Tumor-specific T cell activation by recombinant immunoreceptors:: CD3ζ signaling and CD28 costimulation are simultaneously required for efficient IL-2 secretion and can ge integrated into one combined CD28/CD3ζ signaling receptor molecule

Recombinant immunoreceptors with specificity for the carcinoembryonic Ag (CEA) can redirect grafted T cells to a MHC/Ag-independent antitumor response. To analyze receptor-mediated cellular activation in the context of CD28 costimulation, we generated: 1) CEA(+) colorectal tumor cells that express simultaneously B7-1 and B7-2, and 2) CEA-specific immunoreceptors that harbor intracellularly the signaling moities either of CD28 (BW431/26-scFv-Fc-CD28), CD3 xi (BW431/26-scFv-Fc-CD3 xi), or Fc epsilon RI gamma (BW431/26-scFv-Fc-gamma). By retroviral gene transfer, we grafted activated T cells from the peripheral blood with these immunoreceptors. T cells that express the Fc epsilon RI gamma or CD3 xi signaling receptor lysed specifically CEA(+) tumor cells and secreted high amounts of IFN-gamma upon receptor cross-linking, whereas anti-CEA-CD28 receptor-grafted T cells did not, indicating that CD28 signaling alone is not sufficient for efficient T cell activation. CD28 costimulation did not affect cytolysis by T cells equipped with gamma- or xi -signaling receptors, but enhanced both IFN-gamma secretion and proliferation. CD28 costimulation, however, was required for efficient IL-2 secretion of anti-CEA-gamma receptor-grafted T cells. Both purified CD4(+) and CD8(+) T cells grafted with immunoreceptors required CD28 costimulation for complete T cell activation. We integrated both CD28 and CD3 xi signaling domains into one combined immunoreceptor molecule (BW431/26-scFv-Fc-CD28/CD3 xi) with dual signaling properties. T cells grafted with the combined CD28/CD3 xi signaling receptor secreted high amounts of IL-2 upon Ag binding without exogenous B7/CD28 costimulation, demonstrating that both MHC-independent cellular activation and CD28 costimulation for complete T cell activation can be delivered by one recombinant receptor molecule.