CD28-mediated linear and parallel costimulatory signaling cooperatively regulate CAR-T cell functions via CAR-CD28 microclusters.

Various improvements have been made to chimeric antigen receptor (CAR)-T cells to enhance their antitumor effects and expand their indications to various cancer types. Although second-generation CARs directly induce second signals in a linear pathway via their own costimulatory domain such as CD28, CAR-T cells themselves express costimulatory receptors, which induce additional second signals in parallel. To clarify the differences between these two types of second signals, we here reveal CD28-mediated signalosomes using high-resolution imaging. CAR constructed using CD3ζ and CD28 (CD28ζ.CAR)-T cells demonstrate intense and persistent accumulation of the CD28 downstream kinase protein kinase C θ (PKCθ) at CAR signalosomes, and endogenous CD28 on CAR-T cells prolongs the CD28-CAR association. This assembling of PKCθ is correlated with IL-2 production and in vivo tumor suppression by CAR-T cells. These results indicate that the development and improvement of CARs requires analysis of intrinsic T-cell responses, which can be effectively assessed from signalosome dynamics evaluated by molecular imaging.