Inhibitory role of the transcription Gfi1 in the generation of thymus-derived regulatory T cells

Lewis Zhichang Shia, Nishan S. Kalupahanaa, Meghan E. Turnisa, Geoffrey Nealeb, Hanno Hockc, Dario A. A. Vignalia, and Hongbo Chia,1

aDepartment of Immunology and bHartwell Center for Bioinformatics and Biotechnology, St. Jude Children’s Research Hospital, Memphis, TN 38105; and cCancer Center and Center for Regenerative Medicine, Massachusetts General Hospital, Harvard Medical School, and Harvard Stem Cell Institute, Boston, MA 02114

Edited by Remy Bosselut, National Cancer Institute, Bethesda, MD, and accepted by the Editorial Board July 14, 2013 (received for review January 21, 2013)

+ + Foxp3 regulatory T (Treg) cells are essential for the maintenance of T-cell subsets (14–18), but it is dispensable for CD8 T cell- self-tolerance and immune homeostasis. The majority of Treg cells mediated immune responses in vivo (16). These results indicate is generated in the thymus as a specific subset of CD4+ T cells, an important but cell context-dependent function for Gfi1 in the known as thymus-derived or natural Treg (nTreg) cells, in response immune system. to signals from T-cell receptors, costimulatory molecules, and cyto- Whereas a role for Gfi1 in early thymocytes and peripheral kines. Recent studies have identified intracellular signaling and T cells has been described, its function in the development of transcriptional pathways that link these signals to Foxp3 induc- nTreg cells is unclear. We have previously found that thymic tion, but how the production of these extrinsic factors is controlled development of nTreg cells is orchestrated by S1P1 (19), which is remains poorly understood. Here, we report that the transcription under the control of Klf2 (20) that can be further regulated by fi repressor growth factor independent 1 (G 1) has a key inhibitory Gfi1 (13), but the roles of Gfi1innTreg cells are poorly under- role in the generation of nTreg cells by a noncell-autonomous stood. Therefore, we generated T cell-specificGfi1-deficient mechanism. T cell-specific deletion of Gfi1 results in aberrant ex- mice and had a surprising finding that Gfi1 deletion enhanced pansion of thymic nTreg cells and increased production of cyto- nTreg development through a noncell-autonomous mechanism. kines. In particular, IL-2 overproduction plays an important role Additional analysis revealed an exuberant production of IL-2 by fi in driving the expansion of nTreg cells. In contrast, although G 1 Gfi1-deficient thymocytes as the main mechanism, thereby high- fi fi de ciency elevated thymocyte apoptosis, G 1 repressed nTreg lighting a previously unrecognized mechanism in which IL-2 pro- generation independently of its prosurvival effect. Consistent with duced by conventional T cells shapes thymic microenvironment to fi fi an inhibitory role of G 1 in this process, loss of G 1 dampens direct nTreg development. Furthermore, Gfi1 function in T cells antitumor immunity. These data point to a previously unrecog- was required for optimal antitumor immunity, consistent with its nized extrinsic control mechanism that negatively shapes thymic effects at inhibiting nTreg generation and function. Finally, al- generation of nTreg cells. though Gfi1deficiency increased thymocyte apoptosis, Gfi1re- pressed generation of nTreg cells independently of its prosurvival + ormal development of Foxp3 regulatory T (Treg) cells is effect. These data point to an extrinsic control mechanism that Ncritical for maintaining self-tolerance and preventing exu- negatively shapes thymic generation of nTreg cells. berant immune responses (1). T cells are produced mainly in reg Results the thymus, known as thymus-derived or natural T (nT ) reg reg fi fi cells, and they require expression of the G 1Deciency Promotes the Generation of nTreg Cells. To in- fi fi Foxp3. T-cell (TCR) specificity to self-antigens seems to vestigate the function of G 1 in T-cell development, we rst an- fi fi −/− be a primary determinant for nT lineage commitment in the alyzed mice with germ-line deletion of G 1(G 1 mice) (12). reg fi thymus, with c-Rel being an important factor that links TCR Loss of G 1 altered thymic populations, most notably a higher engagement and Foxp3 expression (2, 3). Costimulatory factors frequency of CD8 single-positive (CD8SP) cells, consistent with (such as CD28) and cytokines, predominantly IL-2, also play crucial roles for the induction of Foxp3 and thymic development Significance of nTreg cells (2, 3). In a two-step model of nTreg development, TCR engagement leads to the expression of the high-affinity IL- Thymic development of regulatory T cells (Treg) is a crucial 2Rα that subsequently responds to IL-2 stimulation for the in- event for immune homeostasis. Despite extensive descriptions duction of Foxp3 expression and nTreg lineage commitment (4, 5). of molecular pathways functioning within Treg cells, little is However, the cellular source of IL-2 is unclear (6). Moreover, known how Treg development is shaped by the control of ex- whereas much emphasis has been placed on T cell-intrinsic con- trinsic factors. Here, we show that the transcription factor fi trol of nTreg development, how the production of these extrinsic growth factor independent 1 (G 1) restrains thymic generation of T cells. Loss of Gfi1 led to aberrant production of the cy- factors is controlled to shape the nTreg pool remains poorly reg understood. tokine IL-2. Elevated IL-2 then drove the expansion of Treg cells. Growth factor independent 1 (Gfi1), a transcription repressor, Accordingly, deficiency of Gfi1 dampened antitumor immunity. has emerged as an important regulator of hematopoietic and Our results establish that Gfi1 actively controls thymocyte IL-2 immune system cells. Gfi1 is required for the normal de- production, thereby shaping thymic generation of Treg cells. velopment and homeostasis of hematopoietic stem cells and both fi Author contributions: L.Z.S. and H.C. designed research; L.Z.S., N.S.K., and M.E.T. per- myeloid and lymphoid progenitors (7, 8). Speci cally, loss of formed research; H.H. contributed new reagents/analytic tools; L.Z.S., N.S.K., M.E.T., Gfi1 impairs the development of neutrophils and B cells while G.N., H.H., D.A.A.V., and H.C. analyzed data; and L.Z.S. and H.C. wrote the paper. – expanding the monocyte and myeloid populations (9 11). In the The authors declare no conflict of interest. fi T-cell lineage, G 1 expression is dynamically regulated (12), and This article is a PNAS Direct Submission. R.B. is a guest editor invited by the its deficiency diminishes double-negative (DN) cell generation Editorial Board. + but increases the differentiation of CD8 T cells in the thymus 1To whom correspondence should be addressed. E-mail: [email protected]. fi (13). In the periphery, G 1 has been implicated in the differ- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. + entiation and in vivo function of CD4 effector and regulatory 1073/pnas.1300950110/-/DCSupplemental.

