yap is essential for treg-mediated suppression of antitumor … · 8(8); 1026–43. ©2018 aacr. 1...

YAP Is Essential for Treg-Mediated Suppression of Antitumor Immunity Xuhao Ni 1 , 2 , Jinhui Tao 1 , 3 , 4 , Joseph Barbi 1 , 5 , Qian Chen 3 , 6 , Benjamin V. Park 1 , Zhiguang Li 7 , Nailing Zhang 3 , Andriana Lebid 1 , Anjali Ramaswamy 1 , Ping Wei 1 , Ying Zheng 1 , Xuehong Zhang 7 , Xingmei Wu 1 , 8 , Paolo Vignali 1 , Cui-Ping Yang 1 , 9 , Huabin Li 8 , Drew Pardoll 1 , Ling Lu 2 , Duojia Pan 3 , and Fan Pan 1

RESEARCH ARTICLE

Research. on March 5, 2020. © 2018 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from

Published OnlineFirst June 15, 2018; DOI: 10.1158/2159-8290.CD-17-1124

http://cancerdiscovery.aacrjournals.org/

August 2018 CANCER DISCOVERY | 1027

ABSTRACT Regulatory T cells (Treg) are critical for maintaining self-tolerance and immune homeostasis, but their suppressive function can impede effective antitumor

immune responses. FOXP3 is a transcription factor expressed in Tregs that is required for their func-tion. However, the pathways and microenvironmental cues governing FOXP3 expression and Treg func-tion are not completely understood. Herein, we report that YAP, a coactivator of the Hippo pathway, is highly expressed in Tregs and bolsters FOXP3 expression and Treg function in vitro and in vivo. This potentiation stemmed from YAP-dependent upregulation of activin signaling , which amplifi es TGFβ/SMAD activation in Tregs. YAP defi ciency resulted in dysfunctional Tregs unable to suppress antitumor immunity or promote tumor growth in mice. Chemical YAP antagonism and knockout or blockade of the YAP-regulated activin receptor similarly improved antitumor immunity. Thus, we identify YAP as an unexpected amplifi er of a Treg-reinforcing pathway with signifi cant potential as an anticancer immu-notherapeutic target.

SIGnIFICAnCE: Tregs suppress antitumor immunity, and pathways supporting their function can be novel immunotherapy targets. Here, the selective expression of YAP by Tregs, its importance for their function, and its unexpected enhancement of pro-Treg Activin/SMAD signaling are reported, as are validations of potential cancer-fi ghting antagonists of YAP and its regulatory targets. Cancer Discov; 8(8); 1026–43. ©2018 AACR.

1 Immunology and Hematopoiesis Division, Department of Oncology, Bloomberg-Kimmel Institute, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland. 2 Translational Medicine Research Center, Affi liated Jiangning Hospital, and Liver Transplantation Center, First Affi liated Hospital, Nanjing Medical University, Nanjing, China . 3 Department of Molecular Biology and Genet-ics, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland. 4 Department of Rheumatology & Immunol-ogy, The First Affi liated Hospital of University of Science and Technology of China, Hefei, Anhui, China. 5 Department of Immunology, Roswell Park Comprehensive Cancer Center, Buffalo, New York. 6 Thorgene Co., Ltd., Beijing, China. 7 Center of Genome and Personalized Medicine, Institute of Cancer Stem Cell, Dalian Medical University, Dalian, Liaoning, China. 8 Department of Otolaryngology, Head and Neck Surgery, Affi liated Eye, Ear, Nose and Throat Hospital, Fudan University, Shanghai, China. 9 Depart-ment of Gastroenterology, Rujin Hospital North, Shanghai Jiaotong Univer-sity School of Medicine, Shanghai, China. note: Supplementary data for this article are available at Cancer Discovery Online (http://cancerdiscovery.aacrjournals.org/). X. Ni, J. Tao, and J. Barbi contributed equally to this article. Current address for X. Wu: First Affi liated Hospital of Sun Yat-sen Uni-versity, Guangdong Sheng, China; current address for P. Vignali: University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; and current address for D. Pan: UT Southwestern Medical Center, Dallas, Texas. Corresponding Author: Fan Pan, Johns Hopkins University School of Medi-cine, CRB1 Room 452, Baltimore, MD 21287; Phone: 443-287-7264; Fax: 410-614-0549; E-mail: [email protected] ; Duojia Pan, Department of Physi-ology, Howard Hughes Medical Institute, UT Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9040; E-mail: [email protected] ; and Ling Lu, 300 Guangzhou Street, Surgery Building, 21 Floor, Nanjing, Jiangsu, China; E-mail: [email protected] doi: 10.1158/2159-8290.CD-17-1124 ©2018 American Association for Cancer Research.

INTRODUCTION Regulatory T cells (Treg) play critical roles in promoting

immunologic self-tolerance and immune homeostasis by sup-pressing aberrant or excessive immune responses that could

give rise to autoimmune diseases ( 1 ). However, their ability to dampen the activation of other leukocytes can also pose a major barrier to effective antitumor immunity and the sterile cure of chronic infections ( 2 ). The signature forkhead family transcription factor FOXP3 anchors the gene expression profi le that is responsible for the characteristic suppressive function of Tregs. Clearly demonstrating the importance of this factor, mutations to the gene encoding FOXP3 can lead to fatal auto-immune disorders in Scurfy mice and in human patients with IPEX alike ( 3, 4 ). Despite the undeniable importance of FOXP3 for Treg function and immune control, our grasp of the factors and mechanisms governing its expression remains incomplete.

The signaling pathways triggered in response to certain cytokines (e.g., IL2 and TGFβ) can be critical for induc-tion and maintenance of FOXP3 expression in Tregs ( 5 ). TGFβ potently induces FOXP3 expression in vitro and in vivothrough activation of SMAD signaling molecules, critical facilitators and regulators of TGFβ-initiated signaling events and downstream gene activation ( 6, 7 ). TGFβ signaling has also been reported to be critical for maintaining FOXP3 expression and Treg function ( 8, 9 ). Likewise, SMAD2 and SMAD3 are also apparently needed for the optimal pheno-typic stability of Tregs ( 10 ). Importantly, mechanisms for the augmentation or amplifi cation of TGFβ/SMAD signaling in Tregs can stabilize or enhance the suppressive function of these cells ( 11 ) and may be crucial determinants of Treg per-formance in a variety of microenvironmental niches.

YAP is a transcriptional coactivator that developmentally regulates organ size ( 12, 13 ). YAP is frequently elevated in a number of cancer types such as lung, colorectal, ovarian, liver, and prostate cancers, where it acts as a powerful tumor promoter, and its activation is a frequent event in tumor pro-gression ( 14 ). The Hippo pathway is believed to be the major regulator of YAP nuclear localization, activity, and tumori-genic potential ( 15–17 ). However, the physiologic role of YAP in the immune system is unknown.




Ni et al.RESEARCH ARTICLE

1028 | CANCER DISCOVERY August 2018 www.aacrjournals.org

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Unexpectedly, we found YAP to be highly expressed by Tregs. In this report, we characterize the role of YAP in these important cellular mediators of immune control. Our stud-ies revealed that in the absence of YAP, Tregs failed to sup-press immune activation in vitro as well as in vivo. We also found that YAP potentiates the signaling events triggered by dimeric members of the TGFβ cytokine superfamily known as activins by activating expression of a key signaling compo-nent of the activin receptor complex. Interestingly, we found that not only is this signaling axis active in Tregs, it could also effectively amplify TGFβ/SMAD signaling and the pro-motion of Treg differentiation and function. Moreover, dis-rupting this YAP/activin/SMAD axis dramatically slowed the growth of tumors in mice, including a highly aggressive mela-noma model. This experimental treatment also enhanced the antitumor efficacy of an antitumor vaccine, suggesting that the targeting of this YAP/activin/SMAD axis can be used to improve anticancer immunotherapy efficacy.

RESULTSYAP Expression Is Induced by T Cell–Receptor Signaling, Is Highly Expressed by Tregs, and Supports Their Function

YAP is a transcriptional coactivator known for its role in the Hippo signaling pathway (13). As such, its importance in tumorigenesis and organ size determination is well recognized

(14). However, little is known about the role of the Hippo pathway and YAP in immune cells. Reports of cross-talk between the Hippo and TGFβ signaling pathways (18, 19) led us to speculate that elements of the former may have a role in the mechanisms governing immune activation and tolerance.

We therefore screened YAP expression across different sub-sets of murine CD4+ T cells in order to assess the likelihood that Hippo signaling plays a role in these functionally distinct T-cell lineages. Little to no Yap mRNA was detected in naïve CD4+ T cells, but, notably, YAP expression was uniquely induced during the early stages of the in vitro induced (iTreg) differentiation. Meanwhile, other T effector subsets (Th0, Th1, Th2, and Th17 cells) failed to markedly upregulate Yap mRNA (Fig. 1A). Interestingly, transient Yap message accu-mulation was noted during Th17 skewing. However, 12 hours after stimulation, Yap transcript levels returned to baseline in these T cells (Fig. 1A; Supplementary Fig. S1A). Impor-tantly, considerable levels of YAP protein were found in cells of the iTreg subset and not other Thelper lineages (Fig. 1B). Human Tregs isolated from the peripheral blood of healthy donors also displayed higher levels of Yap mRNA than their conventional CD4+ (non-Treg) counterparts (Fig. 1C). These results implicate YAP as a transcription factor preferentially expressed by developing and established Tregs of mice and humans.

