Constitutive Nuclear Localization of NFAT in Foxp3 + Regulatory T Cells Independent of Activity

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published April 4, 2012, doi:10.4049/jimmunol.1102376 The Journal of Immunology

Constitutive Nuclear Localization of NFAT in Foxp3+ Regulatory T Cells Independent of Calcineurin Activity

Qiuxia Li,*,1 Arvind Shakya,*,1 Xiaohua Guo,*,1 Hongbo Zhang,† Dean Tantin,* Peter E. Jensen,* and Xinjian Chen*

Foxp3 plays an essential role in conferring suppressive functionality to CD4+/Foxp3+ regulatory T cells (Tregs). Although studies showed that Foxp3 has to form cooperative complexes with NFAT to bind to target genes, it remains unclear whether NFAT is available in the nucleus of primary Tregs for Foxp3 access. It is generally believed that NFAT in resting cells resides in the cytoplasm, and its nuclear translocation depends on calcineurin (CN) activation. We report that a fraction of NFAT protein constitutively localizes in the nucleus of primary Tregs, where it selectively binds to Foxp3 target genes. Treating Tregs with CN inhibitor does not induce export of NFAT from the nucleus, indicating that its nuclear translocation is independent of CN activity.

Consistently, Tregs are resistant to CN inhibitors in the presence of IL-2 and continue to proliferate in response to anti-CD3 Downloaded from stimulation, whereas proliferation of non-Tregs is abrogated by CN inhibitors. In addition, PMA, which activates other tran- scription factors required for activation but not NFAT, selectively induces Treg proliferation in the absence of ionomycin. TCR interaction with self-MHC class II is not required for PMA-induced Treg proliferation. Tregs expanded by PMA or in the presence of CN inhibitors maintain Treg phenotype and functionality. These findings shed light on Treg biology, paving the way for strategies to selectively activate Tregs. The Journal of Immunology, 2012, 188: 000–000. http://www.jimmunol.org/ he naturally occurring CD4+/Foxp3+ regulatory T cells unclear how NFAT becomes accessible to Foxp3 in the nucleus of (Tregs) are absolutely required for the maintenance of Tregs; as in resting cells, NFAT proteins are located in the cyto- T immunological tolerance to self-Ags (1). In both animal plasm where they are heavily phosphorylated through synergistic models and human patients, lack of Tregs is associated with ful- actions of three kinase families: , glycogen syn- minant, lethal autoimmune syndromes (2–6). Even in adult animals thase kinase 3, and dual-specificity tyrosine phosphorylation- in which a normal immune repertoire is already established, acute regulated kinase (17–19). Phosphorylation results in masking of depletion of Tregs gives rise to similarly catastrophic autoimmune a nuclear localization sequence, exposure of a nuclear export outcomes (7), demonstrating that, under physiological conditions, sequence, and NFAT cytoplasmic localization. When cells are Tregs are constantly active in suppressing autoreactive lymphocytes stimulated, intracellular Ca2+ concentrations increase, and the by guest on September 29, 2021 present in a normal immune repertoire. Both the suppressor func- -dependent phosphatase calcineurin (CN) is activated, tion, as well as the phenotype, of Tregs depends on the forkhead which dephosphorylates NFAT, exposing the nuclear localization Foxp3 (8, 9), which binds to and regulates sequence and masking the nuclear export sequence, resulting in hundreds of target genes (10, 11). Foxp3 binds to DNA targets in the nuclear translocation (20, 21). When the stimulus is removed, form of cooperative complexes with other transcription factors, in- nuclear NFAT is rapidly exported back to the cytoplasm as a result cluding NFATc2 and RUNX1 (12–15). Structure-guided mutations of the constitutive kinase activity that rephosphorylates NFAT expected to disrupt the interaction between Foxp3 and NFATc2 in- (22–24). Thus, in resting/nonstimulated cells, the equilibrium terfere with the ability of Foxp3 to repress expression of IL-2, up- of the dephosphorylation/rephosphorylation and nuclear import/ regulate expression of the Treg markers, such as CD25 and CTLA-4, export favors rephosphorylation and cytoplasmic NFAT localiza- or confer suppressor function (12), indicating that the interaction tion (25). Consistent with this notion, it was reported that, in between Foxp3 and NFATc2 is essential for Treg functionality. Foxp3-transduced or -cultured Tregs, the binding of Foxp3:NFAT Although the binding of Foxp2 and Foxp3 to NFATc2 is well complexes to Foxp3 target genes is observed only upon stimula- characterized structurally by crystallography (13, 16), it remains tion of cells with PMA plus ionomycin or ionomycin alone (16). Other studies using Foxp3-transfected T cells or T cell lines also found that changes in expression of Foxp3-bound genes, such as *Department of Pathology, University of Utah, Salt Lake City, UT 84132; and Il2, Ctla4, and Il2ra, occurred only upon TCR stimulation in a † Department of Forensic Sciences, Xi’an Jiaotong University, Xi’an, Shaanxi, CN-dependent manner (11, 26). 710049, China The finding of a requirement for in vitro activation to induce 1Q.L., A.S., and X.G. contributed equally to this work. Foxp3 binding to its target genes has raised a fundamental question Received for publication August 16, 2011. Accepted for publication March 2, 2012. regarding the role of Foxp3 in conferring the Treg phenotype (27, Address correspondence and reprint requests to Dr. Xinjian Chen and Dr. Dean Tantin, 28). The phenotype of the naturally occurring Tregs, such as ex- Department of Pathology, University of Utah, 30 North 1900 East, EEJ 1250, Salt Lake City, UT 84124. E-mail addresses: [email protected] (X.C.) and dean. pression of high levels of CD25 and CTLA-4 and inability to [email protected] (D.T.) produce IL-2, is constitutive, requiring no in vitro activation. If The online version of this article contains supplemental material. Foxp3 plays an essential role in conferring the Treg phenotype Abbreviations used in this article: ChIP, chromatin immunoprecipitation; CN, calci- (28, 29), Foxp3 binding to at least some targets should be con- neurin; CsA, cyclosporine A; GVHD, graft-versus-host disease; MHCII, MHC class stitutive. In addition, studies suggested that NFAT is required for II; nTreg, naturally occurring ; Treg, regulatory T cell. Foxp3 expression (30). Consistent with these observations, Zheng Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 et al. (10) found that Foxp3 is constitutively bound to ∼700 target

