KLF2 is a rate-limiting that can be targeted to enhance regulatory T-cell production

Sudheer K. Pabbisettya, Whitney Rabacala, Damian Masedaa, Delphine Cendrona, Patrick L. Collinsb, Kristen L. Hoeka, Vrajesh V. Parekha, Thomas M. Aunea,c, and Eric Sebzdaa,1

Departments of aPathology, Microbiology, and Immunology, and cMedicine, Vanderbilt University School of Medicine, Nashville, TN 37232; and bDepartment of and Immunology, Washington University School of Medicine, St. Louis, MO 63110

Edited by Tak W. Mak, The Campbell Family Institute for Research at Princess Margaret Cancer Centre, Ontario Cancer Institute, University Health Network, Toronto, Canada, and approved May 22, 2014 (received for review December 17, 2013)

+ Regulatory T cells (Tregs) are a specialized subset of CD4 T cells Results that maintain self-tolerance by functionally suppressing autoreactive Treg Frequencies Are Maintained in Klf2 -Targeted Mice. KLF2 . The Treg compartment is composed of -derived is a -finger transcription factor that has a documented role in Tregs (tTregs) and peripheral Tregs (pTregs) that are generated in controlling naive T-cell migration patterns (12–14). Its expression secondary lymphoid organs after exposure to antigen and specific is promoted by Foxo1, a PKB-regulated transcription factor that cytokines, such as TGF-β. With regard to this latter lineage, pTregs has also been reported to control T-cell circulation (9, 10). [and their ex vivo generated counterparts, induced Tregs (iTregs)] Moreover, Foxo1 and Foxo3 promote Treg development and offer particular therapeutic potential because these cells can be function; mice lacking these transcription factors have reduced + raised against specific antigens to limit autoimmunity. We now percentages of FoxP3 T cells (8), especially at 3 wk of age (7), report that transcription factor Krüppel-like factor 2 (KLF2) is and fail to develop functional Tregs. To determine whether Foxo1 necessary for the generation of iTregs but not tTregs. Moreover, and Foxo3 were acting through Klf2 to control Treg biology, we Klf2 drugs that limit KLF2 proteolysis during T-cell activation en- examined regulatory T cells in gene-targeted animals. Using Lck-cre transgenic mice that specifically excised floxed alleles of hance iTreg development. To the authors’ knowledge, this study fl/fl fl/fl Klf2 (Klf2 ) within the T-cell compartment (Lck-cre; Klf2 ), we identifies the first transcription factor to distinguish between + +

found no obvious Treg defects, as determined by CD4 CD25 IMMUNOLOGY i/pTreg and tTreg ontogeny and demonstrates that KLF2 is a + therapeutic target for the production of regulatory T cells. FoxP3 T-cell frequencies in primary and secondary lymphoid tissues (Fig. 1A). Likewise, Treg frequencies in the thymus of neonates seemed normal (Fig. 1B), which would suggest that egulatory T cells (Tregs) are a vital component of self- Foxo-mediated Treg development was not dependent upon KLF2. Rtolerance (1, 2); however, the signaling events that govern Recent reports have demonstrated that S1P1 suppresses gener- Treg development are not well defined. Treg development and ation of Tregs (15, 16), and because KLF2 promotes S1P ex- + 1 function is dependent upon the forkhead-winged helix tran- pression in the CD4 T-cell lineage (12–14), we decided to scription factor FoxP3, which is coregulated by signaling mole- examine the relationship between KLF2 and S1P1 relative to Treg cules downstream of T-cell receptors and cytokine receptors. At development. Using Vav-cre transgenic mice to thoroughly excise the same time, robust antigen and cytokine receptor floxed alleles within the T-cell compartment, including early stages stimulation activates PI3K, which negatively regulates FoxP3 of development, we found that S1P1 gene-targeted mice Vav-cre; S1P fl/fl Vav-cre; S1P fl/fl;Klf2fl/fl expression (3, 4). This occurs when PI3K activates ( 1 or 1 ) expressed increased frequencies of Tregs, whereas this was not present in kinase B (PKB), which in turn phosphorylates and inactivates fl/fl animals lacking KLF2 alone (Vav-cre; Klf2 ) (Fig. 1C). From forkhead-box O (Foxo) transcription factors that promote these results we conclude that KLF2 does not contribute to the FoxP3 expression (5–7). Consistent with this model, condi- overt Treg homeostatic defects observed in Foxo1/3 or S1P1 gene- tional of Foxo1 and Foxo3 results in reduced numbers targeted mice. of thymic- and peripheral-derived Tregs (7, 8). Moreover, the Tregs that eventually populate Foxo1/3-deficient animals are Significance hyperproliferative, skew toward differentiated effector line- ages, and lack suppressive functions. Within the context of + CD4 T-cell biology, a critical molecule regulated by Foxo1 is Regulatory T cells (Tregs) are crucial for preventing autoim- Krüppel-like factor 2 (KLF2) (9, 10), a zinc-finger transcrip- munity, and thus discovering an efficient means of generating tion factor that is also inactivated in a PI3K-dependent manner antigen-specific Tregs is a medical priority. To this end, we (11). KLF2 maintains T-cell homeostasis, in part by promoting demonstrate that transcription factor Krüppel-like factor 2 (KLF2) is necessary for the generation of antigen-induced Tregs expression of sphingosine-1-phosphate receptor 1 (S1P1)(12– 14). Surprisingly, S1P suppresses expression of FoxP3, as evi- and their in vivo counterpart, peripheral Tregs. Moreover, 1 pharmaceutical drugs that stabilize KLF2 protein levels during denced by increased numbers of thymus-derived Treg (tTregs) + − + + + the transition from CD4 CD25 to CD4 CD25 FoxP3 and induced Treg (iTreg) cells after T cell-specific excision in Treg augment production of these tolerizing lymphocytes. S1P gene-targeted animals (15, 16). On the one hand, studies 1 Results from this study indicate that KLF2 is a viable target for using Foxo1/3-deficient mice suggest that KLF2 promotes Treg de- altering Treg development, which may significantly impact velopment and/or function, whereas reports using S1P -deficient 1 patients prescribed statins. animals predict an opposing outcome. To clarify this apparent