E3198–E3205 | PNAS | Published online August 5, 2013 www.pnas.org/cgi/doi/10.1073/pnas.1300950110 Downloaded by guest on September 30, 2021 − − PNAS PLUS a previous report using Gfi1 / mice (13). Detailed analysis of We determined the cellular mechanisms by which Gfi1 −/− T-cell subsets indicated that the Gfi1 CD4 single-positive restrains nTreg development. We reasoned that the increased (CD4SP) compartment contained a markedly increased frequency frequency of nTreg cells in Gfi1-deficient mice could be caused by + of thymic Foxp3 nTreg population (Fig. S1A). Given a prominent differential regulation of cell expansion or survival between − role of Gfi1 in the development of multiple hematopoietic Foxp3+ and Foxp3 CD4SP T cells. To assess the effects of Gfi1 lineages (8, 21), it remained possible that the expansion of deficiency on cell proliferation, we first used BrdU incorporation −/− Gfi1 nTreg cells was secondary to defective early development assays, which label cells that have synthesized new DNA and – and/or influences by nonlymphoid cells. Hence, we crossed loxP- progressed into the S phase of the cell cycle. Whereas Foxp3 fl fl flanked Gfi1 alleles (Gfi1 / ) with two Cre lines, the hCD2-iCre CD4SP T cells from wild-type (WT) and Gfi1CD4 mice incor- and CD4-Cre transgenic mice, in which Cre expression was ini- porated BrdU to a comparable extent, a greater percentage of tiated at the early and late DN stages, respectively (called Foxp3+ T cells from Gfi1CD4 mice incorporated BrdU (Fig. 1D). Gfi1hCD2 and Gfi1CD4) (22, 23). Gfi1 was efficiently deleted in Similar results were obtained from splenic Foxp3+ T cells from CD4 double-positive (DP) and single-positive (SP) cells from both Gfi1 mice (Fig. S1F). Moreover, Ki-67 staining, which lines (Fig. S1B). More efficient recombination was achieved in denotes proliferating cells at all active phases of the cell cycle, Gfi1hCD2 mice, possibly because of the codon improvement of also revealed an increased frequency of proliferating Foxp3+ the Cre recombinase in hCD2-iCre mice (22). As expected, de- cells in the thymus and spleen of Gfi1CD4 mice (Fig. S1G). We letion of Gfi1 in both systems resulted in an elevated ratio of next analyzed apoptosis of different populations of CD4SP cells. CD8SP/CD4SP T cells (Fig. 1 A and B). More importantly, loss Freshly isolated CD4SP T cells from WT and Gfi1CD4 mice of Gfi1 considerably up-regulated the frequency of nTreg cells showed a similar degree of Annexin V binding and caspase ac- − among CD4SP T cells (Fig. 1 A and B), although the absolute tivity (two hallmarks of apoptotic cell death) in both the CD25 + number of nTreg cells remained largely unaltered because of the and CD25 compartments (Fig. 1E). Therefore, the increased CD4 reduction of total thymocytes and especially, CD4SP cells (Fig. nTreg population in Gfi1 mice is associated with an en- S1C). Greater proportions of Foxp3+ cells among CD4 T cells hanced rate of cell proliferation but not survival. were also observed in the spleen (Fig. 1C and Fig. S1D), liver, CD4 blood, and even DP thymocytes from Gfi1 mice (Fig. S1E). Gfi1Deficiency Enhances nTreg Function and Impairs Antitumor Comparable results derived from these different deletion sys- Immunity. Having shown a role of Gfi1innTreg development, tems indicate a specific role for Gfi1 in restraining nTreg gener- we next examined whether Gfi1 affected the immunosuppressive ation. For the purpose of clarity and consistency, we mainly used function of nTreg cells. Gfi1-deficient nTreg cells exhibited a Gfi1CD4 mice in our analyses. stronger inhibitory capacity against the proliferation of conventional