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102376 2 NFAT IN Foxp3+ TREGs genes in freshly isolated, nonstimulated naturally occurring Tregs. Results These latter findings suggest that Foxp3-transduced cells or cell A fraction of NFAT protein is constitutively localized in the lines may differ from primary naturally occurring Tregs in terms nucleus of primary Tregs independent of CN activity of the status of Foxp3 binding to its target genes. Given the Foxp3- To examine subcellular NFAT localization in primary Tregs, containing cooperative complexes that require NFAT for DNA whole-cell lysates and cytoplasmic and nuclear fractions were pre- binding, the constitutive binding of Foxp3 suggests that NFAT 2 pared from CD25+ and CD25 CD4 T cells freshly isolated from might be present in the nucleus of primary, naturally occurring spleens and lymph nodes of B6 or BALB/c mice. The samples Tregs. were analyzed by Western blot for NFATc1 and NFATc2, along In this study, we examined NFAT localization in freshly isolated with c-Fos and c-Rel, which are the components of two other major primary Tregs and found that a fraction of NFATwas constitutively transcription factors AP-1 and NF-kB (Fig. 1). The total quantities localized in the nucleus where it selectively binds to Foxp3 target of NFATc1, NFATc2, and c-Fos (AP-1) in whole-cell lysates were genes. NFAT nuclear translocation was independent of CN activity, 2 similar between the CD25+ and CD25 T cell subsets, but ∼2–3- and Tregs are consistently resistant to CN inhibition. In addition, fold more c-Rel protein was detected in CD25+ cells compared Tregs can be selectively activated and induced to proliferate from 2 with CD25 cells in multiple experiments (Fig. 1, lanes 1 and 2). a mixed cell population, dominated by non-Tregs, by phorbol ester 2 In CD25 cells, all four transcription factors were detected in the in the absence of CN activator ionomycin. Our findings provide cytoplasm but not the nucleus (Fig. 1, lanes 3 and 6), as previously important insight into Treg biology. reported (23). Similarly, in the CD25+ cells, NFATc1, NFATc2, c- Rel, and c-Fos proteins were also largely present in the cytoplasm Downloaded from Materials and Methods (Fig. 1, lane 4). More c-Rel protein was identified in the cytoplasm Western blot analysis in the CD25+ CD4 T cells compared with the CD252 CD4 T cells, CD25+ and CD252 CD4 T cells were isolated from splenocytes using the consistent with the results from whole-cell lysates (Fig. 1, lane 2). CD25+CD4+ Regulatory T cell Isolation Kit (Miltenyi Biotec), following However, a trace amount of c-Rel protein was repeatedly detected 2 the manufacturer’s instructions, as previously described (31). To isolate in the nucleus of the CD25+, but not the CD25 , cells (Fig. 1, lanes + + + Foxp3 GFP cells, CD4 T cells were first isolated from splenocytes of 5 and 6). In addition, a fraction of NFATc1 (20–40%) and NFATc2 Foxp3EGFP mice (32) by negative selection using a CD4 T Cell Isolation + http://www.jimmunol.org/ + (10–30%) proteins was also detected in the nucleus of the CD25 , Kit (Miltenyi Biotec), followed by sorting of GFP cells by FACS (33). All 2 of the isolated cells were rested in culture or treated with cyclosporine but not the CD25 , CD4 T cells in multiple experiments (Fig. 1, A (CsA) for 30 min before being processed for protein extracts. For whole- lanes 5 and 6). The presence of NFAT and c-Rel protein in the cell lysates, isolated cells were lysed with radioimmunoprecipitation nuclear extracts was not the result of cytoplasmic contamination, assay buffer (50 mM NaCl, 25 mM Tris [pH 7.5], 1 mM EDTA, 0.1% because tubulin, a cytoplasmic protein, was not detected in the SDS, 1% w/v deoxycholate, and 1% Nonidet P-40) 0.5 mMphenyl- methanesulfonylfluoride or phenylmethylsulfonyl fluoride plus protease nuclear extracts, although it was detected in the cytoplasm extracts inhibitor, Complete Mini Pill (Roche) per 10 ml lysis buffer. Nuclear and and whole-cell lysates (Fig. 1). cytoplasmic extracts were obtained using NE-PER Nuclear and Cyto- To rule out the possibility that the nuclear translocation of NFAT plasmic Extraction Reagents (Thermo Scientific), following the manu- +

in CD25 cells was the result of inadvertent activation of the by guest on September 29, 2021 facturer’s instructions, except that CER I and CER II were diluted with cells by the anti-CD25 Ab used during cell isolation, Western blot PBS at 1:1 and 1:4 ratios, respectively, to avoid overlysis. A total of 0.5–2 + 3 106 cell equivalents of protein was loaded per lane. Western blots were analysis was also performed with Tregs isolated from the Foxp3 / + revealed with Abs specific for NFATc1, NFATc2 (both from Thermo GFP mice (32) using a fluorescent cell sorter and no anti-CD25 Scientific), c-Rel, and c-Fos (both from Santa Cruz Biotechnology). Abs. The flow-sorted Foxp3+/GFP+ cells were rested in culture for 30 min before being processed for preparation of nuclear extracts. Chromatin immunoprecipitation-PCR analysis Previous studies showed that resting activated non-Treg cells in Foxp3+GFP+ Tregs and Foxp32GFP2 non-Treg CD4 T cells were isolated culture for 30 min completely exports the nuclear NFAT to the from Foxp3EGFP mice by FACS sorting and rested in culture. A portion of cytoplasm (23, 25). Resting the Foxp3+/GFP+ cells did not export the non-Tregs was stimulated with PMA plus ionomycin for 30 min and used as positive control. Chromatin immunoprecipitation (ChIP) was per- formed as described previously (34). Abs against NFATc1 (MA3-024) and NFATc2 (MA1-025) (Thermo Scientific) were used together to precipitate NFAT–DNA complexes. PCR was performed with primer sets specific for five previously identified Foxp3 targets, including Il2 (16), Pde3b-20, Irf4, miR-155, and Fasl (10), and five genes not identified as Foxp3 targets (10), including promoters of Il-4 (35, 36), Il-12a (37–39), Il-17 (40), Pde3b, and an enhancer of Il-4 (41). The primer sequences that are not published in previous studies and designed to amplify the NFAT binding site in non- Foxp3 targets include the following: Pde3b-F: 59-TGGTGAAAAATAGG- TCTGTTTGTGG-39 and Pde3b-R: 59-TGCTGAATGAAAGGGATTGCC- 39; Il12a-F: 59-TGGGTTAGGGACTGTGTCTGGTG-39, Il12a-R: 59-GG- GGACAAGGGACAATGGATG-39;andIl17-F: 59-CTACCTTCGAGA- CAGATGTTGCCC-39, Il17-R: 59-TGATCAGCTGGTGCTGGGTG-39.