Because YAP is a major component of the Hippo pathway, we assessed levels of several Hippo signaling factors known to

Figure 1. Expression of YAP mRNA and protein by Thelper subsets. Naïve CD4+ T cells (CD4+ CD25−CD62L+) were isolated from the spleen and lymph nodes of WT C57BL/6 mice and activated under polarizing conditions to generate the indicated Thelper subset. The cells were harvested at different time points, and mRNA or protein levels of YAP were assessed by (A) qRT-PCR and (B) western blot. C, Human Tregs (CD3+/CD4+/CD8−/CD25hi/CD127lo/CD39+) and non-Treg CD4+ T cells were obtained from the peripheral blood of healthy donors by FACS after Ficoll–Paque PLUS gradient centrifuga-tion and magnetic bead enrichment of CD4+ T cells. Yap mRNA was measured by qRT-PCR. For A and B, shown are representative findings from at least 3 independent experiments (mean ±SEM of triplicates for A). For C, mean expression of Yap mRNA is shown for 10 healthy human donor samples. The data were analyzed using the Student t test and considered significant if *, P < 0.05; **, P < 0.01; ****, P < 0.001.




YAP Modulates Regulatory T-cell Activity RESEARCH ARTICLE


be upstream of the transcription factor across T-cell subsets to determine if these are also expressed preferentially by Tregs. Interestingly, we found that LATS1/2 and MST1/2, unlike YAP, were not upregulated by iTreg-skewing condi-tions (Supplementary Fig. S1B). These findings suggest that unlike other Hippo pathway factors, YAP is uniquely upregu-lated in developing Tregs, and they imply a role for YAP in the biology of these cells outside of its traditional role.

In order to dissect the potential role of YAP in the biology of CD4+ T cells, including Tregs, we crossed Yapfl/fl mice to CD4-cre transgenic mice to generate animals with a T cell–specific deletion of Yap. These conditional knockout mice (Yap cKO) developed normally without apparent defects in T-cell development or peripheral immune cell populations (Supplementary Fig. S2). Additionally, no obvious spontane-ous immune pathologies were noted in these mice. Likewise, the lung, kidney, liver, small intestine, and stomach of mice with Treg-specific YAP deficiency (generated by crossing Yapfl/fl mice to FOXP3Cre+ transgenic mice) appeared comparable to wild-type (WT) littermates (Supplementary Fig. S3). We used both strains to assess the impact of YAP deficiency on Thelper cytokine production and lineage commitment.

To this end, we isolated naïve CD4+ T cells from Yap cKO and WT mice for activation under different helper CD4+ T cell (Th) polarizing conditions for 72 hours. Yap cKO CD4+ T cells express moderately higher levels of IL2 and IFNγ upon unbiased activation (Th0 conditions; Fig. 2A). Yap cKO CD4+ T cells also express a greater amount of IL17A than WT CD4+ T cells under Th17 polarizing conditions (Fig. 2B), and, con-sistently, Yap cKO CD4+ T cells expressed higher levels of Il17a mRNA than WT cells (Fig. 2C). A modest decrease in FOXP3+ cells was also seen in Yap cKO-derived T cells cultured under Th17 conditions (Fig. 2B). These observations, coupled with our earlier discovery that YAP is upregulated in iTregs, led us to suspect that YAP positively affects the generation of iTregs in vitro over other CD4+ T-cell fates. In line with this, the per-centages of FOXP3+ cells induced from naïve Yap cKO T cells activated under iTreg skewing conditions were modestly, yet significantly, lower than those seen in polarized WT CD4+ T cells (Supplementary Fig. S4). Naïve CD4+ T cells isolated from Yapfl/fl FOXP3Cre+ mice were also consistently less able to upregulate FOXP3 than WT controls in response to activation and various concentrations of the Treg-promoting cytokine TGFβ. Here, YAP deficiency specifically in T cells having already “turned on” FOXP3 expression reduced the intensity of signal for the Treg transcription factor (Fig. 2D). Taken together, these findings suggest that YAP likely plays an important role in the initiation or maintenance of Treg differentiation.

In addition to FOXP3 induction, we also hypothesized that YAP might contribute to the suppressive function of Tregs as well. Indeed, an in vitro suppression assay showed that whereas WT Tregs readily dampened the proliferation of naïve T cells, Yap cKO Tregs were much less effective suppressors (Fig. 2E). In all, these findings implicate YAP as a Treg-associated factor with a role in both the generation and function of these cells.

YAP-Deficiency Enhances Anti-Melanoma Immunity

Although Tregs are necessary to maintain immune homeo-stasis, they pose an obstacle in mounting effective antitumor

immune responses, and their suppressive function dampens the efficacy of anticancer immunotherapies (20). For these reasons, therapies aimed at inhibiting Treg activity are prom-ising additions to the cancer immunotherapy arsenal (21). We hypothesized that the apparent loss of Treg-suppressive function seen in the absence of YAP could enhance antitu-mor immune responses. To test this, WT and Yap cKO mice were challenged with B16 melanoma, an aggressive “nonim-munogenic” cancer model. Tumor growth was measured in these mice over time, and, strikingly, we found that Yap cKO mice controlled the subcutaneous growth of the implanted melanoma cells whereas tumors grew robustly in WT mice (Fig. 3A and B). In line with our in vitro findings, the activa-tion of CD4+ and CD8+ tumor-infiltrating lymphocytes (TIL) from Yap cKO mice was apparently much less restrained than that of their WT counterparts. Intracellular cytokine stain-ing revealed these cells produced significantly higher levels of IFNγ and TNFα (Fig. 3C) compared with those from WT tumors. These results suggest that in the absence of YAP in T cells, a more robust antitumor immune response is mounted.

Tumor challenge of mice with Treg-restricted YAP defi-ciency yielded similar results. Although WT controls expect-edly permitted rapid tumor development, Yapfl/fl FOXP3Cre+ mice maintained small tumors infiltrated by elevated popula-tions of inflammatory cytokine-producing leukocytes. Spe-cifically, producers of the tumoricidal Th1 cytokine IFNγ were found at higher frequencies and in greater numbers in the tumors of Yapfl/fl FOXP3Cre+ mice than those of WT controls (Fig. 3D and E). Analysis of FOXP3 expression by CD4+ TILs revealed that deletion of YAP in Tregs reduces the frequency of suppressive FOXP3+ Tregs in the tumor micro-environment (Fig. 3F, left and middle). The relative balance (i.e., the ratio) of Tregs and potential effector CD8+ T cells was similarly shifted in the tumors of mice with Treg-specific YAP deficiency compared with those of WT controls (Fig. 3F, right). Treg-specific YAP deficiency also slowed the growth of tumors caused by implanted MC38 adenocarcinoma cells (Supplementary Fig. S5A–S5B). Not only were MC38 tumors much smaller in Yapfl/fl FOXP3Cre+ mice 21 days after injec-tion, the relative proportions of FOXP3+ Tregs among tumor-infiltrating T cells were reduced compared with WT tumors. In contrast, the frequencies of intratumoral producers of IFNγ and TNFα were elevated in the absence of Treg-specific YAP expression (Supplementary Fig. S5C–S5D). Corroborat-ing results were seen in the injectable EL4 thymoma model in which Treg-restricted Yap knockout resulted in dramatically stunted tumor growth relative to WT mice. As with other tumor models, this derailed tumor progression was concur-rent with reduced Treg proportions and an elevated presence of proinflammatory cytokine-producing T cells in the tumor microenvironment (Supplementary Fig. S6A–S6D). These experiments make a strong case for YAP’s role as both a facili-tator of Treg presence in the tumor niche and a potent and broadly active driver of Treg-enforced inhibition of endog-enous antitumor immunity.

