Engagement of the T‐cell antigen receptor by anti‐CD3 monoclonal antibody causes a rapid increase in lymphocyte F‐actin

Abstract

Activation of protein kinase C (PKC) causes a rapid and sustained increase in the F‐actin of T lymphocytes. Because the phosphatidylinositol pathway and the cytoskeleton play a role in lymphocyte activation, we examined the relationship between signal transduction and the F‐actin increase in human blood T cells. Anti‐CD3 monoclonal antibodies (mAbs) initiate signals which result in activation of T lymphocytes through the T‐cell receptor (TCR), involving the phosphatidylinositol pathway, activation of PKC, and increasing intracellular calcium (Ca_i²⁺). The fluorescent probe NBD‐phallacidin was used to examine the conformational state of actin following stimulation of T lymphocytes with anti‐CD3 mAb. Each of three different murine anti‐CD3 mAbs caused rapid increases in lymphocytic F‐actin content, which was enhanced by cross‐linking with a goat anti‐mouse IgG. A maximally effective dose of the mAb Leu 4 caused a rise in cellular F‐actin of 1.8‐fold at 2 minutes and a three‐fold increase in Ca_i²⁺. Ionomycin, 100 nM, caused a Ca_i²⁺ rise similar in magnitude to that caused by anti‐CD3 mAb but had no effect on F‐actin content. Inhibitors of PKC, 1(5‐isoquinolinylsulfonyl)‐2methylpiperazine (H7), sphingosine, and sphinganine lowered the resting cellular F‐actin and partially blocked the increase in F‐actin caused by either anti‐CD3 mAb or ionomycin; however, they had no effect on the rise in Ca_i²⁺. Cells leached of Ca²⁺ with EGTA and ionomycin exhibited no Ca_i²⁺ increase in response to anti‐CD3 mAb or ionomycin; such cells retained the F‐actin increase caused by anti‐CD3 mAb. We conclude that stimulation of human T lymphocytes via the TCR causes an early rapid increase in F‐actin content. Activation of PKC may play a role but the concomitant Ca_i²⁺ increase is neither sufficient nor necessary for the F‐actin increase.