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. Author manuscript; available in PMC: 2021 Mar 15.
Published in final edited form as: Sci Signal. 2020 Sep 15;13(649):eaau4518. doi: 10.1126/scisignal.aau4518

Fig. 9. Acute in vitro blockade of PD-1 does not restore killing or cytoskeletal polarization.

Fig. 9.

(A) Fluorescence microscopy analysis of the killing of Renca+HA, Renca+HA PD-L1−/−, and Renca +HA PD-L1-GFP cells in vitro by HA-specific CTLs and TILs in the presence or absence of antibody against PD-1, as indicated. Average Renca+HA cell death rates with means ± 95% confidence interval are from at least four independent experiments. (B) Microscopy analysis of HA-specific CTLs and TILs interacting with HA peptide–pulsed Renca+HA cells in the presence or absence of antibody against PD-1, as indicated. The percentage of cells that translocated away from the contact site are means ± 95% confidence interval of 138 CTLs, 137 TILs (from Fig. 2, F to H), 35 CTLs treated with anti-PD-1, and 24 TILs treated with anti-PD-1 from two independent experiments. (C) Fluorescence microscopy analysis of F-tractin-GFP–expressing HA-specific CTLs and TILs interacting with HA peptide–pulsed Renca+HA cells in the presence or absence of antibody against PD-1, as indicated. Data showing the accumulation of F-tractin-GFP data at the center of the immune synapse are means ± SEM of 60 CTLs, 51 TILs (from Fig. 3D), 41 CTLs+anti-PD-1, and 28 C TILs+anti-PD-1 from two independent experiments. (D) Fluorescence microscopy analysis of Fura-2–loaded, HA-specific CTLs with Renca+HA cells in the presence or absence of antibody against PD-1, as indicated. Normalized Fura-2 emission data are means ± SEM of 50 CTLs (from Fig. 5D) and 52 CTLs treated with anti-PD-1 from two independent experiments. *P < 0.05, **P < 0.0045 (to account for Bonferroni correction), ***P < 0.001, and ****P < 0.0001; by Kruskal-Wallis test with comparison to ‘Renca+HA’ (A, CTL), Kruskal-Wallis test and one-way ANOVA because it is difficult to satisfy all assumptions for either test (A, TIL) proportion’s z-test (B), one-way ANOVA (C), or Student’s t-test (D).