Some of the most promising immunotherapeutic agents (i.e., PD-1 and CTLA4 antagonist antibodies) show even greater antitumor effect when administered in concert (22–24) or alongside tumor vaccine strategies (25–28). We






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Figure 2. The effects of YAP deficiency on CD4+ T-cell subsets. A–C, Naïve CD4+ T cells (CD4+ CD25−CD62L+) were isolated from WT Yapfl/fl CD4Cre− or Yapfl/fl CD4Cre+ (Yap cKO) mice (n = 5/group/experiment) and were activated under the indicated polarizing conditions for 4 days. The cells were harvested and signature cytokines and transcription factors for each Th subset were assessed by flow cytometry and qRT-PCR. Shown in A and B are representative flow cytometry results and the mean percentage of cells ± SEM from at least 3 independent experiments. C, Relative expression level of Il17a transcript during the early stages of Th17 cell differentiation for WT and Yap cKO derive cells ± SEM. D, iTreg differentiation of WT (Yapfl/fl FOXP3Cre−), and FOXP3Cre-driven Yap knockout mice (Yapfl/fl FOXP3Cre+). Naïve CD4+ T cells were isolated from the indicated mice as above before activation in the presence of IL2 and varying concentrations of TGFβ. Treg differentiation was assessed by intracellular staining for FOXP3 and flow cytometry analysis. Shown are representative histograms (left) and the mean fluorescence intensity (MFI) of FOXP3 staining was found in at least 3 independent experiments; average MFI ± SEM are shown. E, The suppressive function of WT or Yap cKO-derived Tregs (CD4+ CD25hi T cells FACS isolated from lymph node and spleen cell suspensions) was determined using an in vitro suppression assay. Naïve CD4+ T cells (responders) and Tregs were isolated from the indicated mice (n = 5/group/experiment). WT responders were prestained with CFSE and cocultured with WT and Yap cKO-derived Tregs at the indicated ratios. The cultures were activated with anti-CD3/anti-CD28–conjugated beads at a cell-to-bead ratio of 1:1. The percentage of proliferating (CFSElo) responder cells in each culture was determined by flow cytometry. Shown are representative histograms (top) and the mean per-centages of proliferating cells ± SEM over at least 3 independent experiments (bottom). For A-E, significant differences were determined by the Student t test (*, P < 0.05; **, P < 0.02; ***, P < 0.002; ns, not significant).






Figure 3. The impact of T cell– and Treg-restricted YAP deficiency and YAP inhibition on the antitumor response. WT (n = 5) or Yapfl/fl CD4Cre+ (Yap cKO; n = 5) mice were challenged with 5 × 105 B16 melanoma cells (s.c.), the tumor dimensions were measured every 2 days, and tumor volume was calcu-lated (A, B). On day 21, the mice were euthanized and TILs were isolated from the excised tumors. C, TILs were gated on CD4+ and CD8+ T cells, and effec-tor cytokines IFNγ and TNFα levels were measured by flow cytometry. D, Tumor challenge of Yapfl/fl FOXP3Cre+ mice (n = 4) and WT controls (n = 6) was carried out as above, and the frequencies of IFNγ- and IL17-producing leukocytes within the B16 TILs of these mice were determined by flow cytometry (E, left). Mean frequencies of IFNγ+ TILs as well as the number of IFNγ+/CD4+ and IFNγ+/CD8+ per gram of tumor tissue (± SEM) are also shown (E, middle and right, respectively) from at least 3 independent experiments. F, Proportions of FOXP3+ Tregs within the TILs of WT and Yapfl/fl FOXP3Cre+ mice (left) were also found by intracellular staining followed by flow cytometry analysis. Average Treg frequencies amongst CD4+ TILs were found and the ratio of tumor CD8+ T cell to FOXP3+ Treg numbers are also shown (center and right, respectively). G, Targeting YAP improves the antitumor effects of immuno-therapies. C57BL/6 mice were challenged with B16 melanoma cells and tumor progression was monitored as mentioned above. Cohorts of mice were treated with i.p. injected VP, GM-Vac, anti–PD-1 antibody, VP and anti–PD-1, or VP and GM-Vac beginning day 7 after tumor injection. Control mice were left untreated (n = 5/group). Shown are the mean tumor volumes for the groups ± SEM. H, Characterization of TILs from treated and control mice were also determined by flow cytometry. The frequencies of IFNγ-producing CD4+ and CD8+ T cells as well as the ratio of tumor CD4+ and CD8+ T-cell numbers to FOXP3+ Treg numbers were also shown. For A, D, and G, the mean tumor volumes for the groups are shown over time ± SEM. Bar graphs in C, E, F, and H depict the mean frequency (%), ratio, or absolute number/gram tumor of the indicated immune cell subset ± SEM in 3 independent experiments. All other findings are representative of at least two independent experiments. Statistically significant differences were determined by t test for all panels except for G, where a two-way ANOVA was used. *, P < 0.05; **, P < 0.01; ***, P < 0.002; ****, P < 0.001.

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therefore tested the therapeutic potential of YAP target-ing as an immunotherapeutic approach to combat cancer. Administration of a known YAP inhibitor, verteporfin (VP; ref. 29), to melanoma-bearing mice resulted in modest reduction in tumor size (Fig. 3G). Treatment of melanoma-bearing Yapfl/fl FOXP3Cre+ mice with VP, on the other hand, failed to alter the already stunted progression of tumors in these mice (Supplementary Fig. S7A), suggesting that poten-tial off-target effects of this drug or any direct effects on tumor cells are not likely contributing to these in vivo obser-vations. We also tested the effects of combining VP with the proven immunotherapeutic agents anti–PD-1 antibody and GM-Vac (irradiated GM-CSF–producing B16 cells). Both anti–PD-1 and GM-Vac treatments were able to slow tumor growth somewhat as monotherapies. Notably, combinato-rial treatment with VP and anti–PD-1 neutralizing antibody suppressed tumor progression to a greater extent than any monotherapy tested. Even more dramatic were the synergistic effects of VP and GM-Vac, which prevented the development of tumors beyond a barely detectable size (Fig. 3G). The decidedly improved antitumor efficacy seen upon combina-tion of either anti–PD-1 or GM-Vac treatment with VP was associated with enhanced proportions of IFNγ-producing CD8+ and CD4+ T cells and a compromised Treg presence in the tumor microenvironment (Fig. 3H; Supplementary Fig. S7B–S7E). These findings strongly suggest a major role for Treg-derived YAP in crafting the immunosuppressive nature of the tumor microenvironment. They also suggest the potential of immunotherapeutic approaches that include YAP-targeting agents.

YAP Potentiates Expression of Genes Involved in TGFa/SMAD and Activin Signaling

To gain insight into the mechanism by which YAP contrib-utes to Tregs and their enforcement of immune suppression, we isolated Tregs from mice lacking YAP in these cells (Yap cKO) and subjected them to RNA-sequencing (RNA-seq) analysis along with WT Tregs and naïve CD4+ T cells from both mice. The results of this analysis revealed that YAP-deficient Tregs display reduced expression of several genes known to be important in the signaling pathway triggered by the anti-inflammatory cytokine TGFβ. Interestingly, one of the genes most downregulated in the absence of YAP was that encoding the signaling component of the activin receptor complex known as Acvr1c (Fig. 4A; Supplementary Fig. S8A). Confirming a role for YAP in potentiating Acvr1c expres-sion, we found that WT CD4+ T cells display considerable upregulation of the transcript for this receptor subunit dur-ing in vitro Treg differentiation, whereas their YAP-deficient counterparts did not. Interestingly, freshly isolated nTregs expressed modest levels of Acvr1c. However, upon activation, these Tregs dramatically activated activin receptor expres-sion in a YAP-dependent manner (Supplementary Fig. S8B). Indeed, neither nTreg nor differentiating iTregs from Yapfl/fl FOXP3Cre+ mice expressed considerable Acvr1c mRNA levels. Thus YAP-mediated activin responsiveness may have consid-erable influence over the biology of multiple Treg popula-tions.

It has been suggested that activin can promote TGFβ signaling. Pathway analysis of our RNA-seq results showed

that the gene expression patterns most affected by YAP deficiency in Tregs were highly relevant to immune control and the diverse autoimmune pathologies resulting from the breakdown of such control. Among these, the genes associ-ated with the TGFβ signaling cascade were markedly altered (Supplementary Fig. S8C). Furthermore, RT-PCR analysis also showed reduced transcript levels for several known TGFβ-responsive genes in Tregs from YAPfl/fl FOXP3Cre+ mice (Supplementary Fig. S8D). In light of these find-ings, we suspected that YAP contributes to Treg-mediated immune control at least in part by bolstering TGFβ/SMAD signaling through the activin/AcVR1C axis in these sup-pressor cells.

Although activin mRNA levels were low in naïve CD4+ T cells, in vitro differentiating Tregs (naïve CD4+ T cells acti-vated with anti-CD3/CD28 in the presence of IL2 and TGFβ) upregulated activin expression over time (Supplementary Fig. S9A). The kinetics of this upregulation paralleled the appearance of FOXP3 expression in these cells (Supplemen-tary Fig. S9B). qRT-PCR analysis also showed that expression of the activin receptor (AcVR1C) was similarly low in naïve CD4+ T cells, but was robustly upregulated under in vitro culture conditions that generate iTreg (Supplementary Figs. S8B and S9C). We went on to dissect which Treg-inducing stimuli were chiefly responsible for inducing expression of YAP and elements of activin/ACVR1C signaling. To this end, naïve CD4+ T cells were activated in vitro with anti-CD3/CD28 antibodies, either alone or in the presence of IL2, TGFβ, or IL2 and TGFβ. As expected, activation alone failed to induce upregulation of these genes or the canonical Treg transcription factor FOXP3. The cytokine TGFβ did trigger significant expression of FOXP3, as expected, but YAP as well. Exposure to IL2 along with TGFβ (but not IL2 alone) greatly augmented expression of YAP and FOXP3. Of the conditions tested, those upregulating robust YAP also brought about expression of activin and ACVR1C (Supplementary Fig. S9D–S9G). These findings further align the upregulation of YAP expression and activin signaling with the Treg lineage and shed some light on the largely unknown cast of molecular characters regulating these processes in T cells.

