Zfp335-Mediated Regulation of T cell Development

Abstract

T cells are a critical arm of the adaptive immune system which function to coordinate and orchestrate complex immune reactions, as well as, kill damaged and infected cells. Production of a diverse peripheral T cell compartment requires massive expansion of the bone marrow progenitors that seed the thymus. There are two main phases of expansion during T cell development, following T lineage commitment at the DN2 stage and following successful rearrangement and selection for functional TCRβ chains in DN3 thymocytes, which promotes development of DN4 cells to the DP stage. Signals driving expansion of DN2 thymocytes are well studied, however, factors regulating the proliferation and survival of DN4 cells remain poorly understood. E proteins are transcription factors which have been shown to play essential non-redundant roles throughout T cell development. The functions of E proteins in T cell development include, enforcing T lineage commitment, promoting proper TCR rearrangements, regulating developmental progression and functional checkpoints, and coordinating complex transcriptional networks underpinning developmental progression. Due to the large number of genome-wide binding sites and massive number of genes regulated by E proteins, their numerous functions are poorly understood. The goal of this dissertation is to determine the role of the E protein-regulated transcription factor Zfp335 in T cell development. We utilized conditional deletion models to determine the role of Zfp335 in early and late stages of conventional and unconventional T cell development. Through these efforts we uncovered we uncover an unexpected link between the transcription factor Zfp335 and control of the cGAS/STING pathway for sensing cytosolic DNA in post-β-selection DN4 thymocytes. The absence of Zfp335 drives cGAS/STING-dependent death of DN4 cells. Zfp335 controls survival by sustaining expression of Ankle2, which in turn regulates the activity of Baf to suppress cGAS/STING-dependent cell death. Additionally, genetic ablation of Zfp335 precludes the development of unconventional iNKT cells due to STING-independent cell death following lineage commitment along with preventing effector differentiation of surviving cells. Our studies also uncovered an additional cGAS/STING-independent role in the terminal maturation of conventional αβ T cells. The absence of Zfp335 prevents the establishment of a naïve T cell compartment, inhibits differentiation of CD4 T cells and promotes the developmental acquisition of an effector program in CD8 T cells. Using in vivo and ex vivo genetic manipulation combined with detailed bioinformatic analyses we show that Zfp335 functions to promote T cell development, maturation, and effector differentiation through the regulation of a small but essential set of genes.To our knowledge, these studies detail the first described role for cGAS/STING in T cell development and strongly suggest a transcriptional mechanism downstream of Zfp335 which coordinates genome-wide alterations to chromatin compaction required for proper establishment of conventional and unconventional T cell pools. Together, these studies will provide a novel framework for understanding the life and death of developing T cells and may uncover novel pathways for enhancing the efficacy of T cell-based therapeutics.