Phosphatase PP2A is essential for TH17 differentiationQin Xua,b,1, Xuexiao Jina,b,c,1, Mingzhu Zhenga,b, Deepak Rohilaa,b, Guotong Fua,b, Zhuoyu Wena,b, Jun Loua,b,Songquan Wud, Richard Sloanc,e, Lie Wanga, Hu Huf, Xiang Gaog,2, and Linrong Lua,b,c,h,i,2

aInstitute of Immunology, and Department of Dermatology and Rheumatology in Sir Run Run Shaw Hospital, Zhejiang University School of Medicine,Hangzhou 310058, P. R. China; bProgram in Molecular and Cellular Biology, Zhejiang University School of Medicine, 310058 Hangzhou, P. R. China;cZhejiang University–University of Edinburgh Institute, Zhejiang University School of Medicine, 310058 Hangzhou, P. R. China; dSchool of Medicine, LishuiUniversity, Lishui 323000, Zhejiang Province, P. R. China; eDivision of Infection and Pathway Medicine, The University of Edinburgh, EH16 4SB Edinburgh,Scotland, United Kingdom; fDepartment of Pathology and Pathophysiology, Zhejiang University School of Medicine, 310058 Hangzhou, P. R. China; gKeyLaboratory of Model Animals for Disease Study of the Ministry of Education, Model Animal Research Center, Nanjing University, 210061 Nanjing, P. R.China; hInnovation Center for Cell Signaling Technology Network, Zhejiang University School of Medicine, 310058 Hangzhou, P. R. China; and iDr. Li DakSum & Yip Yio Chin Center for Stem Cell and Regenerative Medicine, Zhejiang University School of Medicine, 310058 Hangzhou, P. R. China

Edited by Masato Kubo, Tokyo University of Science, Research Institute for Biological Science, Noda, Japan, and accepted by Editorial Board MemberTadatsugu Taniguchi November 26, 2018 (received for review May 6, 2018)

Phosphatase PP2A expression levels are positively correlated tothe clinical severity of systemic lupus erythematosus (SLE) andIL17A cytokine overproduction, indicating a potential role of PP2Ain controlling TH17 differentiation and inflammation. By generat-ing a mouse strain with ablation of the catalytic subunit α of PP2Ain peripheral mature T cells (PP2A cKO), we demonstrate that thePP2A complex is essential for TH17 differentiation. These PP2A cKOmice had reduced TH17 cell numbers and less severe disease in anexperimental autoimmune encephalomyelitis (EAE) model. PP2A de-ficiency also ablated C-terminal phosphorylation of SMAD2 but in-creased C-terminal phosphorylation of SMAD3. By regulating theactivity of RORγt via binding, the changes in the phosphorylationstatus of these R-SMADs reduced Il17a gene transcription. Finally,PP2A inhibitors showed similar effects on TH17 cells as were ob-served in PP2A cKO mice, i.e., decreased TH17 differentiation andrelative protection of mice from EAE. Taken together, these datademonstrate that phosphatase PP2A is essential for TH17 differenti-ation and that inhibition of PP2A could be a possible therapeuticapproach to controlling TH17-driven autoimmune diseases.

TH17 | TGFβ | PP2A | SMAD2/SMAD3 | EAE

T helper type 17 (TH17) cells, a subset of CD4+ T cells definedby IL17, IL22, and IL21 production, are essential for control

and clearance of extracellular bacterial and fungi (1, 2). How-ever, excessive TH17 responses are involved in chronic inflammationand development of many human autoimmune diseases (3). Uponencountering antigen in the context of a local cytokine milieu in-cluding transforming growth factor β (TGFβ) and IL6, naïve CD4+

T cells undergo differentiation into effective TH17 cells. TGFβ is theprincipal, essential factor promoting the differentiation of TH17cells (4, 5).Through two related transmembrane Ser/Thr kinase receptors,

TGFβ induces Ser/Thr signal cascades in activated T cells. Re-cent work including work from our laboratory has revealed theregulatory roles of some other Ser/Thr kinases in this process.For example, both MEKK2/3 and MINK1 suppress TH17 differ-entiation through direct phosphorylation of the TGFβ signalingcomponents SMAD2 and SMAD3 (6, 7). Precise regulation ofSMAD2/3 Ser/Thr phosphorylation status is thus important indriving TH17 differentiation (6–8). Dephosphorylation of SMAD2/3 is equally critical in this process, but the specific phosphatases thatcatalyze SMAD2/3 dephosphorylation remains unknown.As one of the major Ser/Thr phosphatases in eukaryotes,

phosphatase PP2A is critical for many cellular functions in-cluding cell survival, proliferation, activation, and differentiation(9). It has been reported that elevated PP2A expression levelsare linked to the up-regulation of IL17A production by CD4+