To gain further insight into the mechanism of YAP-mediated ACVR1C upregulation, we explored the potential involvement of a known YAP-collaborating factor. Mature YAP protein is known to contain a TEAD-binding domain, and prior studies (largely conducted in non-T cells) have identified numerous target genes controlled by the coop-eration of these factors. Suggesting that transcription at the AcVR1c locus is activated through YAP–TEAD interaction, the promoter sequence of this gene was found to contain two TEAD consensus binding sites (Fig. 5A). To test the importance of TEAD binding for YAP-dependent AcVR1c expression, we prepared luciferase-based reporter constructs under the control of WT murine AcVR1c promoter sequence. Mutant constructs having either or both of the TEAD sites ablated were also designed (Fig. 5B). Each AcVR1c-luciferase reporter construct was delivered into Jurkat T cells along with an expression vector encoding YAP (“YAP1WT”) or a mutant version of this transcription factor unable to interact with TEAD (“YAP1mut”) owing to an S-to-A mutation at residue 94 (“S94A”). In this system, expression of TEAD1 or YAP1WT






Figure 4. RNA-seq analysis of WT and YAP-deficient Treg transcriptomes. Genes that had their expression level significantly changed by Yap knockout in naïve CD4+ T cells, unstimulated (unstim) Tregs, or stimulated (stim) Tregs were determined by RNA-seq. Results are presented as a heat map. Genes are arranged based on the fold change in expression between WT and YAP-deficient Tregs (genes with a fold change of more than 3 are shown). The color representation from green to red denotes log2-transformed FPKM from −2 to 2.

WT Yap cKO

Expression_level−2 0 −5 −10 −15−1 0

IpwOlfr1350

Pan2Jmjd7Casp4

XistAtp10dKat2a

Gm10825Gpr137b-ps

Rab6bAcvr1c

Rfx3Gm9781

Unc5clAlg11

Zfp948Fam174b

Cnnm2Zfand2b

Phc3Tanc1Ptprs

Zfp182Uhrf1Sqrdl

Zfp612Tnip1Itga7Bmf

Gm14085

DutChka

Dcpp2Rab4a

Atp13a2Ccnb1

Tmem194bGet4

GzmbErdr1

Dynlt1bTtbk2

Anxa2Prc1Nfyc

Tbx21Eef2k

Hist1h4fPlac8

Dnmt1SordNkg7

Hist1h3iAkr1c18Gas2I3

CcI4Galnt6

Hist1h2bhHist1h2ab

Lass6

Dmxl2Bcl2I1Gvin1Baat1

Col18a1Slc37a2

Tmem184bAdam8

Jak3

Lamc3Pstpip2Prune2

Hist1h2blGatm

lcmtEno1

Hist1h4iHist1h2bp

Mid1H2-T10Fbxo34Dcpp1

Spt1

Fst

Ifng

Gen

e

Smurf1

1 2

WT WTYap cKO Yap cKO

Naïve T cells Unstim nTreg Stim nTreg0 5 10 15

Log2 (fold change) KO Treg/WT Treg






Figure 5. YAP drives AcVR1c transcription through molecular cooperation with TEAD. A, Shown is a portion (1.2 kb) of the murine AcVR1c promoter with TEAD binding sites determined by transcription factor prediction software. B, Design schema of luciferase-based AcVR1c reporter constructs; C, AcVR1c reporter assays. The mAcVR1c-WT reporter construct was cotransfected into Jurkat T cells along with the indicated YAP and TEAD expres-sion constructs or an empty vector control. Cells were cultured with or without PMA/ionomycin (iono) activation for 8 hours prior to harvest and cell lysis. Luciferase activity was determined as previously described (47). D, As in C, YAP and TEAD expression were delivered to Jurkat T cells, except cells received variants of the mAcVR1c reporter possessing one, both, or none of the identified TEAD binding sites. Reporter activity was determined as in C. For both C and D, the mean relative luciferase values ± SEM are shown for the results of 3 independent experiments. E, A chromatin immunoprecipi-tation assay was carried out in iTregs generated from WT- and Yap cKO-derived naïve CD4+ T cells. The ability of antibodies against TEAD1 and YAP to pull down the indicated factors along with the AcVR1c promoter region was calculated based upon qPCR relative to a control IgG. The relative enrich-ment for each factor over 3 experiments is shown (±SEM). Significant differences for all experiments shown were determined by the Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.002; ****, P < 0.001.

TEAD Consensus binding sites: CATTCC

Partial mouse AcVR1c promoter sequenceLuc mAcVR1c-Luc WT

mAcVR1c-Luc Mut1

: TEAD binding site

: TEAD binding site 1 mutated

mAcVR1c-Luc Mut2

: TEAD binding site 2 mutated

mAcVR1c-Luc Muts

: both TEAD binding sites mutated

Luc

Luc

Luc

TGAGCAAT//TACCTCCCTCCTCCAGTTGCTAACTCTGTAACTGCAGAGATACCTTCTACGTGTCTGTGTCTGTGTCTGGTCCTTTACTTCCATTCCTCATTATAGATGGCTATGCCACCTGGCACAGTTGTCTCAGCTTTGTGCATCGTTTTTATAGGGCACATGAAGTCTTGAGATGAGGACAGGAATGAATGGAGTTTGTTTGGTAAAACAGAGCTGTGTGGTCTGAAGGCAATGGTGCTTGGGGTCCTTTTCAGGGAGTCACAGAGAGAGAGAGC//TGTCAGGGTGGGGCAGGTCTGGAGGAACAGCCTGTGGTGTTGT

A B

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Rel

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YAP1 MutS94A

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+

_ _ _ _

____

_ _ _ _

_

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PMA/Iono

Rel

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e lu

cife

rase

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ue

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AcVR1c-Luc Mut1

AcVR1c-Luc Mut2

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+

− + − −− − + −

− − − +

− − −

**

***

***

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5

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25

Rel

ativ

e en

richm

ent

IgG TEAD1 YAP1 IgG TEAD1 YAP1

WT Yap cKO

**

**

**






alone induced only modest activation of AcVR1c expression. In contrast, robust luciferase signal was detected when WT YAP and TEAD were coexpressed. Mutation of YAP’s TEAD interaction site, however, resulted in far less reporter activity (Fig. 5C), supporting the notion that YAP–TEAD coopera-tion is necessary for optimal AcVR1c expression. Similarly, loss of a single TEAD binding site in the promoter sequence reduced YAP-induced transcription whereas mutation of both sites resulted in a significant and near-complete loss of reporter signal (Fig. 5D). These results clearly implicate a molecular partnership between YAP and TEAD in the potentiation of activin signaling through AcVR1c expression. This point was further supported by chromatin immunopre-cipitation (ChIP) assays showing both YAP1 and TEAD1 are enriched at the AcVR1c locus in WT iTregs (Fig. 5E). Notably, in Yap cKO-derived iTregs, TEAD1 was still found interact-ing with the AcVR1c locus despite the absence of YAP (Fig. 5E). These findings illuminate the mechanism behind YAP’s activation of activin signaling.

Activin Enhances SMAD/TGFa Signaling and Treg Differentiation

Because activin has been reported to promote SMAD sign-aling in non-T cells (30), we tested whether activin signaling in T cells could have a similar effect. SMAD activity was assessed by western blot analysis of SMAD phosphorylation. Indeed, we found that whereas untreated CD4+ T cells did not contain discernible levels of active (phosphorylated) SMAD molecules, treatment with 5 or 10 ng/mL of activin A resulted in elevation of phospho-SMAD levels. As expected, TGFβ treatment (0.5 or 2 ng/mL) also induced SMAD phospho-rylation. Importantly, combined activin and TGFβ treatment resulted in even further activation of the SMAD signaling pathway (Fig. 6A). These findings suggest that activin signal-ing can augment signaling along the TGFβ/SMAD axis—a signaling pathway crucial for multiple aspects of Treg biology and immune tolerance (7).

TGFβ/SMAD-mediated events are important during the upregulation of FOXP3 and the generation of Tregs from naïve CD4+ T-cell precursors. We next investigated whether YAP-dependent activin signaling can participate in the driv-ing of this process. Having shown that YAP plays an impor-tant role in promoting or maintaining FOXP3 expression induced in the presence of various TGFβ concentrations (Fig. 2D), and having implicated the transcription factor in the regulation of TGFβ-sensitive genes (Fig. 4; Supplemen-tary Fig. S8), we therefore postulated that YAP-mediated upregulation of AcVR1C and SMAD signaling might provide a crucial amplification of this important Treg-supporting signaling pathway that allows for more robust or sustained FOXP3 expression.