Cell culture FIGURE 1. Western blot analysis of transcription factors in CD25+ and To examine Treg sensitivity to CN inhibition, isolated Foxp3+GFP+ cells or 2 lanes 1 2 lanes 2 2 CD25 CD4 T cells. Whole-cell lysates ( and ), cytoplasm ( Foxp3 GFP CD44hi and CD44lo CD4 T cells were isolated using a flow 3 and 4), and nuclear extract (lanes 5 and 6) of isolated CD252 (neg) and m cytometric cell sorter and cultured with anti-CD3 Ab (2C11; 1 g/ml), IL- CD25+ (pos) CD4 T cells were analyzed by Western blot for the presence 2 (20 U/ml), and irradiated splenic APCs (1200 rad) in the presence or of NFATc1, NFATc2, c-Rel, and c-Fos. Tubulin and MCM2, a constitutive absence of CsA (1 mM) for 4–5 d. Alternatively, unfractionated spleno- nuclear protein (77), were used as markers for cytoplasmic and nuclear cytes were labeled with CFSE, as previously described (31), and cultured + + with 2C11, with or without IL-2 (20 U/ml), in the presence or absence of proteins, respectively. Foxp3 /GFP CD4 T cells were rested (lane 7)or 1 mM CsA. In PMA-mediated culture, PMA was used at 10–20 ng/ml, with treated with 1 mM CsA in culture for 30 min (lane 8), and their nuclear or without ionomycin (500 ng/ml), as indicated in the figures. IL-2–neu- extracts were blotted for NFATc1, tubulin, and MCM2. The results are tralizing Ab was used at 20 mg/ml. representative of multiple experiments. The Journal of Immunology 3

NFAT from the nucleus (Fig. 1, lane 7, Supplemental Fig. 1a), whether the nuclear NFAT in Tregs was bound to any DNA targets. suggesting that the nuclear localization of a fraction of the NFAT ChIP was performed with anti-NFATc1 and anti-NFATc2 Abs to in the Tregs is unlikely the result of inadvertent activation of the obtain NFAT-bound DNA from freshly isolated and rested Foxp3+ cells during cell isolation. GFP+ CD4 Tregs. For positive and negative controls, anti-NFAT Calcineurin (CN) plays an essential role in dephosphorylating ChIP DNAs were obtained from freshly isolated Foxp32GFP2 NFAT to induce nuclear translocation in non-Tregs (25). To de- CD4 non-Tregs, with and without phorbol ester PMA/ionomycin termine whether NFAT nuclear localization in the Tregs was driven activation. The three ChIP DNA samples were then subjected to by continuous CN activity, the Foxp3+/GFP+ cells were treated PCR amplification with 10 primer sets: 5 specific for the NFAT with the CN inhibitor CsA for 30 min before nuclear extract binding sites of genes previously identified as Foxp3 targets [the preparation. Previous studies demonstrated that CsA completely promoters of Il2, Irf4f, mir155, Fasl, and Pde3b-20 (first intron of abrogates CN activity and induces the export of NFAT from the Pde3b) (10)] (Fig. 2A–E) and 5 specific for genes not identified as nucleus, even when the T cells are in culture with the Ca2+ iono- Foxp3 targets (10) [the promoters of Il4 (35, 36), Il12a (37–39), phore, ionomycin (23). However, treating Foxp3+/GFP+ Tregs did Il17 (40), Pde3b, and an enhancer of Il4] (Fig. 2F–J). The PCR not mobilize the nuclear NFAT proteins out of the nucleus (Fig. 1, products were analyzed by gel electrophoresis. No PCR products lane 8, Supplemental Fig. 1b). These results demonstrate that in were generated with any of the primer sets from ChIP DNA Tregs, a fraction of NFAT, as well as a trace amount of c-Rel samples prepared with the isotype control Abs (except for Pde3b- protein, is constitutively translocated in the nucleus and that the 20, for which weak background PCR products were generated constitutive nuclear localization of NFAT is independent of con- from control Ab-precipitated DNA), ensuring the validity of the tinuous CN activity. assay (Fig. 2, left column of each nine-unit panel). In contrast, Downloaded from To directly visualize NFAT subcellular localization, confocal positive PCR products were obtained with all primer sets using microscopy was performed on FACS-sorted Foxp3/GFP2 and input DNA samples, which serve as positive controls for the pri- Foxp3/GFP+ CD4 T cells (Supplemental Fig. 2). In Foxp3/GFP2 mers (Fig. 2, right column of each nine-unit panel). CD4 T cells, NFAT was visualized only in the cytoplasm (Sup- No PCR product was detected from ChIP DNA isolated from plemental Fig. 2b, 2c). However, in Foxp3/GFP+ Tregs, NFAT was rested Foxp32/GFP2 CD4 T cells with any of the 10 primer sets

