Foxp3+ regulatory T cells (Tregs) are required for immune homeostasis. One notable distinction between conventional T cells (Tconv) and Tregs is differential phosphatidylinositol 3-kinase (PI3K) activity: only Tconv downregulate PTEN, the primary negative regulator of PI3K, upon activation. Here, we show that control of PI3K in Tregs is essential for lineage homeostasis and stability. Mice lacking Pten in Tregs developed an autoimmune-lymphoproliferative disease characterized by excessive TH1 responses and B cell activation. Diminished control of PI3K activity in Tregs led to reduced CD25 expression, accumulation of Foxp3+CD25− cells and ultimately, loss of Foxp3 expression in these cells. Collectively, these data demonstrate that control of PI3K signaling by PTEN in Tregs is critical to maintain their homeostasis, function and stability.

SUMMARY Regulatory T cells (Treg cells) are required for immune homeostasis. Chromatin remodeling is essential for establishing diverse cellular identities, but how the epigenetic program in Treg cells is maintained throughout the dynamic activation process remains unclear. Here we have shown that CD28 co-stimulation, an extracellular cue intrinsically required for Treg cell maintenance, induced the chromatin-modifying enzyme, Ezh2. Treg-specific ablation of Ezh2 resulted in spontaneous autoimmunity with reduced Foxp3+ cells in non-lymphoid tissues and impaired resolution of experimental autoimmune encephalomyelitis. Utilizing a model designed to selectively deplete wild-type Treg cells in adult mice co-populated with Ezh2-deficient Treg cells, Ezh2-deficient cells were destabilized and failed to prevent autoimmunity. After activation, the transcriptome of Ezh2-deficient Treg cells was disrupted, with altered expression of Treg cell lineage genes in a pattern similar to Foxp3-deficient Treg cells. These studies reveal a critical role for Ezh2 in the maintenance of Treg cell identity during cellular activation.

SUMMARYRegulatory T cells (Tregs) are critical for maintaining immune homeostasis, but their presence in tumor tissues impairs anti-tumor immunity and portends poor prognoses in cancer patients. Here, we reveal a mechanism to selectively target and reprogram the function of tumor-infiltrating Tregs (TI-Tregs) by exploiting their dependency on the histone H3K27 methyltransferase enhancer of zeste homolog 2 (EZH2) in tumors. Disruption of EZH2 activity in Tregs, either pharmacologically or genetically, drove the acquisition of pro-inflammatory functions in TI-Tregs, remodeling the tumor microenvironment and enhancing the recruitment and function of CD8+ and CD4+ effector T cells that eliminate tumors. Moreover, abolishing EZH2 function in Tregs was mechanistically distinct from, more potent than, and less toxic than a generalized Treg depletion approach. This study reveals a strategy to target Tregs in cancer that mitigates autoimmunity by reprogramming their function in tumors to enhance anti-cancer immunity.In BriefEZH2 plays an intrinsic role in neoplastic cells as an oncogene, prompting the development of EZH2 inhibitors for cancer therapy. Wang et al. show that disrupting EZH2 function also has immunomodulatory activities and, when blocked in Tregs, promotes potent cancer immunity.

Immunosuppressive regulatory T cells (Tregs) are critical for maintaining immune homeostasis, but their presence in tumor tissues impairs anti-tumor immunity and portends poor prognoses in cancer patients. Targeting Tregs may be a powerful means to unleash more potent immune responses against cancer, but targeting these cells is challenging because their generalized inactivation may incite severe autoimmune toxicities. To selectively target Tregs in tumors, we investigated the role of the H3K27 methyltransferase EZH2 in Tregs and determined that its enhanced activity at tumor sites in mice and humans leads to more robust and stable Tregs. We demonstrate that blocking EZH2 activity, both pharmacologically and genetically, selectively reprograms the function of tumor-infiltrating Tregs without systemically altering Treg function. Genetic disruption of EZH2 in tumor-resident Tregs led to their acquisition of pro-inflammatory functions that remodeled the tumor microenvironment and enhanced the recruitment and function of effector T cells, leading to the complete elimination of tumors. Moreover, abolishing EZH2 function in Tregs was mechanistically distinct from, more potent than, and less toxic than a generalized Treg depletion approach. This study reveals a novel strategy to target Tregs in cancer that mitigates autoimmunity by reprogramming their function in tumors to enhance anti-cancer immunity.

High numbers of tumor infiltrating regulatory T (Treg) cells are indicative of poor outcome in several malignancies, including ovarian cancer, colorectal cancer, and melanoma. Thus, selectively abrogating intratumoral Treg cell function while maintaining systemic immune tolerance remains an attractive, albeit elusive, strategy for cancer immunotherapy. We have identified the epigenetic enzyme Ezh2, an H3K27 methyltransferase, to be a critical mediator of lineage stability and function in activated Treg cells. Consequently, deficiency of Ezh2 in Treg cells strongly impairs their function in non-lymphoid tissues. We hypothesized that this phenomenon might translate to a similar defect that is restricted to the tumor microenvironment. Here we tested this hypothesis in three transplantable syngeneic tumor models in mice (MC38 colon carcinoma, TRAMP-C2 prostate cancer, and B16F10 melanoma). We observed that constitutive deletion of Ezh2 in Treg cells resulted in potent anti-tumor activity, significantly impaired tumor outgrowth, and in many instances, complete tumor rejection. These results were also recapitulated when Ezh2 was temporally deleted in the vast majority of Treg cells at the time of tumor inoculation. Functional analyses at early time points after Ezh2 deletion revealed enhanced functionality of effector CD4 and CD8 populations, as evidenced by increased IFN-γ production. These effects were evident without reduction of intratumoral Treg cell frequencies and despite presence of residual wild type Treg cells. Mechanistic studies showed that Ezh2-deficient Treg cells were destabilized and produced pro-inflammatory cytokines IL-2 and IFN-γ selectively within the tumor microenvironment, suggesting a direct contribution of Ezh2-deficient Treg cells to tumor clearance. To more directly address this hypothesis, we generated mice that harbored both Ezh2-deficient and wild type Treg cells and observed anti-tumor activity similar to mice that exclusively harbored Ezh2-deficient Treg cells. In stark contrast to Ezh2 deficiency, mice whose Treg cells were completely depleted, using a Foxp3-driven diphtheria toxin receptor allele, were unable to reject tumors, further supporting a dominant role of Ezh2-deficient Treg cells in driving anti-tumor immunity. Finally, tumor protection in the presence of Ezh2-deficient Treg cells occurred without significant morbidity due to autoimmune pathologies, which were prevalent in mice with systemic Treg cell depletion. These results suggest that targeted deletion of Ezh2 in Treg cells drives a unique phenotype among tumor infiltrating Treg cells, reprogramming the tumor microenvironment and selectively augmenting the anti-tumor immune response. Cancer therapies that pharmacologically inhibit Ezh2 to target tumor cells are under investigation, going forward it will also be important to assess the potential for beneficial effects via modulating the immune response. Citation Format: David Q. Wang, Jason R. Quiros, Chien-Chun S. Pai, Lawrence H. Fong, Jeffrey A. Bluestone, Michel J. DuPage. Selective impairment of intratumoral regulatory T cells by targeting Ezh2 enhances cancer immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1014. doi:10.1158/1538-7445.AM2017-1014