The phosphorylation kinetics of the key regulators in T cell receptor signaling pathway

Abstract

Abstract T cell receptor (TCR) signaling pathway regulates T cell homeostasis and adaptive immune responses. Clinically, the activation of TCR is indispensable to the success of T cell-based immunotherapy. The engagement of TCR phosphorylates immunoreceptor tyrosine-based activation motifs (ITAM) on CD3, which is followed by a protein phosphorylation cascade. Protein phosphorylation, regulated by kinases and phosphatases, changes protein conformation and determines protein function. A better understanding of TCR signaling phosphorylation events is crucial to both decipher the T cell regulation mechanism and boost the efficacy of T cell immunotherapy. Here, we developed a panel of monoclonal antibodies that target several of the TCR signaling regulators and are applicable for both intracellular flow cytometry (ICFC) and western blot (WB). Using these antibodies, we performed a kinetic analysis of the TCR signaling pathway. Jurkat T cells were cross-linked by soluble anti-CD3 and anti-CD28 antibodies, and the phosphorylation kinetics of the key TCR signaling regulators, such as phospho-ZAP-70 and phospho-PLCg1, were evaluated by ICFC and WB. We show that TCR signaling activation induced by cross-linking CD3 and CD28 is a relatively transient event and the optimal activation of the key TCR signaling regulators varies from 1 to 15 minutes. Our data profiled the phosphorylation kinetics of the key regulators in TCR signaling pathway.