T cells in human systemic lupus erythematosus patients (10).Studies in the PP2Ac transgenic mouse model also demonstratedthe relationship and mechanism linking of PP2A and Il17-dependentimmunopathology (11, 12). PP2A is composed of three polypeptide

chains, the structural A, the regulatory B, and the catalytic C subunits(13). The heterodimer of the A subunit and the C subunit (PP2AA-PP2AC) forms the PP2A core enzyme that associates with one reg-ulatory B subunit, thus determining the substrate specificity of theholoenzyme complex (13).In TGFβ signaling, two related regulatory B subunits, Bα (Ppp2r2a)

and Bδ (Ppp2r2d), opposingly modulate TGFβ/Activin/Nodal sig-naling (14), while carboxy terminal phosphorylation of MAD (theSMAD homolog protein in Drosophila) is negatively regulated bythe PP2A inhibitor okadaic acid (OA) (15). By analogy, these ob-servations suggest that PP2A might be a Ser/Thr phosphorylationmodulator involved in controlling TH17 differentiation.Here, we present data showing that TH17 cell polarization was

largely impaired when Ppp2ca was ablated in mature T cells andrendered resistance toward MOG-induced experimental auto-immune encephalomyelitis (EAE). We also show that PP2Aknockout led to altered activation of R-SMADs (specifically de-creasing SMAD2 activation and increasing SMAD3 activation).This synergistically inhibited RORγt mediated Il17a transcription.This work thus reveals a specific role of PP2A in regulating the

Significance

By using a gene knockout that leads to T cell-specific deletion,we reveal the essential role of Ser/Thr phosphatase PP2A inTH17 differentiation. We also show that this works through theregulation of SMAD2/3 phosphorylation status, which elucidatesmolecular pathways by which PP2A modulates the expression ofTH17 phenotypes. This finding extends our understanding of theclose relationship between PP2A overexpression and inflammatorydisease. PP2A is a Ser/Thr phosphatase shown to be capable ofcontrolling TH17 differentiation via modulating R-SMADs activity.We also demonstrate the translational potential of these findingsby showing a therapeutic effect of PP2A inhibitors in controllingautoimmune disease in the encephalomyelitis model.

Author contributions: Q.X., M.Z., S.W., H.H., X.G., and L.L. designed research; Q.X., X.J.,M.Z., D.R., G.F., Z.W., and J.L. performed research; Q.X., X.J., and L.L. analyzed data; andQ.X., X.J., R.S., L.W., H.H., X.G., and L.L. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. M.K. is a guest editor invited by theEditorial Board.

Published under the PNAS license.

Data deposition: The data reported in this paper have been deposited in the Gene Ex-pression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession no.GSE119836).1Q.X. and X.J. contributed equally to this work.2To whom correspondence may be addressed. Email: gaoxiang@nju.edu.cn orlu_linrong@zju.edu.cn.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1807484116/-/DCSupplemental.

Published online December 28, 2018.

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canonical TGFβ−R-SMADs–RORγt signaling process duringTH17 differentiation and indicates a possible therapeutic ap-proach for controlling TH17-driven autoimmune diseases viainhibition of PP2A.

ResultsNormal T Cell Development in PP2A cKO Mice. To explore thefunction of PP2A in peripheral T cells, we deleted the dominantPP2A Cα isoform of PP2A catalytic subunit (PP2Ac) in T cell bycrossing disLck (dLck) Cre with Ppp2cafl/fl mice (in which exons3–5 of Ppp2ca are loxP flanked) (16) to generate Ppp2cafl/fl dLck-Cre (termed PP2A cKO mice) and Ppp2cafl/+ dLck-Cre orPpp2ca+/+ dLck-Cre mice (collectively called PP2A WT micehere) (SI Appendix, Fig. S1 A and B). The dLck-Cre was drivenby the distal promoter of the lymphocyte protein tyrosine kinase(Lck) gene, enabling investigation of the Ppp2ca deletion afterpositive selection in T cells (17).To assess deletion efficiency, Ppp2ca mRNA and protein

levels were measured and showed clear reduction in peripheralT cells in PP2A cKO mice, while remaining normal in thymicsubsets and splenic B cells (SI Appendix, Fig. S1 C and D). Thecatalytic subunit of PP2A has two isoforms, Cα and Cβ (encodedby Ppp2ca and Ppp2cb, respectively). Notably we didn’t observecompensatory overexpression of Ppp2cb (SI Appendix, Fig. S1C).PP2A activity in cKO CD4+ T cells was reduced to half of thatmeasured in WT controls (SI Appendix, Fig. S1E).Analysis of the numbers and frequencies of different T cell

subsets in these mice showed that cKO mice exhibited normalT cell development in thymus (SI Appendix, Fig. S2 A–C) as wellas in peripheral lymphoid organs (SI Appendix, Fig. S2 A, D, andE). The proportions of naïve/effective T cells in spleen andmesenteric lymph nodes (MLN) were also similar between WTand cKO littermates (SI Appendix, Fig. S2 F and G). The normaldevelopment of peripheral lymphocytes in PP2A cKO mice allowedfurther investigation of the role of PP2A in T cell differentiation.