contradiction and gain further insight into Treg biology, we com- Author contributions: S.K.P., T.M.A., and E.S. designed research; S.K.P., W.R., D.M., D.C., pared gene-targeted mouse models that excised Klf2 within the P.L.C., K.L.H., and V.V.P. performed research; T.M.A. contributed new reagents/analytic T-cell vs. Treg compartments. We now report that (i) KLF2 is tools; S.K.P., D.M., P.L.C., and E.S. analyzed data; and E.S. wrote the paper. selectively required for the generation of iTregs but not tTregs, The authors declare no conflict of interest. (ii)KLF2isnecessaryduringthe inductive phase of iTreg This article is a PNAS Direct Submission. development to promote FoxP3 transcription, and (iii) iTreg pro- 1To whom correspondence should be addressed. E-mail: [email protected]. duction can be augmented by stabilizing KLF2 protein levels with This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. pharmaceutical drugs. 1073/pnas.1323493111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1323493111 PNAS | July 1, 2014 | vol. 111 | no. 26 | 9579–9584 Downloaded by guest on September 30, 2021 β– A Thymus Ms LN Ax LN (18), raising the possibility that IL-4 was suppressing TGF- mediated iTreg generation (19). To address this issue, KLF2- 105 105 105 105 + 11 12 14 12 sufficient and KLF2-deficient CD4 T cells were cocultured 104 104 104 104 control under iTreg differentiating conditions. As shown in Fig. 2C, only 3 3 3 3 10 10 10 10 KLF2-sufficient T cells were capable of producing iTregs, dem- 102 102 102 102 0 0 0 0 onstrating the cell-intrinsic nature of this defect. Moreover,

0102 103 104 105 0102 103 104 105 0102 103 104 105 0102 103 104 105 we did not detect increased expression of the TH2-associated

FoxP3 105 105 105 105 11 10 15 15 transcription factor, GATA3, or elevated production of IL-4 104 104 104 104 + cKO by KLF2-deficient splenic CD4 T cells after antigen stimu- 103 103 103 103 lation (Fig. 2D), indicating that this cytokine was not responsible 102 102 102 102 0 0 0 0 for the iTreg defect in the present study. To determine when 0102 103 104 105 0102 103 104 105 0102 103 104 105 0102 103 104 105 KLF2 was necessary during iTreg generation, we used a ta- fl/fl moxifen-inducible cre system (T2-cre; Klf2 ) to excise Klf2 after CD25 TGF-β–induced FoxP3 expression. As shown in Fig. 2E, Klf2 B control Klf2 cKO excision did not affect the stability of FoxP3 expression, in- 4.9 4.5 5.0 5.1 dicating that KLF2 was required solely at the induction stage of iTreg generation.

In Vivo Development of iTregs Is KLF2-Dependent. We next addressed FoxP3 the physiological relevance of KLF2 by examining peripheral Treg (pTreg) populations under homeostatic conditions. Reports in- CD25 dicate that Helios (an Ikaros transcription factor family member)

C control S1P1 cKO Klf2 cKO dKO

5 5 5 5 10 11.3 10 23.2 10 9.4 10 24.7

10 4 10 4 10 4 10 4 αCD3/CD28 ABαCD3/CD28 αCD3/CD28 ++ + + + TGF

3 3 3 3 10 10 10 10 TGF (ng/ml) 01 5 10 105 105

800 4 4 FoxP3 10 0.4 10 800 600 2 2 2 2 600 10 10 10 10 600 3 3 control 600 0.9 2.1 12.7 18.2 10 10 400 400 0 0 0 0 400 400 54.6 control 102 102 200 200 200 200 0 0 0102 10 3 10 4 10 5 0102 10 3 10 4 10 5 0102 10 3 10 4 10 5 0102 10 3 10 4 10 5

2 3 4 5 2 3 4 5 0 0 0 0 010 10 10 10 010 10 10 10 2 3 4 5 2 3 4 5 2 3 4 5 010 10 10 10 0102 10 3 10 4 10 5 010 10 10 10 010 10 10 10 5 5 800 FoxP3 10 10 1000 800 800

800 4 0.3 4 0.9 600 40 CD25 0.6 0.8 600 1.3 600 1.2 10 10 600 400 cKO 400 400 cKO 103 103 400 Relative cell number 200 200 200 200 102 102

0 0 0 0 0 0 30 control 0102 10 3 10 4 10 5 0102 10 3 10 4 10 5 0102 10 3 10 4 10 5 0102 10 3 10 4 10 5 0102 103 104 105 0102 103 104 105 FoxP3 CD25 S1P1 cKO C CFSE+ CFSE- 60 P<0.0001

20 10 5 10 5 0.9 1.0 60 10 4 10 4 P=0.005 8 40 50 Klf2 cKO CD3 8

α T cells) 3 3 10 10 40 + +αCD28 6 6 30 10 2 10 2

20 20 0 0 10 T cells) 4

dKO + 10 0102 10 3 10 4 10 5 0102 10 3 10 4 10 5 4 to CD4

% iTregs (relative % iTregs 0 10 5 10 5 % FoxP3+ CD25+ cells

FoxP3 2 0 (relative to CD4+ T cells) T (relative to CD4+ 5.1 0.6 2 10 4 10 4

αCD3 to CD4 cKO cKO

% iTregs (relative % iTregs 0 12 0 3 3 0 10 10 control control +αCD28 CFSE+ CFSE- 10 2 10 2 + TGF αCD3/CD28 ++++ 0 0

2 3 4 5 2 3 4 5 TGF (10ng/ml) ++ Ms LN Ax LN 010 10 10 10 010 10 10 10 Thymus Spleen CD25

