Differential regulation and functions of the ZFP36-family RNA binding proteins in CD8⁺ memory T cells

Abstract

CD8⁺ T cells target infected or malignant cells via cytotoxicity and the production of pro inflammatory cytokines and chemokines. In addition to antigen-specific responses mediated by the TCR, pro-inflammatory cytokines such as IL-12 and IL-18 can induce antigen-independent bystander activation of memory T cells. The ZFP36-family of RNA binding proteins, including ZFP36 and ZFP36L1, contribute to the regulation of mRNA stability, protein translation and therein, T cell function. Appropriate regulation of T cell-mediated immunity is critical to enable effective yet restrained responses and limit pathogenesis. The extent of non-redundancy between ZFP36 and ZFP36L1 functions in CD8⁺ T cells remains to be fully established. Furthermore, the mechanisms that choreograph their differential expression are incompletely defined. Our studies utilise in vitro differentiated murine OTI memory T cells for functional and molecular analyses. We utilise western blot and novel fusion reporter proteins, mAmetrineZFP36 and mCherryZFP36L1, which enable further characterisation of ZFP36 and ZFP36L1 expression by flow cytometry in response to a range of stimuli. By selectively inhibiting TCR-induced signalling pathways, we elucidate the roles of multiple key pathways that regulate ZFP36 and ZFP36L1 expression. Flow cytometry was also applied to compare cytokine production by CD4-Cre conditional Zfp36 and/or Zfp36l1 KO memory-like T cells. Regulation of Zfp36 and Zfp36l1 mRNA expression and stability was analysed by qPCR. ZFP36 and ZFP36L1 exhibit distinct TCR-induced expression kinetics; rapid and transient expression of ZFP36 is followed by delayed and sustained ZFP36L1 expression. We demonstrate a central role of calcium signalling in orchestrating the chronological transition between ZFP36 and ZFP36L1 expression following antigen stimulation. We find that pro-inflammatory cytokines IL-12 and IL-18, commonly associated with innate-like bystander activation of memory CD8⁺ T cells, also induce ZFP36 and ZFP36L1 expression that is primarily driven by IL-18. Functionally, we identify ZFP36 as a suppressor of IFN-γ production by CD8+ memory T cells in response to innate-like bystander activation. Our findings characterise the highly regulated expression of ZFP36 and ZFP36L1 in CD8⁺ memory T cells. We demonstrate functional suppression of cytokine production by ZFP36 in antigen-independent contexts. Together, this highlights ZFP36 and ZFP36L1 as intricately integrated regulators of CD8+ T cell responses which function to promote appropriately moderated immune responses.