Investigating the Role of RAC1 in TIM-3-Dependent Suppression of Dendritic Cells and its Impact on Stem-Like T Cells

Abstract

T cell stemness is essential for sustaining long-term immune responses in chronic infections, cancer, and autoimmunity. Dendritic cells (DCs) are known to associate with stem-like T cells niches, but the mechanisms by which DCs regulate T cell stem-like populations remain unclear. This study investigates the role of TIM-3, an inhibitory receptor expressed on DCs, in modulating T cell stemness through its interaction with the small GTPase RAC1. Using bone marrow–derived dendritic cells (BMDCs), CRISPR-mediated gene editing, and multiple in vivo models—including MC38-OVA, B16-OVA tumors, and experimental autoimmune encephalomyelitis (EAE)—this work suggests that TIM-3 deletion in DCs enhances their activation and promotes the expansion of TCF1⁺PD1⁺ stem-like T cells in both the CD8⁺ and CD4⁺ compartments. Mechanistically, TIM-3 and RAC1 were shown to physically associate, and deletion of RAC1 in TIM-3–deficient DCs reversed the enhanced cytokine production, T cell proliferation, and tumor control observed in the TIM-3 knockout condition. Moreover, among several candidate TIM-3 interactors identified by mass spectrometry and CRISPR screening, only RAC1 showed a functional rescue effect, reinforcing its specific role downstream of TIM-3. These findings reveal a previously uncharacterized TIM-3–RAC1 signaling axis in DCs that regulates the induction of stem-like T cells and shapes adaptive immune responses. This work not only advances mechanistic understanding of DC–T cell crosstalk but also opens new avenues for targeting the TIM-3–RAC1 pathway to enhance T cell–based immunotherapies.