800 500

αCD3/CD28 600 + + 400 D αCD3/CD28 E 600 Fig. 1. Treg frequencies in Klf2 gene-targeted mice. (A) Percent FoxP3 CD25 + TGF 300 400 100 400 10.1 11.1 5 5 0.6 + fl/fl 10 10 200 control 200 80 200 100 Tregs relative to CD4 T cells in tissues harvested from Klf2 (control) vs. Lck- 10 4 10 4

60 0 0 0 fl/fl 3 3 2 3 4 5 2 3 4 5 2 3 4 5 10 10 control 010 10 10 10 010 10 10 10 010 10 10 10 cre; Klf2 (cKO) littermates (6 wk of age). This experiment was repeated four 40

2 2 600 20 10 10 800 800 0 4.7 0 0.9 0 600 times. Ms LN, mesenteric lymph nodes; Ax LN, axillary lymph nodes. (B)Percent 600 2345 400 010 10 10 10 0102 10 3 10 4 10 5 0102 10 3 10 4 10 5 12.0 iKO + 0.6 400 11.6 fl/fl fl/fl IL-17 400 100

Relative cell number 200 Tregs relative to CD4 in 3-wk-old Klf2 (control) or Lck-cre; Klf2 10 5 10 5 200 200 80 4 4 10 10 0 0 0 2 3 4 5 2 3 4 5 2 3 4 5 (cKO) littermates. This experiment was performed three times. (C) Proportion of 60 cKO 010 10 10 10 010 10 10 10 010 10 10 10 10 3 10 3 fl/fl fl/fl fl/fl Relative cell number 40 FoxP3 Tregs present in Klf2 (control), Vav-cre; S1P1 (S1P1 cKO), Vav-cre; Klf2 20 10 2 10 2 0 6.1 0 0.6 +++ 0 αCD3/CD28 fl/fl fl/fl 2345 010 10 10 10 (Klf2 cKO), and Vav-cre; S1P1 ;Klf2 (dKO) littermates (n = 2 mice per group). 0102 10 3 10 4 10 5 0102 10 3 10 4 10 5 TGF -++ + + --+ FACS plots: flow cytometric analysis of CD4 T cells in the spleen. % FoxP3 GATA-3 IL-4 tamoxifen CD25+ Tregs are indicated. Bar graph: average frequency of Tregs relative to + Fig. 2. KLF2 is required for the ex vivo generation of iTregs. (A) Ex vivo CD4 T cells in lymphoid organs. Mice were 7 wk old. This experiment was re- + − generation of iTregs from splenic CD4 CD25 T cells derived from Klf2fl/fl peated twice (excluding S1P cKO littermates, which were analyzed once). + 1 (control) or Lck-cre; Klf2fl/fl (cKO) mice. Histograms display FoxP3 T-cell frequencies. This experiment was repeated four times. (B) iTreg development using CD4+CD8−CD25− thymocytes from Klf2fl/fl (control) vs. Lck-cre; Klf2fl/fl KLF2 Is Required for the Ex Vivo Generation of iTregs. We have (cKO) mice. Representative contour plots (Upper) and a chart of accumulated previously demonstrated that KLF2 differentially impacts lym- data (Lower) from four mice per cohort are shown. This experiment was re- + phocyte homeostasis in a lineage-specific manner (17). There- peated three times. (C) Ex vivo generation of iTregs using cocultured CD4 − + − fore, we decided to examine iTreg and tTreg development CD25 T cells harvested from Klf2fl/fl (CFSE )andLck-cre; Klf2fl/fl (CFSE )mice. independent of one another. Strikingly, we found that KLF2 was Contour plots and pooled results for Treg induction (αCD3+αCD28+TGFβ)are + necessary for the ex vivo generation of iTregs. Antigen stimula- shown. This experiment was repeated three times. (D)CD4 TcellsfromKlf2fl/fl tion in the presence of TGF-β induced regulatory T-cell pro- (control) vs. Lck-cre; Klf2fl/fl (cKO) mice were evaluated for their propensity to + fl/fl Lck-cre; Klf2 skew toward the TH2 lineage and secrete IL-4. (Left) Histogram overlays of duction; however, CD4 T cells from mice did + not differentiate into FoxP3-expressing iTregs under these con- GATA-3 expression after culturing conditions that favor CD4 T-cell differen- + − tiation toward a neutral (solid gray), TH2 (gray line), or iTreg lineage (black ditions (Fig. 2A). Similarly, KLF2-deficient CD4 CD25 (single- + + line). (Right) Generation of intracellular cytokines after CD4 T-cell activation positive) thymocytes were unable to differentiate into FoxP3 + (Left) vs. iTreg differentiation (Right). This experiment was performed twice. CD25 cells, demonstrating that defective iTreg development (E) Tamoxifen-induced excision of Klf2 after ex vivo generation of iTregs using was not secondary to aberrant T-cell migration (Fig. 2B). A pub- + − fl/fl + CD4 CD25 TcellsfromT2cre vs. T2cre; Klf2 animals. T cells from T2cre; lished report indicated that KLF2-deficient CD4 T cells secrete Klf2fl/fl mice excised >95% Klf2 as measured by RT-PCR. This experiment was elevated levels of IL-4 after T-cell receptor (TCR) stimulation repeated twice.

