RORγt plays vital roles in development and differentiation of multiple immune cell types with pleiotropic functions, including thymocytes, LTi cells, ILC3, γδ T-cells and Th17 cells. Moreover, RORγ, the isoform of RORγt, was reported to promote cancer cell survival in castration-resistant prostate cancer. RORγ is expressed in tissues like muscle, liver and brain, but its function remains unclear. To address the mechanisms by which RORγt/RORγ contributes to diverse cell fates and differentiation programs, we are exploring RORγt post-translational modifications, RORγt/RORγ-associated protein complexes and downstream signals. To identify RORγt/RORγ-associated protein complexes, we generated knock-in mice expressing Strep-tagged RORγt/RORγ, which had IL-17A inducing activity similar to that of non-tagged RORγt in Th17 cells polarized in vitro and allowed for highly specific isolation of protein complexes. Strep-tagged RORγt proteins will be precipitated from thymocytes and polarized pathogenic or nonpathogenic Th17 cells, followed by mass-spectrometry to identify RORγt-associated protein partners and analysis of their functions in RORγt expressing cells. Moreover, due to its nonoverlapping expression patterns with RORγt and obscure functions, RORγ is very interesting to investigate in terms of associated protein complexes and roles in other tissues. RORγt mutations were employed to dissect its roles in cell fate determination and differentiation. We identified a RORγt mutant by random mutagenesis, designated as MUT, which dramatically lost transcriptional activity when expressed in 293T-cells and in vitro polarized Rorc-KO Th17 cells. Mice harboring MUT mutation had normal thymocyte differentiation and development of secondary lymphoid tissues. No lymphomas were detected in MUT mice, unlike in C57BL/6 RORγt-KO mice that develop T-cell lymphomas. Strikingly, T-cells and ILCs differed in proportions and numbers according to tissues. In secondary lymphoid organs, including draining lymph nodes and spleen, no differences were detected in proportions or numbers of CD4 or CD8 T-cells. In large intestine, however, the proportions and numbers of γδ T-cells and Th17 cells were significantly reduced in MUT mice, while those of Th2 cells were markedly increased. Meanwhile, hyper-proliferation of RORγt-negative T helper cells and Treg cells was detected in large intestine of MUT mice. ILCs from large intestine, including ILC3 with high expression of RORγt, were similar to wild type mice in both proportions and numbers. A different phenotype was detected in small intestine, where T-cells and ILCs were markedly reduced. Given the different requirement or expression stage of RORγt for development of T-cells and ILCs, the results probably reflect the effects of RORγt-associated functions on microenvironments. Analyses of immune cells and microbiome at different ages may help to address the mechanism. The functions of MUT RORγt-expressing immune cells will be explored in homeostatic or pathogenic settings with SFB colonization or the EAE model. Moreover, comparison of transcriptomes of wild-type and MUT RORγt expressing cells may reveal essential targets for differentiation and function, which will be validated by transfer of bone marrow or T-cells engineered by CRISPR-mediated gene knockout or retroviral overexpression. Wild-type and MUT RORγt-associated protein complexes will be compared to interrogate potential mechanisms in directing cell differentiation. Detected by mass-spectrometry and biochemical technologies, the majority of RORγt proteins are phosphorylated in naïve thymocytes and in vitro polarized Th17 cells. Mice with respective mutations in RORγt were generated by CRISPR technology. Similar analysis as indicated above will be performed to interrogate roles of RORγt in cell fate determination and differentiation.
Citation Format: Hao Xu, Dan Littman, Wendy Huang. Dissection of RORγt functions in lymphoid cell fate determination and differentiation [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B198.