Extended Data Fig. 6: Role of PD-L1 as a mediator of p95HER2-dependent tumor immune suppression.
(a) Flow cytometry analyzing the impact of PD-L1 knockout (KO) on cell surface PD-1 binding. Control or PD-L1 KO PB2 cells were treated with 10 ng/mL of IFN-γ for 24 h. Cells were then incubated with PD-1-Fc chimeric protein and PE conjugated F(ab’)2-anti-human IgG Fc 2nd antibody and analyzed by flow cytometry. Results demonstrate loss of PD-1 binding to cells harboring knockout of the PD-L1 locus. (b) PD-L1 KO does not impact cell proliferation in culture. Cell proliferation was monitored in real time using the IncuCyte imaging system. N = 6 replicate wells per cell line; data are expressed as mean ± SEM; results are representative of n = 3 biological repeats. (c) Flow cytometry analysis of single cells isolated from the same p95HER2+ PB2 tumors as shown in Fig. 6c. N = 9 control and n = 11 PD-L1 KO tumors, respectively. Data represent mean ± SEM. Statistical analyses by unpaired, two-tailed Student’s t test. No significant alterations were observed for B-cells, dendritic cells, or M1/M2 macrophages. (d) Western blot verification of PD-L1 depletion by shRNA in PB2 cells. (e) shRNA-mediated PD-L1 knockdown slows growth of p95HER2+ PB2 tumorgrafts in syngeneic, immunocompetent C57BL/6 mice. Control shRNA n = 7, PD-L1 shRNA n = 7. Data represent mean ± SEM. Statistical analysis by unpaired, two-tailed Student’s t test (p = 0.0344). (f) Flow cytometric analysis of single cells isolated from the tumors depicted in (e) (Control shRNA n = 7, PD-L1 shRNA n = 7). Boxes span the upper to lower quartiles with a median center line. Whiskers extend from the minimum to the maximum values in the dataset. Statistical analyses by unpaired, two-tailed Student’s t test. P values are indicated within the plots.