seen both in the cytoplasm and nucleus (Supplemental Fig. 2d). (Fig. 2, top middle grid of each nine-unit panel), consistent with http://www.jimmunol.org/ Treating Foxp3/GFP+ cells with CsA did not induce export of the Western blot results (Fig. 1) and previous reports (23) that, in nuclear NFAT (Supplemental Fig. 2e), indicating that its nuclear resting non-Tregs, NFAT is localized in the cytoplasm and not translocation is independent of CN activity. Culturing Foxp3/ bound to DNA. In contrast to the resting non-Tregs, robust PCR GFP2 CD4 T cells with ionomycin resulted in NFAT nuclear amplicons were generated from ChIP DNA isolated from rested translocation (Supplemental Fig. 2f). However, when CsA was Foxp3+GFP+ CD4 Tregs using primer sets specific to Foxp3 target added to the ionomycin-containing culture, all NFAT protein was genes, including Il2, Irf4, mir155, and Pde3b-20 (Fig. 2A–C, 2E, seen only in the cytoplasm (Supplemental Fig. 2g), indicating that middle row), indicating that NFAT is constitutively bound to these CsA abrogates CN-dependent NFAT nuclear translocation in non- four genes. No amplicons were generated from the Fasl promoter, Tregs, despite the continuous presence of a CN-activating signal. although it was also identified as a Foxp3 target in a previous by guest on September 29, 2021 Therefore, the results of confocal microscopy are consistent with study (10). Despite prevalent binding to the Foxp3 targets, NFAT the results of Western blots, demonstrating that, in Tregs, a frac- was not bound to any of the five tested non-Foxp3 target genes in tion of NFAT proteins is constitutively localized in the nucleus in Tregs, as indicated by a lack of DNA amplicons using the primer a CN-independent manner. sets specific for these five NFAT binding sites (Fig. 2F–J, middle row), which were the Il4, Il12a, Il17, and Pde3b promoters and the NFAT is selectively bound to Foxp3 target genes in Tregs Il4 enhancer. The lack of PCR products from the non-Foxp3 In activated T cells, NFAT complexes with AP-1 to bind to com- binding sites was not due to the inability of NFAT to bind these posite NFAT–AP-1 DNA sites (22, 42). Because no c-Fos (AP-1) sites or of the PCR primers to amplify these regions from ChIP protein was detected in the nucleus of Tregs (Fig. 1), we examined DNA, because all five primer sets generated robust PCR products

FIGURE 2. ChIP analysis of NFAT-bound genes in Tregs and non-Tregs. Chromatin DNA precipitated with isotype control (Ctl; left column of each nine-unit panel) or anti-NFATc1 and -NFATc2 Abs (middle column of each nine-unit panel) from freshly isolated, rested Foxp32/GFP2 non-Tregs (top row of each nine- unit panel) and Foxp3+/GFP+ Tregs (middle row of each nine-unit panel) or PMA plus ionomycin-acti- vated Foxp32/GFP2 CD4 T cells (bottom row of each nine-unit panel) were subjected to PCR amplification with primer sets specific for NFAT binding sites in five known Foxp3 target genes [Il2 (A), Irf4 (B), mir155 (C), Fasl (D), and the first intron of Pde3b (Pde3b-20) (E)] and five non-Foxp3 target genes [Il4 (F), Il12a (G), Il17 (H), Pde3b (I), and Il4 enhancer (J)]. Input DNA of each source for chromatin precipitation was used as positive control (right column of each nine- unit panel). Images of PCR products resolved using agarose gel electrophoresis are shown. The results are representative of two separate experiments with DNA isolated from cells of 20 mice. 4 NFAT IN Foxp3+ TREGs from ChIP DNA samples isolated from PMA/ionomycin-activated Western blot and confocal analyses showing that CN inhibitors non-Tregs (Fig. 2F–J, bottom row). In addition to the non-Foxp3 did not export nuclear NFAT in Tregs. Studies reported CN- targets, NFAT bound to three of the Foxp3 targets, including independent pathways of T cell activation (77); it is possible that the promoters of Il-2, Irf4, and Pde3b-20 in PMA/ionomycin- such pathways might be involved in Treg activation. The sus- activated non-Tregs (Fig. 2A, 2B, 2E, bottom row). NFAT bind- ceptibility of CD44+ memory T cells to inhibition by CsA is ing to Foxp3 targets in activated non-Tregs is consistent with the consistent with the previous report that, although total NFAT is previous report showing NFAT:Foxp3 binding to the Il2 promoter more abundant in memory CD4 T cells than in naive CD4 T cells, in Tregs (16) but NFAT:AP-1 binding in activated T cells (25). The NFAT resides in the cytoplasm, but not in the nucleus, in the forkhead protein-binding sequence resembles the consensus AP-1 absence of activation (45). binding site (43). In activated non-Tregs, NFAT and AP-1, along The lack of significant inhibition of Tregs by CsA could be due with other transcription factors, are translocated into the nucleus; to degradation of CsA by Tregs. To rule out this possibility, whole as a result, NFAT can form a cooperative complex with AP-1 to splenocytes that contained ∼5% Foxp3+ cells (Fig. 3M) were bind to Foxp3 target sites in the absence of Foxp3. ChIP analysis cultured with anti-CD3 Abs in the presence or absence of CsA. was also performed using anti-NFATc1 or anti-NFATc2 Ab sepa- In the absence of CsA, TCR stimulation induced vigorous T cell rately on resting Foxp3/GFP+ and Foxp3/GFP2 CD4 T cells using proliferation dominated by Foxp32 CD4 and CD8 non-Tregs PMA/ionomycin-activated Foxp3/GFP2 CD4 T cells as control. (Fig. 3N, 3R), resulting in proliferative Treg to non-Treg CD4 The ChIP-DNA samples were analyzed by quantitative PCR, and and CD8 T cell ratios of 0.1 and 0.05, respectively. In the presence the results (Supplemental Fig. 3) were in complete agreement with of CsA, the proliferation of all three cell subtypes was abrogated those obtained with the two Abs combined. Both NFATc1 and (Fig. 3O, 3S). However, this result was not unexpected because Downloaded from NFATc2 in Foxp3/GFP+ Tregs bound to three of the four tested CsA inhibits IL-2 production by non-Treg CD4 cells, and Tregs Foxp3 targets (Il2, Irf4, and miR155 but not Fasl or the non-Foxp3 depend on paracrine IL-2 to survive and proliferate (46–48). When target Pde3b). In contrast, neither NFATc1 nor NFATc2 in Foxp3/ exogenous IL-2 was supplemented in cultures containing CsA, GFP2 non-Tregs bound to any of the tested genes before activa- Foxp3+ Tregs preferentially proliferated, resulting in proliferative tion. Only after activation did NFATc1 and NFATc2 differentially Treg to non-Treg CD4 and CD8 cell ratios of 2 and 0.5, respec-