A B Thymus CD4SP * Thymus CD4SP ** 8.03 89 3.95 6.28 91.8 4.05 WT WT WT WT CD8/CD4 ratios 1.56 1.04 CD8/CD4 ratios ** 6.78 88.1 12.8 * 5.32 90.1 11.4 hCD2 CD4 Gfi1 Gfi1 /CD4SP (%) /CD4SP (%) + 4.02 + Foxp3 2.97 Foxp3 CD4 CD4 CD8 CD8 CD25 CD25 hCD2 CD4

Foxp3 WT Gfi1 WT Gfi1 Foxp3

C CD4+ D CD4SP E CD4SP CD4SP 15.7 3.61 0.092 2.24 0.17 2.86 0.4

* IMMUNOLOGY cells + WT WT WT WT WT

CD8/CD4 ratios 3.59 1.86 1.27

** of Foxp3 + 25.6 (%) 3.79 0.52 2.99 0.33 2.38 0.7 + CD4 CD4 CD4 /CD4 + % BrdU Gfi1 Gfi1

6.99 WT Gfi1CD4 Gfi1

Foxp3 3.35

BrdU 2.59 Caspase CD4 Annexin V CD25 Foxp3 WT Gfi1 Foxp3 CD25

hCD2 Fig. 1. Gfi1deficiency promotes the generation of nTreg cells. (A and B) Flow cytometry analysis of thymic populations in (A) WT controls and Gfi1 or (B) Gfi1CD4 mice for expression of CD4 and CD8 and frequency of Foxp3+ cells among gated CD4+TCR+ thymocytes (CD4SP). (Right) Ratio of CD8SP/CD4SP T cells (Upper) and frequency of Foxp3+ cells (Lower). (C) Frequency of Foxp3+ cells in gated CD4+ splenocytes of WT and Gfi1CD4 mice and ratio of CD8+/CD4+ cells (Upper Right). (D) BrdU staining in thymocytes of WT and Gfi1CD4 mice at 18 h after injection with BrdU. (E) Annexin V staining (Left) and caspase activity (Right; by FITC-VAD-FMK (carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone) binding) in CD4SP thymocytes. Data are representative of at least five independent experiments. Error bars indicate SEM. *P < 0.05; **P < 0.01.

Shi et al. PNAS | Published online August 5, 2013 | E3199 Downloaded by guest on September 30, 2021 T cells in standard in vitro Treg suppression assays based on reconstituted chimeras derived from Gfi1-deficient BM cells carboxyfluorescein succinimidyl ester (CFSE) dilution and thy- (Fig. S3A). In the mixed chimeras, Gfi1CD4-derived thymocytes midine incorporation (Fig. 2A and Fig. S2A). We then examined retained the higher CD8SP/CD4SP ratio compared with WT- the expression of the integrin molecule CD103 and the ectonu- or spike-derived cells in the mixed chimeras (Fig. 3A), in- fi fi + cleotidase CD73, effector molecules associated with nTreg cell dicative of a cell-autonomous effect of G 1de ciency on CD8 suppressive activity (24, 25). Whereas Gfi1 has been shown to differentiation. regulate these molecules in discrete contexts (17, 26), the extent In contrast, the frequency of thymic and splenic Foxp3+ cells fi CD4 to which this regulation is operative in nTreg cells is poorly de- derived from G 1 donor cells in the mixed BM chimeras was fined. Compared with the WT counterparts, deletion of Gfi1 indistinguishable from the frequency of Foxp3+ cells from WT resulted in prominent up-regulation of CD103 (Fig. 2B)and donors or the cotransferred CD45.1+ spike cells (Fig. 3B and CD73 (Fig. 2C)onnTreg cells from both the thymus and pe- Fig. S3B). Thus, the increased nTreg population in the un- fi CD4 riphery. Thus, Gfi1 negatively controls nTreg function, which is manipulated G 1 mice seemed to be a noncell-autonomous associated with its effects to down-modulate expression of or bystander defect. However, the up-regulation of CD103 ex- selective suppressive molecules (24, 25). pression among nTreg cells persisted in Gfi1-deficient donor cells Immune tolerance mediated by nTreg cells is a major compo- (Fig. S3C), and this cell-intrinsic effect on CD103 expression is in nent that prevents productive immune responses against tumors agreement with previous studies identifying CD103 as a direct fi (27–29). Given the inhibitory effects of Gfi1 on the nTreg pool G 1 target (17). To further investigate the possibility of and suppressive activity, we tested whether lack of Gfi1 affected a bystander effect on the nTreg population caused by Gfi1- antitumor immunity using a well-established B16 melanoma deficient cells, we set up mixed BM chimeras, in which WT or model. Gfi1CD4 mice failed to restrain the late phase of tumor Gfi1CD4 (CD45.2+) progenitors were a majority and WT spike growth (Fig. 2D), despite slightly increased proportions of IFN-γ+ (CD45.1.2+) cells were a minority (at a 20:1 ratio). As expected, cells among tumor-infiltrating CD4+ and CD8+ cells (Fig. S2B). the Foxp3+ population was increased in Gfi1-deficient cells, but CD4 Associated with impaired antitumor immunity in Gfi1 mice was more strikingly, the nTreg percentage derived from spike thy- an increased number of Foxp3+ cells in tumor-infiltrating cells mocytes was also elevated in the predominant Gfi1-deficient – (Fig. 2E) and a greater Foxp3+/Foxp3 ratio (Fig. 2F). Further- thymus, even with a greater degree that probably reflected more, CD103 and CD73 expression levels on tumor-infiltrating a better survival fitness of these cells (see below) (Fig. S3D). CD4 nTreg cells were elevated in Gfi1 mice (Fig. 2G). These results Similar observations were obtained in the spleen (Fig. S3D). indicate that Gfi1 is required for optimal antitumor immunity, The up-regulation of Foxp3 in WT spike cells indicates that fi fi likely through its inhibitory effects on Treg generation and function. G 1de ciency in T cells exerts a cell-extrinsic effect on the nTreg population. Noncell-Autonomous Effects of Gfi1Deficiency on nTreg Development. We noted that an analogous bystander up-regulation of thymic We next investigated whether the increased thymic nTreg pop- chemokine receptor expression in Klf2-deficient mice results in ulation in Gfi1-deficient mice was a cell-autonomous defect. To an age-related accumulative effect (30). We, therefore, examined this end, we generated mixed bone marrow (BM) chimeras by whether Gfi1deficiency affected the kinetics of nTreg generation −/− reconstituting alymphoid Rag1 mice with a 1:1 mixture of WT by analyzing thymic nTreg cells in mice of different ages. Linear or Gfi1CD4 (CD45.2+; donor) and WT (CD45.1+; spike) BM regression analysis indicated that the enhancing effect of Gfi1 cells. As a comparison, we also generated chimeras that con- deficiency on nTreg cells had a significant positive correlation tained WT or Gfi1CD4 BM cells exclusively and observed the (P = 0.0001) with the age of mice (Fig. 3C). The age-dependent expected increase of the thymic and splenic nTreg population in accumulation of nTreg cells likely reflects an extended exposure

