γδTCR immunoglobulin constant region domain exchange in human αβTCRs improves TCR pairing without altering TCR gene-modified T cell function

摘要

The adoptive genetic transfer of T cell receptors (TCRs) has been shown to be overall feasible and offer clinical potential as a treatment for different types of cancer. However, this promising clinical approach is limited by the serious potential consequence that exogenous TCR mispairing with endogenous TCR chains may lead to the risk of self-reactivity. In the present study, domain-exchange and three-dimensional modeling strategies were used to create a set of chimeric TCR variants, which were used to exchange the partial or complete constant region of αβTCR with corresponding γδTCR domains. The expression, assembly and function of the chimeric TCR variants were examined in Jurkat T cells and peripheral mononuclear blood cells (PBMCs). Genetically-encoded chimeras were fused with a pair of fluorescent proteins (ECFP/EYFP) to monitor expression and the pairing between chimeric TCRα chains and TCRβ chains. The fluorescence energy transfer based on confocal laser scanning microscopy showed that the introduction of γδTCR constant sequences into the αβTCR did not result in a global reduction of mispairing with endogenous TCR. However, the TCR harboring the immunoglobulin-like domain of the γδTCR constant region (i.e., TCR∆IgC), showed a higher expression and preferential pairing, compared with wild-type (wt)TCR. The function analysis showed that TCR∆IgC exhibited the same levels of interferon-γ production and cytotoxic activity, compared with wtTCR. Furthermore, these modified TCR-transduced T cells retained the classic human leukocyte antigen restriction of the original TCR. The other two chimeric TCRs, had either exchange of the cp+tm+ic domain or exchange of the whole C domain (). Ultimately, exchange of these domains demonstrated defective function in the transduced T cells. Taken together, these findings may provide further understanding of the γδTCR constant domain with implications for the improvement of TCR gene transfer therapy.Schematic representation of chimeric TCR variants used in the present study. wtTCRs were isolated from tumor-infiltrating lymphocytes of patients as control TCRs. Three chimeric TCRs constructs were generated by replacement of the either IgC, cp+tm+ic, or C regions of αβTCR (white) by corresponding γδTCR (black). The amino acid boundaries of the domains were as follows: TCR∆IgC α chain (aa132-221) replaced by δ chain (aa140-231), β chain (aa136-263) replaced by γ chain (aa140-248); TCR∆IgC retained the connecting peptide domains of the α and β chains. TCR∆cp+tm+ic α chain (aa222-271) replaced by δ chain (aa232-292), β chain (aa264-313) replaced by γ chain (aa249-311), TCR∆C α chain (aa132-271) replaced by δ chain (aa140-292), β chain (aa136-313) replaced by γ chain (aa140-311). TCR, T cell receptor; wt, wild-type; Ig, immunoglobulin-like; cp+tm+ic, connecting peptide, transmembrane and intracellular; C, complete region.