bind to the DNA targets (Supplemental Fig. 3). These results tively (Fig. 3P, 3T). Given the very low frequency of Tregs in the http://www.jimmunol.org/ further confirm constitutive NFAT nuclear localization and selec- starting population of input cells, the dominant proliferation of tive binding to Foxp3 targets in primary Tregs. Tregs suggests that the Tregs proliferated ∼20-fold more than did Taken together, the results of ChIP-PCR analysis demonstrate the non-Treg CD4 cells. It is possible that CsA might have in- that NFAT in primary Tregs is constitutively bound to some Foxp3 duced more of the non-Treg than Tregs. To rule out the targets but none of the tested non-Foxp3 targets. These results are possibility that the observed Treg resistance to CsA was due to the consistent with the Western blot results (Fig. 1), indicating that unique physical properties associated with CsA, another CN in- a fraction of NFAT is constitutively localized in the nucleus of hibitor, , was tested in total splenocyte culture. Similar primary Tregs. The differential binding of NFAT to the Foxp3 results were obtained (Fig. 3U). Conversion of non-Tregs did not targets in Tregs is consistent with the idea that nuclear localization contribute to the proliferative Foxp3+ cell pool, because the culture by guest on September 29, 2021 of NFAT in Tregs is not the result of inadvertent activation, be- of isolated Foxp32GFP2 non-Tregs in the presence of CN plus IL- cause NFAT in activated cells binds selectively to different DNA 2 did not generate GFP+ cells in the culture (Fig. 3I, 3J). In ad- targets. Our finding of constitutive NFAT binding to Foxp3 targets dition, almost all of the proliferating Foxp3+ cells expressed high in primary Tregs suggests that primary Tregs are different Foxp3- levels of Helios (Fig. 3W), a marker for naturally occurring, but transduced T cells or cultured cell lines, and significant binding of not inducible, Tregs (49). Therefore, the results of culture with Foxp3 to the DNA targets is detected only after the cells are unfractionated splenocytes are consistent with the results of sorted stimulated in vitro (11, 16, 26). Foxp3+GFP+ and Foxp32GFP2 cells (Fig. 3C–K), excluding the possibility that CsA resistance exhibited by the isolated Tregs Tregs are resistant to CN inhibition in the presence of IL-2 (Fig. 3K, 3L) was due to Treg-mediated degradation of CsA in the Full T cell activation is the result of nuclear translocation of culture. These results also demonstrate that Tregs were preferen- a variety of transcription factors, including NFAT, AP-1, and NF- tially expanded in the presence of CsA plus IL-2, even when they kB (44). Given the results of constitutive NFAT nuclear locali- were outnumbered by effector T cells. Based on the extent of CFSE zation in Tregs independent of CN activity, we next examined dilution and the fold expansion of the proliferative cells (Fig. 3P), it whether Treg activation is resistant to CN inhibitors. Foxp3+GFP+ could be inferred that the Foxp3+ CD4 T cells had divided at least cells (Fig. 3B) were stimulated with anti-CD3 (2C11) and IL-2 in two to eight times. The continuous proliferation of Tregs in the the absence or presence of the CN inhibitor CsA. CD442 naive presence of CN inhibitors provides further support that the relo- and CD44+ memory non-Tregs (Fig. 3A) were treated similarly cation of NFAT to the nucleus is independent of CN activity. Given and served as controls. In the absence of CsA, all three subsets of the essential role of NFAT in T cell activation and proliferation, T cells proliferated extensively (Fig 3F–H), with the intensity of these results also suggest a CN-independent mechanism to drive expansion of non-Tregs (∼40-fold for CD44+ and 30-fold for nuclear translocation of NFAT in Tregs to allow the cells to un- CD442 cells, respectively) exceeding that of Tregs (20-fold) (Fig. dergo activation and proliferation in the presence of IL-2. 3L). Despite the relative hyperreactivity of the non-Tregs to anti- CD3 stimulation, the addition of CsA (1 mM) almost completely Tregs can be selectively activated by the phorbol ester PMA in abrogated the proliferation of both CD442 and CD44+ non-Tregs the absence of ionomycin (Fig. 3I, 3J) but only mildly attenuated Foxp3+GFP+ cell prolifer- Although TCR stimulation is a classic way to activate T cells, ation (Fig. 3K, 3L). Thus, CN-dependent NFAT activation is not T cells can also be activated by the synergistic activity of the Ca2+ required for Treg activation and proliferation, although activation of channel ionophore ionomycin and a phorbol ester, such as PMA CN in the absence of CN inhibitor can further augment Treg pro- (50). PMA mimics diacylglycerol to activate protein kinase C u liferation. These findings, although not proving CN-independent and other protein kinase C u-unrelated target proteins (51), re- NFAT nuclear localization, are consistent with the results of the sulting in activation of transcription factors necessary for T cell The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 3. Flow cytometric analysis of impact of CsA on proliferation of Treg and non-Tregs. Foxp32/GFP2 CD44 low or high (A) or Foxp3+/GFP+ CD4 T cells (B) were isolated and cultured with APCs plus IL-2 (C–E), APCs plus IL-2 and anti-CD3 Ab 2C11 (F–H), or APCs plus IL-2, 2C11, and 1 mM CsA (I–K) for 4 d and analyzed by flow cytometry. (L) Summary of the average of triplicate cell counts of each culture. CFSE-labeled unfractionated splenocytes containing CD4 (M) and CD8 (Q) T cells were cultured with 2C11 (N, R), 2C11 plus CsA (O, S), or 2C11 plus CsA and IL-2 (P, T) for 4 d and analyzed by flow cytometry. (U) Summary of the fold expansion of Tregs, as well as non-Treg CD4 and CD8 T cells cultured with 2C11 in the presence or absence of tacrolimus with or without IL-2. The results are representative of more than three experiments. The input (M, V) and proliferative CD4 T cells in the culture containing 2C11 plus CsA and IL-2 (P, W) were analyzed for Foxp3 and Helios expression. activation other than NFAT. In contrast, ionomycin increases the continued to grow: by 10-fold on culture day 5 (Fig. 4H) and up to intracellular Ca2+ concentration to activate NFAT through CN, 800-fold in 2 wk (data not shown). Conversion of Foxp32 cells complementing PMA-induced signaling to give rise to full T cell (inducible Tregs) did not contribute to the expanded Foxp3+ T cell activation and proliferation (50, 52). PMA alone is incapable of pool. Congenic cultures of low-frequency CD45.1+CD25+ CD4 activating primary T cells (53, 54). T cells (7%) with a dominant population of CD25-depleted The constitutive nuclear localization of NFAT in Tregs and their CD45.2+ splenocytes (Fig. 4I) cultured with PMA resulted in an insensitivity to inhibition by CN inhibitor raised the possibility that expanded pool of Foxp3+ CD4 T cells that were almost exclu- Tregs might be selectively activated by stimuli that do not activate sively CD45.1+ (Fig. 4J). Similar to experiments with CsA (Fig. NFAT. To test this possibility, unfractionated splenocytes con- 3O), PMA-induced Treg proliferation depended on paracrine IL-2 taining ∼5% Foxp3+ Tregs (Fig. 4A) were cultured with PMA in produced by the few proliferative non-Treg CD4 T cells (Fig. 4D), the presence or absence of ionomycin. As expected, PMA plus because CsA in the absence of exogenous IL-2 diminished Treg ionomycin induced vigorous T cell proliferation composed of proliferation (Fig. 4E). Supplementing the CsA-containing cul- Foxp32 CD4 and CD8 non-Tregs as early as day 3 (Fig. 4C), tures with exogenous IL-2 restored Treg proliferation comparable whereas PMA alone only elicited mild Foxp3+ CD4 T cell pro- to cultures without CsA (Fig. 4F). These results further support the liferation (Fig. 4B). However, by culture day 5, robust T cell findings that PMA-induced Treg activation and proliferation are proliferation was observed, deriving primarily from the prolifer- independent of CN activity but require IL-2. ation of Foxp3+ CD4 cells, with few proliferative Foxp32 non- Some studies reported that the TCRs expressed by Tregs are Treg CD4 T cells (Fig. 4D). Not only did the non-Treg CD4 and more self-reactive than are those of non-Tregs (55–57). Because CD8 T cells fail to proliferate (Fig. 4D, 4G), they also died during TCR signaling could increase intracellular Ca2+ to activate CN, culture, resulting in a continuous decline in the number of Foxp32 the chronic TCR interaction of Tregs with self-MHC class II CD4 and CD8 T cells, whereas the number of Foxp3+ CD4 cells (MHCII) might cause continuous NFAT activation and nuclear 6 NFAT IN Foxp3+ TREGs

