T lymphocyte antigen activation is facilitated by clustering of membrane glycosphingolipid-enriched microdomains (GEMs, lipid "rafts") at the T cell/APC contact that is linked to changes in actin cytoskeleton and is one major mechanism of CD28 costimulation. Ligation of CD28 alone, or ligation of the CD28-like molecules CTLA-4 (CD152) and ICOS (CD278) induces actin polymerization with cell elongation and generation of lamellipodia and filopodia in T cells. These changes are dependent on Src, PI3-kinase, Vav, and Rho family GTPases. Whereas CD28 and CTLA-4 have been shown to be functional and physically associated with lipid rafts, the presence of ICOS in lipid rafts or its effect in raft clustering is not known. In this work, we have activated the T cell line D10 with anti-ICOS antibodies, alone or combined with anti-CD3 antibodies, bound or unbound to polystyrene microbeads or glass coverslips. The possible relationship of ICOS-induced changes in actin cytoskeleton to the ICOS localization in membrane rafts was then analyzed by fluorescence microscopy, or by immunoblot of detergent insoluble ("raft") or soluble ("non-raft") fractions of cell lysates. Our data show that ICOS promotes TCR/CD3 induction of raft clustering at the site of activation. However, ICOS, which, on its own, can induce accumulations of polymerized actin, is undetectable in membrane rafts, even when using CD3 or ICOS, ligands capable of inducing clear changes in the actin cytoskeleton.
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