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TGF-β1 maintains suppressor function and Foxp3 expression in CD4+CD25+ regulatory T cells

TGF-β1 maintains suppressor function and Foxp3 expression in CD4+CD25+ regulatory T cells BRIEF DEFINITIVE REPORT TGF-1 maintains suppressor function and Foxp3 expression in CD4 CD25 regulatory T cells 1 3 1 Julien C. Marie, John J. Letterio, Marc Gavin, 1,2 and Alexander Y. Rudensky 1 2 Department of Immunology and Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195 Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute, Bethesda, MD 20892 Transforming growth factor (TGF)-1 is a major pluripotential cytokine with a pronounced immunosuppressive effect and its deficiency results in lethal autoimmunity in mice. However, mechanisms of its immunosuppressive action are not completely understood. Here, we report that TGF-1 supports the maintenance of Foxp3 expression, regulatory function, and homeostasis in peripheral CD4 CD25 regulatory T (T reg) cells, but is not required for their thymic development. We found that in 8–10-d-old TGF-1–deficient mice, peripheral, but not thymic, T reg cells are significantly reduced in numbers. Moreover, our experiments suggest that a defect in TGF-–mediated signaling in T reg cells is associated with a decrease in Foxp3 expression and suppressor activity. Thus, our results establish an essential link between TGF-1 signaling in peripheral T reg cells and T reg cell maintenance in vivo. CD4 CD25 regulatory T (T reg) cells play a syndrome and succumbing to disease at 3–4 CORRESPONDENCE Alexander Y. Rudensky: major role in the maintenance of immune toler- wk of age (10, 14). TGF-1 is a widely dis- [email protected] ance to self and in the control of autoimmunity tributed immunomodulatory cytokine. TGF-1 (1, 2). T reg cells have been shown to inhibit signals through a heterodimeric receptor com- autoimmunity in a number of experimental plex formed by TGF-RI and TGF-RII models, including diabetes and inflammatory subunits. Binding of TGF-1 to the receptor bowel disease in rodents (3, 4). They are also complex activates the intracellular kinase do- involved in regulating T cell homeostasis (1, 5). main, which leads to the phosphorylation and Additional studies have demonstrated their role activation of members of the Smad protein in organ transplant tolerance and in modulation family and subsequent regulation of TGF-– of immune responses to pathogens (6, 7). This dependent gene expression (15). CD4 T cell subset constitutes 5–10% of pe- An important role of TGF-1 in down- ripheral CD4 T cells and is capable of inhib- modulation of T cell–mediated immune re- iting the responses of CD4 CD25 and CD8 sponses and in controlling autoimmunity has T cells in vitro and in vivo (1). been clearly established (15). Mice deficient in Recently, Foxp3, a member of the fork- TGF-RII receptor or expressing a dominant head winged helix protein family of transcrip- negative form of TGF-RII receptor (DN– tion factors, has been identified as a specific TGF-RII) encoded by a transgene under a T molecular marker for T reg cells and its ex- cell–specific promoter exhibit inflammatory infil- pression is essential for programming T reg cell tration in multiple organs and uncontrolled T cell development and function (8–11). The Foxp3 proliferation (16, 17) with features similar to that gene is highly conserved, and the function of of the Tgf-1 mice. Notably, several recent Foxp3 appears to be similar in both humans reports suggested that TGF-1 produced by T and mice, as Foxp3 mutations result in a fatal reg cells, and possibly decorating their plasma autoimmune pathologies affecting multiple or- membrane via binding to T reg cell TGF- re- gans in both species (9, 10, 12, 13). ceptors, may serve as an effector mechanism of The phenotype of Foxp3 knockout mice suppression (18, 19). However, a subsequent in closely resembles that of animals deficient in vitro study failed to reproduce this finding (20). TGF- 1 (Tgf-1 ), with both mutants devel- Therefore, we revisited the role that TGF- oping a lethal lymphoproliferative autoimmune 1 plays in T reg cell biology by directly ex- JEM Vol. 201, No. 7, April 4, 2005 1061–1067 www.jem.org/cgi/doi/10.1084/jem.20042276 1061 The Journal of Experimental Medicine Figure 1. Decrease in peripheral CD4 CD25 T cells in Tgf-1 (gray line) or littermate controls (black line) were stained for CD44 and / / mice. Thymocytes (a) and splenocytes (b and c) from 8–10-d-old Tgf-1 CD62L. Thymocytes and splenocytes from 8–10-d-old Tgf-1 mice or mice or littermate controls were counted and stained for CD4 and CD8 and littermate controls were stained for CD4 and CD25 and analyzed by flow cy- analyzed by flow cytometry. Peripheral CD4 T cells from Tgf-1 mice tometry (d). Proportion of CD4 CD25 cells among CD4 T cells (e, n  20). amining potential effect of TGF-1 deficiency on T reg 1–neutralizing antibodies or a soluble form of TGF-RII cells. This was achieved by analyzing the T reg cell subsets in abrogated T reg cell–mediated suppression in vitro and in young Tgf-1 mice. We have found that TGF-1 pro- vivo (18, 19). To reexamine a role for TGF-1 in T reg cell duced by T reg cells is unlikely to play a role as a nonredun- development and function, we used mice deficient for Tgf- dant effector molecule mediating T reg cell suppressor func- 1 expression, which are known to develop an early onset tion. However, Foxp3 expression, the size of peripheral T lethal lymphoproliferative autoimmune syndrome (14). To reg cell compartment, and suppressive activity are dependent avoid potential artifacts due to pathology observed in af- on signals induced by TGF- in T reg cells. fected Tgf-1–deficient mice, we examined the T cell com- partment in 8–10-d-old Tgf-1 mice before the onset of RESULTS AND DISCUSSION lymphoproliferation and clinical symptoms. In these young TGF-1 is required for peripheral T reg cell homeostasis mice, the analysis of thymus and spleen did not reveal any Although a role of TGF-–dependent signals has been well difference in the proportion or absolute number of single documented in the control of T cell activation and inflam- positive or double positive thymocytes, and the peripheral mation in vivo (16), a connection between TGF-1 and T CD4 and CD8 T cell compartments were similar in Tgf- reg cell function in vivo and in vitro has been controversial. 