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Updated: Jun 30, 2025
Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection
Published on: September 2, 2014
An optimized non-T cell transfection system based on HEK293FT cells for CD3ζ phosphorylation and ubiquitination
Jiaqi Zheng1, Yuchuan Zhang2, Yiting Cai3
1Institute of Immunology, Zhejiang University School of Medicine, Hangzhou 310058, Zhejiang, China; Department of Central Laboratory, Affiliated Hangzhou First People's Hospital, School of Medicine, Westlake University, Hangzhou 310006, Zhejiang, China.
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.
Insights
Researchers developed a novel HEK293FT cell system to study CD3ζ signaling. This system effectively mimics T cell receptor (TCR) CD3ζ phosphorylation and ubiquitination, aiding immune response research.
Area of Science:
- Immunology
- Molecular Biology
- Cell Signaling
Background:
- The CD3ζ chain is a crucial component of the T cell receptor (TCR)/CD3 complex, essential for T cell activation and antigen recognition.
- Studying CD3ζ regulation is vital for understanding T cell-mediated immunity, but delivering genes into T cells for analysis is challenging.
Purpose of the Study:
- To establish and validate a non-T cell transfection system for analyzing CD3ζ phosphorylation and ubiquitination.
- To identify key regulatory proteins involved in CD3ζ signaling pathways.
Main Methods:
- Development of a transfection system using HEK293FT cells, optimized with high confluency and PolyJet™ reagent.
- Stimulation with pervanadate (PV) to induce CD3ζ tyrosine phosphorylation.
- Investigation of E3 ligase Cbl-b and kinases Lck and Zap70 in regulating CD3ζ ubiquitination and phosphorylation.
Main Results:
- The HEK293FT system successfully mimicked PV-induced CD3ζ phosphorylation and activation-dependent ubiquitination.
- Cbl-b was identified as the most effective E3 ligase for CD3ζ ubiquitination in this system.
- Lck and Zap70 kinases were shown to enhance CD3ζ phosphorylation levels.
Conclusions:
- A PV-treated HEK293FT cell system provides a viable model for studying CD3ζ signaling, phosphorylation, and ubiquitination.
- This model system facilitates the investigation of T cell activation mechanisms and regulatory protein functions.
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