| Popis: |
Nonspecific TCR stimulation by αCD3 Ab is not suitable for selective reprogramming of tumor-reactive T cells. A, A summary table of top 10 frequent TCRβ sequences in the starting cells, that is, expanded TIL of patient 4069, indicating the CDR3β amino acid sequence, Vβ family and frequency. The most frequent TCR was the patient's neoantigen-specific TCR (indicated in red). B, A summary table of TCRβ sequences of established TIL-iPSCs by αCD3 Ab stimulation, indicating the CDR3β amino acid sequence, Vβ family and the number of TIL-iPSC clones established. C, A summary table of six candidate TCR pairs including five newly found TCRs (NF TCR 1–5) and one PIR-TCR which were sequenced individually from master tube 6 containing 14 individual iPSC clones. The table indicates TCR pairs, CDR3β sequence, Vβ family, CDR3α sequence, and Vα family. NF, newly found TCR (unknown reactivity); PIR, preidentified reactive TCR. D and E, CD137 (4-1BB) upregulation assay of T cells transduced with candidate TCRα and TCRβ pairs identified from TIL-iPSCs (Fig. 2C). T cells were cocultured with autologous DC pulsed with wild-type (WT) peptide (16 mer or 9 mer) or mutant peptide (9 mer). D shows the representative FACS plots and E shows the percentage of 4-1BB+ cells in each condition. Representative data of three independent experiments from 3 different healthy donor T cells. Mock indicates empty vector transduced T cells used as negative control. F and G, CD137 (4-1BB) upregulation assay of T cells transduced with candidate TCRα and TCRβ pairs identified from TIL-iPSCs (Fig. 2C). T cells were cocultured with autologous, or HLA-matched allogeneic tumor cells derived from the PDXs model. F shows the representative FACS plots and G shows the percentage of 4-1BB+ cells in each condition. Representative data of three independent experiments from 3 different healthy donor T cells. Mock indicates empty vector transduced T cells used as negative control. H, A graph showing the real-time cell growth monitoring (cell count/image) of RFP-overexpressed PDX-derived tumor cell line cultured with T cells transduced with PIR-TCR analyzed by the Incucyte live imaging and analysis system (effector-to-target ratio of 2:1). Empty vector transduced T cells (Mock) and tumor cell alone (TC) were used as negative controls. The results are shown as mean ± SD (N = 4). |