1 mice and littermate controls (Fig. 1, a and b). In addi- Two recent in vitro studies have shown no demonstrable tion, peripheral Tgf-1 CD4 T cells expressed the same role for TGF-1 produced by T reg cells in the suppressor level of the activation markers CD44 and CD62L as cells function (20, 21). However, another set of studies suggested from WT littermate controls (Fig. 1 c). However, in 12–14- that TGF-1 produced by T reg cells is an essential effector d-old mice, the number of peripheral CD4 T cells was in- molecule because, in these experiments, either anti–TGF- creased and some of these cells exhibited an activated pheno- 1062 ROLE OF TGF-1 IN T REG CELL MAINTENANCE | Marie et al. BRIEF DEFINITIVE REPORT type (i.e., increased CD44 and diminished CD62L expres- sion as compared with T cells in WT littermate controls; unpublished data). Thus, we have chosen to study the T reg cell subset in 8–10-d-old Tgf-1 mice. Flow cytometric analysis revealed the peripheral CD4 CD25 T cell com- partment reduced by 2.5–3-fold in Tgf-1 mice as com- pared with the WT littermate controls (Fig. 1, d and e). However, the CD4 CD25 thymocyte subset in these ani- mals was similar in size to that of littermate controls (Fig. 1, d and e). Thus, these results indicate that TGF-1 plays a role in the peripheral T reg cell maintenance. In contrast with these data, no difference was previously found in the CD25 CD4 T cell subsets not only in the thymus but also in the periphery in 5–7-d-old TGF-1–deficient mice (20). This apparent discrepancy is most likely due to the fact that the numbers of peripheral T reg cells in 5–7-d-old mice are very small (unpublished data) and the vast majority of these cells are recent arrivals from the thymus exhibiting normal Figure 2. Decrease in Foxp3 level in Tgf-1 CD4 CD25 T cells. levels of Foxp3 characteristic of thymic T reg cells. Thymocytes and splenocytes from 8–10-d-old Tgf-1 mice or littermate controls were stained for CD4 and CD25 followed by anti-Foxp3 intracellular TGF-1 is required to maintain Foxp3 expression in T reg cells staining and analyzed by flow cytometry. Foxp3 staining in CD4 CD25 T Next, we investigated whether TGF-1 can regulate Foxp3 cells (black line) and in CD4 CD25 (gray line) are shown. Isotype control staining is shown (dashed line). These results are representative of three expression in T reg cells. First, we analyzed Foxp3 ex- different experiments. pression by intracellular staining of thymic and splenic CD4 CD25 T cells isolated from Tgf-1 mice or WT littermate controls (Fig. 2). No significant difference in Foxp3 expression was observed in CD4 CD25 thymocytes of TGF-1 (Fig. 3 b), led to an increase in Foxp3 expression in mutant versus WT mice, whereas peripheral Tgf-1 in T reg cells comparable to that of WT littermate control T CD4 CD25 T cells expressed significantly diminished level reg cells. Furthermore, transfer of CD4 CD25 T cells from of Foxp3 compared with the control. Thus, the absence of littermate control mice into recipients treated with the anti– TGF-1 results in diminished Foxp3 expression in periph- TGF- antibody resulted in somewhat diminished Foxp3 eral CD4 CD25 T cells in addition to a substantial de- expression in those cells after 4 d. Altogether, these results, crease in size of this T cell compartment. Nevertheless, some involving both genetic modifications and in vivo adoptive peripheral regulatory T cells in TGF-1 null mice still main- transfer approaches, show that TGF-1 maintains Foxp3 ex- tain Foxp3 expression. The latter is likely due to normal pression in peripheral T reg cells. Furthermore, the engage- level of Foxp3 expression in thymocytes being preserved in ment of TGF- signaling pathway in T reg cells cultured in recent thymic emigrants. Provision of small amounts of the presence of TGF-1 resulted in induction of Smad2 TGF-1 by mother via breastfeeding and potential compen- phosphorylation and concomitant increase in Foxp3 expres- satory role of TGF-2 and TGF-3 can also contribute to sion (Fig. 3 c). This observation further confirms that TGF- maintaining Foxp3 expression in some regulatory T cells in maintains Foxp3 high expression in T reg cells. By inference the knockout mice. This result strongly suggests that TGF- from recent observations of acquisition of T reg cell pheno- 1 is required for the maintenance of Foxp3 levels in pe- type and suppressor activity by CD4 CD25 T cells upon ripheral T reg cells. retroviral transduction with Foxp3 (9, 10), the loss of pe- To test this hypothesis, CD4 CD25 T cells from either ripheral T reg cells and their diminished suppressor activity / / 8–10-d-old Tgf-1 mice or littermate control mice were in Tgf-1 mice can be a direct consequence of decreasing adoptively transferred into lymphopenic TCR/-deficient levels of Foxp3. host treated with the neutralizing anti–TGF- antibody or isotype control IgG and analyzed 4 d later by flow cytome- TGF-1 is required to maintain T reg cell suppressive function try (Fig. 3 a). As expected, the majority of Tgf-1 After establishing a decreased level of Foxp3 expression in CD4 CD25 T cells transferred into anti–TGF-–treated T reg cells in the absence of TGF-1, we investigated recipient mice continued to exhibit decreased Foxp3 levels, whether