Characterizing Zap70 signaling mutants in primary T cells using CRISPR/Cas9

Abstract

Abstract T cell activation is controlled by a complex series of signaling events that originate from the T cell receptor (TCR) at the plasma membrane. These signaling events are both spatially and temporally regulated to ensure that T cells activate only in a correct and specific context. A key aspect of T cell activation is the ability of the TCR to recognize peptides with specific affinities while simultaneously discounting a large proportion of MHC-TCR interactions with lower affinities. Our lab has identified Zap70 interaction dynamics with phosphorylated motifs in the TCR as a key mediator of T cell activation thresholds. However, studying these signaling events within primary T cells remains challenging because manipulating the signaling threshold through germline mutations will cause abnormal T cell development and selection in the thymus. To investigate the role of Zap70 in regulating the activation threshold in mature, peripheral T cells, we have developed a CRISPR/Cas9 expression system in primary murine T cells. Using a retroviral construct, we can simultaneously knock down endogenous Zap70 and replace its expression with ectopic Zap70 mutants with differing TCR interaction dynamics and signal transduction. Ectopic Zap70 is expressed up to 41 days after infection and 33 days after adoptive transfer to Rag1−/− mice. In addition, the primary cells are sensitive to antigenic stimulation comparable to uninfected primary T cells. Using this system, we can both dissect the mechanisms by which Zap70-TCR dynamics regulate signal transduction and determine whether manipulating Zap70-TCR dynamics can affect T cell activity under various pathological conditions.