9580 | www.pnas.org/cgi/doi/10.1073/pnas.1323493111 Pabbisetty et al. Downloaded by guest on September 30, 2021 and Neuropilin 1 (Nrp1, a membrane-bound surface receptor) are not significantly impact nontransgenic Treg numbers. Moreover, + preferentially but not exclusively expressed by tTregs relative to low frequencies of CD4 T cells expressing a TH2-like profile fl/fl pTregs (20–25). To determine whether either of these molecules were identified in the GALT of Lck-cre; Klf2 mice (Fig. S2), were appropriate surrogates for pTreg identification, we initially a phenotype that has previously been associated with a lack of characterized expression patterns within the entire regulatory T- + + pTregs in vivo (28). To confirm that KLF2 was necessary for in- cell compartment. Most FoxP3 CD4 thymocytes belong to the + + duction but not maintenance of FoxP3 expression within the pTreg fl/fl tTreg lineage, whereas peripheral FoxP3 CD4 T cells are com- compartment, we next examined Helios expression in Klf2 posed of both tTregs and pTregs, with a partial preference for mice bred onto a FoxP3-cre transgenic background (FoxP3-cre; pTregs in mucosal-associated lymph nodes. Consistent with this fl/fl low + + Klf2 ). Consistent with our ex vivo results, we found similar fre- phenotype, few Helios FoxP3 CD25 T cells were detected in low quencies and numbers of Helios cells in SLO harvested from the thymus, whereas increased frequencies were found in sec- fl/fl FoxP3-cre; Klf2 mice and littermate controls (Fig. 3C). These ondary lymphoid organs (SLO) and after ex vivo Treg induction (Fig. S1). Although Nrp1low stains also identified iTregs in culture, data indicate that under normal physiological conditions, KLF2 is necessary during the inductive stage of pTreg development. De- expression did not strictly correlate with known pTreg tissue dis- Lck-cre; Klf2fl/fl tribution patterns found under steady-state conditions. For this spite the lack of pTregs, mice were healthy and did reason we focused on Helios expression as a proxy to distinguish not exhibit signs of autoimmunity, including spontaneous T-cell high low A between tTreg (Helios ) and pTreg (Helios ) populations. As activation or pathogenic TH1/TH17 differentiation (Fig. S3 ). + + shown in Fig. 3A, most FoxP3 CD4 thymocytes expressed high Moreover, KLF2-deficient T cells maintained their effector func- + + levels of Helios, whereas FoxP3 CD4 T cells in SLO of wild-type tions (Fig. S3 B–E), suggesting that autoimmunity was actively re- animals contained a spectrum of Helios expression. In contrast, pressed by a functional Treg compartment. Direct analysis of Tregs + + fl/fl fl/fl the majority of FoxP3 CD4 TcellsinLck-cre; Klf2 mice were from Lck-cre; Klf2 mice confirmed that KLF2-deficient Tregs Helioshigh, both in primary and secondary lymphoid tissues. These retained their suppressive properties (Fig. S3F). Therefore, we high + + Helios FoxP3 CD25 T cells were phenotypically quiescent conclude that tTregs are sufficient to prevent autoimmunity under (Fig. S1D), thus excluding the possibility that KLF2-deficient homeostatic conditions and posit that pTregs are necessary to limit Tregs were transiently expressing Helios owing to aberrant tissue damage associated with an inflammatory environment. activation (22). Significantly fewer Helioslow Tregs were fl/fl present in the SLO of Lck-cre; Klf2 mice relative to litter- KLF2 Directly Regulates FoxP3 Expression Within the iTreg Lineage. mate controls, consistent with an in vivo defect in pTreg Foxp3 expression is orchestrated by transcription factors that generation. To directly assess de novo generation of pTregs, selectively bind to key genetic elements, including the IMMUNOLOGY KLF2 gene-targeted mice were crossed onto a TCR trans- region and three conserved noncoding sequences (CNS1–3) genic background (OT2) that recognizes ovalbumin peptide (p323- + + downstream of the transcriptional start site (29). These enhancer 339, OVA) in the context of H2-IAb. OVA-specific (Vα2 /Vβ5 ) + + elements are differentially used by iTregs and tTregs; for in- CD4 FoxP3 Tregs are absent in OT2 animals; however, OT2 stance, CNS1 is necessary for the generation of iTregs, whereas it mice placed on OVA-infused drinking water develop transgenic is dispensable for tTreg production. To determine whether KLF2 pTregs in gut-associated lymphoid tissues (GALT; e.g., mesenteric bound to these regulatory regions, ChIP assays were performed lymph nodes, spleen) (26, 27). In agreement with surrogate Helios + fl/fl using DNA from CD4 T cells that were TCR-stimulated in the staining results, OT2; Lck-cre; Klf2 mice were unable to generate presence (iTregs) or absence (mock control) of TGF-β.Asshown + OVA-specific FoxP3 Tregs (Fig. 3B), although this defect did in Fig. 4A, KLF2 bound to the promoter and all three enhancer

A 100 100 Thymus Spleen 0.3 0.008 0.002 0.0001 0.06 <0.0001 <0.0001 <0.0001 80 80 &!" 40 40 30 60 60 )

4 %#" 40 40 30 30 20 20 20%!"

0 0 $#" 0102 10 3 10 4 10 5 0102 10 3 10 4 10 5 20 20 100 100 Ms LN Ax LN $!" 80 80 10 10 10 % Helios-low #" 60 60 numbers (x10 Helios-low Treg (CD4+ FoxP3+) Regulatory T cells

40 40 0 !" thymus spleen Ms LN Ax LN

Relative cell number 0 0 '()*+," -./001" 2,"34" 56"34" Fig. 3. KLF2 is required to establish a pTreg compartment 20 20 Thymus Spleen Ms LN Ax LN Thymus Spleen Ms LN Ax LN

0 0 2 3 4 5 2 3 4 5 in vivo. (A) Expression of Helios within the Treg compart- 010 10 10 10 010 10 10 10 + + + fl/fl Helios ment (CD4 FoxP3 CD25 )ofKlf2 (gray histograms) vs. B Lck-cre; Klf2fl/fl (open histograms) mice (n = 4 mice per 30 )

) P=0.3 group). Representative histogram overlays are displayed.

