Fig. 3: EMT mediates cell-autonomous resistance to direct cytolytic T cell killing. | Nature Communications

Fig. 3: EMT mediates cell-autonomous resistance to direct cytolytic T cell killing.

From: Plasticity-induced repression of Irf6 underlies acquired resistance to cancer immunotherapy in pancreatic ductal adenocarcinoma

Fig. 3

Flow cytometric analysis of immune populations in s.c. implanted 4662 parental (P, n = 6) vs. Esc (E1 and E2, n = 4 per line) (a) and 4662 parental EV (n = 5) vs. Zeb1/Snail OE (n = 5) tumors (b) on day 18 post inoculation. c Representative αCD3 IHC images (left) and quantitation (right) from s.c. implanted parental and Esc tumors (n = 4 and 6 respectively). Scale bars, 250 µm. Mice that had been transplanted with 4662 parental cells and achieved CR or near CR following combination therapy (black arrows) were treated with control IgG (n = 11), depleting αNK1.1 Ab (n = 10), or αCD4 and αCD8 Abs (n = 13), starting from day 50 (blue arrows) post tumor inoculation, and monitored for tumor recurrence (d). The corresponding survival curves are shown in (e). n.s., non-significant. f Tumor growth of s.c. inoculated OVA-transduced 4662 parental and Esc tumors in NOD/SCID mice, with or without adoptive transfer of activated OVA-specific CD8+ T cells (OT-I) on day 14 (arrow) (n = 6 for parental and 8 for the other groups). g OVA-tdTomato+ 4662 parental and Esc tumors were co-cultured with non-activated or activated OT-I by αCD3 and αCD28 Abs overnight, at indicated tumor to effector (T:E) ratios. Each dot represents biological replicates. Two days later, AnnexinV and 7-AAD expression on tumor cells were determined by flow cytometry. h OVA+ 4662 parental and Esc tumors were additionally transduced with YFP or CFP expression constructs, plated separately or mixed, and co-cultured with activated OT-I. AnnexinV and 7-AAD on each tumor were measured 2 d after co-culture. The percentages of 7-AAD+ cells on co-cultured OVA+ 4662 parental EV vs. Zeb1/Snail OE tumors (i) and 4662 Esc EV vs. Zeb1-/-Snail-/- tumors (j) with or without activated OT-I for 2 d (n = 3). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.0001 by Student’s t test (a, b, g–j), Mann-Whitney t test (c), log-rank (Mantel-Cox) test (e), and one-way ANOVA (f). Data represent two independent experiments. Source data and exact P value are provided as a Source Data file.

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