TGF-1 is also involved in the maintenance of high / whereas few Foxp3 cells were observed. The latter may their regulatory function. Purified T reg cells from Tgf-1 be due to incomplete antibody-mediated TGF-1 deletion mice or littermate control mice were cocultured with re- in addition to the aforementioned compensatory role of sponder CD4 CD25 T cells from control mice in the TGF-2 and TGF-3. However, transfer of these cells into presence of Con A and T cell–depleted splenic APCs iso- control IgG1-treated recipients, expressing normal amounts lated from either Tgf-1 or littermate control mice (Fig. JEM VOL. 201, April 4, 2005 1063 deficient DO11.10 TCR transgenic mice can prevent colitis mediated by WT CD4 CD25 T cells transferred into SCID recipient mice. However, this suppression was abro- gated in the presence of neutralizing antibodies specific for TGF-1 leading to disease progression (22). We also ob- served that T reg cells from Tgf-1 mice were able to / / suppress autoimmunity developing in Tgf-1 RAG mice upon transfer of Tgf-1 CD4 CD25 T cells (23). These findings are consistent with our notion that Tgf-1 T reg cells require TGF-1 provided by other cells to pre- serve their functionality. Although the types of accessory cells supplying TGF-1 for T reg cells in vivo remains to be determined, our analysis of in vitro TGF-1 production us- ing ELISA implicated APCs, such as dendritic cells, as the likely candidates (unpublished data). TGF-1–mediated signaling in T reg cells is required to maintain suppressor function However, these results do not address the question as to whether T reg cells require TGF-1 signaling to maintain their functionality or whether T reg cells are “armed” with the receptor-bound TGF-1 produced either endogenously or secreted by APCs. The latter possibility was raised by two reports suggesting that receptor-bound TGF-1 displayed on the surface of T reg cells may function as an effector mol- ecule in T reg cell–mediated suppression as assessed in an in vitro assay (19, 24). To address these two possibilities, we transduced T reg cells isolated from B6 mice with the Mig- R2 retroviral vector encoding a dominant negative form of Figure 3. TGF-1 is required to maintain Foxp3 in CD4 CD25 the TGF-1 receptor (DN–TGF-RII) and IRES-driven T cells. T reg cells purified from spleen of 8–10-d-old Tgf-1 mice or tail-less human CD2 as a reporter. DN–TGF-RII was gen- littermate controls were transferred into TCR/-deficient mice injected erated upon deletion of the TGF-RII kinase domain. As a every other day with anti–TGF-1 antibody or isotype control IgG. 4 d control, T reg cells were transduced with the MIG-R2 vec- after transfer, cells from spleen and lymph nodes were stained for CD4 tor without insert. Transduced T reg cells were purified by and CD25 followed by anti-Foxp3 intracellular staining and analyzed by magnetic bead sorting using anti–human CD2 antibody. flow cytometry (a). Results are representative of three different experiments. Serum TGF-1 in treated animals was measured by ELISA (b). Western blot Suppressor activity of DN–TGF-RII– and empty vector– analysis of Smad2 phosphorylation and Foxp3 expression in purified wild- transduced T reg cells was tested in an in vitro suppression type T reg cells either freshly isolated or cultured for 24 h in the presence assay. The regulatory capacity of T reg cells expressing DN– or absence of 100 pg/ml TGF-1 (c). TGF-RII was diminished, but not completely abolished, as compared with the control T reg cells (Fig. 4 b). This is likely due to the fact that T reg cells expressing DN–TGF- 4 a). All combinations of WT and mutant T cells and APCs RII remain still sensitive to TGF-, albeit require an 10- resulted in a comparable suppression except for a signifi- fold higher dose to induce Smad2 phosphorylation at a level comparable to that of control MigR2-transduced cells (un- cantly diminished suppression by Tgf-1 T reg cells ob- served only in the presence of Tgf-1 APCs. Thus, these published data). Importantly, although expression of DN– data demonstrate that TGF-1 production by T reg cells is TGF-RII inhibits TGF-–mediated signaling, it is ex- pected to increase ability of T reg cells to capture TGF- on dispensable for their suppressor function because APCs can produce sufficient amounts of TGF-1 to support T reg the plasma membrane. The analysis of suppressor function of cell–mediated suppression. T reg cells transduced with DN–TGF-RII suggests that impairment in suppressor function in the absence of TGF- Although relative contribution of TGF-1 produced by T reg cells versus other cellular sources to T reg cell function 1 is due to a signaling defect and not due to lack of recep- in vivo remains to be studied further, our results suggest a tor-mediated display of TGF- on the T reg cell surface. requirement for paracrine TGF-1 to maintain T reg cell Therefore, our results are consistent with the role of TGF- not as an effector molecule of suppression, but as a mediator function in the periphery. This observation is in agreement with recent experiments by Powrie et al., who showed that of signaling in T reg cells required to maintain their suppres- an adoptive transfer of T reg cells isolated from TGF-1– sor function. Thus, our findings provide novel important in- 1064 ROLE OF TGF-1 IN T REG CELL MAINTENANCE | Marie et al. BRIEF DEFINITIVE REPORT Figure 4. TGF- signaling is required to maintain regulatory func- results are shown as mean cpm of [ H]thymidine incorporation in tripli- tion T reg cells. (a) Analysis of suppressor activity of T reg cells isolated cate cultures  SD. (b) T reg cells transduced with DN–TGF-RII–MigR2 from Tgf-1 (gray symbols) or WT littermates (Lit, black symbols) in (MigR2 DN) or empty vector control (MigR2 Ev) were purified and cocul- cocultures with freshly