FIGURE 4. Flow cytometric analysis of the impact of PMA on Treg and non-Treg proliferation. CFSE-labeled unfractionated splenocytes (A) were cultured with PMA or PMA plus ionomycin for 3 d (B, C), or with PMA (D), PMA plus CsA (E), or PMA plus CsA and IL-2 (F) for 5 d and analyzed by flow cytometry. Cells in (A)–(F) were gated on CD4+ cells. (G) CD8+ cells in day-5 culture of splenocytes with PMA. (H)Av- erage cell counts of Foxp32 CD4 and CD8 T cells and Foxp3+ CD4 cells in splenocyte culture with PMA in triplicates. Data are representative of more than three experi- ments. CD25+ CD4 T cells isolated from CD45.1+ B6 mice were added at a 1:10 Downloaded from ratio to CD25-depleted CD45.2+ spleno- cytes (I) and cultured for 5 d (J). (K) Iso- lated Foxp3+/GFP+ CD4 T cells were cultured with PMA plus IL-2, PMA plus IL-2 and syngeneic APCs, PMA plus IL-2 and MHCII-deficient APCs, or PMA plus

IL-2 and anti-CD28 for 5 d. Data represent http://www.jimmunol.org/ the average Treg counts in each culture condition in triplicates from three indepen- dent experiments. Unfractionated splenocytes (L) were cultured with PMA (M), control (anti-DR) (N), or anti-B7.1/CD80 (1G10) blocking Ab (O), anti-B7.2/CD86 (GL-1) blocking Ab (P), or the two blocking Abs combined (Q) for 5 d and analyzed by flow cytometry. The displayed cells were derived from CD4+ gates. (R) Summary of the aver- by guest on September 29, 2021 age absolute number of proliferative Foxp3+ cells from culture triplicates.

