The T cell receptor (TCR)-CD3 complex consists of the antigen binding subunit TCRαβ and three CD3 heterodimers, including γε, δε, and ζζ (Dong et al., 2019). Phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) in the CD3 chains initiates T cell activation signaling. The CD3γ, δ, and ε chains each contain one copy of ITAM. The CD3ζ chain, which is encoded by Cd247 gene in Mus musculus and by CD247 gene in Homo sapiens, has three copies of ITAM and is the predominant phosphorylated protein in the TCR-CD3 complex (Love and Hayes, 2010). The phosphorylation of CD3 chains triggers the endocytosis of the TCR-CD3 complex, followed by the ubiquitination of CD3 subunits. CD3ζ is ubiquitinated by E3 ubiquitin ligases, such as Cbl-b, c-Cbl, and Grail (Myers et al., 2005; Naramura et al., 2002; Nurieva et al., 2010; Ouchida et al., 2008). The TCR-CD3 complex lacking the CD3ζ chain, but not the other CD3 subunits, fails to respond to the peptide-major histocompatibility complex (pMHC) conjugation and relay the signals (Sussman et al., 1988). Given the critical role of CD3ζ in T cell activation and subsequent function, it is crucial to develop an efficient cellular model to facilitate the study of its regulatory proteins.

Currently, T cell lines such as Jurkat T cells, 2B4 cells, and some T lymphoma cell lines are used for analyzing T cell activation and function (Cenciarelli et al., 1996; Huang et al., 2010; Kawai et al., 2014; Myers et al., 2006). Recombinant retroviruses and lentiviruses are commonly used to overexpress or silence (through RNA interference) specific genes in T cell lines (Ouchida et al., 2008; Kawai et al., 2014; Meng et al., 2018). Despite the high transfection efficiency in T cell lines, the complex packaging process of the virus particles and the limited number of genes that can be introduced into the cells limit the usage of these expression systems. Although the Jurkat T cells can be simultaneously and efficiently transfected with multiple plasmids through electroporation (Huang et al., 2010), the low survival rate after electroporation and the high cost of the procedure limit the applicability of this model for studying T cell activation. Therefore, it is essential to develop a cell system with high transfection efficiency and easy handling to facilitate studies on CD3ζ signaling.

Non-T cell transfection systems, including the HEK293, HEK293T, and COS-7 cells, have been used for the functional analysis of CD3ζ subunit (Huang et al., 2010; Lee et al., 1998; Van Oers et al., 2000; Wang et al., 2001). These cell lines are highly amenable to transfection using simple and economical transfection reagents. Given that these cell lines lack the T cell-specific kinases and adaptor proteins (Lee et al., 1998; Wang et al., 2001), they can be suitable models for evaluating the role of different regulatory proteins in CD3ζ function. The aim of the present study was to establish and optimize an efficient transfection system using HEK293FT cells wherein CD3ζ can be effectively phosphorylated and further ubiquitinated based on an activation-dependent manner. We found that CD3ζ was phosphorylated and subsequently ubiquitinated in this non-T cell system in response to pervanadate and recombinant E3 ligases, and Cbl-b was the most effective E3 ligase. To the best of our knowledge, our study is the first to compare the role of E3 ligases and corresponding adaptor proteins in activation-dependent ubiquitination of CD3ζ in a non-T cell system. Altogether, we established a convenient, stable, low-cost, and easy to repeatable non-T cell system to screen for candidate regulatory proteins of CD3ζ.