A T cell receptor (TCR) mediates antigen-induced signaling through its associated CD3ε, δ, γ, and ζ, 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 CD3ε 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 CD3ε cytoplasmic domain into a second-generation chimeric antigen receptor (CAR) improved antitumor activity of CAR-T cells. Mechanistically, the Csk-recruiting ITAM of CD3ε reduced CAR-T cytokine production whereas the basic residue rich sequence (BRS) of CD3ε promoted CAR-T persistence via p85 recruitment. Collectively, CD3ε is a built-in multifunctional signal tuner, and increasing CD3 diversity represents a strategy to design next-generation CAR.

AT细胞受体（TCR）通过其相关的CD3ε，δ，γ和ζ介导抗原诱导的信号传导，但是不同CD3链的贡献仍然难以捉摸。使用定量质谱，我们同时定量了TCR刺激后所有CD3链的免疫受体基于酪氨酸的活化基序（ITAM）的磷酸化。由于Lck激酶的选择性，CD3εITAM的一个亚群被单磷酸化，并专门募集抑制性Csk激酶来减弱TCR信号传导，这表明TCR是一种自我抑制的信号传导机制，既包含激活基元又包含抑制基元。此外，我们发现将CD3ε胞质域掺入第二代嵌合抗原受体（CAR）中可改善CAR-T细胞的抗肿瘤活性。机械上，CD3ε的Csk诱导ITAM减少了CAR-T细胞因子的产生，而CD3ε的基本残基富集序列（BRS）通过p85募集促进了CAR-T持久性。总的来说，CD3ε是内置的多功能信号调谐器，而不断增加的CD3多样性则代表了设计下一代CAR的策略。