TH17 Cell Numbers Are Reduced in PP2A cKO Mice. To clarify whetherPP2A is involved in T helper cell lineage commitment, we ana-lyzed the populations of T helper subsets in vivo. Interestingly,CD4+ T cells from PP2A cKO mice only contained half thenumber of TH17 cells comparing to their WT littermates (SIAppendix, Fig. S3 A and B), while the numbers of TH1 and TregCD4+ T cells were not affected in the peripheral lymphoid or-gans (SI Appendix, Fig. S3 A, C, D, and F). The frequency ofFoxp3+ regulatory T cells in the thymus was also comparablebetween PP2A WT and cKO mice (SI Appendix, Fig. S3 E andF). Similarly, subsets analysis of the lamina propria of small in-testine also showed a consistent reduction of TH17 cells (SIAppendix, Fig. S3 G and I). These data demonstrate that PP2A isinvolved in maintaining TH17 cell composition, while other T cellsubsets, including Treg and TH1, appear unaffected.

PP2A Deletion Impairs TH17 Differentiation in Vitro. To investigatewhether reduced levels of TH17 cells in PP2A cKO mice resultfrom impaired TH17 differentiation, we sorted naïve CD4+

T cells from both WT and cKO cells and polarized them underTH1, TH2, TH17, and iTreg conditions to compare their differ-entiation efficiencies. The results showed that only the generationof TH17 cells notably declined with the PP2A deficiency, whileother T helper subsets were not affected (Fig. 1 A and B and SIAppendix, Fig. S4 A and B). The expression levels of Ppp2camRNA and PP2A Cα protein were more abundant in TH17 cellsthan in other T helper subsets (SI Appendix, Fig. S4 G and H).p-PP2Ac (Y307) levels, a negative indicator for PP2A activity,

are lower in TH17 and TH1 than in the other subsets, indicatinghigher PP2A activity in these two subsets (SI Appendix, Fig. S4I).Cytokine production showed similar results, confirming thatPP2A deficiency specifically reduced TH17 differentiation (Fig. 1C

and SI Appendix, Fig. S4C). We further tested the expression ofseveral key TH17 signature genes and found notably decreasedIl17a and Il17f expression and slightly reduced expression of Roraand Il21, while the expression of Rorc, Il22, and Il23r were notsignificantly affected (Fig. 1D). The alteration of the expres-sion pattern of TH17 signature genes (including Il17a and Il17f)induced by PP2A deficiency was also confirmed by an RNAseqanalysis (SI Appendix, Fig. S5 A and B and Datasets S1–S3).The proliferation capacity of WT and cKO T cells was mea-

sured under TH17 conditions and was comparable 2 and 5 d afterstimulation (SI Appendix, Fig. S4D). Upon PI and Annexin Vstaining, WT and cKO cells showed comparable apoptotic rates(SI Appendix, Fig. S4E). Thus, reduced IL17A+ CD4+ T cell levelsand IL17A production were not due to either impaired pro-liferation or increased apoptosis in cKO cells. Furthermore, wedid not observe an increased portion of IFNγ+ or Foxp3+ CD4+

T cells in cKO T cells under TH17 conditions, which ruled out thelikelihood of TH17 cells converting to other cell subsets in thesecircumstances (Fig. 1A and SI Appendix, Fig. S4F). These findingstherefore demonstrate a T cell intrinsic impairment of the TH17polarization program upon PP2A deficiency, which is independentof proliferation, apoptosis, or subset conversion.

Reduced Severity of EAE in Mice with PP2A Deficiency. Given therequired role of PP2A in inducing normal TH17 polarization, thequestion of whether defective TH17 PP2A cKO cells also influ-ence TH17-driven autoimmune disease was investigated in vivousing an EAE model. We therefore immunized PP2A WT andcKO mice with myelin oligodendrocyte glycoprotein peptide ofamino acids 35–55 (MOG35–55) to induce EAE.Clinical scoring showed that PP2A deficiency alleviated

symptoms of autoimmunity (Fig. 2A). Histological examinationshowed significantly less mononuclear cell infiltration and de-myelination in the cerebral and spinal cord of the cKO mice (Fig.2B). Inflammatory cell infiltration into the central nervous sys-tem (CNS) was also greatly diminished in the cKO group (Fig.2C). Although the proportion of CD4+ T cells was unchanged inthe CNS of cKO mice, the total numbers of infiltrating CD4+

and CD8+ cells were significantly reduced (Fig. 2C). IL17A-producing CD4+ T cells were also present in significantly de-creased numbers in both the CNS and draining lymph nodes(DLN), whereas the frequencies of IFN-γ–producing CD4+

T cells were unchanged. (Fig. 2 D–F). These data indicate thatreduced IL17A production upon PP2A cKO results in less severeMOG-induced CNS inflammation. Indeed, in vitro MOG recallanalysis also showed reduced IL17A production by PP2A cKOsplenocytes, while IFN-γ production was unaffected (Fig. 2G).