isolated B6 CD4 CD25 responder T cells in the tured with freshly isolated B6 CD4 CD25 responder T cells in the presence presence of either Tgf-1 or littermate control APCs and Con A. Prolif- of irradiated T cell–depleted splenic APCs and Con A. Proliferation was eration of CD4 CD25 T cells was determined after 72 h of culture by measured as described before. Results are representative of two or three [ H]thymidine incorporation. Proliferation of CD4 CD25 from either different experiments. control or Tgf-1 mice in response to Con A was 300–500 cpm. The sights into the role of TGF-1 in T reg cell function recon- purposes, there is no experimental evidence so far that such ciling previous contradictory observations. high concentrations of TGF-1 are ever attainable in vivo Recently, several in vitro studies have reported acquisi- and that such a conversion of CD4 CD25 into CD4 tion of suppressor activity and Foxp3 expression in the bulk CD25 T cells expressing Foxp3 can occur under physio- populations of cultured murine and human CD4 CD25 T logic conditions or in the course of immune inflammation. cells upon activation in the presence of nanomolar amounts In our earlier experiments, we failed to detect any measur- of recombinant TGF-1 (25, 26). Although these studies able conversion of CD4 CD25 T cells into CD4 CD25 opened up an important avenue for pharmacologic in vitro upon their transfer into newborn Foxp3-deficient mice (10). manipulation of T cell function for potential therapeutic Ramsdell et al. have recently reported high levels of TGF- 1 JEM VOL. 201, April 4, 2005 1065 6 formed after cell lysate separation in 12% SDS-PAGE gels (3 10 cell in Foxp3 mutant mice in addition to highly elevated levels equivalents/lane) as described elsewhere (10). of a number of other cytokines (11). Nevertheless, it will Suppression assays were performed as described (10). In brief, CD4 be of interest to experimentally test whether substantial CD25 T cells (4 10 cells/well) were stimulated for 72 h with 1 g/ml amounts of TGF-1 displayed by some immunoprivileged of Con A in the presence of irradiated (2,000 rad) T cell–depleted spleno- tissues and organs, like the ocular chamber of the eye, can fa- cytes (APCs) (2 10 /well) in 96-well round-bottom plates, with indi- cilitate maintenance, expansion, or de novo generation of cated numbers of CD4 CD25 T cells and pulsed with 1 Ci/well of [ H]thymidine for the final 16 h of culture. Data are shown as mean antigen-specific T reg cells (27). This idea is supported by [ H]thymidine incorporation in triplicate cultures. the recent observation of significant expansion and/or gen- eration of protective T reg cells in pancreatic lymph nodes Retroviral infection. The first 656 bp of the coding sequence of TGF- and islets in NOD mice upon temporal TGF-1 induction RII were subcloned from pcDNA3.1-myc-HisB DN-TGF-RII () into in the -cells (28). MSCV MigR2 retroviral vector. The vector DNA with or without insert In conclusion, our study demonstrates an essential in was transfected into the NX-E packaging cell line using Fugene 6 reagent (Roche) according to the manufacturer’s protocol. Retrovirus-containing vivo role for the TGF-1–mediated signaling in the mainte- supernatant was collected after a 36-h culture of the transfected packaging nance of naturally arising regulatory CD4 CD25 T cell cells at 32 C. Freshly isolated MACS-purified CD4 CD25 T cells were numbers and function, and in the maintenance of Foxp3 ex- activated using plate-bound anti-TCR chain and anti-CD28 antibodies, pression in these cells in the periphery. These results demon- in the presence of 100 U/ml recombinant human IL-2 (Hoffmann–La strate an important role of TGF-1 in T reg cell biology and Roche). After 24 h of activation, CD4 CD25 T cells were infected by re- suggest additional applications for targeting of TGF-1 sig- suspending cells in retrovirus-containing supernatants supplemented with 8 g/ml polybrene and 50 U/ml of recombinant IL-2, followed by centrifu- naling pathway for therapeutic immunomodulation. gation for 90 min at 2,500 revolutions/min. Cells were cultured for 48 h at 37 C in the presence of 200 U/ml IL-2 and 10 pg/ml TGF-1 to maintain MATERIALS AND METHODS CD25 and Foxp3 expression. Mice. C57BL/6 (B6) and TCR/-deficient mice were purchased from Charles River Breeding Laboratories and The Jackson Laboratory, respec- / We thank the members of the Rudensky laboratory for discussions and for reading tively. Tgf-1 mice were previously described (29). All mice were main- the text. tained in a specific pathogen-free animal facility at the University of Wash- This work was supported by grants from International Human Frontier Science ington and National Institutes of Health and handled in accordance with Program (to J.C. Marie) and National Institutes of Health nos. AI34206 and AI061816 the institutional guidelines. (to A.Y. Rudensky). A.Y. Rudensky is a Howard Hughes Medical Institute investigator. The authors have no conflicting financial interests. Antibodies. Anti-Foxp3 IgG was purified on a protein G–Sepharose 4 Fast Flow column from the rabbit antiserum raised against purified His- Submitted: 5 November 2004 tagged recombinant Foxp3 protein. Biotinylated (bio), FITC-, PE-, cy- Accepted: 15 February 2005 chrome (CyC)-, peridinin chlorophyl protein (PerCP)-, and allophycocya- nin-conjugated monoclonal antibodies to CD4 (L3T4), CD8 (53–6.7), REFERENCES CD25 (7D4), CD25 (PC-61), CD62L (MEL-14), CD69 (H1.2F3), and 1. 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JEM VOL. 201, April 4, 2005 1067 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Experimental Medicine Pubmed Central

TGF-β1 maintains suppressor function and Foxp3 expression in CD4+CD25+ regulatory T cells

Marie, Julien C.; Letterio, John J.; Gavin, Marc; Rudensky, Alexander Y.
