Selection of highly responsive T cell receptors by an analysis combining the expression of multiple markers

The clinical success of T cell receptor (TCR) gene–transduced T (TCR‐T) cell therapy is expected as one of the next‐generation immunotherapies for cancer, in which the selection of TCRs with high functional avidity (high‐functional TCRs) is important. One widely used approach to select high‐functional TCRs is a comparison of the EC50 values of TCRs, which involves laborious experiments. Therefore, the establishment of a simpler method to select high‐functional TCRs is desired. We herein attempted to establish a simple method to select high‐functional TCRs based on the expression of T cell activation markers using the mouse T cell line BW5147.3 (BW). We examined relationships between the EC50 values of TCRs in interleukin‐2 production and the expression levels of TCR activation markers on BW cells. In TCR‐expressing BW cells stimulated with antigenic peptides, the CD69, CD137, and PD‐1 expression was differentially induced by various doses of peptides. An analysis of TCRs derived from the tumor‐infiltrating lymphocytes of murine melanoma and peripheral blood T cells of hepatocellular carcinoma patients treated with a peptide vaccination revealed that an analysis combining CD69, CD137, and PD‐1 expression levels in BW cells stimulated with a single dose of an antigenic peptide selected high‐functional TCRs with functional avidity assessed by EC50 values. Our method facilitates the section of high‐functional TCRs among tumor‐reacting TCRs, which will promote TCR‐T cell therapy. The stimulation of BW cells expressing objective TCRs with a single dose of antigenic peptides and analysis combining the expression of CD69, CD137, and PD‐1 allows us to select highly responsive TCRs. The stimulation of BW5147.3 cells expressing objective TCRs with a single dose of antigenic peptides and analysis combining the expression of CD69, CD137, and PD1 allows us to select highly responsive T cell receptors.