4 90 P=0.003 4 NT control Percentage and absolute number of Helioslow regulatory T fl/fl OVA-induced Tregs Non-induced Tregs OT2; Klf2 cells from Klf2fl/fl (gray bars) vs. Lck-cre; Klf2fl/fl (black bars) 20 cells (x10 fl/fl cells (x10 OT2; Lckcre; Klf2 + 60 mice. Error bars (SD) and P values are displayed. This ex- + periment was repeated four times. (B) De novo generation P=0.01 P=0.07 FoxP3 FoxP3

+ of pTregs in non-TCR transgenic control (white bars), OT2; 10 + 30 Klf2fl/fl (black bars), and OT2; Lck-cre; Klf2fl/fl (gray) mice CD4 CD4 + P=0.3 - P=0.5 placed on ovalbumin-infused drinking water. (Left) Absolute + + + OT2 0 OT2 number of OT2 CD4 FoxP3 Tregs isolated from tissues 0 associated with pTreg generation [spleen, mesenteric lymph Spleen Ms LN Ax LN Spleen Ms LN Ax LN nodes (Ms LN)] as well as control tissue [axillary lymph nodes (Ax LN)]. (Right) Absolute number of non-TCR transgenic C − + + 100 100 Thymus Spleen 0.9 0.2 0.5 0.2 0.02 0.6 0.4 0.3 OT2 CD4 FoxP3 Tregs isolated from same animals. This 80 80 '#" 40 5040 40 = 60 60 ) '!" experiment was conducted twice, n 3 mice per cohort. 4

40 40 40 &#" 30 30 30 Error bars (SD) and P values (Student t test) are indicated. (C) 20 20 &!"

30 0 0 %#" 0102 10 3 10 4 10 5 0102 10 3 10 4 10 5 20 Comparison of Helios expression within the Treg compart- 20 20%!" 100 100 20 fl/fl Ms LN Ax LN $#" 80 80 ment of Klf2 (gray histograms, gray bars) vs. FoxP3-cre; 10 10 10$!" 10 % Helios-low 60 60 numbers (x10 fl/fl Helios-low Treg #" = Regulatory T cells

(CD4+ FoxP3+) Klf2 (open black histograms, black bars) mice (n 3 ani- 40 40 00 !" 0 thymusthymus spleen MSMs LN AxAX LN 0 Relative cell number Ax LN ()*+,-" ./0112" 3-"45" Ax67"45" LN 20 20 Thymus Spleen Ms LN Thymus Spleen Ms LN mals per group). Bar graphs display percentage and absolute

0 0 2 3 4 5 2 3 4 5 low 010 10 10 10 010 10 10 10 number of Helios Tregs, including error bars (SD) and Helios P values. This experiment was repeated four times.

Pabbisetty et al. PNAS | July 1, 2014 | vol. 111 | no. 26 | 9581 Downloaded by guest on September 30, 2021 elements of TGF-β–stimulated T cells. Acetylated histone H4 that was most evident after T-cell activation (Fig. 5G). All of (H4Ac) epigenetically marks transcriptionally permissive areas of these results are consistent with KLF2 stability being a key chromatin (30), as demonstrated by H4Ac association with FoxP3 rate-determining step during Treg induction, and for that + regulatory sites after iTreg induction of wild-type CD4 T cells reason we repeated /induction experiments using (Fig. 4B). In contrast, minimal H4Ac was recovered from ChIP a mutant form of KLF2 that is no longer recognized by the E3 assays using KLF2-deficient nuclear extracts. From these assays ubiquitin ligase complex, SCFFBW7 (35). SCFFBW7 is a multi- we contend that KLF2 is necessary to open compacted chromatin subunit RING-finger ligase that targets for 26S pro- associated with the FoxP3 , thus permitting iTreg develop- teasomal destruction, and in this context KLF2 has recently been ment under conditions that favor KLF2 expression. identified as a substrate in endothelial cells (35). Despite the fact that several E3 ubiquitin ligases have a documented role in + Drugs That Stabilize KLF2 Expression Enhance iTreg Production. promoting KLF2 degradation (34–36), we found that CD4 + + + − KLF2 is necessary for the generation of iTregs, raising the CD25 FoxP3 frequencies were enhanced when CD4 CD25 T possibility that this transcription factor can be manipulated to cells were transfected with the mutant form of KLF2 before ex enhance i/pTreg development for clinical purposes. The 3-hy- vivo Treg induction (Fig. 5H). From this we conclude that KLF2 + droxy-3-methylglutaryl CoA reductase inhibitor simvastatin has degradation is a key facet of CD4 T-cell conversion that can previously been shown to enhance Klf2 expression in endo- be modified to enhance the generation of i/pTregs. Of note, thelium (31, 32) and activated T cells (33), which led us to test overexpression of KLF2 using pharmaceutical drugs during this US Food and Drug Administration-approved drug on T the inductive phase of iTreg production did not impair sup- cells undergoing iTreg induction. Consistent with our previous pressive activity or cell viability (Fig. S6 A and B). Likewise, experiments, simvastatin increased production of iTregs using elevated levels of KLF2 did not affect ex vivo Treg suppres- + − cultured CD4 CD25 T cells or thymocytes (Fig. 5A)in sion (Fig. S6C), which suggests that KLF2 and the proteins a manner that paralleled KLF2 expression (Fig. 5B). Further- that interface with this transcription factor are potential im- more, this direct correlation extended to other inhibitory munotherapeutic targets. compounds that suppressed TCR-mediated down-regulation of KLF2 (Fig. 5C and Fig. S4). Analysis of simvastatin-treated Discussion Tregs indicated that this drug enhanced/stabilized KLF2 at the Numerous reports have demonstrated that Foxo1 promotes ex- mRNA and protein level (Fig. S5), raising the possibility that pression of KLF2, which in turn drives expression of S1P1 within one of these factors was responsible for augmented iTreg the T-cell compartment. For this reason, it has been difficult to development. Overexpression of KLF2 using an established + reconcile the dichotomy in Treg development that occurs in mammalian vector did not amplify FoxP3 T-cell frequencies Foxo1 vs. S1P1 gene-targeted animals; in the former, tTreg and (Fig. 5D), which suggests that KLF2 stability rather than tran- pTreg populations are decreased, whereas in the latter, both scription was the primary means of regulating KLF2-mediated Treg lineages are increased. Because KLF2 bridges these two events after T-cell activation. To investigate the potential re- signaling components, we were surprised to discover that KLF2 lationship between KLF2 stability and regulatory T-cell de- + − is not required for the generation of tTregs and is only necessary velopment, iTreg production was analyzed using CD4 CD25 for pTreg (and ex vivo iTreg) development. tTregs arise from + + TcellsfromSma and Mad homologue (SMAD) specific E3 self-reactive CD4 CD8 thymocytes (37), and because these ubiquitin protein ligase 1 (SMURF1) gene-targeted animals. cells do not yet express KLF2 (KLF2 is initially expressed at the In keeping with a previous report that demonstrated that single-positive thymocyte stage), it is evident that other Foxo1/ + + SMURF1 targets KLF2 for ubiquitin-mediated degradation 3-transcribed factors are necessary to establish CD4 CD25 + (34), we found that ex vivo iTreg production was enhanced in FoxP3 thymocytes. Interestingly, S1P1 thymic expression pat- the absence of SMURF1 (Fig. 5E). Importantly, SMURF1 terns are similar to those of KLF2, which leads us to speculate ’ β deficiency did not uncouple iTreg srequirementforTGF- that S1P1 modifies FoxP3 expression after the initial generation (Fig. 5E) or affect the activation status of SMAD molecules of tTregs. If true, this would suggest that KLF2 and its tran- β F that act directly downstream of TGF- R1 (Fig. 5 ). Instead, scriptional target, S1P1, regulate pTreg levels by initiating FoxP3 SMURF1-deficient cells exhibited a defect in KLF2 stability transcription and limiting its continued expression, respectively. This temporal separation in FoxP3 regulation may explain the counterintuitive Treg displayed by Foxo1 and S1P1 gene-targeted animals. AB With regard to the molecular mechanisms that underpin iTreg 0.06 0.004 0.6 0.04

