Role of Tim-3:Bat-3 pathway in inducing tolerogenic DCs and peripheral tolerance

Brigham And Women'S Hospital, Boston MA

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Abstract

PROJECT SUMMARY: First cloned in our laboratory, TIM-3 is a hallmark of T cell exhaustion and dysfunction in murine and human chronic viral infections and cancer. Since its discovery, TIM-3 has been recognized as an important negative regulator of T cell functions, which has formed the basis for checkpoint blockade immunotherapies being developed for cancers. Our recent studies suggest that TIM-3 also antagonizes the expression of TCF1, a crucial transcription factor expressed by a recently discovered subset of T cells with stem-like properties that are the source of effector T cells. However, TIM-3 is also highly expressed on dendritic cells (DCs). Despite the high levels expressed on DCs, the role of TIM-3 in these cells remains unclear. The focus of this renewal is on TIM-3 expression in DCs and how this co-inhibitory molecule regulates DC functions and orchestrates the priming and fate of T cells. To interrogate TIM-3-mediated effects on DC functions, we have generated mice in which TIM-3 deficiency is restricted to DCs. Initial studies with these conditional knockout mice revealed that TIM-3 expressed on DCs is pivotal in immune regulation, including in autoimmune diseases and cancer. While we have known that mice deficient in TIM-3 develop more severe experimental autoimmune encephalomyelitis (EAE) and are better at suppressing tumor growth, our published and preliminary findings indicate that TIM-3 expressed on DCs, has profound effects on these disease outcomes. In these experimental models, we found that mice with DC-specific loss of TIM-3 generated an expanded pool of stem-like CD4+ T cells. Further investigation revealed that multiple pathways, including inflammasome and cGAS-STING, were perturbed in TIM-3- deficient DCs upon activation. These observations led us to hypothesize that TIM-3 curbs the activation of DCs with functional implications for the generation and maintenance of autoreactive stem-like CD4+ T cells. Uncovering the molecular mechanisms by which TIM-3 exerts its effects on DC functions and influences T-cell-mediated immunity has fundamental and therapeutic importance. Thus, to test this hypothesis, we propose two specific aims to: 1) elucidate the functions of TIM-3 in DCs that impact the generation of stem-like autoreactive CD4+ T cells driving CNS autoimmunity, and 2) uncover the molecular mechanisms underlying TIM- 3:Inflammasome crosstalk and its impact on CNS autoimmunity. Using an extensive suite of mutant mouse lines, we outline experiments that will tease apart the TIM-3- mediated molecular interactions between DCs and T cells during CNS autoimmunity and build on the advances made during the previous grant cycle. By combining the power of sequencing and CRISPR/Cas9 genetic screening with in vitro functional assays and in vivo disease models, we will explore how TIM-3 signaling regulates DC function and generation of autoreactive stem-like T cells. The proposed studies will provide a greater understanding of the contribution of cell-intrinsic and -extrinsic effects of TIM-3 in DCs on the development and persistence of autoimmunity.