Fig. 1. PP2A deficiency specifically limits TH17 differentiation in vitro. (Aand B) Flow cytometry (A) and quantification (B) of IL17A staining in naïveCD4+ T cells from PP2A WT and cKO mice differentiated under TH17 polar-izing condition for 5 d. (C) ELISA of IL17A in the culture medium of eachpolarizing condition (n = 3 technical replicates). (D) RT-PCR analysis of TH17signature genes (n = 3 technical replicates). Each symbol represents an in-dividual mouse (n = 8); error bars showmean ± SEM. Data are representativeof at least three independent experiments with similar results. *P < 0.05;**P < 0.01; ***P < 0.001; NS, not significant.

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Intriguingly, Foxp3+ CD4+ T cell proportions were also reduced inthe CNS but not in the DLN (Fig. 2 D–F). This might be explainedby markedly milder inflammation in the CNS, which consequentlyrecruited less regulatory cells. PP2A cKO mice are thus resistantto EAE, and this is strongly correlated with an observed significantreduction in TH17 cell induction.

Intact TCR and IL6 Signaling in PP2A cKO CD4+ T Cells. We next in-vestigated how PP2A deficiency affected intracellular signalingin controlling TH17 cell differentiation. Intact TCR signaling isindispensable for TH17 commitment (18). However, our resultsshowed that PP2A deficiency did not affect normal CD69 up-regulation (SI Appendix, Fig. S6A), cell proliferation (SI Ap-pendix, Fig. S6B), or production of IL2 or IFN-γ (SI Appendix,Fig. S6C) following anti-CD3 stimulation. Interestingly, Westernblot analysis showed up-regulation of pERK and pP38 in PP2A-deficient cells (SI Appendix, Fig. S6D), and these two pathwaysare known to play opposing roles in TH17 differentiation. In-hibition of MEK-ERK signaling enhances TH17 differentiationwhile activation of P38 is critical for optimal TH17 polarization(19–21). We found that use of an MEK inhibitor (inhibiting ERK,U0126) failed to restore TH17 differentiation due to PP2A de-ficiency (SI Appendix, Fig. S6E). Thus, subtle changes in MAPKsactivation are not correlated with decreased TH17 differentiationdue to PP2A deficiency. PP2A overexpression has been previouslyreported to up-regulate Il17a gene transcription by enhancingIRF4 activity (11). However, the observed mRNA, protein, andIRF4 activity did not support involvement of IRF4 in leading todiminished Il17a transcription (SI Appendix, Fig. S5 C–E).

By using the PP2A inhibitor OA, previous work has suggesteda possible regulatory role of PP2A in regulating IL6 signaling,including promoting the stability of IL6 receptor gp130 (CD130)(22) and modulating STAT3 phosphorylation (23). However, ourexperiments did not show any alterations in either IL6 receptorexpression (SI Appendix, Fig. S7 A and B) or STAT3 phos-phorylation (Y705 and S727) after IL6 stimulation in cKOT cells (SI Appendix, Fig. S7C), thus excluding the possibility thatthe inhibition of TH17 differentiation upon PP2A deletion is dueto TCR or IL6 signaling changes.

Differential Modulation of SMAD2/3 Activity by PP2A DeficiencyRestrains TH17 Differentiation. In TGFβ pathway, TGFβ RI andRII, which are reported to be opposingly regulated by two dif-ferent PP2A B subunits (14), were similarly expressed in PP2AcKO T cells (Fig. 3 A and B). Meanwhile, we found that pSMAD2(Ser465/467) level was decreased but pSMAD3 (Ser423/425) levelwas increased after TGFβ stimulation in PP2A cKO cells (Fig. 3C).In accordance, SMAD2 hyperphosphorylation and SMAD3 under-phosphorylation were observed in PP2A Cα overexpressed 293FTcells after TGFβ stimulation (SI Appendix, Fig. S8 A and B). Wenext performed the Co-IP assay in Jurkat cells to explore thebinding of R-SMADs to PP2Ac. We found that PP2Ac can formstable complex with SMAD2 and SMAD3, which does not de-pend on either TCR or TGFβ stimulation (SI Appendix, Fig. S8 Cand D).In addition, in vitro dephosphorylation assay showed that

PP2A can directly dephosphorylate pSMAD3 (SI Appendix, Fig.S8 E–G). Studies on SMAD2 and SMAD3 conditional knock-out mice have revealed the opposite functions of these two

