Interaction of cell surface glycoproteins with endogenous lectins on the cell surface regulates formation and maintenance of plasma membrane domains, clusters signaling complexes, and controls the residency time of glycoproteins on the plasma membrane. Galectin-9 is a soluble, secreted lectin that binds to glycoprotein receptors to form galectin-glycoprotein lattices on the cell surface. Whereas galectin-9 binding to specific glycoprotein receptors induces death of CD4 Th1 cells, CD4 Th2 cells are resistant to galectin-9 death due to alternative glycosylation. On Th2 cells, galectin-9 binds cell surface protein disulfide isomerase (PDI), increasing retention of PDI on the cell surface and altering the redox status at the plasma membrane. Cell surface PDI regulates integrin function on platelets and also enhances susceptibility of T cells to infection with HIV. We find that galectin-9 binding to PDI on Th2 cells results in increased cell migration through extracellular matrix via β3 integrins, identifying a unique mechanism to regulate T-cell migration. In addition, galectin-9 binding to PDI on T cells potentiates infection with HIV. We identify a mechanism for regulating cell surface redox status via a galectin-glycoprotein lattice, to regulate distinct T-cell functions.

The galectin family of lectins regulates multiple biologic functions, such as development, inflammation, immunity, and cancer. One common function of several galectins is the ability to trigger T cell death. However, differences among the death pathways triggered by various galectins with regard to glycoprotein receptors, intracellular death pathways, and target cell specificity are not well understood. Specifically, galectin-9 and galectin-1 both kill thymocytes, peripheral T cells, and T cell lines; however, we have found that galectin-9 and galectin-1 require different glycan ligands and glycoprotein receptors to trigger T cell death. The two galectins also utilize different intracellular death pathways, as galectin-9, but not galectin-1, T cell death was blocked by intracellular Bcl-2, whereas galectin-1, but not galectin-9, T cell death was blocked by intracellular galectin-3. Target cell susceptibility also differed between the two galectins, as galectin-9 and galectin-1 killed different subsets of murine thymocytes. To define structural features responsible for distinct activities of the tandem repeat galectin-9 and dimeric galectin-1, we created a series of bivalent constructs with galectin-9 and galectin-1 carbohydrate recognition domains connected by different peptide linkers. We found that the N-terminal carbohydrate recognition domain and linker peptide contributed to the potency of these constructs. However, we found that the C-terminal carbohydrate recognition domain was the primary determinant of receptor recognition, death pathway signaling, and target cell susceptibility. Thus, carbohydrate recognition domain specificity, presentation, and valency make distinct contributions to the specific effects of different galectins in initiating T cell death.Cell death is an essential factor in T cell development, which regulates selection of functional T cells during development in the thymus, as well as elimination of activated T cells after microbial infection or other exposure to antigen (1, 2). A number of distinct T cell death pathways have been described, including those triggered by members of the galectin family of vertebrate lectins (3-5). Galectin-1 was the first family member described to induce death of developing thymocytes and activated peripheral T cells, and the galectin-1 T cell death pathway is the best characterized to date. Specific glycoprotein receptors involved in galectin-1 death and specific types of O-and N-glycan ligands required for galectin-1 death have been identified, and galectin-1 has been shown to trigger a novel intracellular death pathway (6 -14). Other galectins have also been reported to trigger death of T cell lines and various T cell subsets, including galectin-2, galectin-3, galectin-8, and galectin-9 (15-19). Relatively little is known about the glycoprotein receptors, glycan ligands, and intracellular death pathways used by these galectins. However, our laboratory has found that galectin-1 and galectin-3 kill different subsets of thymocytes, and use distinct sets...

Galectin-1, a ␤-galactoside-binding protein highly expressed in the thymus, induces apoptosis of specific thymocyte subsets and activated T cells. Galectin-1 binds to N-and O-glycans on several glycoprotein receptors, including CD7, CD43, and CD45. Here we show that galectin-1 signaling through CD45, which carries both N-and O-glycans, is regulated by CD45 isoform expression, core 2 O-glycan formation and the balance of N-glycan sialylation. Regulation of galectin-1 T cell death by O-glycans is mediated through CD45 phosphatase activity. While galectin-1 signaling in cells expressing low molecular weight isoforms of CD45 requires expression of core 2 O-glycans (high affinity ligands for galectin-1), galectin-1 signaling in cells expressing a high molecular weight isoform of CD45 does not require core 2 O-glycans, suggesting that a larger amount of core 1 O-glycans (low affinity ligands for galectin-1) is sufficient to overcome lack of core 2 O-glycans. Furthermore, regulation of galectin-1 signaling by ␣2,6-sialylation of N-glycans is not solely dependent on CD45 phosphatase activity and can be modulated by the relative expression of enzymes that attach sialic acid in an ␣2,6-or ␣2,3-linkage. Thus, N-and O-glycans modulate galectin-1 T cell death by distinct mechanisms, and different glycosylation events can render thymocytes susceptible or resistant to galectin-1.

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.

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