RNA binding proteins ZFP36 and ZFP36L1 limit CD8+ T cell differentiation and effector function

Abstract

Post-transcriptional regulation of gene expression is mediated in part by RNA binding proteins (RBPs). The ZFP36 family of RBPs are key regulators of gene expression in the immune system. The founding member ZFP36 is a well-known regulator of cytokine mRNA stability. Paralogs ZFP36L1 and ZFP36L2 act redundantly during thymopoiesis and in developing B lymphocytes, where they limit DNA damage response and cell cycle progression. Furthermore, ZFP36L2 has been shown to repress translation of preformed Ifng mRNA and maintain quiescence in CD8+ memory T-cells. However, roles for the ZFP36 proteins in the differentiation and effector functions of CD8+ T cells remain to be explored. My project utilized mice featuring the OT-1 transgenic T cell receptor to produce cytotoxic T lymphocytes (CTLs) and memory-like CD8+ T cells in vitro. I made use of knockout mouse models with CD4cre-mediated conditional deletion of Zfp36 and Zfp36l1, to determine the role of these RBPs in limiting CD8+ T cell differentiation and effector functions. Using this approach, I demonstrated that ZFP36L1 acts to limit the cytotoxicity of CTLs and, by employing a CRISPR/Cas9 mediated gene knockout system, I demonstrated that this occurs early after initial T cell stimulation. I also demonstrated that upon adoptive transfer, memory-like CD8+ T cells form part of the central memory niche and respond to Listeria monocytogenes-OVA infection by differentiating and proliferating and that cells that lack ZFP36 and ZFP36L1 show more rapid terminal differentiation and improved effector functions. To begin to assay the molecular mechanisms by which ZFP36L1 limits CD8+ T cell differentiation, I made use of a recently published improved individual nucleotide crosslink immunoprecipitation methodology to identify directly bound target mRNAs. I demonstrated that transcripts encoding for factors in the MAPK, PI3K-AKT, and JAK/STAT signaling pathways are bound by ZFP36L1 and transcription factors IRF8 and Notch-1, and multiple cytokines and chemokines, including IFN-γ, TNF-α, IL-2 and CCL3/4. In addition, I describe a novel reporter mouse model of ZFP36L1 with an N-terminal fusion of a red-fluorescent protein expressed from the endogenous Zfp36l1 locus. Using this model, I analyzed the kinetics of ZFP36L1 expression in response to TCR stimulation. These findings demonstrate a role for ZFP36 and ZFP36L1 in coordinating both the differentiation and effector functions of CD8+ T lymphocytes. In the future, I hope to determine the mechanisms of action for ZFP36 RBPs in limiting CD8+ T cell differentiation.