Fig. 2. Loss of PP2A protects mice from EAE by repressing IL17A production.(A) Mean clinical scores for EAE from each group. (B) Representative his-tology of the brain and spinal cord [hematoxylin and eosin (H&E) at Left andluxol fast blue (LFB) at Right] of mice after EAE induction (day 19). Arrow-heads indicate inflammatory infiltration (Left) and demyelination (Right).(Scale bars: 100 μm.) (C) Number and frequency of mononuclear cells orCD4+ or CD8+ T cells infiltrated into CNS. (D) Flow cytometry of IL17A, IFN-γ,and Foxp3 staining from CNS (Left) or DLN (Right) CD4+ T cells. (E and F)Quantification of IL17A+, IFNγ+, and Foxp3+ CD4+ T cells in CNS (E) or DLN(F). (G) Splenocytes were rechallenged with MOG peptide (5 μg/mL) orcontrol vehicle for 3 d, and cytokine production was measured by ELISA.Each symbol represents an individual mouse (n = 4–6); error bars showmean ± SEM. Data are representative of three independent experimentswith similar results. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant.

Fig. 3. Insufficient SMAD2 activation and overactivated SMAD3 under TGFβpathway restrains TH17 differentiation. (A and B) Histograms (A) and meanfluorescence intensity (MFI) quantification (B) of TGFβ receptor I and IIstaining on CD4-gated cells from splenocytes of PP2A WT and cKO mice. (C)Enriched CD4+ T cells from PP2AWT and cKO were stimulated with 10 ng/mLTGFβ as indicated, whole-cell lysates were probed with the indicated anti-bodies in the immunoblots. (D and E) Flow cytometry (D) and quantification(E) of TH17 polarization with ectopic expression of Vector, WT, 2SA, and 2SDof SMAD2 in WT naïve CD4+ T cells. GFP-expressing cells were gated foranalysis on day 3. (F and G) Flow cytometry (F) and quantification (G) of TH17polarization with ectopic expression of Vector, WT, 2SA, and 2SD of SMAD3in WT naïve CD4+ T cells. GFP expressing cells were gated for analysis on day3. Each symbol represents an individual mouse (n = 4). Error bars showmean ± SEM. Data are representative of three independent experiments (C)or two independent experiments with two replicates (D and F). *P < 0.05;**P < 0.01; NS, not significant.

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molecules in inducing TH17 cells (24–27). We thus suspectedthat the altered activation of SMAD2/3 in PP2A cKO cells mightserve as the major contributor toward decreased Il17a tran-scription. Indeed, when we expressed different activation formsof SMAD2/3 (WT, dominant negative form 2SA, constitutiveactive mutants 2SD) in naïve CD4+ T cells and analyzed celldifferentiation in TH17 condition, the results clearly showed thatinsufficient activation of SMAD2 caused defective TH17 differentia-tion, while constitutively activated SMAD2 promoted optimal TH17priming (Fig. 3 D and E). On the contrary, ectopic expression ofSMAD3 dramatically repressed TH17 polarization. Interestingly, aSMAD3-2SA mutant also showed inhibitory activity to TH17 polari-zation although to a lesser degree (Fig. 3 F and G), indicating thatsuppression of TH17 differentiation by SMAD3 depends on bothSMAD3 activation and on its overall expression level. To rule outalterations in iTreg cell skewing condition in the previous experi-ments, we also used a series of TGFβ doses for suboptimal iTregpriming but observed no difference between the PP2A cKO and WTgroups (SI Appendix, Fig. S7D).

Changes in SMAD2/3 Activity Synergistically Down-Regulate Il17aTranscription via Reducing RORγt Activity. We next asked how theshift of SMAD2/3 phosphorylation status affected Il17a tran-scription. We found that the protein expression levels of RORγtand RORα were not different in PP2A WT and cKO TH17 cells(Fig. 4 A and B). More importantly, retrovirus-mediated ectopicexpression of RORγt could not completely restore TH17 po-tentiation in cKO T cells (Fig. 4 C and D). These data supportedthe hypothesis that PP2A-controlled TH17 differentiation is in-dependent of RORγt protein expression. To address whetheroveractivation of SMAD3 and insufficient activation of SMAD2could work cooperatively to suppress RORγt activity, ChIPanalysis of RORγt occupancy of Il17a gene region was carriedout. The result confirmed our hypothesis that with equal RORγtexpression, its activity was largely reduced due to PP2A de-ficiency (Fig. 4E). Phosphorylation changes of SMAD2/3 af-fected their binding ability with RORγt. In cKO TH17 cells,RORγt binded more SMAD3 and less SMAD2 than observed inWT TH17 cells (Fig. 4F).Different activation forms of SMAD2/3 were then cotrans-

fected with RORγt in the 293FT cell line. These experimentsshowed that the constitutively active SMAD2/3 preferentiallyinteracts with RORγt over the inactive forms, and that SMAD3is more accessible to bind RORγt than SMAD2 (Fig. 4G). These

results suggest that phosphorylation changes in SMAD2/3 mayinhibit RORγt activity by affecting the capacity of SMAD2/3 toform complexes with RORγt. Based on this hypothesis, we per-formed rescue experiments with SMAD2-2SD transfection orSMAD3 knockdown. Both approaches significantly improvedTH17 polarization in cKO naïve CD4+ T cells (Fig. 4 H–K).SMAD3 knockdown efficiency by siRNA was verified by West-ern blot and RT-PCR (SI Appendix, Fig. S9 A and B).

