Association of various T cell-surface molecules with the cytoskeleton. Effect of cross-linking and activation

Abstract

The association of various surface molecules with the cytoskeleton in resting peripheral blood T cells was examined by assaying the capacity of detergent to solubilize them. Cytoskeletal association was assessed by staining T cells with a fluorescein-conjugated mAb, resuspending the cells in buffer with or without the nonionic detergent, NP-40, and determining the capacity of the detergent to remove the mAb from the cell surface by using flow microfluorimetry. MAb to CD3, the TCR, and CD45 were completely removed from the cell surface by detergent. In contrast, 7 to 50% of mAb to CD2, CD4, CD8, CD11a/CD18, CD44, and class I MHC molecules were resistant to detergent solubilization, demonstrating that a fraction of these molecules was constitutively associated with the cytoskeleton. The effect of cross-linking these molecules with a mAb and a secondary goat anti-mouse Ig was also examined. Cross-linking CD3 or the TCR induced cytoskeletal association of these molecules. In addition, cross-linking increased the fraction of CD2, CD4, CD8, CD11a/CD18, CD44, and class I MHC molecules that was associated with the cytoskeleton. In contrast, cross-linking CD45 did not induce an association with the cytoskeleton. The effect of T cell activation on the cytoskeletal association of these molecules was also examined. Stimulation of T cells with ionomycin and PMA greatly increased the expression of CD2 and CD44 without increasing the number of molecules associated with the cytoskeleton. Stimulation with PMA alone had no effect on the expression of CD2 or CD44, but was found to decrease the percentage of these molecules associated with the cytoskeleton. Stimulation with ionomycin and PMA increased both the expression of class I MHC molecules and the number of molecules associated with the cytoskeleton proportionally. Finally, stimulation with ionomycin and PMA decreased CD3 expression, but increased the number of CD3 molecules associated with the cytoskeleton. The data establish a pattern of cytoskeletal association of T cell-surface molecules that is a characteristic of each individual molecule and can be altered by cross-linking. Moreover, the results indicate that the association of various T cell surface molecules with the cytoskeleton is a dynamic process that varies with the state of activation and or differentiation of the cells.