A B Thymus Spleen C Thymus Spleen

Teff alone WT WT WT Treg: Teff (1:4) Gfi1CD4 Gfi1CD4 CD4 Gfi1 Treg: Teff (1:4) Cells Cells CFSE CD103 Cells CD73

D WT E F G 2.0 CD103 CD73

) CD4 2

Gfi1 ) 5 * * * * reg * * 1.5 reg

1.0 cells (x10 + /non-T

0.5 reg T Tumor size (mm Tumor 0.0 Foxp3 MFI of tumor nT Time (Day) WT Gfi1CD4 WT Gfi1CD4 WT Gfi1CD4 WT Gfi1CD4

Fig. 2. Gfi1 restrains nTreg function and is required for antitumor immunity. (A) CFSE levels in CFSE-labeled effector T (Teff) cells activated in the presence of + CD4 nTreg cells for 3 d. (B and C) Expression of (B) CD103 or (C) CD73 among Foxp3 cells. (D–G)WTandGfi1 mice were inoculated with B16 melanoma cells, and (D) tumor size was measured. Tumor-infiltrating lymphocytes were isolated at day 16 and analyzed for Foxp3 staining; shown are (E) Foxp3+ numbers, (F)the ratio of Foxp3+/Foxp3– among CD4+ cells, and (G) mean fluorescent intensity (MFI) of CD103 and CD73 among Foxp3+ tumor-infiltrating lymphocytes. Data are representative of (A and D–G) two and (B and C) five independent experiments. Error bars indicate SEM. *P < 0.05.

E3200 | www.pnas.org/cgi/doi/10.1073/pnas.1300950110 Shi et al. Downloaded by guest on September 30, 2021 PNAS PLUS A CD45.1 CD45.2 B CD45.1 CD45.2 9.8 84.5 10.3 85.1 Spike WT Spike Gfi1CD4 7.36 8.9 NS WT WT WT Gfi1CD4 chimera chimera ** 4.4 2.39 NS cells (CD45.2/ CD45.1) 9.09 87.1 6.48 86.7 6.3 7.2 + Gfi1CD4 Gfi1CD4 chimera CD8/CD4 ratios chimera Foxp3 WT Gfi1CD4 Foxp3 2.29 3.59 CD4 CD25 CD8 NS

C D E Thymus Spleen

4.12 14.7 in thymus (%)

+ WT

Y=0.102X+1.057 (p=0.0001) + Gfi1Foxp3 WT WT Gfi1 Foxp3 NS

3.37 12.1 of CD4SP of CD4SP Foxp3 in spleen (%) Relative value of + Gfi1 Fold increase of Foxp3