PP2A Inhibitors Phenocopy Phosphorylation Changes of SMAD2/3 andRestrain TH17 Polarization in Vitro. We observed that administra-tion of PP2A inhibitors in 293FT cells phenocopied the SMAD2/3activation changes in a dose-dependent manner (Fig. 5A). Further,the PP2A inhibitor Cantharidin (CAN) restrained RORγt-mediatedIl17a promoter activation (Fig. 5B). More importantly, when ad-ministrating PP2A inhibitor CAN in TH17 culture medium, wealso observed a dose-dependent inhibitory role at a concentrationthat had no effect on cell proliferation (Fig. 5 C–E). Another twoPP2A inhibitors OA and Fostriecin (FOS) also showed similareffects upon TH17 differentiation (SI Appendix, Fig. S10 A–E).In addition, PP2A cKO T cells did not display suppression effectsby PP2A inhibitors, ruling out the off-target effects might result inTH17 depression (Fig. 5F and SI Appendix, Fig. S10F).

A PP2A Inhibitor Protects Mice from EAE. We examined whetherCAN can block TH17 cell-mediated inflammation in EAE. Asignificant reduction of disease severity was observed in CAN-treated PP2A WT mice compared with PBS-treated WT mice(Fig. 6A). Histological examination also showed significantly lessmononuclear cell infiltration and demyelination in the spinalcord of the CAN-treated WT mice (Fig. 6B). Meanwhile, therewas no observable aggravation of clinical symptoms by applyingCAN in PP2A cKO mice, suggesting that the CAN dose waswithin a safe range (Fig. 6A). Fewer mononuclear lymphocytesand CD4+ T cells infiltrated into the CNS of CAN-treated WTmice than of the PBS WT group at the onset or peak of EAE,while the CD8+ and myeloid population was not significantlychanged (Fig. 6 C and D). Additionally, the ratio of TH17 cellswas lower in CAN-treated WT mice than in PBS-treated WTmice (P = 0.08) (Fig. 6E). The number of infiltrated TH17 cellswas also significantly lower in the CNS. Treg cells and IFN-γ–producing cells were slightly decreased (Fig. 6F). Further-more, CAN treatment in PP2A cKO groups did not alter thenumber of inflammatory lymphocytes infiltrated into CNS (Fig. 6G and H). CAN-treated cKO groups showed no effects to EAE

Fig. 4. Differential modulation of SMAD2/3 inhibits RORγt-mediated Il17a transcription by forming complex with RORγt. (A) Flow cytometry of RORγtstaining from naïve CD4+ T cells primed under TH17 polarizing condition for 2 d. (B) RORα was immunoblotted with whole-cell lysate of TH17 cells. (C and D)Flow cytometry (C) and quantification (D) of TH17 polarization with ectopic expression of Vector or RORγt in PP2A WT and cKO naïve CD4+ T cells. GFP-expressing cells were gated for analysis on day 3. (E) RORγt binding to the sites of the Il17a gene promoter was analyzed by using chromatin immuno-precipitation (ChIP) assay with RT-PCR. (F) Co-IP analysis of binding ability with RORγt among SMAD2/3 in WT and cKO TH17 cells. (G) Co-IP analysis of bindingability with RORγt among WT, 2SA, and 2SD mutant of SMAD2/3 in 293FT cells. (H) Flow cytometry of TH17 polarization with ectopic expression of Vector orSMAD2-2SD in PP2A WT and cKO naïve CD4+ T cells. (I) Ratio of IL17A frequency comparing VEC-cKO or SMAD2-2SD-cKO to VEC-WT. (J) Flow cytometry ofTH17 polarization after suppressing SMAD3 expression by siRNA in naïve CD4+ T cells. (K) The ratio of IL17A frequency in cKO cells transfected with siRNA-SMAD3 or control to the frequency of WT cells transfected with control. Error bars show mean ± SEM. Data are representative of three (A, B, G, H, and J) ortwo (C–F) independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

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symptoms, suggesting the specific targeting of CAN to PP2A inT cells in this experimental setting. However, whether CAN canalso act on other cells and contribute to the therapeutic effects isunclear. Collectively, data above showed that PP2A inhibitorCAN can limit EAE development principally by reducing TH17differentiation.

