SELECTIVE CD28PYMNM MUTATIONS IMPLICATE PHOSPHATIDYLINOSITOL 3-KINASE IN CD86-CD28-MEDIATED COSTIMULATION

CD28 costimulatory signals are required for lymphokine production and T cell proliferation. CD28 signaling recruits the intracellular proteins PI 3-kinase, ITK, and GRB-2/SOS. PI 3-kinase and GRB-2/SOS bind the CD28 cytoplasmic pYMNM motif via SH2 domains. We generated CD28 pYMNM mutants and found that Y-191 mutation (Y(191)CD28F) disrupted both PI 3-kinase and GRB-2 binding, while M(194) mutation (M(194)CD28C) disrupted only PI 3-kinase binding. Both mutants still bound ITK. We have assessed the ability of these selective mutants to support IL-2 production upon TCR zeta/ CD3 ligation in the presence of CHO-CD86 (B7-2) cells. Both Y(191)CD28F and M(194)CD28C mutants failed to generate IL-2. These data directly implicate PI 3-kinase in CD28-mediated costimulation leading to IL-2 secretion. Wortmannin, an inhibitor of PI 3-kinase, induced cell apoptosis and as such was unsuitable for use in this study.