and purified anti-CD3 (145-2C11, NA/LE™), PE-anti–CTLA-4, induction of T has also been suggested in humans (10), reg hamster IgG isotypic control, FITC- or biotinylated antimurine but whether TGF- converts naive T cells to regulatory CD25 (Clone 7D4), FITC–rat IgM, PE- or purified anti-CD25 cells and, most importantly, the underlying molecular (Clone PC61, NA/LE™), and anti-FcRII/III. FITC or PE-anti– mechanisms await revelation. mouse CD4, anti-CD8, and the respective isotypic control In exciting new papers, Foxp3, which encodes a tran- mAbs and streptavidin-FITC, PE, or Tricolor were purchased scription factor that is genetically defective in an autoim- from Caltag. Recombinant human TGF-1, anti–TGF-1, 2,3 mAb, biotinylated chicken anti–TGF-1, and recombinant mu- mune syndrome in humans and mice (11, 12), has been rine IL-10 were purchased from R&D Systems. 7-amino-actino- shown to be not only specifically expressed in professional mycin D (7-AAD) was purchased from Calbiochem. T , but also required for their development (13–15). Nei- reg Cell Preparation. Spleens were gently minced in complete ther naive nor activated CD4 CD25 responder T cells DMEM containing 10% FBS (BioWhittaker), and CD4 T cells express Foxp3, distinguishing Foxp3 from other T associ- reg were purified using a mouse CD4 T cell column system (R&D ated molecules (CD25, CTLA-4, and GITR) that can be Systems; references 20, 21). T cell–depleted or whole spleen cells acquired in CD4 CD25 responder T cells once activated. (irradiated, 3,000 rad) of BALB/c or C57BL6 mice were used as Foxp3/Scurfin-deficient mice develop massive autoim- APCs as indicated. To isolate CD4 CD25 T cells, anti-CD25 mune and inflammatory disease, which shares many patho- antibody (PC61, 1 g/10 cells) was added into the antibody genetic features of mice deficient in CTLA-4 (16, 17) or cocktail and incubated with spleen cells before separation on the CD4 T cell column, to yield a purity of CD4 CD25 T cells TGF- (18, 19). Significantly, gene transfer of Foxp3 con- 90%. For T , the CD4 T cells were incubated with FITC– verts naive CD4 CD25 T cells toward a regulatory T cell reg anti-CD25 (1 g/10 cells; antibody depleted of sodium azide by phenotype similar to that of the professional T (13–15). reg dialysis in PBS overnight) in 2% PBS-FBS for 30 min at 4 C, Although the identification of Foxp3 in T has greatly ex- reg washed, resuspended in X-Vivo 20 serum-free medium (Bio- panded our ability to decipher the development and func- Whittaker), and the T and CD4 CD25 T cells were purified reg tion of this unique population of regulatory T cells, how Plus using a FACStar ™ Cell Sorter (Becton Dickinson). The purity Foxp3 is controlled and by what physiological inducers re- of sorted cells was 95–99%. main mysteries to be solved. Cell Culture and T Cell Proliferation Assay. For normal In this paper, we present evidence that TGF- converts C57BL/6 CD4 T cell primary stimulation, purified CD4 , naive CD4 CD25 T cells into CD4 CD25 anergic/sup- CD4 CD25 , or T were stimulated with 0.5 g/ml anti-CD3 reg pressor T cells in the periphery. These suppressor T cells not in the presence of APCs in complete DMEM at 37 C and 5% CO for 7–10 d and CD4 T cells were isolated for further studies. In only exhibit unresponsiveness to TCR stimulation, but also some studies, 2 ng/ml TGF-1 was included in the cultures as in- suppress normal CD4 T cell activation and Th1 and Th2 dicated. In other experiments, CD4 CD25 or CD25 cells were cytokine production in vitro. Moreover, these suppressor T stimulated with 2 g/ml of platebound anti-CD3 and 2 g/ml cells also inhibit immune responses in vivo, as evident in soluble anti-CD28 in the absence or presence of 0.02, 0.2, 2, or 20 their striking prevention of allergic pathogenesis in a house ng/ml TGF-1, 1 ng/ml IL-10, or 100 U/ml IL-2 for 3 d. dust mite (HDM)–induced mouse asthma model and in their For DO11.10 TCR transgenic mice, spleen cells (2 significant suppression of antigen-specific CD4 T cell pro- cells/ml) were stimulated with 100 g/ml OVA in the absence liferation in an OVA peptide CD4 transgenic T cell adop- or presence of 2 ng/ml recombinant TGF-1 or 1 ng/ml murine tive transfer model. Significantly, we also show that TGF- IL-10 in complete DMEM for 7–10 d. Cells were harvested and conversion of naive CD4 CD25 T cells into CD4 CD25 extensively washed, and dead cells were eliminated by centrifuga- suppressor T cells involves induction of Foxp3 expression, tion over Ficoll-Paque (Amersham Biosciences). For secondary stimulation, CD4 cells purified from the primary cultures were the first identification of an inducer of this pathway, which restimulated with OVA or anti-CD3 in the presence of APCs as offers an opportunity to control tolerance. indicated. For activation with PMA, CD4 T cells were incu- bated with 5 ng/ml PMA and 250 ng/ml ionomycin. Cells were cultured at 37 C in 5% CO for 72 h and pulsed with 1 Ci Materials and Methods 3 [ H]thymidine for the last 6–16 h. Radioactivity incorporated was counted using a flatbed  counter (Wallac). In some experi- Mice. Normal BALB/c and DO11.10 transgenic mice ex- pressing a TCR with specificity for chicken OVA peptide 323- ments, 10 U/ml rIL-2 or 20 g/ml anti–TGF-1, 2,3 mAb and its isotypic antibody control were added at the beginning of the 339 were purchased from The Jackson Laboratory and provided by B. Kelsall (National Institute of Allergy and Infectious Dis- secondary culture. Cytokine Induction and Determination. For cytokine induc- eases, Bethesda, MD). C57BL/6 mice were obtained from The Jackson Laboratory and from in-house breeding at the National tion, cells were cultured with antigens or antibodies as indicated in complete DMEM for 24–96 h. Cell-free supernatants were Institute of Dental and Craniofacial Research. Antibodies and Reagents. Crystallized chicken OVA, hen egg collected for the determination of IL-2, IFN-, IL-4, and IL-13 production by ELISA using paired