Journal: Cancer Research Communications

Article Title: Reprogramming of Tumor-reactive Tumor-infiltrating Lymphocytes to Human-induced Pluripotent Stem Cells

doi: 10.1158/2767-9764.CRC-22-0265

Figure Lengend Snippet: The list of antibodies used in the study

Article Snippet: Anti-Human CD137 (4-1BB) , 130-119-885 , PE , 4B4-1 , Miltenyi Biotec.

Techniques:

Journal: Cancer Research Communications

Article Title: Reprogramming of Tumor-reactive Tumor-infiltrating Lymphocytes to Human-induced Pluripotent Stem Cells

doi: 10.1158/2767-9764.CRC-22-0265

Figure Lengend Snippet: 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 . 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 . 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).

Article Snippet: Anti-Human CD137 (4-1BB) , 130-119-885 , PE , 4B4-1 , Miltenyi Biotec.

Techniques: Sequencing, Clone Assay, Transduction, Mutagenesis, Plasmid Preparation, Negative Control, Derivative Assay, Cell Counting, Cell Culture, Imaging

Journal: Cancer Research Communications

Article Title: Reprogramming of Tumor-reactive Tumor-infiltrating Lymphocytes to Human-induced Pluripotent Stem Cells

doi: 10.1158/2767-9764.CRC-22-0265

Figure Lengend Snippet: TIL stimulated with autologous tumor cell line generated antigen specific iPSC whereas αCD3/28 beads mediated stimulation did not. A, 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 ), 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 and C, CD137 (4-1BB) upregulation assay of T cells transduced with candidate TCRα and TCRβ pairs identified from TIL-iPSCs . 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 + cells in each condition. N = 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. D, 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 . T cells were cocultured with autologous or allogeneic tumor cell lines for 16 hours and culture soup were analyzed for IFNγ by ELISA. N = 3 for each condition. Representative data of four independent experiments. E, 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 ), 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 N = 4 each condition. A representative data of four independent experiments.

Article Snippet: Anti-Human CD137 (4-1BB) , 130-119-885 , PE , 4B4-1 , Miltenyi Biotec.

Techniques: Generated, Clone Assay, Transduction, Plasmid Preparation, Negative Control, Positive Control, Enzyme-linked Immunosorbent Assay, Cell Counting, Imaging

Journal: Cancer Research Communications

Article Title: Reprogramming of Tumor-reactive Tumor-infiltrating Lymphocytes to Human-induced Pluripotent Stem Cells

doi: 10.1158/2767-9764.CRC-22-0265

Figure Lengend Snippet: T cells with tumor-reactive TCRs of extremely low frequency were reprogrammed to TIL-iPSCs. A, Summary of five candidate TCR pairs including four newly found TCRs (NF TCR 1–4) and one PIR-TCR (IV) identified in TIL-iPSCs which were sequenced individually. The table includes CDR3 amino acid sequences of α and β chains, Vα family, Vβ family, and immunosequencing analysis of TCRβ which contains frequency in bulk (before coculture) and in sorted DP population after coculture, and enrichment (DP/Bulk). B and C, CD137 (4-1BB) upregulation assay of T cells transduced with candidate TCRα and TCRβ pairs identified from TIL-iPSCs . 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 + cells in each condition. Representative data of four independent experiments. Control PBL; no transgene, Mock indicates empty vector transduced T cells used as a negative control, TIL; expanded TIL containing tumor-reactive T cells. D, 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). T cells were cocultured with autologous or allogeneic tumor cell lines for 16 hours and culture soup were analyzed for IFNγ by ELISA ( N = 3). Representative data of four independent experiments. E, A graph showing the real-time cell growth monitoring (cell count/image) of RFP-overexpressed tumor cells from pt-3784 cocultured with different TCRs (listed in A), analyzed by the Incucyte live imaging and analysis system in an effector-to-target ratio of 2:1. Mock indicates empty vector transduced T cells used as a negative control; TC only indicates tumor cells alone, and four newly found candidate TCRs (NF TCR 1–4) and a PIR-TCR IV were used for coculture and their antitumor properties were measured over 72 hours time period. The results are shown as mean ± SD of N = 4 each condition. A representative data of four independent experiments.

Article Snippet: Anti-Human CD137 (4-1BB) , 130-119-885 , PE , 4B4-1 , Miltenyi Biotec.

Techniques: Transduction, Control, Plasmid Preparation, Negative Control, Enzyme-linked Immunosorbent Assay, Cell Counting, Imaging