translocation, enabling Tregs to selectively respond to PMA abrogated Treg proliferation when added to the culture together stimulation in the absence of ionomycin. To determine whether (Fig. 4Q, 4R). PMA-induced Treg proliferation depends on Treg interaction with self-MHCII, Foxp3+GFP+ or CD25+ CD4 T cells were isolated Tregs expanded in the absence of CN activation maintain Treg and cultured with PMA and IL-2 in the presence or absence of phenotype and suppressive capacity wild-type or MHCII-deficient APCs. In the absence of APCs, Studies showed that structure-guided mutations of Foxp3 predicted PMA/IL-2 induced only minimal Treg proliferation (Fig. 4K). In to disrupt its interaction with NFAT disrupt Foxp3 function to contrast, an ∼30-fold expansion of Tregs was obtained when ei- upregulate the expression of the Treg markers CTLA4 and CD25 ther wild-type or MHCII-deficient APCs were added to the culture and to confer suppressive function (16). If constitutive NFAT nu- (Fig. 4K). These findings indicate that, although a Treg–APC in- clear localization in Tregs depends on CN activity, Tregs expanded teraction is necessary, MHCII-mediated TCR signaling is not re- in the presence of CN inhibitors or expanded by PMA should lose quired for PMA-induced Treg proliferation, consistent with the expression of CTLA-4 and CD25, as well as suppressive function above results that Tregs could be activated in the presence of CN due to interruption of Foxp3–NFAT interaction, because CN in- inhibitors (Fig. 3). Because APCs could provide costimulation to hibitors prevent NFAT nuclear translocation, and PMA does not Tregs through CD28 to synergize PMA stimulatory effect on activate CN. To examine this possibility, freshly isolated primary Tregs, stimulatory anti-CD28 Ab was added to the culture in place naturally occurring Foxp3+GFP+ cells (nTregs) and Tregs expanded of APCs. This combination of PMA and CD28 stimulation by anti-CD3/CD28 in the absence or presence of CsA (Treg/CsA) resulted in an ∼18-fold Treg expansion (Fig. 4K), suggesting that or expanded by PMA (Treg/PMA) were compared. Expression part of the APC-derived synergistic stimulatory effect can be levels of CTLA-4, CD25, and Foxp3, as well as the suppressive delivered through CD28 but independently of TCR stimulation. To functions, were measured. Both Treg/CsA and Treg/PMA cells examine the role of costimulation, B7.1- and B7.2-blocking Abs expressed slightly higher levels of CTLA-4 (Fig. 5A), CD25 (Fig. were added individually or together to the PMA-stimulated 5B), and Foxp3 (Fig. 5C) compared with freshly isolated primary splenocyte culture (Fig. 4L–P). The two blocking Abs nearly Tregs. The level of CD25 on Treg/CsA cells was also higher than The Journal of Immunology 7 that of Treg/CD3/CD28 cells, whereas CTLA-4 levels were similar. Tregs, suggesting that the Foxp3–NFAT interaction in Treg/CsA However, the level of CTLA-4 expressed by Treg/PMA cells was and Treg/PMA cells is not interrupted by CN inhibitors or the lower than that of Treg/CD3/28 cells, whereas the CD25 levels lack of CN-mediated activation of NFAT. These results are con- were similar. In contrast, CsA inhibited CD25 upregulation on sistent with the finding that treating Tregs with CsA did not result expanded non-Tregs (Fig. 5P). Consistent with the high-level ex- in the export of NFAT from the nucleus (Fig. 1). Nevertheless, pression of the Treg markers, both Treg/CsA and Treg/PMA cells further activation of NFAT through activating CN can enhance Treg were able to suppress anti-CD3–elicited proliferation of non-Treg suppressive capacity. CD4 and CD8 responder cells with similar efficacy as freshly iso- lated nTregs (Fig. 5C, 5D). However, Treg/CD3/CD28 cells were Discussion much more potent in suppression than were the nTregs and Treg/ We show that a fraction of NFATc1 and NFATc2, as well as c-Rel CsA and Treg/PMA cells (Fig. 5C, 5D). These results demonstrate protein, is present in the nucleus of freshly isolated primary Tregs that Tregs expanded in the absence of CN activation do not lose but not non-Tregs. Consistent with this result, ChIP analysis reveals the prototypic Treg phenotype or suppressive function of primary that NFAT in primary Tregs is constitutively bound to genes that are Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 5. Characterization of Tregs expanded in the presence or absence of CN activation. Foxp3+/GFP+ CD4 T cells expanded with anti-CD3/28 in the presence (black) or absence of CsA (green) or with PMA (red) were compared with primary Tregs (nTregs; blue) for their levels of expression of CTLA- 4(A), CD25 (B), or Foxp3 (C). CFSE-labeled CD25-depleted splenocytes were stimulated with 2C11 in the absence of Tregs (D, E), with nTregs (F, G), with Tregs expanded in the presence of 1 mM CsA (H, I), with PMA-expanded Tregs (J, K), or with Tregs expanded in the absence of CsA (L, M) at a 1:1 Treg/non-Treg CD4 T cell ratio, cultured for 3 d, and analyzed for proliferation of non-Treg CD4 and CD8 T cells by flow cytometry. Proliferation of non- Treg CD4 (N) and CD8 (O) T cells stimulated by 2C11 in the presence of each expanded Treg preparation at various Treg/responder T cell ratios is displayed as fractions relative to the number of proliferative cells in the absence of Tregs (proliferation index). (P) CD25 levels on non-Treg CD4 cells expanded in the presence of absence or absence CsA. 8 NFAT IN Foxp3+ TREGs known to be Foxp3 targets, whereas the binding of NFAT to DNA expressed in Tregs, and it plays an important role in Treg devel- in non-Tregs only occurs after the cells are stimulated. Resting opment (28, 61, 62). Pde3b is the most repressed gene among all or treating Tregs with CN inhibitor CsA did not induce export of of the Foxp3-target genes, and its expression in Tregs is delete- NFAT from the nucleus of the Tregs, suggesting that NFAT nuclear rious to the homeostasis of this subset of T cells (29). Therefore, localization in primary Tregs is independent of CN activity. In line the constitutive binding of NFAT to these Foxp3 targets might be with this finding, Tregs are resistant to CN inhibition in the critical in the maintenance of Treg properties. presence of IL-2. Similarly, PMA, which activates transcription Consistent with a previous report (16), NFAT binding can be factors other than NFAT, selectively activates Tregs to proliferate. associated with either target gene repression or activation in Tregs. In addition, the Tregs expanded by PMA or by anti-CD3 in the In addition, NFAT differentially regulates target gene expression presence of CN inhibitors maintain phenotype and functionality between Treg and non-Tregs. In Tregs, NFAT represses Il-2 and Irf4 similar to primary Tregs, suggesting that the Foxp3–NFAT inter- transcription by forming a complex with Foxp3. In contrast, NFAT action is not interrupted by lack of CN activation during prolif- promotes transcription of these two genes in activated non-Tregs, eration. Taken together, these results indicate that a fraction of presumably through cooperation with AP-1 (16). To repress Pde3b NFAT proteins is constitutively present in the nucleus of primary expression, NFAT binds to the first intron of Pde3b (Pde3b-20) (Fig. Tregs, independent of CN activity. 2D) (28), and this seems to prevent NFAT from binding to the Pde3b The finding of constitutive nuclear NFAT localization is con- promoter (Fig. 2H). However, in activated non-Tregs, NFAT binds sistent with the reports that, in primary Tregs, Foxp3 is constitu- to the Pde3b promoter region. In Tregs, NFAT binds to mir155 (Fig. tively bound to hundreds of target genes (10) and that the binding 2E), where its expression is upregulated (28). In contrast, in acti- of Foxp3 to Il2, Ctla4, and Il2ra (CD25) requires Foxp3 to form vated non-Tregs, NFAT no longer binds (Fig. 2E), suggesting that Downloaded from a cooperative complex with NFATc2 (13, 16). In addition to only Foxp3:NFAT complexes are capable of binding to this region. NFATc2, we detected NFATc1 in the Treg nucleus. It is known The complete repertoire of NFAT target genes in primary and ac- that NFATc1, NFATc2, and NFATc3 are coexpressed in T cells, tivated Tregs remains to be determined. In addition to NFAT, a trace and are all activated in response to TCR stimulation. Although we amount of c-Rel protein was also detected in the nucleus of Tregs. only examined NFATc1 and NFATc2, given the high degree of This finding is consistent with the report that c-Rel is required for