0.0035 0.50.05 and pTreg development, current results indicate that KLF2 is 0.030.003 0.40.04 absolutely necessary during the initial stages of Treg induction 0.0025

0.03 0.020.002 0.3 but is redundant once FoxP3 is stably expressed. We propose

0.0015 0.20.02 that KLF2 is incorporated into a signaling complex that provides 0.010.001 0.01

Fraction of Input (%) 0.1 Fraction of Input (%) 0.0005 transcriptional accessibility to the entire FoxP3 locus, after which

0 0 0 0 additional nuclear factors stabilize the open conformation in

Prom. CNS1 CNS2 CNS3 Prom. CNS1 CNS2 CNS3 a KLF2-independent manner. Consistent with a previous report

H4Ac immunoprecipitation (38), this larger signaling complex may include histone acetyl- Klf2 IP: αCD3 WT αCD3 cKO αCD3 transferases that reverse p300/CBP-associated factor-mediated Klf2 IP: αCD3 + TGF + WT αCD3 + TGF cKO αCD3 + TGF Control IP: αCD3 + TGF FoxP3 gene silencing in naïve CD4 T cells. Importantly, we demonstrate that KLF2 is a rate-limiting factor during this in- Fig. 4. KLF2 directly promotes FoxP3 transcription within the iTreg lineage. ductive process that can be targeted with drugs to enhance iTreg (A) ChIP assays using antibody directed against KLF2 after lysis of wild-type + − production via increased KLF2 stability. Perhaps because KLF2 CD4 CD25 T cells cultured under conditions that promote T-cell activation is a transcription factor, its regulation is typically described in (black) vs. iTreg differentiation (white). Isotype control antibody (gray) is included for iTreg ChIP assays. DNA primers are specific for the promoter or terms of mRNA levels and Foxo1-mediated transcription; how- enhancer elements (CNS1-3) of FoxP3. KLF2 binding to the regulatory ever, current results indicate that KLF2 protein levels are far regions of FoxP3 is graphed relative to input amounts of genomic DNA. more relevant when describing biological events in postactivated These experiments were performed three times. (B) Wild-type (WT) and lymphocytes. Case in point, concurrent with TCR stimulation + − KLF2-deficient (cKO) CD4 CD25 T cells were cultured under activating that promotes KLF2 degradation, KLF2 proteolysis is partially inhibited by TGF-β–mediated signaling events that favor iTreg (black, gray) vs. iTreg-inducing conditions (white, blue) before performing + ChIP assays using antibody directed against acetylated histone H4. This ex- production over other CD4 T-cell differentiation outcomes. periment was conducted twice. Moreover, this block in TCR-mediated KLF2 degradation is

9582 | www.pnas.org/cgi/doi/10.1073/pnas.1323493111 Pabbisetty et al. Downloaded by guest on September 30, 2021 A CD3+ CD28 -+++ B CD3+ CD28 ++++ TGF --++ TGF ++ ++ KLF2 simvastatin -- -+ simvastatin ( M) 00.51 2 tubulin

5 5 10 5 10 10 5 10

0.3 10.6 36.5 105 105 105 105 0.1 4 4 10 4 10 10 4 10 1.3 2.5 4.4 12.7 104 104 104 104 1.0 3 3 10 3 10 10 3 10 control

103 103 103 103 2 control 2 2 2 10 10 0.75 10 10 0 0 0 0 102 102 102 102

2 3 4 5 0 0 0 0 2 3 4 5 0102 10 3 10 4 10 5 010 10 10 10 0102 10 3 10 4 10 5 010 10 10 10 0.5 2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5 FoxP3 010 10 10 10 010 10 10 10 010 10 10 10 010 10 10 10 10 5 10 5 10 5 10 5

0.2 0.2 0.3 0.4 FoxP3 105 105 105 105 10 4 10 4 10 4 10 4 1.6 0.8 0.3 1.2 0.25 104 104 104 104 3 3 3 3 10 10 10 10 (relative to tubulin) cKO KLF2 protein levels 103 103 103 103 0 2 2 2 2 cKO 10 10 10 10