mAbs specific for the corre- lysozyme, PMA, and ionomycin were purchased from Sigma- Aldrich. OVA peptide 323-339 (ISQAVHAAHAEINEAGR) was sponding cytokines (BD Biosciences) or the respective ELISA kits TGF- Induces CD4 CD25 Regulatory T Cells through Induction of Foxp3 1876 (Biosource International and R&D Systems). A standard curve RT-PCR for Foxp3 Expression. RT-PCR was performed as was generated using known amounts of the respective purified described previously (13), except the number of cycles was 28 for recombinant murine cytokines. Foxp3. The primer sequences were as follows and synthesized by Coculture of CD4 T Cells. Freshly isolated C57BL/6 or Invitrogen: HPRT, 5 -GTTGGATACAGGCCAGACTTTG- BALB/c CD4 CD25 responder T cells were cultured in TTG-3 and 5 -GATTCAACTTGCTCTCATCTTAGGC-3 ; 96-well plates with anti-CD3, splenic APCs, and indicated and Foxp3, 5 -CAGCTGCCTACAGTGCCCCTAG-3 and 5 - CD4 CD25 cells for 72 h for proliferation assay. Transwell ex- CATTTGCCAGCAGTGGGTAG-3 . Normalized values for Foxp3 periments were performed as described previously (22). In brief, mRNA expression in each sample were calculated as the relative CD4 CD25 responder T cells were cultured in 24-well plates quantity of Foxp3 divided by the relative quantity of hypoxan- with APCs and 0.5 g/ml anti-CD3 in the presence or absence thineguanine phosphoribosyl transferase (HPRT). of TGF-–converted or control CD4 CD25 cells in the Trans- Statistical Analysis. Student’s t tests were used for the signifi- well™ (0.4 M pore size; Costar). For carboxy-fluorescein diac- cance of data comparison. etate succinimidyl ester (CFSE)–labeling assay, 10 cells/ml T Online Supplemental Material. Fig. S1 shows that TGF-– cells were incubated with 3 M CFSE in plain DMEM (without converted CD25 suppressor cells require cell contact to carry phenol red) at 37 C for 15 min. Cells were washed three times out their action, and that IL-10–induced CD25 T cells fail to and resuspended in complete DMEM for cell culture. suppress normal CD4 T cell proliferation in vitro. Fig. S2 shows Flow Cytometry Analysis. T cells were resuspended in PBS increased P-Smad2/3 expression in TGF-–converted CD25 containing 1% BSA (Irvine) and 0.1% sodium azide (Sigma- suppressor cells and that anti–TGF- reverses TGF-–induced Aldrich). For the staining of surface antigens, cells were incubated anergy of CD4 T cells. Fig. S3 shows that cotransfer of TGF-– with FITC-, PE-, or Tricolor-conjugated mAbs or their negative converted CD4 suppressor T cells blocked epithelial cell mucin control antibodies as indicated for 30 min on ice. For surface ac- production in airways in lungs induced by HDM as detected by tive TGF- staining, cells were stained with biotinylated chicken PAS staining. Online supplemental material is available at http:// anti–TGF-1 and FITC–anti-CD25 for 30 min, followed by PE- www.jem.org/cgi/content/full/jem.20030152/DC1. streptavidin incubation for an additional 30 min at 4 C. Intracel- lular staining of CTLA-4 was performed as described previously (23). Intracellular staining of phosphorylated Smad2/3 (P-Smad2/3) Results was performed as described in the figure legend for Fig. TCR and TGF- Costimulation Induces CD4 CD25 T S2 (available at http://www.jem.org/cgi/content/full/jem. 20030152/DC1. Cell Anergy, but Fails to Expand Professional T . Although reg Adoptive Transfer Experiments. Freshly isolated CD4 KJ1- TGF- suppression of normal CD4 T cell activation has 26 transgenic T cells were injected i.p. into unirradiated synge- been validated, it remains unclear whether TGF- action is neic BALB/c recipients. Some mice were cotransferred i.p. with through induction of CD4 CD25 T cell anergy or by pro- control, TGF-–, or IL-10–pretreated naive CD4 KJ1-26 T moting growth of existing T . First, we studied whether reg cells. Animals were immunized s.c. with 100 g of P323-339 TCR and TGF- costimulation could expand a population emulsified in IFA (Difco). Cells from draining lymph nodes (in- of existing T in vitro. As expected, freshly isolated T reg reg guinal) were harvested at indicated time points and stained ex were unresponsive to anti-CD3 stimulation, but could be vivo with Tricolor–anti-CD4, PE–anti-CD25, and FITC–KJ1- coerced to proliferate upon stimulation with anti-CD3 in 26 mAbs to determine the expansion of CD4 KJ1-26 T cells in the presence of a high dose of exogenous IL-2 (100 U/ml; vivo. For in vitro restimulation assay, draining lymph node cells were cultured with OVA peptide 323-339 for 3 d to determine references 1, 2; unpublished data). However, after primary T cell proliferation or for 6 h in the presence of GolgiPlug™ culture with anti-CD3 only or with anti-CD3 and TGF- (BD Biosciences) to determine intracellular cytokines as de- in the presence of APCs for 7 d, most of the T died. There reg scribed previously (24). was no increase in the number of T in TGF-–treated cul- reg HDM-induced Allergic Pathogenesis. Allergen-induced asthma tures, but the surviving anti-CD3 plus TGF-–treated T , reg was induced as described previously (25) with some modifica- similar to only anti-CD3–treated cells, remained unrespon- tions. In brief, 6–8-wk-old C57BL/6 mice were immunized by sive to anti-CD3 restimulation (Fig. 1 A), indicating that ex- i.p. injection of 10 g HDM antigen (Greer Laboratories) in 0.1 ogenous TGF- in vitro in the absence of other factors, such ml PBS or PBS alone (unpublished data) at days 1 and 7, followed as IL-2, was insufficient to expand the original T . reg by intratracheal challenge with 100 g HDM antigen in 40 l Alternatively, to determine whether TGF- could aner- PBS or an equivalent volume of PBS as a control at days 14 and gize CD4 CD25 naive T cells, purified CD4 CD25 T 21, respectively. 