The Journal of Experimental Medicine , Volume 201 (7) – Apr 4, 2005
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Pubmed Central
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Copyright © 2005, Government
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0022-1007
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1540-9538
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10.1084/jem.20042276
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Abstract

BRIEF DEFINITIVE REPORT TGF-1 maintains suppressor function and Foxp3 expression in CD4 CD25 regulatory T cells 1 3 1 Julien C. Marie, John J. Letterio, Marc Gavin, 1,2 and Alexander Y. Rudensky 1 2 Department of Immunology and Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195 Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute, Bethesda, MD 20892 Transforming growth factor (TGF)-1 is a major pluripotential cytokine with a pronounced immunosuppressive effect and its deficiency results in lethal autoimmunity in mice. However, mechanisms of its immunosuppressive action are not completely understood. Here, we report that TGF-1 supports the maintenance of Foxp3 expression, regulatory function, and homeostasis in peripheral CD4 CD25 regulatory T (T reg) cells, but is not required for their thymic development. We found that in 8–10-d-old TGF-1–deficient mice, peripheral, but not thymic, T reg cells are significantly reduced in numbers. Moreover, our experiments suggest that a defect in TGF-–mediated signaling in T reg cells is associated with a decrease in Foxp3 expression and suppressor activity. Thus, our results establish an essential link between TGF-1 signaling in peripheral T reg cells and T reg cell maintenance in vivo. CD4 CD25 regulatory T (T reg) cells play a syndrome and succumbing to disease at 3–4 CORRESPONDENCE Alexander Y. Rudensky: major role in the maintenance of immune toler- wk of age (10, 14). TGF-1 is a widely dis- [email protected] ance to self and in the control of autoimmunity tributed immunomodulatory cytokine. TGF-1 (1, 2). T reg cells have been shown to inhibit signals through a heterodimeric receptor com- autoimmunity in a number of experimental plex formed by TGF-RI and TGF-RII models, including diabetes and inflammatory subunits. Binding of TGF-1 to the receptor bowel disease in rodents (3, 4). They are also complex activates the intracellular kinase do- involved in regulating T cell homeostasis (1, 5). main, which leads to the phosphorylation and Additional studies have demonstrated their role activation of members of the Smad protein in organ transplant tolerance and in modulation family and subsequent regulation of TGF-– of immune responses to pathogens (6, 7). This dependent gene expression (15). CD4 T cell subset constitutes 5–10% of pe- An important role of TGF-1 in down- ripheral CD4 T cells and is capable of inhib- modulation of T cell–mediated immune re- iting the responses of CD4 CD25 and CD8 sponses and in controlling autoimmunity has T cells in vitro and in vivo (1). been clearly established (15). Mice deficient in Recently, Foxp3, a member of the fork- TGF-RII receptor or expressing a dominant head winged helix protein family of transcrip- negative form of TGF-RII receptor (DN– tion factors, has been identified as a specific TGF-RII) encoded by a transgene under a T molecular marker for T reg cells and its ex- cell–specific promoter exhibit inflammatory infil- pression is essential for programming T reg cell tration in multiple organs and uncontrolled T cell development and function (8–11). The Foxp3 proliferation (16, 17) with features similar to that gene is highly conserved, and the function of of the Tgf-1 mice. Notably, several recent Foxp3 appears to be similar in both humans reports suggested that TGF-1 produced by T and mice, as Foxp3 mutations result in a fatal reg cells, and possibly decorating their plasma autoimmune pathologies affecting multiple or- membrane via binding to T reg cell TGF- re- gans in both species (9, 10, 12, 13). ceptors, may serve as an effector mechanism of The phenotype of Foxp3 knockout mice suppression (18, 19). However, a subsequent in closely resembles that of animals deficient in vitro study failed to reproduce this finding (20). TGF- 1 (Tgf-1 ), with both mutants devel- Therefore, we revisited the role that TGF- oping a lethal lymphoproliferative autoimmune 1 plays in T reg cell biology by directly ex- JEM Vol. 201, No. 7, April 4, 2005 1061–1067 www.jem.org/cgi/doi/10.1084/jem.20042276 1061 The Journal of Experimental Medicine Figure 1. Decrease in peripheral CD4 CD25 T cells in Tgf-1 (gray line) or littermate controls (black line) were stained for CD44 and / / mice. Thymocytes (a) and splenocytes (b and c) from 8–10-d-old Tgf-1 CD62L. Thymocytes and splenocytes from 8–10-d-old Tgf-1 mice or mice or littermate controls were counted and stained for CD4 and CD8 and littermate controls were stained for CD4 and CD25 and analyzed by flow cy- analyzed by flow cytometry. Peripheral CD4 T cells from Tgf-1 mice tometry (d). Proportion of CD4 CD25 cells among CD4 T cells (e, n  20). amining potential effect of TGF-1 deficiency on T reg 1–neutralizing antibodies or a soluble form of TGF-RII cells. This was achieved by analyzing the T reg cell subsets in abrogated T reg cell–mediated suppression in vitro and in young Tgf-1 mice. We have found that TGF-1 pro- vivo (18, 19). To reexamine a role for TGF-1 in T reg cell duced by T reg cells is unlikely to play a role as a nonredun- development and function, we used mice deficient for Tgf- dant effector molecule mediating T reg cell suppressor func- 1 expression, which are known to develop an early onset tion. However, Foxp3 expression, the size of peripheral T lethal lymphoproliferative autoimmune syndrome (14). To reg cell compartment, and suppressive activity are dependent avoid potential artifacts due to pathology observed in af- on signals induced by TGF- in T reg cells. fected Tgf-1–deficient mice, we examined the T cell com- partment in 8–10-d-old Tgf-1 mice before the onset of RESULTS AND DISCUSSION lymphoproliferation and clinical symptoms. In these young TGF-1 is required for peripheral T reg cell homeostasis mice, the analysis of thymus and spleen did not reveal any Although a role of TGF-–dependent signals has been well difference in the proportion or absolute number of single documented in the control of T cell activation and inflam- positive or double positive thymocytes, and the peripheral mation in vivo (16), a connection between TGF-1 and T CD4 and CD8 T cell compartments were similar in Tgf- reg cell function in vivo and in vitro has been controversial. 