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. 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.
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•EZH2 expression is elevated in tumor-infiltrating (TI) Tregs•Pharmacological inhibition of EZH2 destabilizes FOXP3 expression and slows tumor growth•Genetic disruption of Ezh2 function in Tregs leads to robust anti-tumor immunity•Blockade of EZH2 in Tregs reprograms TI-Tregs to gain pro-inflammatory activity
EZH2 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.
Foxp3(+) regulatory T cells (Treg cells) are required for immunological homeostasis. One notable distinction between conventional T cells (Tconv cells) and Treg cells is differences in the activity ...of phosphatidylinositol-3-OH kinase (PI(3)K); only Tconv cells downregulate PTEN, the main negative regulator of PI(3)K, upon activation. Here we found that control of PI(3)K in Treg cells was essential for lineage homeostasis and stability. Mice lacking Pten in Treg cells developed an autoimmune-lymphoproliferative disease characterized by excessive T helper type 1 (TH1) responses and B cell activation. Diminished control of PI(3)K activity in Treg cells led to reduced expression of the interleukin-2 (IL-2) receptor α subunit CD25, accumulation of Foxp3(+)CD25(-) cells and, ultimately, loss of expression of the transcription factor Foxp3 in these cells. Collectively, our data demonstrate that control of PI(3)K signaling by PTEN in Treg cells is critical for maintaining their homeostasis, function and stability.
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.
Abstract
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.
Foxp3 super(+) regulatory T cells (T sub(reg) cells) are required for immunological homeostasis. One notable distinction between conventional T cells (T sub(conv) cells) and T sub(reg) cells is ...differences in the activity of phosphatidylinositol-3-OH kinase (PI(3)K); only T sub(conv) cells downregulate PTEN, the main negative regulator of PI(3)K, upon activation. Here we found that control of PI(3)K in T sub(reg) cells was essential for lineage homeostasis and stability. Mice lacking Pten in T sub(reg) cells developed an autoimmune-lymphoproliferative disease characterized by excessive T helper type 1 (T sub(H)1) responses and B cell activation. Diminished control of PI(3)K activity in T sub(reg) cells led to reduced expression of the interleukin-2 (IL-2) receptor alpha subunit CD25, accumulation of Foxp3 super(+)CD25 super(-) cells and, ultimately, loss of expression of the transcription factor Foxp3 in these cells. Collectively, our data demonstrate that control of PI(3)K signaling by PTEN in T sub(reg) cells is critical for maintaining their homeostasis, function and stability.
Foxp3
+
regulatory T cells (T
regs
) are required for immune homeostasis. One notable distinction between conventional T cells (T
conv
) and T
regs
is differential phosphatidylinositol 3-kinase ...(PI3K) activity: only T
conv
downregulate PTEN, the primary negative regulator of PI3K, upon activation. Here, we show that control of PI3K in T
regs
is essential for lineage homeostasis and stability. Mice lacking
Pten
in T
regs
developed an autoimmune-lymphoproliferative disease characterized by excessive T
H
1 responses and B cell activation. Diminished control of PI3K activity in T
regs
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 T
regs
is critical to maintain their homeostasis, function and stability.
In vitro differentiation of human intestinal organoids (HIOs) from pluripotent stem cells is an unparalleled system for creating complex, multicellular three-dimensional structures capable of giving ...rise to tissue analogous to native human tissue. Current methods for generating HIOs rely on growth in an undefined tumour-derived extracellular matrix (ECM), which severely limits the use of organoid technologies for regenerative and translational medicine. Here, we developed a fully defined, synthetic hydrogel based on a four-armed, maleimide-terminated poly(ethylene glycol) macromer that supports robust and highly reproducible in vitro growth and expansion of HIOs, such that three-dimensional structures are never embedded in tumour-derived ECM. We also demonstrate that the hydrogel serves as an injection vehicle that can be delivered into injured intestinal mucosa resulting in HIO engraftment and improved colonic wound repair. Together, these studies show proof-of-concept that HIOs may be used therapeutically to treat intestinal injury.
In vitro differentiation of human pluripotent stem cell (hPSC)-derived organoids (HOs) facilitates the production of multicellular three-dimensional structures analogous to native human tissues. Most ...current methods for the generation of HOs rely on Matrigel, a poorly defined basement membrane derivative secreted by Engelbreth-Holm-Swarm mouse sarcoma cells, limiting the potential use of HOs for regenerative medicine applications. Here, we describe a protocol for the synthesis of a fully defined, synthetic hydrogel that supports the generation and culture of HOs. Modular, cell-encapsulating hydrogels are formed from a four-armed poly(ethylene glycol) macromer that has maleimide groups at each terminus (PEG-4MAL) and is conjugated to cysteine-containing adhesive peptides and cross-linked via protease-degradable peptides. The protocol also includes guidelines for the localized in vivo delivery of PEG-4MAL hydrogel-encapsulated HOs to injured mouse colon. The PEG-4MAL hydrogel supports the engraftment of the HOs and accelerates colonic wound repair. This culture and delivery strategy can thus be used to develop HO-based therapies to treat injury and disease. Hydrogel and tissue preparation and subsequent encapsulation can be performed within 2.5-3.5 h. Once HOs have been cultured in synthetic hydrogels for at least 14 d, they can be prepared and delivered to the mouse colon in under 5 h.
In response to injury, epithelial cells migrate and proliferate to cover denuded mucosal surfaces and repair the barrier defect. This process is orchestrated by dynamic crosstalk between immune cells ...and the epithelium; however, the mechanisms involved remain incompletely understood. Here, we report that IL-10 was rapidly induced following intestinal mucosal injury and was required for optimal intestinal mucosal wound closure. Conditional deletion of IL-10 specifically in CD11c-expressing cells in vivo implicated macrophages as a critical innate immune contributor to IL-10-induced wound closure. Consistent with these findings, wound closure in T cell- and B cell-deficient Rag1-/- mice was unimpaired, demonstrating that adaptive immune cells are not absolutely required for this process. Further, following mucosal injury, macrophage-derived IL-10 resulted in epithelial cAMP response element-binding protein (CREB) activation and subsequent synthesis and secretion of the pro-repair WNT1-inducible signaling protein 1 (WISP-1). WISP-1 induced epithelial cell proliferation and wound closure by activating epithelial pro-proliferative pathways. These findings define the involvement of macrophages in regulating an IL-10/CREB/WISP-1 signaling axis, with broad implications in linking innate immune activation to mucosal wound repair.
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