Phosphotyrosine-Dependent Coupling of Tim-3 to T-Cell Receptor Signaling Pathways

The transmembrane protein Tim-3 has been shown to negatively regulate T-cell-dependent immune responses and was recently demonstrated to be associated with the phenomenon of immune exhaustion, which can occur as a consequence of chronic viral infection. Unlike other negative regulators of T-cell function (e.g., PD-1), Tim-3 does not contain any obvious inhibitory signaling motifs. We have found that ectopic expression of Tim-3 in T cells leads to enhancement of T-cell receptor (TCR)-dependent signaling pathways, which was observed at the level of transcriptional reporters and endogenous cytokine production. We have exploited this observation to dissect what elements within the cytoplasmic tail of Tim-3 are required for coupling to downstream signaling pathways. Here we have demonstrated that two of the more membrane-proximal cytoplasmic tail tyrosines are required for Tim-3 signaling to T-cell activation pathways in a redundant fashion. Furthermore, we show that Tim-3 can directly bind to the Src family tyrosine kinase Fyn and the p85 phosphatidylinositol 3-kinase (PI3K) adaptor. Thus, at least under conditions of short-term stimulation, Tim-3 can augment T-cell activation, although this effect can be blocked by the inclusion of an agonistic antibody to Tim-3. These findings should help further the study of Tim-3 function in other physiological settings, such as those that lead to immune exhaustion. During the expansion phase of an immune response to acute infection, newly activated antigen-specific T cells expand rapidly and acquire effector functions. This is then followed by a period of contraction, where all but 5 to 10% of these effector T cells succumb to apoptosis. The remaining T cells constitute the memory pool—multifunctional T cells that persist in the host in an antigen-independent manner with the ability to respond quickly upon reexposure to viral antigen. However, during chronic viral infection, effector CD8 T cells generated during the expansion phase fail to develop into memory CD8 T cells. Instead, these effector CD8 T cells appear to become exhausted (2, 33). T-cell exhaustion is characterized as the progressive and stepwise loss of the ability to secrete interleukin 2 (IL-2), tumor necrosis factor alpha (TNF-), and gamma interferon (IFN-) in response to antigenic stimulation, culminating (in the most extreme cases) in apoptosis (33). This system of clonal deletion has been documented under conditions of persistent antigen stimulation, such as high-grade chronic viral infections in both mouse and human and, most recently, in patients with advanced melanoma. Exhausted CD8 T cells have a distinct molecular signature that resembles that of effector T cells more than that of memory T cells (34). Of the several hundred genes differentially upregulated in exhausted CD8 T cells, some are inhibitory receptors, such as CTLA-4,