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. 2021 Nov 16;9(11):e003213. doi: 10.1136/jitc-2021-003213

Figure 2.

Figure 2

Cytotoxic activity of opT cells. (A) CFSE (carboxyfluorescein succinimidyl ester) labeled PBMC and opT cell proliferation were co-cultured with autologous tumor organoids for 4 days and changes in percentage of CFSE-low T cell population shown. Mean±SEM from three independent experiments shown. N.S., not significant. **, p<0.01. ***, p<0.001. P value calculated using two-tailed, unpaired t-test. (B) Time lapse image of fluorescently labeled organoids (green) incubated with unlabeled opT cells over a period of 20 hours. Scale bar, 100 µm. (C) Changes in levels of epithelial cell-specific caspase-cleaved cytokeratin 18 (CK18) fragments containing the CK18Asp396 (‘M30’) neo-epitope in the media, quantitated by ELISA. Mean±SEM from three independent experiments shown. Each dot represents the mean of three technical replicates from independent experiments. N.S., not significant. *, p<0.05. **, p<0.01. ***, p<0.001. P value calculated using two-tailed, unpaired t-test. (D) Interferon gamma (IFN-γ) and granzyme B produced by PBMC or opT in the presence of autologous tumor organoids at different time points for Pt3 and Pt38. Mean±SEM from three independent experiments shown. Each dot represents the mean of at least two technical replicates from independent experiments. N.S., not significant. *, p<0.05. **, p<0.01. P value calculated using two-tailed, unpaired t-test. (E) FasL expression in opT cells from Pt3 and Pt38 by immunoblot or flow cytometry. (F) Relative M30 production from the supernatants of Pt38 or Pt3 co-cultured in the presence or absence of anti-FasL blocking antibody for 72 hours. NS, not significant. **, p<0.01. P value calculated using two-tailed, unpaired t-test. FasL, Fas-Fas ligand; h, hours; opT, organoid-primed T; PBMCs, peripheral blood mononuclear cells; Pt, patient.