Fig. 1: PD-1 signaling is negatively correlated with γc level.
a γc level is negatively correlated with PD-L1 level in human NSCLC tumor biopsies. Representative images from IHC staining of γc and PD-L1 in human NSCLC tumor biopsies are shown (left panels). Quantification of PD-L1 and γc staining intensities was performed by semi-quantitative scoring (right panel). n = 127 independent samples, R = –0.2966. Correlation coefficients were calculated using the Pearson test. Two-sided P-value was given. Scale bars, 100 μm. b PD-1 blockade increases γc level in tumor tissues. B16F10 cells (5 × 105) were subcutaneously injected into C57BL/6J mice. CT26 cells (5 × 105) were subcutaneously injected into Balb/c mice. Mice were intraperitoneally injected with control or anti-PD-1 antibody (100 μg per mouse) every three days (four times in total) five days after inoculation of cells. After 17 days, tumor-bearing mice were euthanized and tumor tissues were analyzed. Representative images from IHC staining of γc in tumor sections are shown. Scale bars, 100 μm. c PD-1 blockade increases γc level in tumor-infiltrating CD8+ T cells. TILs were isolated from B16F10 tumor tissues (left panels) or CT26 tumor tissues (right panels), stained with the indicated antibodies and analyzed by flow cytometry. Graph shows mean ± SEM, n = 8 independent samples. Data were analyzed using Student’s unpaired t-test with GraphPad Prism 8. MFI, median fluorescence intensities. d PD-1 ligation down-regulates γc level. Human CD8+ T cells or PD-1-expressing Jurkat (Jurkat-PD-1) cells were stimulated with plate-bound anti-CD3 (1 μg/mL) and solubilized anti-CD28 (5 μg/mL) in the presence of plate-bound hPD-L1-Fc fusion protein or control hIgG1 (2 μg/mL) for 3 days. The cells were stained with the indicated antibodies and analyzed by flow cytometry. Graph shows mean ± SEM, n = 3 technical repeats. Data were analyzed using two-way ANOVA with GraphPad Prism 8. e The level of γc is down-regulated after PD-1 ligation. Human CD8+ T cells were stimulated with plate-bound anti-CD3 (1 μg/mL) and solubilized anti-CD28 (5 μg/mL) in the presence of plate-bound hPD-L1-Fc fusion protein or control hIgG1 (2 μg/mL) for 3 days before immunoblotting analysis with the indicated antibodies. f PD-1 ligation down-regulates γc level. Left panels: Jurkat cells transduced with empty vector (Jurkat-EV) or Jurkat-PD-1 cells were analyzed by immunoblots with the indicated antibodies. Middle panels: Jurkat-EV and Jurkat-PD-1 cells were stimulated with plate-bound anti-CD3 (1 μg/mL) and solubilized anti-CD28 (5 μg/mL) in the presence of plate-bound hPD-L1-Fc fusion protein or control hIgG1 (2 μg/mL) as indicated for 3 days before immunoblotting analysis with the indicated antibodies. Right panels: Jurkat-PD-1 cells were stimulated with PHA (50 ng/mL) in the presence of plate-bound hPD-L1-Fc fusion protein or control hIgG1 (2 μg/mL) for the indicated times before immunoblotting analysis with the indicated antibodies. g Effects of PD-1 ligation on transcription of γc. Human CD8+ T cells or Jurkat-PD-1 cells were stimulated with plate-bound anti-CD3 (1 μg/mL) and solubilized anti-CD28 (5 μg/mL) in the presence of plate-bound hPD-L1-Fc fusion protein or control hIgG1 (2 μg/mL) for 3 days before qPCR analysis of mRNA levels of the indicated genes. Graph shows mean ± SEM, n = 3 independent samples from one representative experiment. Data were analyzed using two-way ANOVA with GraphPad Prism 8. The experiments in c–g were repeated for at least two times with similar results.
