title: TCR mispairing in genetically modified T cells was detected by fluorescence resonance energy transfer.
abstract: Adoptive transfer of T lymphocytes genetically modified with antigen-specific T cell receptor (TCR) constitutes a promising approach for the treatment of malignant tumors and virus infections. One of the challenges in this field of TCR gene therapy is TCR mispairing defining the incorrect pairing between an introduced TCR α or β chain and an endogenous TCR β or α chain, which results in diluted surface expression of the therapeutic TCR αβ. Although there is currently no clinical evidence for TCR mispairing-induced autoreactivity, the generation of autoreactive TCRs upon TCR mispairing cannot be excluded. So it is important to detect TCR mispairing to evaluate the efficiency of TCR gene therapy. Currently there is no available quantitative assay for the measurement of TCR mispairing. Fluorescence resonance energy transfer (FRET) is a powerful approach for identifying biologically relevant molecular interactions with high spatiotemporal resolution. In this study, we described the method of FRET for the measurement of TCR mispairing. It was found that the average FRET efficiency was 12.2 ± 7.5% in HeLa cells and 8.4 ± 3.3% in Jurkat cells (P = 0.026605). The reduction of FRET efficiency in lymphocytes reflected the presence of mispaired TCRs, indicating there were ~30% TCR mispairing in lymphocytes. This study provides a quantitative intracellular assay that can be used to detect TCR mispairing in genetically modified T lymphocytes.
authors.affiliation: School of Life Science and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, 510006, People's Republic of China.
authors.forename: Hongwei
authors.initials: H
authors.lastname: Shao
authors.forename: Wenfeng
authors.initials: W
authors.lastname: Zhang
authors.forename: Qinglian
authors.initials: Q
authors.lastname: Hu
authors.forename: Fenglin
authors.initials: F
authors.lastname: Wu
authors.forename: Han
authors.initials: H
authors.lastname: Shen
authors.forename: Shulin
authors.initials: S
authors.lastname: Huang
mesh_terms: D002462:Cell Membrane / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells; D008164:Luminescent Proteins / Q000378:metabolism; D011485:Protein Binding; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D013601:T-Lymphocytes / Q000378:metabolism*
chemical_list: D008164:Luminescent Proteins; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins