Multiple Signaling Roles of CD3ε and Its Application in CAR-T Cell Therapy

A T cell receptor (TCR) mediates antigen-induced signaling through its associated CD3 epsilon, delta, gamma, and zeta, but the contributions of different CD3 chains remain elusive. Using quantitative mass spectrometry, we simultaneously quantitated the phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) of all CD3 chains upon TCR stimulation. A subpopulation of CD3 epsilon ITAMs was mono-phosphorylated, owing to Lck kinase selectivity, and specifically recruited the inhibitory Csk kinase to attenuate TCR signaling, suggesting that TCR is a self-restrained signaling machinery containing both activating and inhibitory motifs. Moreover, we found that incorporation of the CD3 epsilon cytoplasmic domain into a second-generation chimeric antigen receptor (CAR) improved antitumor activity of CAR-T cells. Mechanistically, the Csk-recruiting ITAM of CD3 epsilon reduced CAR-T cytokine production whereas the basic residue rich sequence (BRS) of CD3 epsilon promoted CAR-T persistence via p85 recruitment, Collectively, CD3 epsilon is a built-in multifunctional signal tuner, and increasing CD3 diversity represents a strategy to design next-generation CAR.