0 0 0 0 102 102 102 102 CD3+ CD28 ++++ 0 0 0 0 0102 10 3 10 4 10 5 0102 10 3 10 4 10 5 0102 10 3 10 4 10 5 0102 10 3 10 4 10 5

2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5 TGF ++++ 010 10 10 10 010 10 10 10 010 10 10 10 010 10 10 10 CD25 simvastatin ( M)0 0.5 1 2 P<0.0001 CD25 P=0.09 80 &!" KLF2 P<0.0001 60 %!" P=0.7 tubulin TGF -induced TGF -induced TGF -induced TGF -induced C 1.0 40 $!" Activated TGF -induced + LY + LY + PD + statin + rapamycin 0.75 20 #!" 0 4 55 Thymocytes 68 45 47 0.5 0 !" %CD4+CD25+FoxP3+ '()*'(#&" +,-." /012" 344" FoxP3 0.25 CD3+ CD28 ++++ -- ++ 0

TGF (relative to tubulin) KLF2 protein levels simvastatin - + - + CD25 CD3+ CD28 +- +++++ TGF --+++++ KLF2 supplement D tubulin LY statin LY+PD 18 <0.0001 rapamycin empty vector 0.5 g 1.0 g 2.0 g 4.0 g E 75 14 14 13 12 12 8 F 6 50 0.0005 FoxP3 4 control

T cells T SMURF1-/- (relative to tubulin) 2 KLF2 protein levels 25 0 CD25

%CD4+CD25+FoxP3+ 0.07 Transfected 0 0.5 1 24 TGF - ++ KLF2 vector ( g) 0 H Vector Empty Empty KLF2 0.5 g CD3+ CD28 ++ + control SMURF1-/- 105 105 105 TGF ng 0510 0.6 8.2 5.6 KLF2 4 4 4 G 10 10 10 tubulin 103 103 103

2 2 2 1.5 FoxP3 10 10 10 0 0 0 phospho-Smad2/3

2 3 4 5 2 3 4 5 2 3 4 5 1.0 010 10 10 10 010 10 10 10 010 10 10 10 WT SMURF1-/- CD25 0.5 WT+TGF SMURF1-/- TGF +++ + +TGF (relative to tubulin) IMMUNOLOGY KLF2 protein levels 0 Vector KLF2-dm 0.5 g KLF2-dm 1 g KLF2-dm 1.5 g KLF2-dm 2 g CD3+ CD28 --++++ 105 105 105 105 TGF --++ -- 12.2 13.5 17.2 27.2 104 104 104 104

103 103 103 103

2 2 2 2 FoxP3 10 10 10 10 0 0 0 0

0102 103 104 105 0102 103 104 105 0102 103 104 105 0102 103 104 105 CD25

Fig. 5. Increased KLF2 expression correlates with enhanced generation of iTregs. (A) Ex vivo generation of iTregs using CD4+CD25− T cells from Lck-cre (control) vs. Lck-cre; Klf2fl/fl (cKO) littermates in the presence of simvastatin. (Upper) Contour plots of iTregs (FoxP3+CD25+) after induction of splenic T cells. (Lower) Same performed in triplicate using CD4+CD8−CD25− thymocytes from control (gray) vs. cKO (black) mice. Statistical differences between control groups (gray numbers) and KLF2-deficient populations (black numbers) are shown. Both sets of experiments were repeated three times. (B and C) A com- + − parison between iTreg induction and KLF2 protein levels. (B)(Left) FACS analysis of KLF2-replete (control) vs. KLF2-deficient (cKO) CD4 CD25 T cells cultured under suboptimal iTreg conditions with or without increasing concentrations of simvastatin. (Right) Immunoblots of KLF2/tubulin and corresponding den- + − sitometry plots using total cell lysate from CD4 CD25 T cells cultured (12 h) as specified. Both sets of experiments were repeated three times. (C)(Left) Contour plots of iTregs using wild-type CD4+CD25− T cells cultured with the indicated compounds (LY, LY294002; PD, PD98059; statin, simvastatin). Activated, αCD3 + αCD28; TGFβ-induced, αCD3 + αCD28 + TGFβ.(Right) Western blots of KLF2/tubulin and corresponding densitometry data from CD4+CD25− T cells + − cultured for 12 h under the specified conditions. These experiments were repeated four times. (D) iTreg production after transfection of wild-type CD4 CD25 T cells with KLF2 vector. Left panel: densitometry plot of KLF2 protein levels (relative to tubulin) 24 h after transfection with pcDNA-KLF2. (Right) Contour plots of T cells after KLF2 transfection (24 h, vector concentrations indicated) and 48 h iTreg induction. This experiment was repeated twice. (E) Quantification + − of ex vivo iTreg production using splenic CD4 CD25 T cells harvested from wild-type (gray bars) vs. SMURF1-deficient (black bars) animals. Error bars (SD) and P values are shown. n = 2 experiments, performed in triplicate. (F) Histogram overlays displaying Smad2/3 phosphorylation after TGF-β stimulation of CD4+ CD25− T cells harvested from wild-type vs. SMURF1-deficient mice. This experiment was performed once, in triplicate. (G) KLF2/tubulin immunoblot and + corresponding densitometry plot of 12-h cultured CD4 T cells harvested from wild-type vs. SMURF1-deficient mice. This experiment was repeated twice. (H) + Contour plots of CD4 T cells after transfection [24 h with pcDNA3 (empty), pcDNA3-KLF2 (KLF2), or pcDNA3-KLF2-dm (KLF2-dm)] and 48 h iTreg induction. Percentages of iTregs are included. This experiment was repeated twice.

