An optimized non-T cell transfection system based on HEK293FT cells for CD3ζ phosphorylation and ubiquitination

Abstract

CD3ζ is part of the T cell receptor (TCR)/CD3 complex that plays a critical role in antigen recognition and subsequent T cell activation. Understanding the mechanisms that regulate CD3ζ can provide new insights into the T cell-mediated immune responses. However, it is challenging to deliver exogenous genes into T cells for functional and mechanistic analyses. To this end, we established a non-T cell transfection system based on HEK293FT cells to screen for candidate regulatory proteins. The transfection was optimized using relatively high confluent cultures and the transfection reagent PolyJet™. Pervanadate (PV) treatment sustained tyrosine phosphorylation of CD3ζ, and facilitated the subsequent activation-dependent ubiquitination by E3 ligase Cbl-b in the HEK293FT system. Lck and Zap70 kinases enhanced the levels of phosphorylated CD3ζ in the presence of PV. We compared the effects of E3 ligases and the corresponding adaptor proteins on activation-dependent ubiquitination of CD3ζ in the PV-stimulated cells, and found that Cbl-b was most effective. Taken together, we have demonstrated that a non-T cell transfection system based on PV-treated HEK293FT cells could effectively mimic CD3ζ phosphorylation and ubiquitination and is a promising model for studying the role of CD3ζ signaling in T cell activation.