4 d after the last challenge, mice were killed, and tissues were harvested for immunohistopathologic analysis or in cells from spleens of normal C57BL/6 (Fig. 1) or BALB/c vitro cultures. The mucin expression in the airways was deter- (not depicted) mice were cultured with anti-CD3 and APCs mined with periodic acid schiff (PAS) staining (25). Where indi- in the presence and absence of TGF- for 1 wk, harvested, cated, TGF-–anergized or control CD4 T cells were injected washed, and restimulated with anti-CD3 and APCs. Consis- i.v. into the mice on day 1 and again on day 14. tent with previous papers (1, 2), control CD4 T cells, pre- Western Blot Analysis. Western blot analysis was performed cultured with anti-CD3 and APCs (neutral stimulation as described previously (24) with an antibody to P-Smad2/3 (rab- condition), proliferated vigorously in response to TCR re- bit polyclonal IgG, 1:200) or to Smad2/3 (goat polyclonal IgG, stimulation (Fig. 1 A). In contrast, anti-CD3 plus TGF-– 1:200; Santa Cruz Biotechnology, Inc.) followed by horseradish pretreated cells were unresponsive to TCR restimulation peroxidase–conjugated goat anti–rabbit IgG or donkey anti–goat (Fig. 1 A) without increased apoptosis (not depicted), consis- IgG (Santa Cruz Biotechnology, Inc.) as recommended by the tent with TGF- induction of CD4 CD25 T cell anergy. manufacturer. Chen et al. 1877 Figure 1. Costimulation of TCR and TGF- induces CD4 CD25 T cell anergy, but fails to expand existing T . (A) reg C57BL/6 CD25 naive cells or T (5 reg 10 ) were cultured (primary) with anti-CD3 and APCs (2 10 ) in the absence (CD3 med) and presence (CD3  TGF-) of 2 ng/ml TGF- for 7 d. 3 10 harvested viable CD4 responder T cells or 5 T were restimulated with anti-CD3 and reg APCs for 72 h to monitor their prolifera- tion. The data are representative of three separate experiments. (B–F) TGF- induces OVA TCR transgenic CD4 T cells (KJ1- 26 ) anergy. 2 10 cells/ml spleen cells were cultured with OVA in the presence or absence of TGF- for 7–10 d (primary). Viable CD4 T cells were purified and re- stimulated with 100 g/ml OVA, 100 g/ml hen egg lysozyme (HEL), 1 g/ml anti- CD3 mAb, or 10 U/ml IL-2 as indicated in the presence of BALB/c APCs or with PMA and ionomycin (secondary stimulation). The values are expressed as mean SD of triplicate wells for H incorporation (CPM, 10 T cells) (B and C) or of duplicate wells of the ELISA (D–F, 2 10 T cells). (B) TGF- induces transgenic CD4 TCR-specific anergy. (C) Inclusion of exogenous IL-2 in primary cultures blocks TGF-–induced CD4 T cell anergy. (D–F) TGF- induces both Th1 and Th2 cell anergy. Cytokine levels of IL-2 (D), IFN- (E), and IL-4 (F) in secondary culture supernatants (after 24–48 h) were determined by ELISA. The data shown were repeated from two to six times with similar results. Extending this study to CD4 T stimulation with a spe- of T cell proliferation (Fig. 1 B) and Th1 and Th2 cytokine cific peptide antigen in TCR transgenic mice, similar results production in TGF-–anergized T cells (Fig. 1, D–F). To were obtained. Using TCR transgenic mice DO11.10 in study IL-2 effects on the induction phase of the TGF-– which the majority of the peripheral CD4 T cells express induced anergy, IL-2 was included in the primary culture clonotypic TCR (KJ1-26 , V 8.2) recognizing the OVA with OVA plus TGF- for 7 d, and cells were washed and peptide 323-339, of TGF- inhibited OVA-specific activa- restimulated with OVA or anti-CD3 in the absence of IL-2. tion of these TCR transgenic T cells in primary culture in a Under these conditions, TGF-–induced suppression of dose-dependent manner (unpublished data). When TGF- TCR-specific T cell proliferation was abrogated (Fig. 1 C), was included in primary spleen cell cultures with OVA for 1 documenting that IL-2 not only reverses TGF-–induced wk and washed, and the purified transgenic CD4 KJ1-26 anergy, but also abolishes the development of TGF-– T cells were rechallenged to monitor their secondary re- induced CD4 T cell anergy. Thus, in two discrete sys- sponses, the TGF-–pretreated CD4 T cells exhibited tems, TGF- was uniquely able to induce CD4 CD25 profound antigen-specific unresponsiveness to OVA or cells into an anergic state. anti-CD3 in the presence of syngeneic (BALB/c) APCs TCR and TGF- Converts CD4 CD25 T Cells to (Fig. 1 B), in contrast to the vigorous proliferation of con- CD4 CD25 Suppressor T Cells In Vitro. We studied trol cells (Fig. 1 B, OVA  Medium). However, these whether the TGF-–anergized CD4 T cells were func- TGF-–treated transgenic CD4 T cells proliferated nor- tionally active and able to suppress responder T cell prolif- mally to signals from PMA and ionomycin stimulation (Fig. eration. Freshly isolated CD4 CD25 naive T cells were 1 B). Both OVA-specific production of Th1 (Fig. 1, D and cultured with anti-CD3 and APCs in the absence and E, IL-2 and IFN-) and Th2 (Fig. 1 F, IL-4) cytokines were presence of TGF-. After 1 wk, viable CD4 T cells blunted in TGF-–pretreated transgenic CD4 T cells. were stained with FITC–anti-CD25 and sorted with flow This antigen-specific T cell unresponsiveness was not due to cytometry into four populations (Fig. 2 A): Control cell death because TGF-–treated transgenic T cells mani- CD4 CD25 (control 25 ) and CD4 CD25 (control fested similar numbers of apoptotic cells compared with 25 ), and TGF-–treated CD4 CD25 (TGF- 25 ) and control as determined by DNA dye 7-AAD (20, 21) after CD4 CD25 (TGF- 25 ). The individual populations 16-h restimulation with OVA and APCs (7-AAD OVA  were restimulated with anti-CD3 and APCs (Fig. 2 B). Medium [13%] vs. OVA  TGF- [17%]). These data sug- Both control 25 ( H uptake mean CPM 35,661) and gest that TGF-1 induces both antigen-specific Th1 and control 25 (mean CPM 65,057) cells proliferated vig- Th2 cell anergy in transgenic CD4 T cells. orously (Fig. 2 B). In contrast, TGF- 25 exhibited re- Because exogenous IL-2 reportedly blocks T cell anergy duced TCR-triggered proliferation (mean CPM 14,684) (26), we included IL-2 in secondary restimulation cultures as did TGF- 25 cells (Fig. 2 B, mean CPM 2,471). and found that it reversed the antigen-specific suppression Most importantly, when these individual