Fig. 7: Pitavastatin calcium potentiates anti-tumor immunity triggered by combination therapy of IL-2 and PD-1 blockade. | Cell Research

Fig. 7: Pitavastatin calcium potentiates anti-tumor immunity triggered by combination therapy of IL-2 and PD-1 blockade.

From: PD-1 signaling negatively regulates the common cytokine receptor γ chain via MARCH5-mediated ubiquitination and degradation to suppress anti-tumor immunity

Fig. 7

a PC treatment up-regulates the level of γc. Human CD8+ T, mouse CD8+ T, Jurkat, HBP-ALL or CTLL2 cells were treated with PC (0, 0.5, 1, 2 μM) for 24 h before immunoblotting analysis with the indicated antibodies. The γc band intensities relative to the corresponding β-actin bands were shown in the histograph. b MARCH5-deficiency impairs PC-induced up-regulation of γc. Control or MARCH5-deficient Jurkat cells were treated with PC (0, 0.5, 1 μM) for 24 h before immunoblotting analysis with the indicated antibodies. The immunoblots were repeated for two times with similar results. c PC treatment suppresses tumor growth. C57BL/6J mice were subcutaneously injected with 5 × 105 of MC38 or B16F10 cells. On day 3 (MC38) or 5 (B16F10) after tumor cell implantation, mice were intraperitoneally injected with control or PC (5 mg/kg/day). Tumor sizes were measured every two days by caliper. Tumor-bearing mice were euthanized on day 13 (MC38) or day 15 (B16F10). Tumor weights were measured by Analytical Balance. Graph shows mean ± SEM, n = 6. Data were analyzed using Student’s unpaired t-test with GraphPad Prism 8. d Effects of PC on the level of γc in TILs. TILs were isolated from the MC38 tumor tissues in c. TILs were stained with the indicated antibodies and analyzed by flow cytometry. Graph shows mean ± SEM, n = 6 independent samples. Data were analyzed using a Student’s unpaired t-test with GraphPad Prism 8. MFI, median fluorescence intensities. e PC treatment increases tumor infiltrating CD8+ cytotoxic T cells. TILs were isolated from the MC38 tumor tissues in c. TILs were stained with the indicated antibodies and analyzed by flow cytometry. Graph shows mean ± SEM, n = 6 independent samples. Data were analyzed using Student’s unpaired t-test with GraphPad Prism 8. f PC potentiates the anti-tumor efficacy of IL-2 and PD-1 combination. C57BL/6J mice were subcutaneously injected with MC38 cells (5 × 105). On day 5 after tumor cell implantation, mice were intraperitoneally injected with control, PC (5 mg/kg), IL-2 (50,000 IU per mouse) or anti-PD-1 (100 μg per mouse) (Supplementary information, Fig. S10b). Tumor sizes were measured every two days by caliper from day 5. Graph shows mean ± SEM, n = 8. Data were analyzed using two-way ANOVA with GraphPad Prism 8. g PC promotes the survival rate of mice treated with IL-2 and PD-1 blockade. C57BL/6J mice were subcutaneously injected with MC38 cells (5 × 105). On day 5 after tumor cell implantation, mice were intraperitoneally injected with control, PC (5 mg/kg), IL-2 (50,000 IU per mouse) or anti-PD-1 (100 μg per mouse) (Supplementary information, Fig. S10b). Mice were sacrificed when the tumor size is bigger than 15 mm of the mean tumor diameter, tumor volume exceeded 2000 mm3, or tumor had ulcers with diameter reached 10 mm. Statistical analysis was performed using the GraphPad Prism 8 software, n = 8. Kaplan–Meier survival curves and corresponding log-rank (Mantel-Cox) tests were used to evaluate the statistical differences between groups in survival studies. There is a significant difference when the P < 0.05.

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