similarity among the Rel-homology region of the different NFAT Foxp3 expression (30, 63). Therefore, in addition to NFAT, c-Rel http://www.jimmunol.org/ family members and the fact that the Rel-homology region confers may be constitutively present in the nucleus for Tregs to keep their common DNA-binding specificities and partner interactions (58), phenotype and functionality. it is possible that multiple members of the NFAT family, including Our finding that Tregs are resistant to CN inhibitors in the pres- NFATc3, are involved in partnership with Foxp3. ence of exogenous IL-2 could have important clinical implications. Interestingly, the ChIP analysis revealed that NFAT in Tregs CN inhibitors, such as CsA and tacrolimus, are classic drugs used for selectively binds to Foxp3 target genes but not to the NFAT binding the treatment and prophylaxis of allograft rejection and graft-versus- sites of the non-Foxp3 targets tested. The reasons for lack of binding host disease (GVHD). Although some patients respond to CsAwell, a to the non-Foxp3 targets are not clear. Although this selectivity high percentage (up to 50%) of patients, especially those receiving could be due to the limited number of genes examined, other more unrelated or mismatched bone marrow grafts, develop severe GVHD by guest on September 29, 2021 likely possibilities include lack of other transcription partners, such when treated with CsA (64, 65). The reason for the lack of thera- as AP-1, in the nucleus of primary Tregs (Fig. 1). In effector cells, peutic effect in these patients is not well understood. Our results NFAT forms cooperative complexes with AP-1 to bind to NFAT– (Fig. 3), along with the data from other investigators (66, 67), AP-1 composite sites present in the promoter region of genes in- suggest that CsA may harm Tregs by IL-2 deprivation. Loss of Tregs duced during T cell activation (25, 59). In Tregs, NFAT forms in bone marrow recipients is reported to increase the risk for GVHD analogous cooperative complexes with Foxp3 to regulate expres- (68, 69). These findings raise the possibility that CN inhibitors might sion of Foxp3 target genes, as suggested in a previous report (16). be more efficacious if used in conjunction with IL-2, because IL-2 Therefore, the absence of AP-1 and the presence of Foxp3 in the selectively allows Treg cells to survive and propagate in the pres- nucleus of Tregs could prevent NFAT from binding to the non- ence of CN inhibitors (Fig. 3). Early studies reported a positive Foxp3 targets but allow NFAT to bind to Foxp3 targets. Further impact of IL-2 on protection against GVHD (70, 71). studies are needed to elucidate the mechanisms. However, in ac- Clearly, a fundamental question that has arisen from this study is tivated non-Tregs, both NFAT and AP-1 are translocated into the how NFAT becomes translocated into the nucleus in primary Tregs. nucleus along with other transcription factors, allowing NFAT to It has been generally believed that CN is the only phosphatase that form cooperative complexes with AP-1 to bind to the non-Foxp3 dephosphorylates NFAT (72, 73). Our results show that CN activity targets (Fig. 2). It is also possible that the non-Foxp3 targets of is not required for constitutive NFAT nuclear translocation in Tregs, NFAT binding sites are inaccessible in Tregs as a result of DNA suggesting that other mechanisms might activate NFAT. Studies hypermethylation and chromatin condensation at these regions. suggested the potential role for CsA-resistant protein phosphatases Although further studies are needed to determine the mechanisms, in dephosphorylating NFAT (74, 75). Another possibility is that the finding of differential binding of NFAT to Foxp3 targets sup- the activity of NFAT kinases, such as glycogen synthase kinase ports the idea that the nuclear localization of NFAT in Tregs is not 3b, which rephosphorylate NFAT, in Tregs is attenuated, shifting an experiment-induced artifact of T cell activation. the dynamic equilibrium between CN and kinase activities toward In addition to the Il2 promoter, which is a previously identified formation of dephosphorylated NFAT and NFAT nuclear retention. Foxp3:NFAT target (16), we identified three additional Foxp3 A recent study reported that inhibition of glycogen synthase kinase targets, Irf4, mir155, and Pde3b-20 (10), which are bound by 3b activity enhances Tregs functionality (76). It is not clear NFAT. All four genes are known to serve important functions in whether NFAT proteins can remain in the nucleus of Tregs, re- maintaining the phenotype and function of Tregs. Repression of gardless of its phosphorylation status. Given the extensive protein– Il-2 transcription by Foxp3 is a hallmark of Tregs and is closely protein interaction interface between NFAT and Foxp3 (13, 16), related to their inhibitory capacity (16). Foxp3-dependent ex- there is a possibility that the interaction with Foxp3 facilitates pression of IRF4 is required by Tregs to suppress Th2 autoim- NFAT nuclear retention in Tregs in the form of Foxp3:NFAT munity (60). mir155 is a direct target of Foxp3. It is highly complexes. All of these possibilities are yet to be investigated. The Journal of Immunology 9

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