populations were TGF- Induces CD4 CD25 Regulatory T Cells through Induction of Foxp3 1878 contact because no inhibition occurred when TGF-–con- verted suppressor T cells were separated from the responder cells in Transwell™ plates (Fig. S1, E and F). If TGF- was replaced with IL-10, another potent immunoregulatory cy- tokine, in parallel cultures of naive CD4 CD25 T cells for 7 d, the resultant CD4 CD25 T cells (IL-10 25 ) ex- hibited no suppression to normal CD4 T cell proliferation in vitro (Fig. S1, G and H). The data support a role for TGF- in the conversion of CD4 CD25 naive/re- sponder into CD4 CD25 anergic/suppressor T cells. Phenotype of TGF-–induced Anergic/Suppressor CD4 T Cells. We determined whether TGF-–induced anergic/ suppressor T cells exhibited a phenotype comparable to /low that of T (e.g., CD25 , CD45RB ) and intracellular reg CTLA-4 (1, 23). OVA TCR transgenic CD4 T cells were stimulated with OVA in the presence or absence of TGF-. After 1 wk, TGF-–anergized viable CD4 trans- genic T cells were found to be smaller in size (Fig. 3 A), to express CD25 (Fig. 3 B), and to show reduced CD45RB /low (Fig. 3 C, CD45RB ). Next, we focused on the expression of CTLA-4 associ- ated with T (23, 27, 28). After primary culture for 7 d, reg viable transgenic CD4 T cells were isolated and main- tained in complete DMEM for up to 56 h without addition of any growth factors or cytokines to rest the T cells com- pletely. The CD4 T cells were doubly stained with anti– CTLA-4 antibody together with KJ1-26, and both surface and intracellular CTLA-4 levels were examined. Although Figure 2. TGF- converts naive CD4 CD25 T cells to CD4 almost all surviving control CD4 transgenic T cells lost CD25 anergic/suppressor T cells. (A) Schematic for the experi- ment. Freshly isolated B6 CD4 CD25 T cells were stimulated with their intracellular (8%) and surface (1.5%) CTLA-4 as typi- anti-CD3 and APCs in the absence and presence of TGF- for 1 wk. cal resting T cells (Fig. 3 D), TGF-–anergized CD4 Viable CD4 T cells were stained with FITC–anti-CD25 mAb, and four KJ1-26 T cells retained intracellular CTLA-4 (56%), al- populations of cells (control 25 , control 25 , TGF- 25 , and TGF- Plus beit not on the surface (2.2%; Fig. 3 D and not depicted). 25 ) were sorted by FACStar ™ Cell Sorter. (B) The individual popu- 4 5 lations (5 10 ) were restimulated with anti-CD3 and APCs (2 10 ) Similar results were obtained when normal B6 and BALB/c to monitor their proliferative response. The data are shown as mean of CD4 T cells were cultured with anti-CD3 plus TGF- 3 4 duplicate wells of H incorporation (CPM). (C) 1.5 10 freshly iso- (unpublished data). Thus, TGF-–induced anergic/sup- lated CD4 CD25 responder T cells were stimulated with anti-CD3 pressor CD4 T cells exhibit a similar phenotype to T . and APCs in the absence (naive CD25 alone) or presence of the four reg individual populations of cells (5 10 ) to examine their suppressive TGF-–induced Anergic/Suppressor CD4 T Cells Express ability for naive responder T cell activation. The data are representative Cell Membrane–bound Active TGF-. Previous papers have of three experiments. shown that the T express cell membrane–bound TGF-, reg which is involved in T cell contact–dependent immuno- reg suppression (references 29, 30; unpublished data). We ex- cocultured with freshly isolated CD4 CD25 naive re- amined whether TGF-–induced anergic/suppressor T sponder T cells, only the TGF- 25 cells inhibited the cells express increased TGF-. Naive C57BL/6 CD4 anti-CD3–induced responder T cell proliferation (Fig. 2 C; CD25 T cells were stimulated with anti-CD3 and APCs Fig. S1 H, available at http://www.jem.org/cgi/content/ in the presence or absence of TGF- for 3 d, washed full/jem.20030152/DC1). Neither control 25 nor control extensively, and stained doubly with antibody to active 25 had any suppressive action (Fig. 2 C; Fig. S1 H). Inter- TGF- and anti-CD25. As expected, most of the CD4 T estingly, TGF-–treated cells that remained 25 cells also cells (90%) expressed CD25 after TCR stimulation for 3 d. lacked suppressive action to normal T cell proliferation, de- The striking finding was that the majority of TGF-– spite their anergy to TCR stimulation (Fig. 2 B). Similar treated CD25 T cells (50–80%) exhibited cell surface ac- results were obtained in normal BALB/C mice (unpub- tive TGF-, whereas only a few CD25 T cells (3–5%) in lished data). When CD4 CD25 responder T cells were control cultures (anti-CD3 alone) were positive for surface labeled with CFSE and cocultured with TGF- 25 or TGF-, determined by flow cytometry (Fig. 3 E) and im- control 25 T cells, only TGF- 25 , but not control 25 munofluorescence microscopy (Fig. 3 F). To establish a link T cells, blocked CFSE-labeled reduction as a marker of between their increased surface TGF- and functional an- proliferation in CD25 responder cells (Fig. S1, A–D). The ergy to TCR stimulation, anti-CD3 restimulation showed immunosuppressive action in vitro was dependent on cell increased phosphorylation of Smad2/3, which are key me- Chen et al. 1879 Figure 3. Phenotype of TGF- –induced anergic/suppressor T cells. (A–D) DO11.10 TCR transgenic spleen cells were stim- ulated with OVA in the absence (OVA  Med) and presence of TGF- (OVA  TGF-) for 7 d. CD4 T cells were purified and stained with PE–anti-CD4 and FITC–anti-CD25, or FITC– anti-CD45RB. CD4 T cells (98% KJ1-26 ) were gated, and histogram profiles of cell size on FSC (A) and CD25 (B) are displayed. Profile of dual CD4 and CD45RB expression (C) CD4 T cells purified at day 7 after the primary cultures were rested in complete DMEM for 56 h. Viable CD4 T cells were stained with FITC-anti–KJ1-26 and intracellular PE-anti– CTLA-4. (D) TGF-–induced anergic/suppressor T cells express membrane-bound TGF- (E and F). B6 CD4 CD25 T cells were cultured with anti-CD3 and APCs in the absence (panels a and b, CD3  Med) or pres- ence of TGF- (panels c and d, CD3  TGF-) for 3 d. After extensive