DiscussionBy using peripheral T cell-specific KO mice, we have establishedthe positive regulatory role of PP2A in TH17 differentiation as wellas in inflammatory autoimmune diseases. We have also proved thatunderactivated SMAD2 and overactivated SMAD3 downstream ofTGFβ signaling collectively reduced RORγt-mediated Il17a tran-scription in PP2A cKO mice. PP2A inhibitors also reduced TH17polarization, indicating a promising therapeutic avenue for treatingTH17 cell-mediated autoimmune diseases.PP2A is one of the most abundant phosphatases and crucial

for many key cellular events. A total knockout of the PP2Acatalytic subunit a in mice is lethal (16). To our surprise, PP2Adeficiency in T cell did not lead to fundamental changes in basicprocesses such as cell survival and proliferation. This is eitherbecause of the residual PP2A expression in our cKO mice or thestage and cell-specific functions of PP2A.As shown by RNAseq and RT-PCR analysis, the transcrip-

tional changes of TH17 signature genes induced in PP2A cKOwere limited. Obvious changes were observed in Il17a and Il17f,but not in Rorc. Expression of RORγt was intact, which excludethe possibility that PP2A is involved in the pathways leading toRORγt induction. Therefore, intracellular signaling eventsdownstream of RORγt and closely related to Il17a transcriptionmight be the candidate targets of PP2A-mediated inhibition ofTH17 differentiation.

SMAD2, SMAD3, and SMAD4 are all critical for TGFβ sig-naling and participate in TH17 or iTreg cell priming to inducebalanced expression of Foxp3 and RORγt (24–26, 28–30). It isintriguing that SMAD2 knockout mice show reduced TH17 celldifferentiation and ameliorated EAE, while a deficiency in SMAD3has the opposite effects (24, 26–28). Furthermore, overexpressionof SMAD2 and RORγt augments the differentiation of TH17 cells.However, SMAD3 binds to RORγt in Co-IP experiments and de-creases its transcriptional activity (26). Moreover, R-SMAD acti-vation is mainly via the phosphorylation of the C-terminal SSXSmotif, which is critical for R-SMAD function. The active form ofSMAD3 might enhance its binding affinity with RORγt, and it isknown that SMAD3 can compete with SMAD2 for binding withRORγt (27). These results suggest that active SMAD2 plays apositive role and active SMAD3 plays a negative role in TH17 celldifferentiation likely via dynamic interaction with RORγt (26, 27).Our study confirmed that altered SMAD2/3 activation by

PP2A affects TH17 differentiation, resulting in decreased Il17atranscription via forming complex with RORγt and reducing itsactivity and, thus, affecting TH17 differentiation. Importantly,this is a study that identifies PP2A as the critical phosphataseresponsible for Ser/Thr dephosphorylation on R-SMADs andnecessary for efficient TH17 differentiation.It is important to know the precise dephosphorylation site on

R-SMAD and the responsible kinases. Our previous work haselucidated the importance of threonine residue T324 in theα-helix 1 region of SMAD2 for regulation during TH17 differ-entiation (6). It is thus likely that PP2A up-regulates SMAD2C-terminal phosphorylation via modulating MINK1 activity.However, the function of PP2A appears to be broader andmore dominant than this single phosphorylation event, since wealso observed increased SMAD3 phosphorylation in cKO CD4+

Fig. 5. PP2A inhibitors lead the same change of SMAD2/3 activation underTGFβ pathway and limit TH17 priming in vitro. (A) SMAD2/3 was overex-pressed in the 293FT cells, and PP2A inhibitors were added in serum-freemedium for 2 h followed by TGFβ (3 ng/mL) stimulation for 3 h. Western blotanalysis using phospho-specific SMAD2/3 (p-SMAD2/3) and FLAG-tag (total-SMAD2/3) antibodies. (B) Il17a promoter and SMAD2/3 with or withoutRORγt was transfected into 293FT cells and then treated with PP2A inhibitorsand TGFβ as A. Luciferase activity was measured and normalized based onRenilla luciferase gene. (C) Sorted naïve CD4+ T cells were polarized underTH17 priming condition with the indicated concentration of CAN for 3 d, andIL17A+ population was analyzed using flow cytometry on day 5. (D) Theconcentration of IL17A in the culture supernatant was measured by ELISA.(E) Histogram of CFSE fluorescence staining of cells in C. (F) WT naïve CD4+

T cells cultured with CAN (5 μM) for the first 3 d in TH17 priming conditionand analyzed IL17A+ population by flow cytometry on day 5. Error bars showmean ± SEM. Data are representative of two independent experiments withsimilar results.