1 mice and littermate controls (Fig. 1, a and b). In addi- Two recent in vitro studies have shown no demonstrable tion, peripheral Tgf-1 CD4 T cells expressed the same role for TGF-1 produced by T reg cells in the suppressor level of the activation markers CD44 and CD62L as cells function (20, 21). However, another set of studies suggested from WT littermate controls (Fig. 1 c). However, in 12–14- that TGF-1 produced by T reg cells is an essential effector d-old mice, the number of peripheral CD4 T cells was in- molecule because, in these experiments, either anti–TGF- creased and some of these cells exhibited an activated pheno- 1062 ROLE OF TGF-1 IN T REG CELL MAINTENANCE | Marie et al. BRIEF DEFINITIVE REPORT type (i.e., increased CD44 and diminished CD62L expres- sion as compared with T cells in WT littermate controls; unpublished data). Thus, we have chosen to study the T reg cell subset in 8–10-d-old Tgf-1 mice. Flow cytometric analysis revealed the peripheral CD4 CD25 T cell com- partment reduced by 2.5–3-fold in Tgf-1 mice as com- pared with the WT littermate controls (Fig. 1, d and e). However, the CD4 CD25 thymocyte subset in these ani- mals was similar in size to that of littermate controls (Fig. 1, d and e). Thus, these results indicate that TGF-1 plays a role in the peripheral T reg cell maintenance. In contrast with these data, no difference was previously found in the CD25 CD4 T cell subsets not only in the thymus but also in the periphery in 5–7-d-old TGF-1–deficient mice (20). This apparent discrepancy is most likely due to the fact that the numbers of peripheral T reg cells in 5–7-d-old mice are very small (unpublished data) and the vast majority of these cells are recent arrivals from the thymus exhibiting normal Figure 2. Decrease in Foxp3 level in Tgf-1 CD4 CD25 T cells. levels of Foxp3 characteristic of thymic T reg cells. Thymocytes and splenocytes from 8–10-d-old Tgf-1 mice or littermate controls were stained for CD4 and CD25 followed by anti-Foxp3 intracellular TGF-1 is required to maintain Foxp3 expression in T reg cells staining and analyzed by flow cytometry. Foxp3 staining in CD4 CD25 T Next, we investigated whether TGF-1 can regulate Foxp3 cells (black line) and in CD4 CD25 (gray line) are shown. Isotype control staining is shown (dashed line). These results are representative of three expression in T reg cells. First, we analyzed Foxp3 ex- different experiments. pression by intracellular staining of thymic and splenic CD4 CD25 T cells isolated from Tgf-1 mice or WT littermate controls (Fig. 2). No significant difference in Foxp3 expression was observed in CD4 CD25 thymocytes of TGF-1 (Fig. 3 b), led to an increase in Foxp3 expression in mutant versus WT mice, whereas peripheral Tgf-1 in T reg cells comparable to that of WT littermate control T CD4 CD25 T cells expressed significantly diminished level reg cells. Furthermore, transfer of CD4 CD25 T cells from of Foxp3 compared with the control. Thus, the absence of littermate control mice into recipients treated with the anti– TGF-1 results in diminished Foxp3 expression in periph- TGF- antibody resulted in somewhat diminished Foxp3 eral CD4 CD25 T cells in addition to a substantial de- expression in those cells after 4 d. Altogether, these results, crease in size of this T cell compartment. Nevertheless, some involving both genetic modifications and in vivo adoptive peripheral regulatory T cells in TGF-1 null mice still main- transfer approaches, show that TGF-1 maintains Foxp3 ex- tain Foxp3 expression. The latter is likely due to normal pression in peripheral T reg cells. Furthermore, the engage- level of Foxp3 expression in thymocytes being preserved in ment of TGF- signaling pathway in T reg cells cultured in recent thymic emigrants. Provision of small amounts of the presence of TGF-1 resulted in induction of Smad2 TGF-1 by mother via breastfeeding and potential compen- phosphorylation and concomitant increase in Foxp3 expres- satory role of TGF-2 and TGF-3 can also contribute to sion (Fig. 3 c). This observation further confirms that TGF- maintaining Foxp3 expression in some regulatory T cells in maintains Foxp3 high expression in T reg cells. By inference the knockout mice. This result strongly suggests that TGF- from recent observations of acquisition of T reg cell pheno- 1 is required for the maintenance of Foxp3 levels in pe- type and suppressor activity by CD4 CD25 T cells upon ripheral T reg cells. retroviral transduction with Foxp3 (9, 10), the loss of pe- To test this hypothesis, CD4 CD25 T cells from either ripheral T reg cells and their diminished suppressor activity / / 8–10-d-old Tgf-1 mice or littermate control mice were in Tgf-1 mice can be a direct consequence of decreasing adoptively transferred into lymphopenic TCR/-deficient levels of Foxp3. host treated with the neutralizing anti–TGF- antibody or isotype control IgG and analyzed 4 d later by flow cytome- TGF-1 is required to maintain T reg cell suppressive function try (Fig. 3 a). As expected, the majority of Tgf-1 After