washes, cells were stained with FITC–anti-CD25 and biotinylated chicken anti– TGF-1, followed by streptavi- din-PE. Cells were analyzed on flow cytometry and under im- munofluorescence microscopy. diators of TGF- signaling (6, 7) and which is indicative of CD25 naive/responder T cells, switching them toward a TGF- signal transduction, by Western blot analysis (Fig. regulatory phenotype. S2 A) or intracellular immunofluorescence staining (Fig. S2 Freshly purified (99%) T (CD25 ), but not CD4 reg B). Furthermore, when anti–TGF- neutralizing monoclo- CD25 (CD25 ) naive responder T cells, in normal nal antibody was included in secondary restimulation cul- C57BL/6 mice express Foxp3 (Fig. 4 A; references 13–15). tures of these anergic T cells, anti–TGF-, but not isotypic When naive responder T cells were stimulated with anti- control antibodies, reversed their proliferation (Fig. S2 C), CD3 and anti-CD28, the Foxp3 gene was not up-regulated as well as IL-2, IFN-, and IL-4 production (not depicted). (Fig. 4 B), consistent with previous findings (13–15). Signif- Anti–TGF- had no apparent effects on control CD4 T icantly, stimulation of naive responder T cells with anti- cells (Fig. S2 C, OVA  Med), consistent with their lack of CD3 and anti-CD28 (Fig. 4 B) or with anti-CD3 and APCs surface TGF- (Fig. 3, E and F). Thus, TGF-–induced (Fig. 4 C) in the presence of active TGF- dramatically in- anergic/suppressor CD4 T cells expressed increased TGF-, duced Foxp3 expression, which was dependent on the levels which likely mediates their own anergic state, as well as of TGF- (Fig. 4 D), whereas TGF- treatment alone their suppression of normal T cell activation. failed to elicit Foxp3 (Fig. 4 B). Similar results were ob- TGF- Induces Foxp3 Expression in CD4 CD25 Naive/ served in BALB/c mice (unpublished data), indicating a Responder T Cells. Although TGF- converts peripheral generality of the effect. By comparison, stimulation of naive CD4 CD25 T cells into anergic/suppressor T cells, Foxp3-expressing T with anti-CD3 and anti-CD28 and a reg the molecular mechanisms by which TGF- drives this high dose of IL-2 (100 U/ml), a regimen for their prolifera- transition are unknown. Transcription factor Foxp3 is a re- tion (1), did not increase constitutive Foxp3 (Fig. 4 E; refer- cently described gene associated with the development of ence 13), nor did the addition of TGF- (Fig. 4 E). Separa- regulatory T cells, and retroviral gene transfer of Foxp3 into tion of TGF- and anti-CD3–cultured CD4 CD25 cells naive responder T cells converts them toward a regulatory (day 7) into CD25 suppressor and CD25 nonsuppressor T cell phenotype (13–15). The physiological inducers for cells by FACS (Fig. 2 A and Fig. 4 F) revealed that the Foxp3 gene expression remain a mystery, but we reasoned TGF-–converted CD25 subset had much higher levels of whether TGF- might trigger Foxp3 expression in CD4 Foxp3 than did the CD25 subpopulation (Fig. 4 G). As ex- TGF- Induces CD4 CD25 Regulatory T Cells through Induction of Foxp3 1880 Figure 4. TGF- and TCR costimulation induces Foxp3 expression in CD4 CD25 naive responder T cells. (A) B6 spleen cells were sorted into CD4 CD25 (CD25 ) and T reg (CD25 ) populations. cDNA from each population was sub- jected to nonsaturating PCR using Foxp3 or HPRT-specific primers, and data are presented as Foxp3/HPRT ratio. (B) CD25 cells were cultured with medium or 2 ng/ml TGF-1 (24 h) or stimulated with platebound anti- CD3 and soluble anti-CD28 in the absence or presence of TGF- 1 (72 h) and assessed for the expression of Foxp3 by RT-PCR. (C) CD25 cells were activated with soluble anti-CD3 and APCs with or without TGF- for 3 d and assessed for Foxp3 expression. (D) Dose depen- dence of TGF- and failure of IL-10 on Foxp3 induction in CD25 naive T cells. CD25 cells were cultured as in B in the presence of indicated concentra- tions of TGF- or recombinant murine IL-10. (E) Both TGF- and IL-10 failed to further enhance Foxp3 expression in T . Freshly isolated T were activated with platebound anti-CD3, soluble anti-CD28, and IL-2 reg reg (100 U/ml) with or without TGF- or IL-10, and Foxp3 expression was assessed by RT-PCR. (F and G) Flow cytometry analysis (F) and Foxp3 expression (G) of CD25 or CD25 T cells sorted from TGF-– and anti-CD3–costimulated naive CD25 T cells (day 7). (H) No Foxp3 expression in both CD25 and CD25 subsets purified from control (anti-CD3 only) stimulated CD25 naive cells (day 7). The data in the figure are representative of at least three experiments. pected, both CD25 and CD25 subsets from control cul- levels of CD25 (70%) and preserved their specific unre- tures had undetectable Foxp3 (Fig. 4 H). On the other sponsiveness to OVA peptide 323-339 restimulation in hand, IL-10 failed to induce Foxp3 expression in naive vitro (unpublished data). CD4 KJ1-26 T cells recovered CD4 CD25 cells (Fig. 4 D) or to further enhance Foxp3 from mice receiving Control Cell had much lower levels in T (Fig. 4 E). Thus, costimulation of Foxp3-negative of CD25 (28%) and proliferated vigorously to OVA pep- reg CD4 CD25 naive responder T cells with TCR engage- tide 323-339 (unpublished data). When cotransferred with ment in the presence of TGF- induces de novo expression naive/responder CD4 transgenic T cells, the TGF- of Foxp3, concomitant with conversion to a population of Cell, but not Control Cell, blocked antigen-specific ex- CD4 CD25 Foxp3 T cells phenotypically and function- pansion of naive CD4 KJ1-26 T cells in draining lymph ally indistinguishable from professional T . nodes (Fig. 5, A–C and E). Importantly, the recovered reg TGF-–induced CD4 Transgenic Anergic/Suppressor T CD4 KJ1-26 T cells from the mice injected with naive Cells Suppress OVA-specific T Cell Proliferation In Vivo. CD4 KJ1-26 cells plus TGF- Cell also expressed much Extending these studies in vivo, first we used an adoptive higher levels of CD25 (Fig. 5 c) compared with that in transfer model (31, 32) in which transgenic CD4 KJ1- mice injected with naive CD4 T cells alone (Fig. 5 a) or 26 T cells were transferred into syngeneic BALB/c mice plus Control Cell (Fig. 5 b). Interestingly, in contrast to followed by OVA peptide immunization. The prolifera- their nonsuppressive features in vitro, IL-10–induced tion of the transgenic CD4 T cells in host mice was CD4 transgenic T cells (IL-10 Cell) suppressed antigen- monitored using clonotypic antibody KJ1-26 (Fig. 5). In driven CD4 KJ1-26 T cell proliferation in vivo (Fig. 5, contrast to their anergic state in vitro, the TGF-–con- D and E). However, the recovered CD4 KJ1-26 T cells verted CD4 CD25 transgenic suppressor T cells (TGF- from mice receiving IL-10 cells expressed reduced CD25 Cell) expanded at similar levels as control CD4 trans- (Fig. 5 d) compared with those from mice receiving TGF- genic T cells (Control Cell) in vivo after OVA peptide Cell (Fig. 5 c). 