ADENOSINE-DIPHOSPHATE (ADP)-RIBOSYLATION OF THE GUANOSINE TRIPHOSPHATASE (GTPASE) RHO IN RESTING PERIPHERAL-BLOOD HUMAN T-LYMPHOCYTES RESULTS IN PSEUDOPODIAL EXTENSION AND THE INHIBITION OF T-CELL ACTIVATION

Scrape loading Clostridium botulinum C3 exoenzyme into primary periphe ral blood human T lymphocytes (PB T cells) efficiently adenosine dipho sphate (ADP)-ribosylates and thus inactivates the guanosine triphospha tase (GTPase) Rho. Basal adhesion of PB T cells to the beta 1 integrin substrate fibronectin (Fn) was not inhibited by inactivation of Rho, nor was upregulation of adhesion using phorbol myristate acetate (PMA; 10 ng/ml) or Mn++ (1 mM) affected. Whereas untreated PB T cells adher ent to Fn remain spherical, C3-treated PB T cells extend F-actin-conta ining pseudopodia. Inactivation of Rho delayed the kinetics of PMA-dep endent PB T cell homotypic aggregation, a process involving integrin a lpha L beta 2. Although C3 treatment of PB T cells did not prevent adh esion to the beta 1 integrin substrate Fn, it did inhibit beta 1 integ rin/CD3-mediated costimulation of proliferation. Analysis of intracell ular cytokine production at the single cell level demonstrated that AD P-ribosylation of Rho inhibited beta 1 integrin/CD3 and CD28/CD3 costi mulation of IL-2 production within 6 h of activation. Strikingly, IL-2 production induced by PMA and ionomycin was unaffected by C3 treatmen t. Thus, the GTPase Rho is a novel regulator of T lymphocyte cytoarchi tecture, and functional Rho is required for very early events regulati ng costimulation of IL-2 production in PB T cells.