Post-transcriptional regulation of T lymphocyte responses by RNA-binding proteins

Activation of T lymphocytes induces robust immune responses that lead to the complete eradication of invading pathogens and tumor cells. However, T cell responses must be highly controlled in magnitude and duration to avoid excessive damage that would lead to organ dysfunction and disease, as in the case of autoimmunity. To achieve such sophisticated level of control, T cells exploit an array of transcriptional and post-transcriptional regulatory mechanisms. This enables the acquisition of a phenotype tailored to the incoming stimulus while at the same time restraining unwarranted activation. Post-transcriptional regulation mediated by RNA-binding proteins (RBPs) affects all aspects of RNA biology, from maturation to stability and translation, thereby broadly impacting cellular functions. In the immune system, work from many groups including ours has shown that several RBPs are central to T cell biology and to the regulation of immune responses. However, only a handful of RBPs have been studied in depth, and the role in immunity of most post-transcriptional regulators remains to be uncovered.

The challenge. Hundreds of RBPs are expressed by T cells and are dynamically regulated in response to activation, as shown by systematic profiling of immune cells. The current bottleneck is therefore to ascribe functions to each of these regulators, and to mechanistically define their signal-dependent effects.

The solution. The overarching goal of this project is therefore to fill a large knowledge gap in fundamental immunology, by linking RBPs to human T cell regulation and to disease-relevant mechanisms. A greater mechanistic understanding of the RBP-dependent circuits that control T cell responses may expand our possibilities for therapeutic intervention in immune-related diseases. Specifically, we will pursue the following:

Aim 1. Assigning RBPs to post-translational regulation of T cell function. We performed a large-scale functional CRISPR screen in human CD4+ T helper cells to characterize the role of RBPs in modulating proliferation and production of inflammatory mediators. This led to the identification of RBPs that will be further investigated in this Aim. We will additionally expand our functional CRISPR screen to cytotoxic CD8+ T lymphocytes, providing a comprehensive resource regarding the functional roles of RBPs in human T cells, complemented by the mechanistic knowledge on selected RBPs obtained in Aim 2.

Aim 2. In depth mechanistic analysis of RBPs with a functional role in human T lymphocytes. Selected RBPs prioritized based on results obtained in Aim 1 will undergo detailed mechanistic studies using state-of-the-art methodologies already optimized in the lab. Using a combination of ‘omics’ and targeted approaches we will establish i) the dynamics of mRNA binding profiles in T cells in response to stimulation, ii) the impact on mRNA stability and protein translation and ultimately iii) the impact on functional and regulatory T cell programs, providing crucial mechanistic knowledge on RBP-mediated regulation in T cells.

Aim 3. In vivo control of T cell states by RBPs. We will generate knock-in mouse models genetically modified to express rapidly degradable RBPs in T cells, in order to modulate protein expression in vivo and assess the impact of selected RBPs on T cell differentiation and responses in models of autoimmunity and infection.

Overall, by combining functional studies in human T cells with state-of-the-art mechanistic analyses and in vivo models of disease, this project will bridge a major knowledge gap in the functional characterization of RBPs in the stimulus-dependent, post-transcriptional regulation of adaptive immunity, potentially enabling targeted manipulation of T cells for both autoimmunity and cancer immunotherapy.