Mechanisms of Tim-3 Signal Transduction in the Modulation of Downstream TCR Signaling

T-cell immunoglobulin and mucin 3 protein (Tim-3) is a type-I transmembrane protein known to negatively regulate Th1 and Th17 CD4 T cells, and CD8 T cell mediated immune responses. Tim-3 expression level correlates with the severity of T cell exhaustion under conditions of chronic viral infection and tumor burden and Tim-3 antibodies can reverse this dysfunctional phenotype. Exploring the signal transduction mechanism of Tim-3, I demonstrated that ectopic expression of Tim-3 enhanced NFAT/AP-1, NFAT and NF-κB reporter activity induced by TCR stimulation, and enhanced AP-1 reporter activity independent of TCR stimulation. Two tyrosines in the cytosolic tail of Tim-3 are responsible for the potentiation of signal transduction by TCR in a redundant manner. I demonstrated that the Src family kinase (SFK) Fyn can phosphorylate Y256 and Y263 residues of Tim-3. An SH2 domain screen and co-immunoprecipitation identified p85 PI3K interaction with Tim-3 in a tyrosine phosphorylation-dependent manner. I also found that Tim-3 expression upregulates phosphorylation of PLC-γ1 and MAP kinases ERK1/2, p38, and JNK. Finally, I demonstrated that Tim-3 upregulates phosphorylation of ribosomal protein S6, a downstream target of PI3K and ERK, and IL-2 secretion induced by TCR stimulation. I conclude that potentiation of NFAT/AP1 and NF-κB activity by Tim-3 is mediated by enhancement of the PI3K, PLC-γ1 and ERK pathways. Thus, we have demonstrated a paradoxical activating function of Tim-3 in TCR signaling, while Tim-3 is known as a negative regulator of Th1 and Tc1 T cell mediated immune responses. This augmentation of TCR signaling by Tim-3 may contribute to driving and/or maintaining T cell exhaustion. Alternatively, Tim-3 may have a dual activation and inhibition role depending on the ligands available at a particular stage of T cell activation