To explore the involvement of activin/AcVR1C signaling in the enhancement of Treg differentiation by YAP, the effect of supplemental activin A on in vitro Treg commitment was also investigated. As expected, activation of naïve CD4+ T cells without TGFβ yielded little to no FOXP3 induction regardless of YAP expression. Strikingly, activation of WT cells with exogenous activin A, even in the absence of TGFβ, generated a population of FOXP3+ cells. Although a subopti-

mal concentration of TGFβ resulted in modest upregulation of FOXP3 (mirroring the effects on SMAD activation), com-bined treatment of WT naïve CD4+ T cells with low doses of TGFβ plus activin A resulted in synergistic promotion of FOXP3+ T-cell induction. This induction of Tregs by activin A treatment alone was largely not seen upon FOXP3-driven knockout of Yap, and although dual treatment of differenti-ating Yapfl/fl FOXP3Cre+ iTregs did enhance the generation of FOXP3+ cells, it was to an extent far less than that seen in their WT counterparts (Fig. 6B). These results suggest that activin signaling via YAP-dependent AcVR1C expres-sion on Treg not only augments TGFβ signaling but can also drive the process of FOXP3 upregulation. Supporting this notion, naïve T cells lacking AcVR1c were found to be less sensitive to TGFβ-induced iTreg differentiation than their WT counterparts, particularly when TGFβ concentrations were low (Supplementary Fig. S10A–S10B). Interestingly, exogenous activin supplementation could do little to rescue the deficient FOXP3 induction seen in naïve CD4+ T cells lacking either SMAD2 or SMAD3, or, for that matter, the pronounced defect in iTreg generation seen in T cells geneti-cally lacking both SMAD molecules (Supplementary Fig. S11). This observation confirms that functional SMAD sig-naling is required for activin-mediated enhancement of Treg generation, in agreement with prior studies (31). In all, these findings are very much in line with a role for YAP-driven activin signaling in the augmentation of signaling down the SMAD/TGFβ axis in T cells.

Activin-Mediated Support of Treg Function Is YAP/AcVR1C Dependent

YAP deficiency leads to improved antitumor immunity and a Treg pool that is insensitive to an activator of the TGFβ/SMAD signaling pathway (i.e., activin). We therefore hypothesized that YAP facilitates robust Treg function in vivo through the induction of AcVR1C, which in turn amplifies the pro-Treg signaling cascade. In order to determine if the Treg-promoting effects of YAP were due to the upregulation of AcVR1C, we set out to test whether the defective Treg function seen in Yap knockouts could be restored by ectopic expression of AcVR1C. In an in vitro suppression assay, as expected, Yapfl/fl

FOXP3Cre+-derived Tregs transduced with an empty vector control expressed reduced levels of ACVR1C protein and were much less efficient suppressors of naïve CD4+ T-cell prolifera-tion than their WT counterparts. However, lentiviral-based delivery of an ACVR1C-encoding expression construct into Yapfl/fl FOXP3Cre+-derived Tregs more than rescued receptor expression, which greatly enhanced their suppressive potency beyond even that of WT Tregs (Fig. 6C). These results sup-port the conclusion that activin signaling through AcVR1C (upregulated by YAP) can amplify the suppressive potency of established Tregs as well as the TGFβ-driven differentia-tion of iTregs and potentially other facets of this cytokine’s broadly immunosuppressive action. Importantly, they also suggest that targeting either YAP or activin signaling is likely to undermine the tolerance-promoting attributes of TGFβ and both subsets of FOXP3+ Tregs in the cancer setting. These approaches may provide avenues to enhance antitu-mor immunity either as novel treatments on their own or as






Figure 6. The YAP/activin/ACVR1C pathway enhances SMAD activation, Treg generation and function, and tumor progression. A, Freshly isolated CD4+CD25− T cells were isolated from the lymphoid tissues of WT mice (n = 6/experiment), cultured with plate bound anti-CD3 (2 μg/mL) and soluble anti-CD28 (2 μg/mL) for 24 hours, followed by treatment with different concentrations of activin A and TGFβ as indicated for an additional 12 hours. Cells were harvested and subjected to SDS-PAGE and western blot with the indicated antibodies (left). Band densities indicating protein amount were quantified by using ImageJ software, normalized to β-actin loading controls, and the mean density ± SEM across 3 independent experiments were found (right). B, Naïve CD4+ T cells from Yapfl/fl, FOXP3Cre+, and YapWT/WT, FOXP3Cre+ (WT) mice (n = 6/group/experiment) were stimulated with anti-CD3/CD28 antibodies (1 and 4 μg/mL, respectively) for 3 days in the presence of IL2 (100 U/mL) and the indicated doses of TGFβ and exogenous activin A. Activin was dosed at 50 ng/mL on days 0 and 2. Treg induction was assessed by flow cytometric detection of intracellular FOXP3. Shown are representative FOXP3 stainings (left) and the mean results of 3 independent experiments ± SEM (right). C, Effect of ectopic AcVR1c expression on YAP-deficient Tregs. As before, WT responder T cells and Tregs were isolated from the indicated mice (n = 6/group/experiment). Following lentiviral delivery of an ACVR1C overexpression construct or an empty vector control construct to Yapfl/fl FOXP3Cre+ Tregs (activated ex vivo overnight with anti-CD3/CD28 antibodies and IL2), the func-tional capacity of these cells was assessed in vitro. The transduced Tregs were cocultured with CFSE-stained CD45.1+ naïve CD4+ T cells (responders) at the indicated ratio and antigen-presenting cells (T cell–depleted splenocytes). After 5 days of activation, responder cell proliferation was assessed by flow cytometry. Shown at left are representative plots of responder cell gated (CD45.1+/CD4+) events from 1 of 2 independent experiments with like results. The immunoblot at right confirms expression levels of AcVR1c in transduced Tregs, and the bar graph (lower right) depicts the mean fraction of proliferat-ing responder cells over all experiments ± SEM. Where indicated by asterisks, significant differences were found by the Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.002; ****, P < 0.001.

A

C

B

Cou

nt

CFSE (Teff)

Yapfl/fl

FOXP3Cre+

LV-Ctrl

Yapfl/fl FOXP3Cre+

Yapfl/fl

FOXP3Cre+

LV-AcVR1c

WT

Yapfl/fI FOXP3Cre− Yapfl/fI FOXP3Cre+

AcVR1c

GAPDH

Lv-Ctrl Lv-AcVR1c1:2 1:4 1:8 1:16Treg:Teff

Foxp3

WT

+act./−TGFβ+act./+ TGFβ (0.075 ng/mL)

−act./−TGFβ−act./+ TGFβ (0.075 ng/mL)

+act.

/−TGFβ

+act.

/+TGFβ

−act.

/−TGFβ

−act.

/+TGFβ

FO

XP

3 M

FI

WT

400

300

200

100

0

Yapfl/fl FOXP3Cre+

Yapfl/fl FOXP3Cre+ LV-AcVR1c Yapfl/fl FOXP3Cre+ LV-Ctrl

****

*

ns*

WT

1:2 1:4 1:8 1:160

20

40

60

80

100

% P

rolif

erat

ion

****

ns****

ns ****

** *

****

Activin A

pSMAD2/3

SMAD2/3

β-Actin

TGFβ

0

0 0

5

0.5

0

0.5

5

0

10

2

0

2

10(ng/mL)

Activin A

TGFβ

0

0 0

5

0.5

0

0.5

5

0

10

2

0

2

10

(ng/mL)0

50

100

150

*

*

pSM

AD

2/3/

Act

in

14.5 85.5 17.3 82.7 24.3 59.7 40.375.7

41.2 58.8 53.1 46.9 60.2 39.8 65.8 34.2

8.06 91.9 12.8 87.2 16.7 83.3 21.0 79.0






potent enhancers of other promising immunotherapeutic agents.

Activin Blockade or AcVR1c Knockout Inhibits Tumor Growth

As an instigator of an apparent feed-forward loop capable of amplifying TGFβ/SMAD activity, YAP presents a tempt-ing target for those aiming to break tolerance in the cancer setting. However, the targeting of YAP in patients with cancer may prove problematic owing to the molecule’s intracellular location and the chemical drawbacks of known inhibitors (e.g., VP has noted solubility issues; ref. 29). Therefore, the activin/AcVR1C interaction is likely to serve as a desirable alternative strategy. Having demonstrated the positive effects of activin signaling on the TGFβ/SMAD signaling pathway and the processes of Treg generation and function, which can oppose immune-mediated tumor cell killing, we suspected that disrupting activin function should enhance antitumor immunity. We therefore tested the potential of activin tar-geting as an immunotherapeutic approach to combat can-cer. Administration of anti-activin monoclonal antibody to mice injected subcutaneously with B16 melanoma markedly stunted the development of tumors relative to an inert iso-type control (Fig. 7A). We also tested the value of combining anti-activin blocking antibody treatment with the anticancer vaccine GM-Vac. Treatment with GM-Vac alone was able to partially slow the growth of tumors to an extent similar to anti-activin monotherapy. However, combining anti-activin treatment with GM-Vac was able to arrest tumor growth at a barely detectable size (Fig. 7A). Anti-activin treatment also successfully reduced the frequency of FOXP3+ Tregs among TILs, and although GM-Vac–receiving mice displayed some reduced Treg presence in their tumors, combined GM-Vac and activin blockade resulted in dramatic loss of these suppressor T cells from the tumor microenvironment (Fig. 7B). The effect of blocking activin on Tregs coincided with increased frequencies of IFNγ-producing CD8+ and CD4+ T cells, an observation even more prominent upon combina-tion of GM-Vac and anti-activin treatments (Fig. 7C). These results demonstrate the susceptibility of the YAP/AcVR1c/activin axis to therapeutic targeting at multiple points.