323-339 immunization (Fig. 5 E and not depicted; with- When draining lymph node cells were restimulated in out immunization, recovered CD4 KJ1-26 T cells were vitro with OVA peptide 323-339, the antigen-specific T 0.1 10 in draining lymph nodes in all groups). Signif- cell proliferation from mice cotransferred with TGF- icantly, the recovered CD4 KJ1-26 T cells from drain- Cell was significantly inhibited compared with that of ing lymph nodes receiving TGF- Cell expressed high mice receiving naive CD4 KJ1-26 T cells alone (Fig. 5 Chen et al. Figure 5. TGF-–converted CD4 sup- pressor T cells inhibited antigen-specific expansion of transgenic CD4 T cells in vivo. Freshly isolated CD4 KJ1-26 T cells (DO11.10 CD4 cells) or with equal number of OVA only (Control cell), plus 2 ng/ml TGF- (TGF- cell) or plus 1 ng/ml IL-10– converted (IL-10 cell) CD25 KJ1-26 T cells were injected i.p into normal BALB/c mice. Some mice were injected with 2.5 10 of Control cell, TGF- cell, or IL-10 cell alone. Mice were immunized with Peptide 323-339 emulsified with IFA on day 2. On day 11, draining lymph nodes (inguinal) were harvested, and cells were stained with Tricolor–anti-CD4, FITC– KJ1-26, and PE–anti-CD25 and analyzed on FACSCalibur™. (A–D) Live cells were gated, and the profiles between CD4 versus KJ1-26 are displayed. The numbers repre- sent CD4 KJ1-26 cell frequency within lymph node cells. The numbers in the paren- theses are injected cells. Each group con- tained two mice and the lymph node cells were pooled before staining. (a–d) CD4 KJ1- 26 T cells in corresponding A–D were gated, and CD25 frequency is shown. (E) Total CD4 KJ1-26 T cells in draining lymph nodes per mouse (percentage of CD4 KJ1-26 the number of total lymph node cells/2). (F) 2 10 lymph node cells labeled with 3 M CFSE were cultured with 1 g/ml Peptide 323-339 in 24-well plates for 72 h. Cells were stained with Tricolor–anti-CD4 and PE–KJ1-26 and analyzed on FACSCalibur™. CD4 KJ1- hi 26 cells were gated, and the CFSE cells are shown. The marker was set according to CFSE fluorescence of the live CD4 KJ1- 26 T cells in parallel cultures with medium only. (G) 2 10 lymph node cells were cultured with Peptide 323-339 in the presence of GolgiPlug™ for 5–6 h. Cells were stained with Tricolor–anti-CD4 and FITC– KJ1-26 antibodies before being fixed and intracellularly stained for IL-4 or IFN- cytokines with respective antibodies (PE-anti–IL-4 or PE-anti–IFN-, 0.5 g/10 cells). 40,000–80,000 cells were acquired on FACSCalibur™, and percentages of KJ1-26 IL-4 or IFN- cells within CD4 T cells are shown. The experiments were repeated twice with similar results. F). The antigen-specific IL-4 and IFN- production in sence or presence of TGF- for 3–4 d, washed extensively, recovered CD4 KJ1-26 T cells was also inhibited in and rested in complete medium containing IL-2 for an ad- mice cotransferred with TGF- suppressor cells (Fig. 5 ditional 3 d. Viable cells were harvested and delivered in- G). The recovered CD4 KJ1-26 T cells from mice travenously into mice immunized and challenged with cotransferred with IL-10 Cell exhibited less inhibition of HDM as depicted in Fig. 6 A. After HDM intraperitoneal OVA-specific T cell proliferation and cytokine produc- immunization and intratracheal challenge, massive inflam- tion (Fig. 5, F and G). Clearly, TGF-–converted CD25 matory cell infiltrates were evident with mucus obstruction T cells have regulatory ability for antigen-induced naive of the airway in the lung (Fig. 6 C), compared with the CD4 T cell activation in vivo, consistent with the func- lung tissues in naive or PBS-injected mice (Fig. 6 B and tional features of T (31, 32), but distinct from IL-10– not depicted). Administration of control CD4 T cells reg induced CD25 T cells. (Control Cell) pretreated with anti-CD3 alone failed to re- TGF-–induced CD4 Anergic/Suppressor T Cells Prevent duce the inflammatory cell infiltration significantly, al- HDM-induced Allergic Pathogenesis In Vivo. The immuno- though mucus was less obvious in the airways (Fig. 6 D). In regulatory effect of TGF-–induced anergic/suppressor contrast, administration of TGF-–converted CD4 sup- CD4 T cells in vivo was further assessed in an antigen- pressor T cells (TGF- cell) dramatically reduced HDM- induced allergic model. C57BL/6 CD4 CD25 naive T induced inflammatory cell infiltration and preserved the cells were cultured with anti-CD3 and APCs in the ab- integrity of airway structures (Fig. 6 E). Cotransfer of TGF- Induces CD4 CD25 Regulatory T Cells through Induction of Foxp3 Figure 6. TGF-–converted-CD4 sup- pressor T cells prevent HDM-induced allergic pathogenesis in vivo. B6 CD4 CD25 T cells were cultured with anti-CD3 and APCs in the absence (Control cell) and presence of TGF- (TGF- cell) for 3–5 d, extensively washed and rested in DMEM complete medium containing 10 U/ml of IL-2 for an additional 3 d. Viable cells were harvested, washed, and resuspended in PBS for injection. For induction of allergic immune responses in lungs, B6 mice were injected with HDM allergen as depicted in schematic plan (A). 