Recent studies have elucidated the molecular mechanism of RORγT transcriptional regulation of Th17 differentiation and function. RORγT was initially identified as a transcription factor required for thymopoiesis by maintaining survival of CD4+CD8+ (DP) thymocytes. While RORγ antagonists are currently being developed to treat autoimmunity, it remains unclear how RORγT inhibition may impact thymocyte development. In this study, we show that in addition to regulating DP thymocytes survival, RORγT also controls genes that regulate thymocyte migration, proliferation, and T cell receptor (TCR)α selection. Strikingly, pharmacological inhibition of RORγ skews TCRα gene rearrangement, limits T cell repertoire diversity, and inhibits development of autoimmune encephalomyelitis. Thus, targeting RORγT not only inhibits Th17 cell development and function but also fundamentally alters thymic-emigrant recognition of self and foreign antigens. The analysis of RORγ inhibitors has allowed us to gain a broader perspective of the diverse function of RORγT and its impact on T cell biology.

Prostaglandin E₂ (PGE₂), an arachidonic acid pathway metabolite produced by cyclooxygenase (COX)-1/2, has been shown to impair anti-tumor immunity through engagement with one or more E-type prostanoid receptors (EP_1-4). Specific targeting of EP receptors, as opposed to COX-1/2 inhibition, has been proposed to achieve preferential antagonism of PGE₂–mediated immune suppression. Here we describe the anti-tumor activity of MF-766, a potent and highly selective small-molecule inhibitor of the EP₄ receptor. EP₄ inhibition by MF-766 synergistically improved the efficacy of anti-programmed cell death protein 1 (PD-1) therapy in CT26 and EMT6 syngeneic tumor mouse models. Multiparameter flow cytometry analysis revealed that treatment with MF-766 promoted the infiltration of CD8⁺ T cells, natural killer (NK) cells and conventional dendritic cells (cDCs), induced M1-like macrophage reprogramming, and reduced granulocytic myeloid-derived suppressor cells (MDSC) in the tumor microenvironment (TME). In vitro experiments demonstrated that MF-766 restored PGE₂-mediated inhibition of lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α production in THP-1 cells and human blood, and PGE₂-mediated inhibition of interleukin (IL)-2-induced interferon (IFN)-γ production in human NK cells. MF-766 reversed the inhibition of IFN-γ in CD8⁺ T-cells by PGE₂ and impaired suppression of CD8⁺ T-cells induced by myeloid-derived suppressor cells (MDSC)/PGE_2. In translational studies using primary human tumors, MF-766 enhanced anti-CD3-stimulated IFN-γ, IL-2, and TNF-α production in primary histoculture and synergized with pembrolizumab in a PGE₂ high TME. Our studies demonstrate that the combination of EP₄ blockade with anti-PD-1 therapy enhances antitumor activity by differentially modulating myeloid cell, NK cell, cDC and T-cell infiltration profiles.