Thymus Spleen Foxp3 Age (weeks) CD4 CD25 Foxp3 WT Gfi1

Fig. 3. Gfi1deficiency affects nTreg generation by a noncell-autonomous mechanism. (A and B) Mixed BM chimeras were generated by reconstituting − − sublethally irradiated Rag1 / mice with a 1:1 mixture of spike (CD45.1+) and WT or Gfi1CD4 (CD45.2+) BM cells and analyzed later for (A) CD8/CD4 ratios among thymocytes and (B) frequency of Foxp3+ cells among CD4SP thymocytes. (C) Linear regression analyses of fold increases of Foxp3+ frequency among CD4SP thymocytes in Gfi1CD4 mice relative to the ages of experimental mice. Each dot represents one independent experiment. (D) Gfi1 deletion efficiency in Foxp3 + + Foxp3 Gfi1 thymic or splenic nTreg cells. (E) Frequency of Foxp3 cells among CD4SP thymocytes or CD4 splenocytes from WT and Gfi1 mice. Data are representative of at least (A–C) five and (D and E) three independent experiments. Error bars indicate SEM. **P < 0.01; NS, not significant.

to nTreg-stimulating factors that are differentially produced by velopment and expansion (32–34). However, the cellular source Gfi1-deficient T cells. responsible for IL-2 production in this process remains poorly To further dissect the noncell-autonomous effects of Gfi1on defined (6), because IL-2 can be produced by dendritic cells and fl/fl YFP-Cre nTreg development, we crossed Gfi1 mice with Foxp3 additional cells other than T cells (35). Compared with WT cells, mice, in which the Cre recombinase was knocked into the CD4SP and CD8SP T cells from Gfi1CD4 mice expressed a endogenous Foxp3 locus (31), to delete Gfi1 selectively in nTreg higher level of Il2 mRNA under resting conditions as well as cells (called Gfi1Foxp3 mice). Gfi1 expression was considerably after phorbol 12-myristate 13-acetate (PMA) and ionomycin stim- + diminished in Foxp3 nTreg cells in the thymus, and complete de- ulation (Fig. 4A). The increased Il2 was restricted to the non-Treg letion was achieved in the periphery (Fig. 3D), which was in line population of Gfi1-deficient CD4SP cells, because IL-2 expres- YFP-Cre with the timing of Cre expression in Foxp3 mice (31). sion in the mutant nTreg cells was unaltered (Fig. S4A). Also, Consistent with a direct role of Gfi1 in CD103 expression, CD103 splenic CD4+ cells or thymic dendritic cells of Gfi1CD4 mice wasup-regulatedinGfi1-deficient nTreg cells, especially in splenic showed normal expression of IL-2 (Fig. S4 B and C). Consistent nTreg cells that had more efficient Gfi1deletion(Fig. S3E). Ac- with mRNA analysis, intracellular cytokine staining revealed an Foxp3 fi fi cordingly, nTreg cells from Gfi1 mice had a small increase of increased proportion of G 1-de cient CD4SP T cells capable of fi producing IL-2 (Fig. 4B). A similar trend of increase was noticed suppressive activity (Fig. S3F). However, the speci c deletion of IMMUNOLOGY + fi fi Gfi1innTreg cells did not affect the frequency of the Foxp3 nTreg in G 1-de cient CD8SP cells, although this increase did not population in the thymus or spleen (Fig. 3E). Taken together, we reach statistical significance in the limited number of mice ana- conclude that the nTreg cell expansion induced by Gfi1deficiency lyzed (Fig. 4B). was caused by a noncell-autonomous mechanism. This expansion We then determined whether augmented IL-2 production was a unique effect, because altered CD8SP/CD4SP ratios and from Gfi1-deficient thymocytes contributed to the nTreg de- CD103regulationinnT cells were intrinsic to the loss of Gfi1 velopment phenotypes. Specifically, we used two approaches to reg − − function. These findings further highlight that deletion of Gfi1in block IL-2 function. First, we crossed Gfi1CD4 mice with Il2 / fi CD4 −/− total T cells altered the thymic environment important for nTreg mice to generate G 1 Il2 double KO mice. To avoid the generation, and the underlying mechanism was further ex- confounding effects caused by an autoimmune syndrome man- − − plored in the following studies. ifested in adult Il2 / mice (34), we analyzed these mice at a young age (5–6 wk). Deletion of IL-2 blocked the higher fre- + CD4 Gfi1AffectsnTreg Development by Modulating IL-2 Production. quency of Foxp3 cells in the thymus of Gfi1 mice (Fig. 4C). CD4 Thymic nTreg development is dependent on TCR, costimu- Second, we treated WT and Gfi1 mice with blocking anti- lation, and cytokine signaling (2, 3). A noncell-autonomous ef- bodies for IL-2 or IL-2R (36). After administration of either + fect mediated by Gfi1onnTreg development prompted us to test neutralizing antibody, the frequency of Foxp3 cells in the thy- whether Gfi1deficiency altered cytokine production. We first mus between WT and Gfi1CD4 mice became comparable, whereas focused on IL-2, a key cytokine capable of driving nTreg de- the difference remained evident in PBS-treated control groups