4 d after the last HDM intratracheal challenge, mice were killed, and lungs were immediately fixed with 10% PBS-Formalin for immunohistological staining (H&E). One representative lung from each group (three to five mice) is shown. The magnification of the images is 20. (B) Naive lung. (C) HDM-injected lung. (D) HDM-injected lung coinjected 6 5 with Control cell (10 on day 1 and 5 on day 14, i.v.). (E) HDM-injected lungs on day 1 coinjected with TGF- cell (10 and 5 10 on day 14, i.v.). (inset) Boxed images are mucin staining in airways by PAS (40 ). The red staining represents mucin positive cells. (F–H) Spleen cells were harvested and pooled from three to five mice per group. 4 10 spleen cells were cultured with 100 g/ml HDM (white bars) or 0.5 g/ml anti-CD3 (black bars) for 24–96 h, the cell-free supernatants were collected to measure IFN- (F, 48 h), IL-4 (G, 24 h), and IL-13 (H, 96 h), respec- tively, by ELISA. IL-4 levels are shown as OD (OD value of test supernatants  OD value of culture medium; OD 0.107). n.d., not done. Discussion TGF-–converted CD4 suppressor T cells blocked epi- thelial cell mucin production in airways induced by HDM Of many unresolved questions regarding T , where and reg as detected by PAS staining, whereas control CD4 T cells how they are generated remains significant (2, 33). In this re- did not (Fig. 6, B–E; Fig. S3, available at http://www. port, we have provided evidence that T can be induced/ reg jem.org/cgi/content/full/jem.20030152/DC1). Spleen T converted from peripheral CD4 CD25 naive responder T cells from mice treated with HDM and TGF-–induced cells through costimulation of TCR and TGF- signaling. anergic/suppressor CD4 T cells produced much lower Several important conclusions can be drawn from the current levels of HDM-specific, as well as anti-CD3 driven, Th1 work. First, TGF- treatment in the presence of TCR stim- (IFN-) and Th2 (IL-4 and IL-13) cytokines compared ulation converts naive CD4 CD25 responder T cells to an- with mice receiving HDM alone (Fig. 6, F–H). In contrast, ergized CD4 CD25 T cells, but fails to promote growth of spleen T cells from mice treated with HDM and control existing T , at least in vitro. The effect of TGF- on the fate reg CD4 T cells produced higher levels of IFN- (Fig. 6 F), of T cell activation is determined not only by TGF- con- although these cells produced reduced levels of IL-4 and centration, but also by the level of IL-2. Importantly, exoge- IL-13 (Fig. 6, G and H). Thus, TGF-–induced anergic/ nous IL-2 not only reverses unresponsiveness to TCR restim- suppressor CD4 T cells dampen inflammation and associ- ulation of TGF-–anergized CD4 T cells but also ated mucin production in allergic immune responses in antagonizes the induction of CD4 T cell anergy by TGF-. vivo. Second, TGF-1–converted anergic CD4 T cells are Chen et al. 1883 suppressor cells. Phenotypically, TGF-–converted aner- cells under conditions involving either anti-CD3 and anti- gic/suppressor T cells retain surface CD25 and exhibit CD28 costimulation in the absence of APCs or with anti- /low CD45RB , similar to professional T (2, 34). Another CD3 in the presence of APCs, suggesting the dispensability reg key finding is that TGF-–anergized CD4 CD25 T cells of APCs. The Foxp3 induction is dependent on the levels express intracellular CTLA-4 as do T (23, 27, 28). TGF- of TGF-, suggesting the causal influence of TGF-. Sec- reg regulation of intracellular CTLA-4 in CD4 T cells does ond, in the absence of TCR stimulation, TGF- itself fails not appear to involve induction of CTLA-4 synthesis, but to induce or enhance Foxp3 expression, indicating the ne- rather prevention of its degradation (unpublished data). cessity of TCR engagement. Moreover, when CD25 and Functionally, when cocultured with normal CD4 T cells, CD25 subsets were isolated from TGF-– and TCR- TGF-1–anergized T cells inhibit proliferation and cyto- costimulated cultures, CD25 T cells exhibited much higher kine production of the target T cells. This suppressive ac- levels of Foxp3 than the CD25 subset, supporting the no- tivity is mainly attributed to the CD25 subpopulation of tion that Foxp3 may be involved in T immunoregulation reg anergized cells, because TGF-–anergized cells lacking (38). Lastly, TGF- had no further enhancement on exist- CD25, at least in vitro, have no obvious suppressive ability. ing Foxp3 gene in professional T , consistent with the ev- reg Neither CD25 nor CD25 T cells from control cultures idence that Foxp3 expression is stable in T (13). The fail- reg (without TGF-) have suppressive activity, consistent with ure of IL-10 to induce Foxp3 in CD25 naive T cells previous observations (1, 2, 34, 35). The level of immuno- indicates that IL-10 is not a physiological inducer for Foxp3 suppression is dependent on the cell number and reversible expression. Although mechanistically elusive, the observa- by exogenous IL-2. Consistent with T , TGF-–con- tion that IL-10–induced CD25 T cells lack characteristic reg verted CD25 suppressor T cells also require cell contact to suppression in vitro, but are functional in vivo suggests that carry out their suppression, which can be mediated by cell these cells are distinct from TGF-–converted CD25 reg- membrane–bound TGF- (29, 30). ulatory cells. The data are consistent with the notion that The data that TGF-–converted CD4 CD25 T cells IL-10 is more likely a switch for CD4 T cells into Trl cells express cell surface TGF- in active form provide further (39). These discriminating results not only provide a mo- evidence that the professional T can be converted by lecular mechanism for the TGF-–driven transition of na- reg TGF- and TCR costimulation from peripheral naive ive T cells toward regulatory T cells, but also unveil the CD4 T cells. Although not unanimous, increasing evi- first physiological inducer for Foxp3 expression in CD4 dence has demonstrated that T produce/express higher naive/responder T cells, which may enable manipulation