Fig. 6. A PP2A inhibitor suppresses EAE development. (A) PP2AWT and cKOmice were immunized with MOG(35-55) peptide. PP2A inhibitor CAN (0.6 μg/g)was administered i.p. daily from day 10 to day 12 and then was given onceevery 2 d. Mean clinical scores for EAE from each group. (B) Representativehistology of the spinal cord (H&E at Left and LFB at Right) of mice after EAEinduction (day 19). Arrowheads indicate inflammatory infiltration (Left)and demyelination (Right). (Scale bars: 100 μm.) (C) Quantification of totalmononuclear cells, CD4+ T cells, CD8+ T cells, and myeloid cells that infil-trated into the CNS of WT mice at peak of the disease. (D) Ratio of CD4+

and CD8+ T cells (gated at CD45hi CD11b−) in the CNS of WT groups. (E andF) Ratio (E ) and number (F ) of IL17A+ or Foxp3+ or IFNγ+ CD4+ cells in theCNS of the WT groups at disease peak. (G) Quantification of total mono-nuclear cells, CD4+ T cells, CD8+ T cells, and myeloid cells that infiltratedinto the CNS of the cKO groups. (H) Ratio of CD4+ and CD8+ T cells (gatedat CD45hi CD11b−) that infiltrated into the CNS of the cKO groups. Eachsymbol represents an individual mouse (n = 5–7 per genotype); error barsshow mean ± SEM. Data are representative of two independent experi-ments with similar results.

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T cells, which also contributes to deceased TH17 differentiation.This observation is in accordance with a previous finding that thePP2A structural subunit PR65 could interact with SMAD3 (31).It is also likely that PP2A directly regulates the phosphoryla-

tion status of RORγt. A recent study showed that two functionalphosphorylation sites identified on RORγt played opposite rolesin TH17 polarization. IKKα was discovered as an upper streamregulator for the phosphorylation change (32). Whether PP2Aparticipates in the interaction with IKKα or RORγt in regulatingRORγt activity remains to be elucidated.The classical TGFβ pathway is also critical for iTreg differ-

entiation (33). SMAD2 and SMAD3 double knockout miceshowed a dramatic loss of Foxp3 induction (28). Then why isiTreg differentiation not affected by PP2A deficiency? It is mostlikely because of the redundant roles of SMAD2 and SMAD3 inTGFβ-induced iTreg plasticity (28). Overactivated SMAD3 maycompensate for insufficient activation of SMAD2 in Foxp3 in-duction. In a recent study, PP2A was reported to be indispens-able for the maintenance of the suppressive function of Tregcells via regulating the activity of the mTORC1 complex. Specificablation of PP2A in Treg cells by Foxp3-YFP-Cre leads to au-toimmunity with similar clinical features of scurfy mice (34). Wealso observed the same Treg phenotype in dLck-driven PP2AcKO mice, but the overall outcome of PP2A defect in peripheralT cells is dominated by TH17 differentiation impairment, whichis demonstrated by their reduced susceptibility to autoimmunediseases induction.Finally, in addition to finding the importance of PP2A to TH17

differentiation, we demonstrated the translational potential of

this pathway by showing the therapeutic effect of PP2A inhibitorin controlling autoimmune diseases in the EAE model.

Materials and MethodsMice. Ppp2ca floxed mice were provided by X.G. Mice with Cre recombinasedriven by the distal promoter of the gene encoding the kinase Lck werebought from the Jackson Laboratory. The experimental protocols were ap-proved by the Review Committee of Zhejiang University School of Medicineand followed institutional guidelines.

EAE Induction. EAE was induced as described (35). Briefly, mice aged 6–8 wkwere immunized with 200 mg of MOG35–55 (Sangon, MEVGWYRSPFSRVVH-LYRNGK) in an equal amount of Complete Freund’s Adjuvant (Chondrex,Inc.) and received 200 ng of pertussis toxin (List Biochemicals) i.v. on days0 and 2 after induction. Clinical evaluation was assigned daily using a five-point scale: 1, flaccid tail; 2, impaired righting reflex and hindlimb weakness;3, hindlimb paralysis; 4, hindlimb and forelimb paralysis; 5, moribund. De-tailed materials and methods are presented fully in SI Appendix, SI Materialsand Methods.

Statistical Analysis. Statistical analysis was performed using GraphPad Prism.The data were analyzed by Student’s t test. All P value less than 0.05 wasconsidered significant (*P < 0.05; **P < 0.01; ***P < 0.001).

ACKNOWLEDGMENTS. We thank Dr. Dawang Zhou for providing plasmidsin luciferase assay; Dr. Xinhua Feng for providing ALK5(202D)-HA plasmid;Youfa Zhu, Yanwei Li, Jiajia Wang, and Yingying Huang from the corefacilities (Zhejiang University School of Medicine) for technical assistance inhistology and FACS analysis. This work was supported by National NaturalScience Foundation of China Grants 31770954 and 31530019 (to L.L.),81570013 (to S.W.), and 31500708 (to M.Z.); National Key R&D Program ofChina Grant 2018YFC1105102 (to L.L.); and Fundamental Research Funds forthe Central Universities Grant 2018XZZX001-12 (to L.L.).

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