Along this line, B16 tumor growth was also markedly slower in AcVR1c knockout mice than in WT controls (Fig. 7D). Cor-respondingly, the TILs from AcVR1c-deficient mice contained fewer FOXP3+ Tregs than their WT counterparts and displayed a selective elevation of IFNγ-producing T cells (Fig. 7E and F). As with chemical YAP inhibition and antibody-mediated activin blockade, administering GM-Vac to AcVR1c knockout mice enhanced the already considerable antitumor effect of genetic AcVR1c ablation (Supplementary Fig. S12). From these results, it is clear that disrupting any of the several ele-ments of the YAP/activin/SMAD axis can undermine immune suppression and oppose tumor progression in mice.

In all, our findings support the conclusion that signaling along the YAP-regulated activin/ACVR1C axis can support Treg generation and function and potentially other broadly immune-suppressing effects of the TGFβ/SMAD pathway. Importantly, they also suggest that targeting this axis is likely to undermine the immune suppressive attributes of TGFβ and FOXP3+ Tregs in the cancer setting—either alone

or in combination with other promising immunotherapeutic agents (e.g., immune checkpoint–blocking antibodies and anticancer vaccines).

DISCUSSIONTregs are indispensable for restraining potentially lethal

self-directed (autoimmune) responses or overexuberant ones mounted against normally harmless commensal microbes (inflammatory bowel disease; ref. 1). However, in patients with cancer, Tregs can be greatly enriched within tumors, sometimes systemically (32). The suppressive function of these cells in this setting dampens the effectiveness of tumor-directed immunity and is a major obstacle for developing effective anticancer immunotherapies (21).

As part of an ongoing effort to identify precise mechanisms of Treg generation, maintenance, and function in the context of cancer, we have made the surprising discovery that YAP, a transcription factor critical in developmental regulation of organ size, is in fact an important factor in the generation and function of Tregs. Deletion of Yap1 in T cells some-what enhances both Th1 and Th17 development but most impressively diminishes generation of iTregs under condi-tions of limited TGFβ. YAP deficiency also negatively affects the suppressive function of Tregs. The inability of Tregs to suppress immunity in vivo in the absence of YAP was dramati-cally illustrated by our B16 melanoma tumor model experi-ments (Fig. 3). The poorly immunogenic tumor failed to grow in mice with Treg-specific Yap deletion, which displayed markedly enhanced indicators of proinflammatory antitu-mor immunity compared with WT controls. This improved deployment of antitumor immunity was seen alongside a markedly diminished Treg presence in the tumor microenvi-ronment (Fig. 3E and F)—observations also seen upon Treg-specific YAP deficiency across other, distinct tumor models as well. These findings strongly suggest that YAP is important for the accumulation and suppressive function of Tregs in the tumor microenvironment. Furthermore, they imply that tar-geting YAP should be a potent means of overcoming immune suppression in the cancer setting and improving the efficacy of endogenous and therapeutically induced tumor killing by leukocyte. Further characterization of YAP expression by Treg subsets found in different healthy and diseased tissues (including tumors) should more clearly define this factor’s role in immune control in specific physiologic contexts.

Here, we present a body of data strongly suggesting a Treg-specific role for YAP in promoting the immune sup-pression capable of allowing the persistence and progression of tumors in the cancer setting. Indeed, YAP-expression pat-terns and the dramatically stunted tumor growth seen in Yapfl/fl FOXP3Cre+ mice support this. However, comparing the degree of antitumor effect resulting from T cell– and Treg-driven YAP deficiency, it appears that a slightly more dramatic effect is seen in the former case. Although the bulk of the effect seen in Fig. 3A is phenocopied by the more restrictive deletion of YAP in only FOXP3+ cells (Fig. 3D), it is possible that YAP may play a tumor-abetting role in some other T-cell population capable of inducing the factor in the cancer setting. Although such YAP expression appears to have relatively minor consequences next to Treg-derived






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7 9 11 13 15 17 19 21 23 25

IgG1

Anti-activin A

Anti-activin A+GM-Vac

GM-Vac

**

*

105

104

103

−103

105

104

103

−103

−103 103 104 1050

0

105

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−103 103 104 1050

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0

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105

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−103 103 104 105

0

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−103 1030

0 51.1

0 48.9 0 20.3

0 79.7

104 105 −103 1030 104 105

0

105

104

103

−103

−103 1030 104 105

0

105

104

103

−103

−103 1030 104 105

0

0 38.3

0 61.7

0 5.27

0 94.7

105

104

103

−1030*

CD3+ TIL

*

ns**

ns

ns

*

% F

OX

P3+ o

f CD

4+

lgG1Anti-activin AGM-VacAnti-activin A+GM-Vac

CD3+ CD8+ CD3+ CD4+0

20

40

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% IF

Nγ *

*

ns

ns

ns ns

****

******

ns *

F

IFN

γ

CD4

IFN

γ

CD8

lgG1 Anti-activin A GM-Vac Anti-activin A+GM-Vac

CD3+CD4+ TIL

FO

XP

3

CD4

FO

XP

3

CD4

lgG1 Anti-activin A

GM-Vac Anti-activin A+GM-Vac

% IF

Nγ+

WTAcVR1c KO

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*

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20

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80

CD3+CD4+ CD3+CD8+

CD3+ TIL

IFN

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TNFα

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0

WT AcVR1c KO

*

% F

OX

P3+ o

f CD

4+

WT AcVR1c KO

FO

XP

3

IL17

WT AcVR1c KOCD4+

13 15 17 19 21 230

500

1,000

1,500

Tum

or v

olum

e (m

m3 )

*

**

nsns

ns

WT

AcVR1c KO

Days

*

80

60

40

20

0

IgG1

Anti-a

ctivin

A

Anti-a

ctivin

A+G

M-V

ac

GM-V

ac

8.16 13.7 5.04

75.5

53.7 2.76

41.9 1.61

5.73

3.21

84.8 3.85

37.0 4.67

56.2 2.18

40.4

57.2

1.16 29.4 0.54

68.6 1.471.25






YAP, at least in the tumor models used in our study, future work may bring to light additional layers of YAP’s protumor effects involving cells beyond FOXP3+ Tregs (such as aner-gized or exhausted T cells, non–FOXP3-expressing TR1 Tregs, etc.). These too may be susceptible to YAP-targeting strate-gies, which, based on our results, clearly should have potent antitumor effects.

Indeed, using a known YAP antagonist with modest inhibi-tory activity (29), we confirmed the potential of YAP as a target for Treg-undermining immunotherapies. Although inhibiting YAP alone slightly decreased tumor growth, we observed strong synergy in antitumor activity and immunity-boosting effects when the drug was combined with a tumor vaccine and checkpoint inhibitor treatment that alone possess much less potent effects. These findings suggest that YAP-targeting approaches should increase the efficacy of current immuno-therapies, potentially by enhancing the presence of activated effector leukocytes in the tumor microenvironment.

Analysis of the downstream targets of YAP activity in Treg identified ACVR1C led to the finding that the activin–activin receptor signaling axis plays a major role in the augmentation of TGFβ/SMAD signaling and Treg gen-eration and function (summarized in Supplementary Fig. S13). This pathway is highly important for the induction of extrathymic FOXP3+ T cells from naïve CD4+ precursors, as SMADs bind critical enhancer regions for the FOXP3 gene (6, 33). It is also important for sustaining FOXP3 expres-sion and suppressive function in Tregs (7), and TGFβ has been implicated as a promoter of survival and phenotypic stabilization of thymic Tregs (34, 35). With such reliance on TGFβ and SMAD signaling, it stands to reason that Tregs use mechanisms to optimize or amplify the downstream signaling events and resultant gene regulation triggered by this pathway. Such amplification mechanisms can be important for maintaining the gene expression and phe-notype traits underlying the suppressive function of Tregs. Documented examples include the enzymatic conversion of latent TGFβ to its active form (36) and the triggering of SMAD activation by galectin and CD44 (11). The upregula-tion of YAP and subsequently ACVR1C—the receptor for a known enhancer of SMAD signaling (i.e., activin)—may serve as an additional mechanism for amplifying this decid-edly pro-Treg cascade. Herein activin/ACVR1C signaling can enhance the downstream signaling events triggered by TGFβ. Reports of activin expression in several tumor types (37, 38) support the notion that tumor-accumulating Tregs

benefit particularly from activin/ACVR1C signaling facili-tated by YAP induction.

