FIGURE 4 from Reprogramming of Tumor-reactive Tumor-infiltrating Lymphocytes to Human-induced Pluripotent Stem Cells

Abstract

<p>TIL stimulated with autologous tumor cell line generated antigen specific iPSC whereas αCD3/28 beads mediated stimulation did not. <b>A,</b> Summary of three candidate TCR pairs identified in TIL-iPSC lines established by different stimulation methods. Bead Stim TCR: The most dominant clone in the TIL-iPSCs established by αCD3/28 beads stimulation, Tumor Stim TCR: The TCR identified in the TIL-iPSC clones established by coculture with autologous tumor cells (TCR 4 in <a href="#fig3" target="_blank">Fig. 3B</a>), and PIR-TCR III: preidentified reactive TCR clone detected in the TIL-iPSC clones established by coculture with autologous tumor cells. The table includes TCR type, CDR3β amino acid sequences, Vβ family, CDR3α amino acid sequences, Vα family are described. <b>B</b> and <b>C,</b> CD137 (4-1BB) upregulation assay of T cells transduced with candidate TCRα and TCRβ pairs identified from TIL-iPSCs (<a href="#fig4" target="_blank">Fig. 4A</a>). T cells were cocultured with autologous or allogeneic tumor cell lines. B shows the representative FACS plots and C shows the percentage of 4-1BB<sup>+</sup> cells in each condition. <i>N</i> = 3 for each condition. Representative data of four independent experiments. Mock: empty vector transduced PBL as a negative control, PIR-TCR III as a positive control. <b>D,</b> A bar graph showing the results of ELISA IFNγ production assay of T cells transduced with candidate TCRα and TCRβ pairs identified from TIL-iPSCs (<a href="#fig4" target="_blank">Fig. 4A</a>). T cells were cocultured with autologous or allogeneic tumor cell lines for 16 hours and culture soup were analyzed for IFNγ by ELISA. <i>N</i> = 3 for each condition. Representative data of four independent experiments. <b>E,</b> A graph showing the real-time cell growth monitoring (cell count/image) of RFP-overexpressed tumor cells from pt. 1913 cocultured with different TCRs (listed in <a href="#fig4" target="_blank">Fig. 4A</a>), analyzed by the Incucyte live imaging and analysis system at effector-to-target ratio of 2:1. Mock indicates empty vector transduced T cells used as a negative control. The results are shown as the mean ± SD of <i>N</i> = 4 each condition. A representative data of four independent experiments.</p>