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 ϩ T cells generated during the expansion phase fail to develop into memory CD8 ϩ T cells. Instead, these effector CD8 ϩ 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 ϩ 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 ϩ T cells, some are inhibitory receptors, such as CTLA-4, LAG-3, and PD-1. Several studies have confirmed that PD-1 is upregulated on exhausted CD4 ϩ and CD8 ϩ T cells (4, 6, 7, 10). However, blocking the interaction between PD...

The transmembrane immunoglobulin and mucin domain (TIM) family was identified more than a decade ago. Although the founding member of the family was first described in a rat model of ischemia reperfusion injury (IRI), much of the recent interest in the TIM family members has focused on their potential roles in immunity. There are now a large number of genetic studies that have investigated the possible association of various TIM1 and TIM3 polymorphisms with different diseases. Here we review this body of literature, and highlight some of the most interesting studies.

Expression of the transmembrane protein Tim-3 is increased on dysregulated T cells undergoing chronic activation, including during chronic infection and in solid tumors. Thus, Tim-3 is generally thought of as an inhibitory protein. We and others previously reported that under some circumstances, Tim-3 exerts paradoxical costimulatory activity in T cells (and other cells), including enhancement of the phosphorylation of ribosomal S6 protein. Here, we examined the upstream signaling pathways that control Tim-3–mediated increases in phosphorylated S6 in T cells. We also defined the localization of Tim-3 relative to the T cell immune synapse and its effects on downstream signaling. Recruitment of Tim-3 to the immune synapse was mediated exclusively by the transmembrane domain, replacement of which impaired the ability of Tim-3 to costimulate T cell receptor (TCR)–dependent S6 phosphorylation. Furthermore, enforced localization of the Tim-3 cytoplasmic domain to the immune synapse in a chimeric antigen receptor still enabled T cell activation. Together, our findings are consistent with a model whereby Tim-3 enhances TCR-proximal signaling under acute conditions.

T-cell immunoglobulin and mucin 3 protein (Tim-3) is a type-I transmembrane protein expressed on murine T-helper type 1 (Th1) cells which regulates Th1 cell proliferation and development of tolerance. Tim-3 has six tyrosine residues in the cytosplasmic tail, two and three tyrosines residues are clustered in proximal and distal region of the cytoplasmic tail respectively. Using a motif scan program, we have predicted that two tyrosine residues from each cluster may be phosphorylated by Src family kinases, and three of the five residues may interact with SH2 domains. We generated serial deletion and point mutation constructs of Tim-3 and demonstrated that deletion of the cytoplasmic tail region containing the distal tyrosine residues (Y250, Y251, Y253) slightly increased NFAT-Luciferase reporter activity, while deletion of the region containing distal and proximal (Y235, Y242) tyrosine residues abolished the reporter activity to basal level in Jurkat cells. Point mutation of both Y235 and Y242 decreased NFAT-Luc reporter activity close to basal level while single point mutation of Y235 or Y242 did not result in significant changes in reporter activity. Therefore we conclude that phosphorylation of Y235 and Y242 residues of Tim-3 are responsible for potentiation of signal transduction initiated by activation of TCR, upstream of NFAT/AP1 pathways, in a redundant manner.

Circadian rhythms refer to biologic processes that oscillate with a period of approximately 24 hours. These rhythms are sustained by a molecular clock and provide a temporal matrix that ensures the coordination of homeostatic processes with the periodicity of environmental challenges. We demonstrate the circadian molecular clock controls the expression and function of toll like receptor 9 (TLR9). In a vaccination model using TLR9 ligand as adjuvant, mice immunized at the time of enhanced TLR9 responsiveness presented weeks later with an improved adaptive immune response. In a TLR9-dependent mouse model of sepsis, we found that disease severity was dependent on the timing of sepsis induction, coinciding with the daily changes in TLR9 expression and function. These findings unveil a direct molecular link between the circadian and innate immune systems with important implications for immunoprophylaxis and immunotherapy.