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β-Galactoside-binding lectin 9 (galectin-9) is a tandem repeat-type member of the galectin family. It was initially characterized as an eosinophil chemoattractant and an inducer of apoptosis in thymocytes. Subsequently, galectin-9 was identified as a ligand for transmembrane immunoglobulin mucin domain 3 (Tim-3), a type I glycoprotein induced on T cells during chronic inflammation. Work in autoimmune diseases and chronic viral infections have led to the current hypothesis that the function of Tim-3 is to limit immune responses. However, it is still not known to what degree these effects are due to the galectin-9/Tim-3 interaction. In this study, we show that galectin-9 is not limited to the role of a pro-apoptotic agent, but that it can also induce the production of pro-inflammatory cytokines from T helper cells. This effect is dose-dependent and does not require Tim-3. These findings suggest that the effects of galectin-9 on T cells are more complex than previously thought and are mediated by additional receptors apart from Tim-3.

Interleukin-2 (IL-2) is a lymphocyte growth factor that is an important component of many immune-based cancer therapies. The efficacy of IL-2 is thought to be limited by the expansion of T regulatory cells, which express the high-affinity IL-2 receptor subunit IL-2Rα. IL-15 is under investigation as an alternative to IL-2. Although both cytokines signal through IL-2Rβγ, IL-15 does not bind IL-2Rα and therefore induces less T regulatory cell expansion. However, we found that transferred effector CD8(+) T cells induced curative responses in lymphoreplete mice only with IL-2-based therapy. Although conventional in vitro assays showed similar effector T cell responsiveness to IL-2 and IL-15, upon removal of free cytokine, IL-2 mediated sustained signaling dependent on IL-2Rα. Mechanistically, IL-2Rα sustained signaling by promoting a cell surface IL-2 reservoir and recycling of IL-2 back to the cell surface. Our results demonstrate that IL-2Rα endows T cells with the ability to compete temporally for limited IL-2 via mechanisms beyond ligand affinity. These results suggest that strategies to enhance IL-2Rα expression on tumor-reactive lymphocytes may facilitate the development of more effective IL-2-based therapies.

CD4+T helper 1 (TH1) cells are important mediators of inflammation and are regulated by numerous pathways, including the negative immune receptor Tim-3. We found that Tim-3 is constitutively expressed on cells of the innate immune system in both mice and humans, and that it can synergize with Toll-like receptors. Moreover, an antibody agonist of Tim-3 acted as an adjuvant during induced immune responses, and Tim-3 ligation induced distinct signaling events in T cells and dendritic cells; the latter finding could explain the apparent divergent functions of Tim-3 in these cell types. Thus, by virtue of differential expression on innate versus adaptive immune cells, Tim-3 can either promote or terminate TH1 immunity and may be able to influence a range of inflammatory conditions.

Tim-3 is a type I transmembrane glycoprotein first identified through a screen for TH1-specific cell surface markers. Since then, it has been detected on a wide variety of cell types. Galectin-9, a β-galactoside binding lectin is the only known ligand of Tim-3 and has been used to study the function of Tim-3 due to the lack of agonistic Tim-3 antibodies. Galectin-9 induces apoptosis and calcium flux in TH1 cells but promotes the maturation of immature monocyte-derived dendritic cells and induces IgE-sensitized mast cells to secrete higher levels of TH2 cytokines. To better understand the regulation of T cell effector function by Tim-3, we treated TH1 cells with varying doses of galectin-9 and found that at an optimal concentration, galectin-9 has the ability to enhance IFN-γ production by TH1 cells without affecting their viability. Then, using luciferase reporter assays, we found that overexpression of Tim-3 in Jurkat T cells can enhance NF-κB and NFAT/AP-1 activity downstream of the T-cell receptor and CD28. Therefore, these findings indicate that Tim-3 can positively affect T cell effector function.

MUC1 (CD227) is a large (>400kDa), heavily glycosylated, type-I transmembrane protein present most notably on the apical surface of ductal epithelia. In epithelial adenocarcinomas MUC1 is overexpressed as a hypoglycosylated protein in a non-polarized fashion. Because of these attributes, MUC1 has been studied foremostly as a tumor-associated antigen. The role of MUC1 in other cell types remained largely unexplored until MUC1 was found to be expressed by activated human T cells from healthy donors. Preliminary studies we have performed on the role of MUC1 in T cells point towards MUC1 as a negative regulator of effector T cell function. Crosslinking of MUC1 limits the proliferative capacity of primary human T cells activated with anti-CD3, as well as the ability of these cells to make IL-2 and upregulate IL-2Rα. Studies using the Jurkat T cell line have yielded intriguing results. When MUC1 is present and crosslinked, Jurkat T cells activated with anti-CD3 do not secrete IL-2. Paradoxically, knockdown of MUC1 in Jurkat T cells with siRNA also renders them unable to produce IL-2 when similarly activated. Because failure to secrete IL-2 and proliferate following TCR ligation are characteristics of T cell hyporesponsiveness, elucidating the exact role of MUC1 in T cell activation in the context of other co-inhibitory or co-activating receptors is important. To do so, we have been employing luciferase reporter assays to determine the effect of MUC1 expression and crosslinking on well-characterized signaling pathways activated during the engagement of TCR/CD3 and CD28. This will add to our current, limited, knowledge on MUC1 signaling in T cells.