Redistribution of specialized molecules in migrating cells develops asymmetry between two opposite cell poles, the leading edge and the uropod. We show that acquisition of a motile phenotype in T lymphocytes results in the asymmetric redistribution of ganglioside GM3-and GM1-enriched raft domains to the leading edge and to the uropod, respectively. This segregation to each cell pole parallels the specific redistribution of membrane proteins associated to each raft subfraction. Our data suggest that raft partitioning is a major determinant for protein redistribution in polarized T cells, as ectopic expression of raft-associated proteins results in their asymmetric redistribution, whereas non-raft-partitioned mutants of these proteins are distributed homogeneously in the polarized cell membrane. Both acquisition of a migratory phenotype and SDF-1␣-induced chemotaxis are cholesterol depletion-sensitive. Finally, GM3 and GM1 raft redistribution requires an intact actin cytoskeleton, but is insensitive to microtubule disruption. We propose that membrane protein segregation not only between raft and nonraft domains but also between distinct raft subdomains may be an organizational principle that mediates redistribution of specialized molecules needed for T cell migration. Cell movement across a two-dimensional substrate requires a dynamic interplay between attachment at the cell front and detachment at the rear cell edge, combined with a traction machinery that pulls the net cell body forward. As adhesion and detachment occur at opposite cell edges, the moving cell must acquire and maintain spatial and functional asymmetry, a process called polarization (1, 2). This asymmetry develops between two opposite cell edges-the leading edge, which protrudes, and the rear (termed uropod in lymphocytes), which retracts.Because of the specialized functions of these compartments, each pole in migrating cells is enriched in specific receptors and signaling molecules but lacks others. In fibroblast-like cells and lymphocytes, the leading edge contains chemokine receptors, several glycosylphosphatidylinositol-linked proteins, such as the urokinase plasminogen activator receptor (uPAR), as well as the machinery that senses the environment and induces localized actin polymerization (1). Whereas the rear edge in fibroblasts appears to be a passive tail, the lymphocyte uropod is a specialized pseudopod-like projection with important functions, including motility and recruitment of bystander cells. Several intercellular adhesion molecules (ICAMs) concentrate at the uropod, including ICAM-1, -2 and -3, CD43, CD44, as well as the actin-binding proteins of the ezrin-radixinmoesin family. In accordance with its importance in lymphocyte migration, crosslinking of molecules located in the uropod is sufficient to trigger neutrophil polarization and motility (3).To understand polarization and chemotaxis processes, the molecular mechanisms involved in the generation and maintenance of the asymmetric distribution of cell-surface components must be...

The immunoregulatory protein T cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) mediates T cell exhaustion and contributes to the suppression of immune responses in both viral infections and tumors. Tim-3 blockade reverses the exhausted phenotype of CD4+ and CD8+ T cells in several chronic diseases including melanoma. Interestingly, natural killer (NK) cells constitutively express Tim-3; however, its role in modulating the function of these innate effector cells remains unclear, particularly in human disease. In this study, we compared the function of Tim-3 in NK cells from healthy donors and patients with metastatic melanoma. NK cells from the latter were functionally impaired/exhausted and Tim-3 blockade reversed this exhausted phenotype. Moreover, Tim-3 expression levels correlated with the stage of the disease and poor prognostic factors. These data indicate that Tim-3 can function as an NK cell exhaustion marker in advanced melanoma and supports the development of Tim-3-targeted therapies to restore antitumor immunity.