Shi et al. PNAS | Published online August 5, 2013 | E3201 Downloaded by guest on September 30, 2021 A B CD4SP CD8SP * WT * CD4 * 3.8 1.54 Gfi1 of CD4SP + WT WT Il2 % IL-2

p=0.06 7.65 3.06 CD4 Relative value of * of CD8SP +

* Gfi1 IL-2 CD4SP CD4SP (P+I) CD8SP CD8SP (P+I) TCR % IL-2 WT Gfi1CD4 C WT Il2 E 4.38 3.61 WT Gfi1CD4 NS NS NS NS Tgfb1 (%) + Gfi1CD4 Gfi1CD4Il2

7.02 2.45 Foxp3 Relative value of WT Gfi1CD4 Il2 Gfi1CD4Il2 CD4SP CD4SP P+I CD8SP CD8SP P+I Foxp3 CD25

D PBS Anti-IL-2 Anti-IL-2R F 3.71 4.26 2.16 WT WT ** Gfi1CD4 Gfi1CD4 WT WT Il10 (%) +

9.34 4.96

2.69 Foxp3 CD4

Relative value of ** Gfi1

Foxp3 CD4SP CD4SP P+I CD25 PBS Anti-IL-2 Anti-IL-2R

CD4 CD4 Fig. 4. Enhanced IL-2 production by Gfi1 thymocytes contributes to the expansion of nTreg cells. (A) Il2 mRNA expression in resting WT and Gfi1 CD4SP or CD8SP thymocytes or those cells stimulated with PMA and ionomycin (P+I) for 4–5h.(B) Intracellular IL-2 production by thymic CD4SP or CD8SP cells of WT and Gfi1CD4 mice after PMA and ionomycin stimulation for 4–5 h in the presence of monensin. (C) Frequency of Foxp3+ cells among CD4SP thymocytes from − − − − WT, Gfi1CD4, Il2 / ,andGfi1CD4 Il2 / mice (6 wk). (D) Analysis of Foxp3+ cells after in vivo neutralization of IL-2 signaling. WT and Gfi1CD4 mice were injected i.v. with neutralizing anti–IL-2 (S4B6) or anti–IL-2R (PC61) antibody and analyzed 2 wk later. (E and F) Expression of (E) Tgfb1 and (F) Il10 in WT and Gfi1CD4 thymocyte populations with or without P+I stimulation (Il10 was nondetectable in CD8SP cells). Data are representative of (A, B, and D–F) four and (C)three independent experiments. Error bars indicate SEM. *P < 0.05; **P < 0.01; NS, not significant.

(Fig. 4D). Thus, an important mechanism by which Gfi1 de- play an important role in nTreg cell generation. Altogether, our ficiency promotes nTreg development is to induce excessive IL-2 results highlight an IL-2–dependent, cell-extrinsic mechanism CD4 production. responsible for the expansion of nTreg cells in Gfi1 mice. Aside from IL-2, the immunosuppressive cytokines TGF-β1 + fi and IL-10 have been implicated in promoting Foxp3 cell gen- Survival-Independent Functions of G 1 in Thymic nTreg Generation. eration under different conditions (37–40). Whereas TGF-β1 Gfi1 mutation is frequently associated with elevated apoptosis expression was comparable between WT and Gfi1CD4 cells (Fig. of multiple cell types (8, 21). Undoubtedly, regulation of apoptosis 4E), increased IL-10 expression was observed in Gfi1CD4 CD4SP is a major component of thymocyte developmental processes, in- cells (Fig. 4F). To test the contribution of the elevated IL-10 cluding nTreg generation (2, 3). Although Gfi1 germ-line deletion fi expression to the phenotypic alteration of nTreg cells, we gen- promoted apoptosis of early DN thymocytes (13), G 1 did not − − erated Gfi1CD4 Il10 / mice. Deletion of IL-10 had minimal seem to be essential for the survival of DP and SP thymocytes effects on the altered Foxp3+ percentage or CD103 expression under steady state conditions (Fig. 1E). However, because apo- CD4 among nTreg cells in Gfi1 mice (Fig. S4 D and E), indicating that ptotic cells can be rapidly cleared in vivo, we performed additional the enhanced IL-10 expression by Gfi1-deficient T cells does not analysis to closely examine the effects of Gfi1lossonthymocyte

