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. Author manuscript; available in PMC: 2016 Sep 10.
Published in final edited form as: Cell. 2015 Sep 10;162(6):1242–1256. doi: 10.1016/j.cell.2015.08.052

Figure 3. Tumor cell-intrinsic PD-1 signaling promotes murine melanoma growth.

Figure 3

(A) Growth kinetics (mean±s.d.) of Pdcd1-OE vs. vector control B16-F10 melanomas in PD-L1(−/−) KO Rag(−/−) KO (n=14 vs. 20 vs. 10) vs. wildtype Rag(−/−) KO recipients (n=14 vs. 14 vs. 8) treated with anti-PD-L1- vs. isotype control monoclonal antibody (mAb). (B) Growth kinetics (mean±s.d.) in C57BL/6 (left) and NSG mice (right) of Pdcd1-OE B16-F10 cells co-transduced with PD-L1 (Cd274, also known as Pdcd1lg1)-shRNA vs. control-shRNA compared to vector controls (n=10 each). (C) Mean number of tumor spheres±s.e.m (left), and immunoblot analysis of p- and total S6, AKT, and ERK in PD-L1 Ig vs. control Ig-treated B16 cultures (right). (D) Immunoblot analysis of p- and total S6, AKT, and ERK in PD-L1 Ig vs. control Ig-treated B16-F10 melanoma cells cultured in the presence of the pharmacologic PI3K inhibitors, wortmannin or LY294002, or the mTOR pathway inhibitors, rapamycin or PP242. (E) Schematic diagram illustrating the introduction of tyrosine to phenylalanine mutations to murine PD-1 signaling motifs via site-directed mutagenesis. (F) Relative Pdcd1 mRNA expression (top, mean±s.d.) and representative flow cytometry plots of PD-1 surface protein expression (bottom) by wildtype Pdcd1-OE vs. Y225F-Pdcd1-OE, Y248F-Pdcd1-OE, Y225F/Y248F-Pdcd1-OE, and vector-control B16-F10 variants. (G) Tumor growth kinetics in C57BL/6 (top, n=10–14 each) and NSG mice (bottom, n=8–10 each), (H) mean number of tumor spheres±s.e.m, and (I) immunoblot analysis of p- and total S6, AKT, and ERK in B16-F10 melanoma variants as in (F). Immunoblot results are representative of n=2 independent experiments, respectively (*P<0.05, **P<0.01, ***P<0.001). See also Figure S5.