of reg levels of TGF-, which plays a significant role in immune this specific population of regulatory cells. regulation in vitro and in vivo (27, 29, 30, 36, 37). That More importantly, these TGF-1–converted CD4 TGF-–converted CD25 suppressor T cells express cell CD25 suppressor cells also inhibit T cell responses surface TGF- not only supports the concept that these when transferred in vivo. Evidence from the OVA trans- suppressor cells possess the same phenotype as the profes- genic adoptive transfer model document that TGF-–con- sional T but also offers an explanation for how profes- verted CD25 suppressor cells resemble T . First, TGF- reg reg sional T are developed. Although how these TGF- sup- –converted CD25 suppressor T cells proliferate in vivo reg pressor T cells acquire surface TGF- remains elusive, it is upon specific antigen immunization, yet preserve their an- conceivable that TGF- enhances its own production as tigen specific anergy when recultured in vitro. Second, evident by exogenous TGF- up-regulation of its own transgenic CD4 KJ1-26 T cells recovered from mice re- mRNA expression in CD4 T cells (reference 7; unpub- ceiving TGF-–converted suppressor cells expressed ele- lished data) and/or TGF- binds onto its cognate receptors vated CD25. Significantly, TGF-–converted KJ1-26 on these suppressor T cells, because TCR stimulation en- CD25 suppressor cells inhibit antigen-driven CD4 T cell hances TGF- receptor expression in CD4 T cells (un- growth in vivo. Again, the recovered CD4 KJ1-26 cells published data). The cell membrane–bound TGF- is from the suppressed draining lymph nodes show high levels likely involved in unresponsiveness to TCR engagement of CD25 and fail to respond to specific antigen stimulation. and suppression of normal T cells by these TGF- anergic/ All these phenotypic and functional features of TGF-– suppressor T cells because restimulation of the suppressor converted suppressor T cells are consistent with those of cells drives a higher level of phosphorylation of Smad2/3, professional T (31, 32). Moreover, TGF-–converted reg and anti–TGF- antibody reverses their anergy and abro- suppressor T cells may carry out their suppression in vivo gates their suppressive action. in a nonspecific manner because TGF-–converted The strongest evidence that TGF- converts CD4 BALB/c CD25 T cells harvested from the primary cul- CD25 naive T cells to CD25 regulatory cells tures exhibit the inhibition of antigen-driven expansion of comes from our surprising findings that TGF- and TCR the transferred CD4 KJ1-26 T cells (unpublished data). costimulation induces transcription factor Foxp3 gene ex- In HDM-induced allergic immune responses in lungs, pression in CD4 CD25 T cells. The observation that TGF-–converted suppressor T cells ameliorate allergen- TGF- induces Foxp3 expression in CD4 CD25 naive T driven inflammatory cell infiltration and mucin production cells has led to several unprecedented conclusions. First, in airways. Anergic/suppressor T cells appear to inhibit TGF- can induce Foxp3 expression in CD4 CD25 T both Th1 and Th2 responses, whereas administration of TGF- Induces CD4 CD25 Regulatory T Cells through Induction of Foxp3 control CD4 cells enhanced Th1 IFN-, which might 7. Chen, W., and S.M. Wahl. 2002. TGF-beta: receptors, sig- naling pathways and autoimmunity. Curr. Dir. Autoimmun. contribute to the inhibition of Th2 cytokines, but failed to 5:62–91. significantly limit the inflammatory response in the lungs, 8. Gorelik, L., and R.A. Flavell. 2000. Abrogation of TGFbeta indicating the complexity of the Th1 effector T cell role in signaling in T cells leads to spontaneous T cell differentiation allergic lung inflammation (40). Interestingly, we also ob- and autoimmune disease. Immunity. 12:171–181. served an increase in TGF- production in the TGF- 9. Lucas, P.J., S.J. Kim, S.J. Melby, and R.E. Gress. 2000. Dis- suppressor cell–treated mice (unpublished data), which re- ruption of T cell homeostasis in mice expressing a T cell–spe- flects previous findings that the professional T require reg cific dominant negative transforming growth factor  II re- TGF- in vivo to carry out their suppressive activity (27), ceptor. J. Exp. Med. 191:1187–1196. and supports our evidence that TGF- and TCR costimu- 10. Yamagiwa, S., J.D. Gray, S. Hashimoto, and D.A. Horwitz. lation convert naive CD4 T cells toward a regulatory T 2001. A role for TGF-beta in the generation and expansion of CD4CD25 regulatory T cells from human peripheral cell phenotype mimicking professional T . reg blood. J. Immunol. 166:7282–7289. In summary, we have provided evidence for TGF-1 as 11. Brunkow, M.E., E.W. Jeffery, K.A. Hjerrild, B. Paeper, L.B. a critical factor in the development of peripheral T . The reg Clark, S.A. Yasayko, J.E. Wilkinson, D. Galas, S.F. Ziegler, present paper has not only demonstrated that TGF- con- and F. Ramsdell. 2001. Disruption of a new forkhead/ verts CD4 CD25 naive T cells toward a suppressor T cell winged-helix protein, scurfin, results in the fatal lymphopro- phenotype similar to that of T but also uncovered that reg liferative disorder of the scurfy mouse. Nat. Genet. 27:68–73. TGF- induces transcription factor Foxp3 expression in 12. Wildin, R.S., F. Ramsdell, J. Peake, F. Faravelli, J.L. CD25 naive T cells to enforce transition to regulatory T Casanova, N. Buist, E. Levy-Lahad, M. Mazzella, O. Goulet, cells. Functionally, TGF-–converted suppressor T cells L. Perroni, et al. 2001. X-linked neonatal diabetes mellitus, not only suppress T cell proliferation and Th1 and Th2 cy- enteropathy and endocrinopathy syndrome is the human equivalent of mouse scurfy. Nat. Genet. 27:18–20. tokine production in vitro but also inhibit antigen-driven 13. Hori, S., T. Nomura, and S. Sakaguchi. 2003. 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The Journal of Experimental Medicine – Pubmed Central

Published: Dec 15, 2003

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