E3202 | www.pnas.org/cgi/doi/10.1073/pnas.1300950110 Shi et al. Downloaded by guest on September 30, 2021 survival. When cultured for 2 d in vitro, Gfi1CD4 CD4SP thymo- sequences of IL-2 blocking. This model is further supported by PNAS PLUS cytes showed modestly impaired survival (Fig. 5A). Moreover, in age-dependent accumulation of thymic nTreg cells observed in a competitive environment introduced by the mixed chimeras, Gfi1CD4 mice and the increased BrdU incorporation among thymic CD4 Gfi1 -derived donor cells had impaired capacity to populate the nTreg cells lacking Gfi1. Altogether, these data establish a pre- thymic CD4SP compartment (Fig. 5B). These results reveal a re- viously unrecognized mechanism for the active control of IL-2 quirement of Gfi1 for the survival fitness of mature thymocytes. production in the thymic microenvironment to shape nTreg To directly address whether this prosurvival effect of Gfi1is development. CD4 a contributing factor to nTreg development, we crossed Gfi1 Our identification of a noncell-autonomous mode of nTreg cell mice with lymphocyte-specific transgenic mice for Bcl-2 that generation echoes the recent findings describing bystander de- potently inhibits thymocyte death (41). As expected, the intro- velopment of innate CD8+ T cells, another nonconventional duction of the Bcl-2 transgene nearly completely rectified the T-cell subset derived from the thymus. Specifically, thymocytes survival defects of Gfi1-deficient thymocytes in vitro (Fig. 5C). deficient in transcription factors such as Klf2, Itk, or Id3 aber- However, Gfi1CD4 Bcl2-TG mice still contained an increased rantly produce IL-4 that mediates the generation of such cells thymic nTreg population (Fig. 5D). Similarly, deletion of Bim (30, 44–46). These studies collectively highlight the importance (encoded by Bcl2l11), a proapoptotic molecule important for and complexity of transcription factor-dependent shaping of thy- thymic-negative selection (42), did not affect the increased nTreg mic cytokine levels to ensure normal development of various cells in Gfi1CD4 mice (Fig. S5). Collectively, Gfi1 controls thymic thymus-derived populations. fi generation of nTreg cells through a mechanism largely inde- Using a B16 melanoma tumor model, we showed that G 1 pendently of cell survival. function is required for efficient antitumor immunity. The un- controlled tumor growth in Gfi1CD4 mice was associated with Discussion increased nTreg cells infiltrating the tumor as well as elevated Thymic development of nTreg cells is orchestrated by signals expression of CD103 and CD73 that endows nTreg cells with transduced from TCRs, costimulation, and cytokines (2, 3). Al- strong suppressive activity (24, 25). Furthermore, nTreg cells though the involvement of nTreg-intrinsic pathways has been deficient in Gfi1 had enhanced suppressive activity in vitro. described extensively, how nTreg development is shaped by ex- These results highlight the important roles of Gfi1 in negatively trinsic factors remains poorly understood (43). Additionally, al- affecting both nTreg generation and suppressive activity. Despite CD4 though a role for IL-2 to promote thymic nTreg development has the impaired antitumor immunity in Gfi1 mice, we observed been appreciated for some time (32–34), the source of IL-2– increased IFN-γ production from effector cells lacking Gfi1. producing cells remains undefined (6). In this study, we have These results indicate that the increased frequency and function fi fi identi ed a noncell-autonomous role of G 1 in inhibiting nTreg of Gfi1-deficient nTreg cells are important factors for the de- generation by down-regulating IL-2 expression from conventional fective in vivo responses, although we could not exclude addi- thymocytes. Our conclusions are derived from the analysis of tional contribution from conventional T cells. mixed BM chimeras and Gfi1Foxp3 mice, the exuberant production Consistent with a prosurvival role of Gfi1 observed in other of IL-2 from Gfi1-deficient thymocytes, and the functional con- cell types (8, 21), deficiency of Gfi1 modestly elevated apoptosis

A B WT Gfi1CD4 WT Gfi1CD4 ** 56.1 31.5 40.5 69

42.1 66.6 53.6 25.5 7-AAD CD45.2 Annexin V CD45.1 CD45.2/CD45.1 ratios

C WT Bcl2-TG D WT Bcl2-TG 1.86 75.5 25.2 5.04 5.92 IMMUNOLOGY

22.2 0.42 72.8 CD4 CD4 Gfi1CD4 Gfi1CD4 Bcl2-TG Gfi1 Gfi1 Bcl2-TG 3.19 86.7 30.8 11.5 11.3 Foxp3 7-AAD 9.9 0.26 64 Annexin V CD25

CD4 Fig. 5. Survival defect of Gfi1-deficient thymocytes does not contribute to the altered nTreg development. (A) CD4SP thymocytes from WT or Gfi1 mice were cultured for 2 d followed by Annexin V and 7-aminoactinomycin D (7-AAD) staining. (B) Competitive fitness of CD4SP thymocytes in vivo. Mixed BM chimeras were set up as described in Fig. 3A, and the contributions of WT or Gfi1CD4 donor (CD45.2+) and spike (CD45.1+) cells were analyzed. (Right) Ratios of CD45.2/CD45.1 (after normalization to nonrelevant B220+ cells). (C) CD4SP thymocytes from WT, Gfi1CD4, Bcl2-TG, or Gfi1CD4 Bcl2-TG mice were cultured for 3 d followed by Annexin V and 7-AAD staining. (D) Frequency of Foxp3+ cells among gated CD4SP cells from WT, Gfi1CD4, Bcl2-TG, or Gfi1CD4 Bcl2-TG mice. Data are representative of (A–C) five and (D) three independent experiments. Error bars indicate SEM. **P < 0.01.