establishing a decreased level of Foxp3 expression in CD4 CD25 T cells transferred into anti–TGF-–treated T reg cells in the absence of TGF-1, we investigated recipient mice continued to exhibit decreased Foxp3 levels, whether TGF-1 is also involved in the maintenance of high / whereas few Foxp3 cells were observed. The latter may their regulatory function. Purified T reg cells from Tgf-1 be due to incomplete antibody-mediated TGF-1 deletion mice or littermate control mice were cocultured with re- in addition to the aforementioned compensatory role of sponder CD4 CD25 T cells from control mice in the TGF-2 and TGF-3. However, transfer of these cells into presence of Con A and T cell–depleted splenic APCs iso- control IgG1-treated recipients, expressing normal amounts lated from either Tgf-1 or littermate control mice (Fig. JEM VOL. 201, April 4, 2005 1063 deficient DO11.10 TCR transgenic mice can prevent colitis mediated by WT CD4 CD25 T cells transferred into SCID recipient mice. However, this suppression was abro- gated in the presence of neutralizing antibodies specific for TGF-1 leading to disease progression (22). We also ob- served that T reg cells from Tgf-1 mice were able to / / suppress autoimmunity developing in Tgf-1 RAG mice upon transfer of Tgf-1 CD4 CD25 T cells (23). These findings are consistent with our notion that Tgf-1 T reg cells require TGF-1 provided by other cells to pre- serve their functionality. Although the types of accessory cells supplying TGF-1 for T reg cells in vivo remains to be determined, our analysis of in vitro TGF-1 production us- ing ELISA implicated APCs, such as dendritic cells, as the likely candidates (unpublished data). TGF-1–mediated signaling in T reg cells is required to maintain suppressor function However, these results do not address the question as to whether T reg cells require TGF-1 signaling to maintain their functionality or whether T reg cells are “armed” with the receptor-bound TGF-1 produced either endogenously or secreted by APCs. The latter possibility was raised by two reports suggesting that receptor-bound TGF-1 displayed on the surface of T reg cells may function as an effector mol- ecule in T reg cell–mediated suppression as assessed in an in vitro assay (19, 24). To address these two possibilities, we transduced T reg cells isolated from B6 mice with the Mig- R2 retroviral vector encoding a dominant negative form of Figure 3. TGF-1 is required to maintain Foxp3 in CD4 CD25 the TGF-1 receptor (DN–TGF-RII) and IRES-driven T cells. T reg cells purified from spleen of 8–10-d-old Tgf-1 mice or tail-less human CD2 as a reporter. DN–TGF-RII was gen- littermate controls were transferred into TCR/-deficient mice injected erated upon deletion of the TGF-RII kinase domain. As a every other day with anti–TGF-1 antibody or isotype control IgG. 4 d control, T reg cells were transduced with the MIG-R2 vec- after transfer, cells from spleen and lymph nodes were stained for CD4 tor without insert. Transduced T reg cells were purified by and CD25 followed by anti-Foxp3 intracellular staining and analyzed by magnetic bead sorting using anti–human CD2 antibody. flow cytometry (a). Results are representative of three different experiments. Serum TGF-1 in treated animals was measured by ELISA (b). Western blot Suppressor activity of DN–TGF-RII– and empty vector– analysis of Smad2 phosphorylation and Foxp3 expression in purified wild- transduced T reg cells was tested in an in vitro suppression type T reg cells either freshly isolated or cultured for 24 h in the presence assay. The regulatory capacity of T reg cells expressing DN– or absence of 100 pg/ml TGF-1 (c). TGF-RII was diminished, but not completely abolished, as compared with the control T reg cells (Fig. 4 b). This is likely due to the fact that T reg cells expressing DN–TGF- 4 a). All combinations of WT and mutant T cells and APCs RII remain still sensitive to TGF-, albeit require an 10- resulted in a comparable suppression except for a signifi- fold higher dose to induce Smad2 phosphorylation at a level comparable to that of control MigR2-transduced cells (un- cantly diminished suppression by Tgf-1 T reg cells ob- served only in the presence of Tgf-1 APCs. Thus, these published data). Importantly, although expression of DN– data demonstrate that TGF-1 production by T reg cells is TGF-RII inhibits TGF-–mediated signaling, it is ex- pected to increase ability of T reg cells to capture TGF- on dispensable for their suppressor function because APCs can produce sufficient amounts of TGF-1 to support T reg the plasma membrane. The analysis of suppressor function of cell–mediated suppression. T reg cells transduced with DN–TGF-RII suggests that impairment in suppressor function in the absence of TGF- Although relative contribution of TGF-1 produced by T reg cells versus other cellular sources to T reg cell function 1 is due to a signaling defect and not due to lack of recep- in vivo remains to be studied further, our results suggest a tor-mediated display of TGF- on the T reg cell surface. requirement for paracrine TGF-1 to maintain T reg cell Therefore, our results are consistent with the role of TGF- not as an effector molecule of suppression, but as a mediator function in the periphery. This observation is in agreement with recent experiments by Powrie et al., who showed that of signaling in T reg cells required to maintain their suppres- an adoptive transfer of T reg cells isolated from TGF-1– sor function. Thus, our findings provide novel important in- 1064 ROLE OF TGF-1 IN T REG CELL MAINTENANCE | Marie et al. BRIEF DEFINITIVE REPORT Figure 4. TGF- signaling is required to maintain regulatory func- results are shown as mean cpm of [ H]thymidine incorporation in tripli- tion T reg cells. (a) Analysis of suppressor activity of T reg cells isolated cate cultures  SD. (b) T reg