Our proof-of-concept experiments demonstrate that this pro-Treg amplification mechanism is susceptible to thera-peutic disruption. Particularly, our findings suggest that antibody-mediated activin blockade may prove a most effec-tive means for the disruption of Treg- and tumor-abetting TGFβ activation in patients with cancer. Additionally, the development and vetting of therapeutic antibodies capable of neutralizing activin, AcVR1C, or blocking its association with AcVR1C in patients with cancer may lead to new and potent immunotherapeutic regimens capable of releasing antitumor immunity from stifling Treg-enforced tolerance. On the other hand, our findings suggest that supplementation of activin or other therapeutic enhancements of the activin/ACVR1C axis could have considerable potential as a strategy to correct inadequate immune regulation in settings of autoimmunity (e.g., multiple sclerosis) or inflammatory disease (e.g., inflam-matory bowel disease). Future application of YAP inhibitors or activin/ACVR1C ablation in mouse models relevant to these and other pathologies of immune dysregulation will shed light on whether this pro-Treg loop is generally impor-tant for immune control or if it is principally operative in the tumor setting.

Our findings are, to our knowledge, the first to implicate YAP as a transcriptional facilitator of Treg differentiation and function. Although this molecule has been previously studied for its regulation of development, organ size, regeneration, and tumorigenesis (39), and its role as a transcriptional effec-tor of gene expression downstream of the Hippo pathway is well established, the importance of the Hippo pathway and its associated cofactors in Tregs and immune control is only beginning to be understood. A recent study showed that the Hippo pathway kinase known as MST1 plays an important role in stabilizing FOXP3 protein levels and supporting Treg function (40). Our present findings reveal that YAP potenti-ates Treg-supporting SMAD activity in T cells through activin signaling. Notably, though, this unexpected role appears to be independent of other Hippo factors (i.e., MST1/2 and LATS1/2), as these, unlike YAP, were not highly upregulated in developing Tregs. Interestingly, another Hippo effector known as TAZ (regarded to be a YAP paralog) was recently identified as a promoter of Th17 differentiation in naïve CD4+ T cells and a negative regulator of FOXP3 function and expression in these cells (41). This role for TAZ in the generation of proinflammatory T cells was also apparently

Figure 7. Activin blockade and AcVR1c deficiency slows B16 tumor growth and enhances the antitumor immune response. A, B16 melanoma cells were injected into individual female C57BL/6 mice (8–12 weeks of age). Tumor-bearing mice were randomly assigned into treatment groups once tumors were palpable ∼7 days after injection. Anti-activin A antibodies (R&D Systems) were administered (100 μg/mouse/injection) intraperitoneally twice a week once to one group. Another group received like doses of control IgG1. Other cohorts of tumor-bearing mice received GM-vaccine [100 μL of 1 × 106 lethally irradiated (150 Gy) B16 GM-vaccine cells or combined anti-activin/GM-vaccine treatment; n = 10 mice per group]. B, Treg frequencies among the TILs of treated mice. Intracellular staining of FOXP3 in CD4+ TILs from the indicated treatment groups were determined by flow cytometry. C, IFNγ-producing CD4+ and CD8+ T cells recovered from tumor cell suspensions were similarly assessed. D, The right flank of 8-week WT and AcVR1c KO female mice (C57BL/6 background; n = 8/group) were injected with 4 × 105 B16 cells in 100 μL PBS. E, The proportions of IFNγ- and TNFα-expressing T cells (CD3+) with the TILs of these mice were determined by flow cytometry (F) as were the frequencies of FOXP3 and IL17+ CD4+ T cells. For A and D, tumor development and changes in tumor volume were recorded for all groups, and the mean volume ± SEM for each are displayed. For B, C, E, and F, representa-tive flow plots from a single mouse from each group are depicted (left) alongside the mean cell frequencies across 3 independent experiments (right). All experiments were repeated at least three times. Significant differences were determined by a Student t test for all panels, except A, where a two-way ANOVA was used. *, P < 0.05; **, P < 0.01; ***, P < 0.002; ****, P < 0.001.






beyond its traditional Hippo-dependent role. Taken together, these newly uncovered immunologic roles played by YAP and TAZ suggest that different molecular players in the Hippo pathway can have functionally opposite and mechanistically distinct roles in determining the balance between inflamma-tion and tolerance. Further dissection of this pathway in T cells should add considerably to our understanding of this balance and, based on our current study’s findings, may lead to potent new immunotherapy approaches.

METHODSMice

C57/BL6 Yapfl/fl mice were generous gifts of Dr. Duojia Pan. C57/BL6 AcVR1c knockout mice were gifts from Dr. Ning Lu. C57/BL6 CD4-cre and FOXP3-YFP-Cre transgenic mice were purchased from The Jackson Laboratory. Smad2−/−, Smad3−/−, and Smad2/3 double knockout mice on a C57BL/6 background were originally obtained from Dr. Se-Jin Lee’s laboratory and were previously described (42). All animal experiments performed were approved by the Johns Hopkins University Institutional Animal Care and Use Committee.

In Vitro T-cell DifferentiationNaïve CD4+ T cells (CD4+ CD25− CD62Lhi) were sorted on a

FACSAria II high-speed sorter. The sorted cells were activated with plate-bound anti-CD3 (1 μg/mL) and soluble anti-CD28 (2 or 4 μg/mL) in a 24-well plate with the following polarizing conditions: Th1 [IL12 (10 ng/mL), anti-IL4 (10 μg/mL)], Th2 [IL4 (10 ng/mL), anti-IFNγ (10 μg/mL), anti-IL12 (10 μg/mL)], Th17 [IL6 (10 ng/mL), TGFβ1 (1.25 ng/mL), IL23 (10 ng/mL), IL1β (10 ng/mL), anti-IFNγ (10 μg/mL), anti-IL4 (10 μg/mL)], Treg [TGFβ1 (5 ng/mL, or as indi-cated), IL2 (100 IU/mL)] typically for 4 days, unless otherwise indicated.

Human T-cell Isolation from Peripheral BloodDeidentified human peripheral blood was obtained from blood

bank in strict accordance with the Johns Hopkins University School of Medicine’s Institutional Review Board guidelines. Samples were obtained from a total of 10 healthy adult volunteers (age range, 30–46 years). Peripheral blood mononuclear cells were extracted from whole blood through a gradient of Ficoll–Paque PLUS (GE Healthcare). CD4+ T cells were enriched using a Dynabeads Untouched CD4 T-cell isola-tion kit (Invitrogen). Tregs were identified and flow sorted via the fol-lowing staining profile: CD3+/CD4+/CD8−/CD25hi/CD127lo/CD39+. Non-Treg CD4+ T cells were sorted as previously described (43).

In Vitro Suppression AssayWT naïve CD4+ T cells (0.1 × 106) were labeled with carboxyfluo-

rescein diacetate succinimidyl ester (CFSE) and cultured in a 96-well bottom plate with anti-CD3/CD28-conjugated beads at a cell-to-bead ratio of 1:1. Serially diluted Tregs (CD4+ CD25hi) were cocul-tured for 72 hours, and cellular proliferation by CFSE was measured by flow cytometry.

Lentivirus Production and TransductionHEK293T cells were purchased from the ATCC in 2015 and were kept

as a frozen stock. This cell line has not been authenticated by the labo-ratory. Recombinant lentiviruses were generated using a three-plasmid system as described previously (44). The AcVR1c cDNA was cloned into the modified pLV lentiviral vector carrying cytomegalovirus-driven Thy1.1 as a transduction efficiency marker. Virus was harvested at 48 and 72 hours after transfection, and titer was determined based on percentages of Thy1.1-positive Jurkat T cells after transduction with

serially diluted viral supernatant. The titer, calculated as transducing units (TU)/mL of supernatant, was from 2 × 106 to 8 × 106 TU/mL. The virus-containing supernatant was concentrated using an Amicon Ultra Concentrator (Millipore) and stored at −80°C. Gene transduc-tion into CD4+CD25− conventional T cells and CD4+CD25+ Tregs was performed by stimulating cells with plate-bound anti-CD3 (10 μg/mL) and soluble anti-CD28 (1 μg/mL) with 60 U/mL human recombinant IL2 for 16 hours. Activated T cells were transduced with viral supernatants supplemented with 60 U/mL IL2 and 8 μg mL-1 polybrene, followed by centrifugation for 1 hour at 2,500 rpm. After transduction, 20 U/mL human recombinant IL2 (eBioscience) was added to the culture. At 40 hours after transduction, Thy1.1+ Tregs were sorted for western blot and/or suppression assay as indicated.

RNA-seq AnalysisSpleen and peripheral lymph nodes were harvested from YapWT/WT;

CD4-Cre-WT and Yap flox/flox (fl/fl); CD4-Cre+ mice (n = 5/group). CD4+ T cells were magnetically enriched, and naïve (CD4+ CD62L+ CD25−) T cells and natural Tregs (nTregs, CD4+ CD62L+/− CD25hi) were flow sorted from each group. For activation con-dition, sorted nTregs were further activated with 2 μg/mL of plate-coated αCD3 and 2 μg/mL of soluble αCD28 with TGFβ1 (5 ng/mL) and IL2 (100 U/mL) for 24 hours. nTregs (2 × 106; no stimulation or stimulation) from WT and Yap cKO mice were har-vested and washed with 1× PBS twice and immediately snap-frozen until further RNA-seq analysis.

