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.

Rapid Assessment of Functional Avidity of Tumor-Specific T Cell Receptors Using an Antigen-Presenting Tumor Cell Line Electroporated with Full-Length Tumor Antigen mRNA

“…Our study also confirms the suitability of 2D3 cells for the analysis of TCR avidity, thanks to their expression of human CD8 co-receptor, the absence of a native TCRαβ, the simplicity to engineer them with an antigen-specific TCR, and the expression of EGFP upon TCR triggering. The lack of endogenous TCR eliminates the possibility of TCR mispairing between endogenous and transgenic TCRs [41]. Therefore, EGFP expression can be directly correlated with the degree of introduced TCR triggering, i.e., the capacity of different APCs to present a peptide and to activate T cells.…”

Transduction of SIV-Specific TCR Genes into Rhesus Macaque CD8+ T Cells Conveys the Ability to Suppress SIV Replication

“…One complication to introducing cloned TCRs into CD8 + T cells that already express α/β TCR complexes is the prospect of forming hybrid dimers between the exogenous and endogenous chains [25], [67], [68], [69]. While we do not know to what extent hybrid complexes are being formed in our TCR-transduced CD8 + T cells, the T cells that we isolated by sorting the transduced populations for high tetramer binding had similar receptor densities to those of typical SIV specific CD8 + T-cell clones.…”

Genome-Edited T Cell Therapies

“…TCR mispairing, where there is heterologous pairing between the endogenous and recombinant TCRs, is capable of generating novel-targeting specificities and inducing autoreactivity [53]. While codon optimization, murinization of human TCRs, and the addition of cysteine residues have been found to reduce mispairing [54], complete knockout of the endogenous TCR would be advantageous.…”