cells transduced with DN–TGF-RII–MigR2 from Tgf-1 (gray symbols) or WT littermates (Lit, black symbols) in (MigR2 DN) or empty vector control (MigR2 Ev) were purified and cocul- cocultures with freshly isolated B6 CD4 CD25 responder T cells in the tured with freshly isolated B6 CD4 CD25 responder T cells in the presence presence of either Tgf-1 or littermate control APCs and Con A. Prolif- of irradiated T cell–depleted splenic APCs and Con A. Proliferation was eration of CD4 CD25 T cells was determined after 72 h of culture by measured as described before. Results are representative of two or three [ H]thymidine incorporation. Proliferation of CD4 CD25 from either different experiments. control or Tgf-1 mice in response to Con A was 300–500 cpm. The sights into the role of TGF-1 in T reg cell function recon- purposes, there is no experimental evidence so far that such ciling previous contradictory observations. high concentrations of TGF-1 are ever attainable in vivo Recently, several in vitro studies have reported acquisi- and that such a conversion of CD4 CD25 into CD4 tion of suppressor activity and Foxp3 expression in the bulk CD25 T cells expressing Foxp3 can occur under physio- populations of cultured murine and human CD4 CD25 T logic conditions or in the course of immune inflammation. cells upon activation in the presence of nanomolar amounts In our earlier experiments, we failed to detect any measur- of recombinant TGF-1 (25, 26). Although these studies able conversion of CD4 CD25 T cells into CD4 CD25 opened up an important avenue for pharmacologic in vitro upon their transfer into newborn Foxp3-deficient mice (10). manipulation of T cell function for potential therapeutic Ramsdell et al. have recently reported high levels of TGF- 1 JEM VOL. 201, April 4, 2005 1065 6 formed after cell lysate separation in 12% SDS-PAGE gels (3 10 cell in Foxp3 mutant mice in addition to highly elevated levels equivalents/lane) as described elsewhere (10). of a number of other cytokines (11). Nevertheless, it will Suppression assays were performed as described (10). In brief, CD4 be of interest to experimentally test whether substantial CD25 T cells (4 10 cells/well) were stimulated for 72 h with 1 g/ml amounts of TGF-1 displayed by some immunoprivileged of Con A in the presence of irradiated (2,000 rad) T cell–depleted spleno- tissues and organs, like the ocular chamber of the eye, can fa- cytes (APCs) (2 10 /well) in 96-well round-bottom plates, with indi- cilitate maintenance, expansion, or de novo generation of cated numbers of CD4 CD25 T cells and pulsed with 1 Ci/well of [ H]thymidine for the final 16 h of culture. Data are shown as mean antigen-specific T reg cells (27). This idea is supported by [ H]thymidine incorporation in triplicate cultures. the recent observation of significant expansion and/or gen- eration of protective T reg cells in pancreatic lymph nodes Retroviral infection. The first 656 bp of the coding sequence of TGF- and islets in NOD mice upon temporal TGF-1 induction RII were subcloned from pcDNA3.1-myc-HisB DN-TGF-RII () into in the -cells (28). MSCV MigR2 retroviral vector. The vector DNA with or without insert In conclusion, our study demonstrates an essential in was transfected into the NX-E packaging cell line using Fugene 6 reagent (Roche) according to the manufacturer’s protocol. Retrovirus-containing vivo role for the TGF-1–mediated signaling in the mainte- supernatant was collected after a 36-h culture of the transfected packaging nance of naturally arising regulatory CD4 CD25 T cell cells at 32 C. Freshly isolated MACS-purified CD4 CD25 T cells were numbers and function, and in the maintenance of Foxp3 ex- activated using plate-bound anti-TCR chain and anti-CD28 antibodies, pression in these cells in the periphery. These results demon- in the presence of 100 U/ml recombinant human IL-2 (Hoffmann–La strate an important role of TGF-1 in T reg cell biology and Roche). After 24 h of activation, CD4 CD25 T cells were infected by re- suggest additional applications for targeting of TGF-1 sig- suspending cells in retrovirus-containing supernatants supplemented with 8 g/ml polybrene and 50 U/ml of recombinant IL-2, followed by centrifu- naling pathway for therapeutic immunomodulation. gation for 90 min at 2,500 revolutions/min. Cells were cultured for 48 h at 37 C in the presence of 200 U/ml IL-2 and 10 pg/ml TGF-1 to maintain MATERIALS AND METHODS CD25 and Foxp3 expression. Mice. C57BL/6 (B6) and TCR/-deficient mice were purchased from Charles River Breeding Laboratories and The Jackson Laboratory, respec- / We thank the members of the Rudensky laboratory for discussions and for reading tively. Tgf-1 mice were previously described (29). All mice were main- the text. tained in a specific pathogen-free animal facility at the University of Wash- This work was supported by grants from International Human Frontier Science ington and National Institutes of Health and handled in accordance with Program (to J.C. Marie) and National Institutes of Health nos. AI34206 and AI061816 the institutional guidelines. (to A.Y. Rudensky). A.Y. Rudensky is a Howard Hughes Medical Institute investigator. The authors have no conflicting financial interests. Antibodies. Anti-Foxp3 IgG was purified on a protein G–Sepharose 4 Fast Flow column from the rabbit antiserum raised against purified His- Submitted: 5 November 2004 tagged recombinant Foxp3 protein. Biotinylated (bio), FITC-, PE-, cy- Accepted: 15 February 2005 chrome (CyC)-, peridinin chlorophyl protein (PerCP)-, and allophycocya- nin-conjugated monoclonal antibodies to CD4 (L3T4), CD8 (53–6.7), REFERENCES CD25 (7D4), CD25 (PC-61), CD62L (MEL-14), CD69 (H1.2F3), and 1. 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Journal

The Journal of Experimental Medicine – Pubmed Central

Published: Apr 4, 2005

References