Shi et al. PNAS | Published online August 5, 2013 | E3203 Downloaded by guest on September 30, 2021 ’ of CD4SP thymocytes and rendered them a competitive disad- Foxp3 and Ki-67 staining was performed per manufacturer s instructions vantage. However, forced expression of the Bcl2 transgene or (eBioscience). For intracellular cytokine staining, T cells were stimulated for 4–5 h with PMA and ionomycin in the presence of monensin before staining deletion of Bim did not affect the generation of nTreg cells in Gfi1CD4 mice. In contrast, in hematopoietic stem cells deficient per manufacturer’s instructions (BD Biosciences). in Gfi1, the survival defect is a central mechanism for impaired stem cell function, because ectopic Bcl-2 expression allows Cell Culture. Lymphocytes were isolated from the thymuses, spleens, and fi fi lymph nodes of mice and sorted on a MoFlo (Beckman-Coulter) or Reflection G 1-de cient stem cells to self-renew and initiate multilineage − fi (i-Cyt) cell sorter as described previously (50). SP thymocytes (CD4+CD8 TCRβ+ differentiation (47). These results reveal that G 1-mediated – + β+ + hi – prosurvival function exerts context-specific physiological effects. and CD4 CD8 TCR ) and peripheral effector T cells (CD4 CD45Rb CD25 ) were sorted based on the defined surface markers. T cells were cultured in In summary, we report here a nonconventional mechanism for Click’s medium supplemented with 10% (vol/vol) FBS and 1% (vol/vol) pen- the regulation of nT development. The transcription repressor reg icillin-streptomycin. For nT suppression assays, effector T cells from CD45.1 Gfi1 restrains production of IL-2 from conventional T cells, reg mice were labeled with CFSE and cocultured with nTreg cells in the presence thereby shaping the development of nTreg cells by a noncell- fi of anti-CD3 (145-2C11) for 72 h for the measurement of CFSE dilution of the autonomous mechanism. Loss of G 1 results in expansion of CD45.1+ cells. Alternatively, unlabeled effector T cells were used, and cell nTreg cells and enhanced suppressive activity that contribute proliferation was assayed by 3H-thymidine incorporation assay. to impaired antitumor immunity. Our studies highlight that Treg cells are regulated by more than Treg-intrinsic pathways RNA Analysis. RNA was extracted with an RNeasy Kit (QIAGEN), and cDNA was that sense and integrate signals from TCR and cytokines. Rather, synthesized with SuperScript III/II Reverse Transcription (Invitrogen). Real- thymic generation of these cells is further shaped by active control time PCR was performed as described previously using primer and probe sets of cytokine production from conventional T cells. purchased from Applied Biosystems (50).

Materials and Methods B16 Tumor Challenge Model. Inoculation with B16 melanoma was carried out − − − − − − Mice and in Vivo Studies. C57BL/6, CD45.1, Rag1 / , Il2 / , Bcl2l11 / , and as previously described with minor modifications (51). Briefly, mice were − − Il10 / mice were purchased from the Jackson Laboratory. CD4-Cre, hCD2- injected with 1.25 × 105 to 5.0 × 105 B16 cells i.d. in the back. Tumor di- Cre-YFP iCre, and Foxp3 mice were as described (22, 23, 31). Details for the ameter was measured every 2–3 d with an electronic caliper and reported as fi fl/fl fl generation of G 1 mice will be described elsewhere. The LoxP anked area (millimeters squared). To isolate tumor-infiltrating lymphocytes, solid fi – region included coding exons for zinc ngers 1 4, which were essential for tumors were excised around 15–16 d, and single-cell suspensions were pre- DNA binding; homozygosity for the excised allele reproduced the pheno- pared by mechanical dissociation followed by density gradient centrifuga- type of the previously published conventional Gfi1-deficient allele (48). All tion on an 80%/40% (vol/vol) Percoll (GE Healthcare) gradient. The purified mice have been backcrossed to the C57BL/6 background extensively. Mixed cells were then subjected to Foxp3 staining to enumerate T and non- BM chimeras were generated by reconstituting sublethally irradiated (4 Gy) reg −/− 6 T cells. alymphoid Rag1 mice with ∼5 × 10 BM cells depleted of T cells as pre- reg viously described (49), with the specified ratios of donor cells from various sources distinguished by congenic markers. To label proliferating cells in Statistical Analysis. P values were calculated with Student t test or ANOVA. < fi vivo, mice were injected with 0.8 mg BrdU/mouse i.p., and BrdU in- P values 0.05 were considered signi cant. corporation was analyzed 18 h later using the BrdU Staining Kit (BD Bio- sciences). To block IL-2 signaling, mice were injected i.v. with anti–IL-2 (clone ACKNOWLEDGMENTS. The authors thank Stuart H. Orkin for help with fi YFP-Cre S4B6) or anti–IL-2R (clone PC61) antibody (1 mg/mouse). Animal protocols G 1-targeted mice; Alexander Rudensky for Foxp3 mice; Maureen fi were approved by the Institutional Animal Care and Use Committee of McGargill for reading of the manuscript and critical scienti c inputs; Caryn Cloer and Nicholas Brydon for animal colony management and other tech- St. Jude Children’s Research Hospital. nical support; and Richard Cross, Greig Lennon, and Stephanie Morgan for cell sorting. This work was supported by National Institutes of Health Grants Flow Cytometry. For flow cytometric analyses of surface markers, cells were AI 101407, AI 094089, NS 064599 (to H.C.), and CA 122726 (to H.H.) and the stained with antibodies (eBioscience) in PBS containing 2% (wt/vol) BSA. American Lebanese Syrian Associated Charities (H.C.).

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