a bottleneck in iTreg production that can be amplified using Materials and Methods a diverse array of chemical inhibitors. Future work will de- Mice. Klf2fl/fl mice were generated as previously described (40). SMURF1 termine whether drugs that stabilize KLF2 protein levels expand gene-targeted mice (41) were obtained from the laboratory of Ying E. pTreg populations in vivo, especially during inflammatory out- Zhang (Center for Cancer Research, National Cancer Institute, Bethesda, breaks encountered in autoimmune diseases. In this regard, it is MD). Lck-cre mice were purchased from Taconic, and all other mice worth noting that statins, which are currently prescribed to ap- were purchased from Jackson Laboratories. All KLF2 gene-targeted proximately one-quarter of all Americans over the age of 45 y animals were back-crossed a minimum of 10 generations onto a C57BL/6 (www.cdc.gov/nchs/hus.htm), have a documented role in allevi- background. Mice were housed in pathogen-free conditions in accor- ating autoimmune symptoms that are independent of their lipid- dance with the Institutional Animal Care and Use Committee at Van- reducing properties (39). At the same time, there are numerous derbilt University. situations in which Tregs hinder desired immune responses, such Antibodies. Anti-Thy1.2 (53-2.1), -CD3e (145-2C11), -CD4 (GK1.5), -CD8α (53- as those directed against tumors; under these circumstances 6.7), -CD25 (PC61.5), -CD28 (37.51), -CD44 (IM7), -CD69 (H1.2F3), -CD71 pharmaceuticals that stabilize KLF2 protein levels would be (C2), -IFN-γ (XMG1.2), -IL4 (11b11), -IL17 (TC11-18H10), -GATA3 (L50-823), predicted to harm patient outcome. Instead, it may prove ad- and –phospho Smad2/3 (072-670) were purchased from BD Biosciences. vantageous to treat the T-cell compartment with drugs that Anti-FoxP3 (FJK-16s) and -CD152 (UC10) were obtained from eBioscience promote KLF2 degradation via E3 ubiquitin ligases such as USA. Anti-Helios (22F6) was purchased from Biolegend. Anti-Neuropilin WWP1 (36), SMURF1 (34), or FBW7 (35). was purchased from R&D Systems. Anti-KLF2 and -Acetyl–Histone H4

Pabbisetty et al. PNAS | July 1, 2014 | vol. 111 | no. 26 | 9583 Downloaded by guest on September 30, 2021 (H4Ac) were purchased from Millipore. Anti-α-tubulin was obtained from an additional 72 h. Klf2 excision was assessed by RT-PCR, and cells were Cell Signaling Technologies. analyzed by flow cytometry.

+ − Flow Cytometry and Cell Sorting. Intracellular and surface staining was per- ChIP Assays. FACS-sorted naïve CD4 CD25 T cells were activated and cul- formed using standard procedures. For cell quantification, CaliBRITE Beads tured for 4 d. ChIP assays were performed using the EZ-ChIP kit (Millipore) + (BD Biosciences) were added to individual tubes before acquisition. CD4 according to the manufacturer’s protocol. Immunoprecipitations were per- + CD25 T cells were isolated using a Treg isolation kit (Miltenyi Biotec) formed using anti-Klf2 antibody, anti-H4Ac or rabbit IgG control antibody. according to the manufacturer’s instructions. The following primer sets were used to amplify the DNA from KLF2 pull- downs: Foxp3 Promoter F 5′-CTC TGT GGT GAG GGG AAG AA-3′; Foxp3- + − Ex Vivo Induction of Tregs. FACS- or MACS-sorted naïve CD4 CD25 cells (2 × Promoter R 5′CGC AGA CCT CGC TCT TCT AA-3′; Foxp3 CNS1 F 5′- ACG TAT 106 cell/mL) were preincubated with or without LY294002 (10 μM), PD98059 CTC TCT AGT GGG TCT GGA-3′; Foxp3 CNS1 R 5′- TGA GGA ACA GTG CAG (10 μM), simvastatin (2 μM), or rapamycin (100 nM), activated with plate- GAC AG-3′; Foxp3 CNS2 F 5′-CTA GCC ACT TCT CGG AAC GA-3′; Foxp3 CNS2 R bound anti-CD3e (2 μg/mL) and soluble -CD28 (2 μg/mL) antibody, then cul- 5′-CAG CTT CCT GCA CTG TCT GTT-3′; Foxp3 CNS3 F 5′- GCC CAC ACC TCT TCT tured with TGF-β (1–10 ng/mL) in complete medium containing IL-2 (5 ng/mL). TCC TT-3′; Foxp3 CNS3 R 5′- GGG ACC CAT AAA CCA CTT CC-3′. After 2–4 d of culture cells were collected and stained for FoxP3. Nucleofection of Quiescent CD4+CD25− T Cells. Transient nucleofection was In Vivo Induction of Tregs. Klf2fl/fl, OT2; Klf2fl/fl, and OT2; Lck-cre; Klf2fl/fl mice performed using the Lonza/Amaxa Mouse T-cell Nucleofector Kit (VPA-1006) were placed on chicken ovalbumin (p323-339)-infused drinking water according to the manufacturer’s instructions. Nucleofection of purified + − (20 mg/mL) for 7 d, after which animals were killed and analyzed for pTreg splenic CD4 CD25 T cells (1 × 106) from C57BL/6J mice was performed with development. Antibodies directed against Vα2 and Vβ5 (BD Biosciences) the indicated amounts of pcDNA3 (empty vector), pcDNA3-KLF2 (40), or were used to identify OVA-specific T cells. pcDNA3-dmKLF2 plasmid (35). Cells were subsequently cultured under iTreg- inducing conditions before FACS analysis. Ex Vivo Excision of KLF2. A tamoxifen-inducible cre transgenic mouse model (T2-cre) (42) was used to excise KLF2 after ex vivo generation of Tregs. After Statistical Analysis. Data were analyzed using a two-tailed Student t test. 4 d of iTreg culture, cells were treated with 100 nM 4-hydroxy-tamoxifen for P values ≤ 0.05 were considered statistically significant.

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