Construction of RNA-seq LibrariesTotal RNA was isolated by TRIzol from naïve CD4+ T cells or

natural Tregs with or without the stimulation anti-CD3/CD28 for 48 hours from WT or Yap cKO mice. RNA quality was monitored on Bioanalyzer. Strand-specific RNA-seq libraries were prepared using the TruSeq Stranded Total RNA LT Sample Prep Kit (with Ribo-Zero Gold, RS-122-2301, Illumina) from 322 ng of total RNA by fol-lowing the manufacturer’s protocols. Briefly, rRNAs were depleted using biotinylated, target-specific oligos combined with Ribo-Zero rRNA removal beads. After purification, RNA was fragmented using divalent cations under elevated temperature, and transcribed into first-strand cDNA using reverse transcriptase and random primers, followed by second-strand cDNA synthesis using DNA Polymerase I and RNase H. A single “A” base was added to these cDNA frag-ments that were subsequently ligated with the adapter. The products were enriched with 12-cycle PCR. The concentration of final cDNA libraries in 30 μL ddH2O reached 24 to 27 ng/μL as determined on Qubit 2.0.

Analysis of RNA-seq DataSequencing was performed on Illumina HiSeq2000 at Beijing

Genomics Institute with the type of paired-end, 100 bp. Data quality was assessed by FastQC software (http://www.bioinformatics.babraham. ac.uk/projects/fastqc/). Mapping to a mouse reference genome (mm10) was conducted by TopHat. Differentially expressed genes were called by Cuffdiff (45). The genes with P value < 0.05 and abso-lute values of log2-transformed fold changes larger than 1.5 between WT and Yap cKO T cells were considered differentially expressed. A heat map was generated in R statistical software using the geom_tile function under ggplot2 package. Clustering was done with the com-plete linkage and Euclidean distance using the hclust function in R statistical software. Pathway analysis (Ingenuity) was carried out as described previously (46).

Flow CytometryFor extracellular staining, harvested cells were washed and incu-

bated in PBS containing 1% FBS containing the below fluorochrome-






conjugated antibodies in a U-bottom 96-well plate. For intracellular cytokine staining, harvested cells were restimulated in PMA and ionomycin in the presence of Golgi-Plug (BD Biosciences). After 5-hour incubation, the cells were fixed/permeabilized (eBioscience) and incubated with antibodies (see Supplementary Table S1A for a comprehensive list). For cellular proliferation, cell Trace CFSE cell proliferation kit (Invitrogen) was used per the manufacturer’s manual.

Quantitative Real-Time PCRRNA was extracted using TRIzol (Invitrogen) followed by cDNA

synthesis reaction using SuperScript III (Invitrogen) in a 20 μL reaction/well. The same amount of RNA was used in each cDNA synthesis reaction measured by NanoDrop Spectrophotometer (ThermoScientific). The same volume of cDNA per sample was pre-pared for real-time PCR analysis using SYBR Green (Pierce) and the indicated primers to assess transcript levels of each gene.

Tumor Growth ExperimentsMurine B16 melanoma, MC38 colon cancer, and EL4 thymoma

cell lines were purchased from the ATCC and kept as frozen stock in 2015. These cell lines have not been authenticated by the labora-tory. Cells were cultured in vitro in DMEM plus 10% heat-inactivated FBS and were detached by trypsinization and washed prior to s.c. injection into the shaved side flank of the indicated strains of female mice between the ages of 6 and 8 weeks on a C57BL/6 background (1 × 105 cells). In some experiments, 1 × 104 to 5 × 104 B16 melanoma cells were injected into each mouse in the footpad. Where indicated, once tumors were palpable (7–10 days after injection), 100 mL of 1 × 106 lethally irradiated (150 Gy) B16 GM-vaccine cells (GM-VAX) were injected s.c. into the contralateral limb. A hybridoma cell line expressing a blocking anti–PD-1 antibody (clone G4) was obtained from Dr. Charles Drake. One hundred microgram/mouse/injection of anti–PD-1 (G4) was injected intraperitoneally twice a week once tumors were palpable (7–10 days) in conjunction with vaccine and verteporfin (USP, USP-1711461) treatments. Verteporfin was dosed at 2 mg/mouse diluted to 200 μL with PBS and injected intraperi-toneally every two days. Activin neutralization antibodies and iso-type control IgG were purchased from R&D Systems. One hundred microgram/mouse/injection of activin-neutralizing antibodies was given intraperitoneally twice a week. For all these experiments, 5 to 10 mice were used per group. Tumor progression was assessed by measuring changes in tumor length (L) and width (W) and tumor volume (V) over time. Tumor volume was calculated using the for-mula (L × W2)/2.

Molecular Cloning and Site-Directed MutagenesisMouse AcVR1c promoter (1.2 kb) was cloned from the genomic

DNA of isolated CD4+ T cells, and the sequence was confirmed. The amplified clones were ligated to SacI/XhoI-digested pGL4.1-Basic Vector (Promega) using the In-Fusion Cloning Kit (Clontech). Site-directed mutagenesis was carried out using the QuikChange Light-ning Kit (Agilent Technologies).

Transient Transfection and Luciferase AssayJurkat T cells (clone E6-1) were purchased from the ATCC in

2016 and were kept as a frozen stock. This cell line has not been authenticated by the laboratory. Jurkat T cells (1.5 × 107) were transfected with 5 μg pGL4.1-AcVR1c, 1 μg of pRL-TK Vector (Promega), and other indicated plasmids by electroporation using Nucleofector II (Amaxa/Lonza). The cells were rested overnight and stimulated with mock or PMA/ionomycin for 8 hours before being harvested and lysed followed by luminescence measurement

using a Dual-Luciferase Assay (Promega) as per the manufacturer’s instructions.

ChIP AssayChIP assay was performed according to the manufacturer’s guid-

ance (Invitrogen MAGnify ChIP system). Briefly, sorted CD4+ iTregs were activated with αCD3/αCD28–conjugated beads overnight and fixed with 2% formaldehyde. Sonicated DNA was immunoprecipi-tated with anti-YAP1 (Cell Signaling Technology), and anti-TEAD1 (Santa Cruz Biotechnology). The immunoprecipitated chromatin was analyzed on Roche LightCycler 480 by SYBR Green using the following primers for AcVR1c promoter: 5′-CATTGACGTCTCTATG GAAG-3′ (forward), 5′-CAAGCACCATTGCCTTCAGAC-3′ (reverse).

Statistical AnalysesValues are presented as means ± SEM where appropriate. Statisti-

cal differences among multiple groups were determined using a two-way analysis of variance (ANOVA) with a Newmane–Keuls multiple comparison test, unless otherwise indicated. Unpaired, two-tailed Student t tests were used for single comparisons. In general, P values <0.05 were considered statistically significant and are indicated as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.002; ****, P < 0.001; ns, not significant. GraphPad Prism 7 was used to calculate P values.

Data AvailabilityRNA-seq dataset has been uploaded to an appropriate online

repository. The GEO accession number is GSE112593.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors’ ContributionsConception and design: D. Pardoll, L. Lu, D. Pan, F. PanDevelopment of methodology: J. Tao, Q. Chen, P. Wei, D. Pardoll, L. Lu, F. PanAcquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): X. Ni, J. Tao, J. Barbi, Q. Chen, B.V. Park, N. Zhang, A. Lebid, A. Ramaswamy, P. Wei, Y. Zheng, X. Wu, P. Vignali, C.-P. Yang, L. Lu, D. PanAnalysis and interpretation of data (e.g., statistical analysis, bio-statistics, computational analysis): X. Ni, J. Tao, J. Barbi, B.V. Park, Z. Li, Y. Zheng, X. Zhang, P. Vignali, D. Pardoll, L. Lu, F. PanWriting, review, and/or revision of the manuscript: X. Ni, J. Barbi, B.V. Park, A. Ramaswamy, H. Li, D. Pardoll, L. Lu, D. Pan, F. PanAdministrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J. Barbi, B.V. Park, L. LuStudy supervision: H. Li, D. Pardoll, L. Lu, F. Pan

AcknowledgmentsF. Pan’s research is supported by the Bloomberg-Kimmel Insti-

tute (Immunometabolism Program & Immune Modulation Pro-gram), the Melanoma Research Alliance, the NIH (RO1AI099300, RO1AI089830, and R01AI137046), and The DoD (PC130767). J. Barbi’s research is supported by the Melanoma Research Founda-tion, Phi Beta Psi, the Roswell Park Alliance Foundation, and NCI grant P30CA016056. The Li Lab was supported by the National Natural Science Committee of China (No. 81725004) and Shanghai Science and Technology Committee (No. 16410723600). L. Lu’s research is supported by the National Natural Science Fund of China (grants 81571564, 1521004, and 81522020) and the Founda-tion of Jiangsu Collaborative Innovation Center of Biomedical Func-tional Materials. D. Pan is an investigator of the Howard Hughes Medical Institute.






Received October 6, 2017; revised April 5, 2018; accepted June 6, 2018; published first June 15, 2018.

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2018;8:1026-1043. Published OnlineFirst June 15, 2018.Cancer Discov Xuhao Ni, Jinhui Tao, Joseph Barbi, et al. ImmunityYAP Is Essential for Treg-Mediated Suppression of Antitumor

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