Figure 5.
Identification of EVs and PD-L1 expression roles on the resistance to chemotherapy in TP53-deficeint B-cell lymphomas. (A) Flow cytometer analysis of shCTRL− and shTP53-EVs bound to polystyrene microspheres and stained for the indicated proteins or the correspondent isotype control (1 representative experiment of 3). (B) Alemtuzumab-mediated ADCP assay with normal hMB cells and J774A.1 macrophages in the presence of EVs derived from shCTRL and shTP53 hMB cells that were incubated (10 minutes) with anti–PD-L1 antibody (atezolizumab) prior to their addition to the coculture. The same concentration of anti–PD-L1 antibody diluted in PBS was used as control. Data shows 1 representative experiment of 3, with 5 replicates. (C) Immunoblotting detection of p53 and PD-L1 protein in shTP53/PD-L1–WT and -KO hMB cells. The corresponding total protein staining of the samples as a protein-loading control is shown. (D) Alemtuzumab-mediated ADCP assay with normal hMB cells with J774A.1 macrophages in the presence of shTP53/PD-L1–WT and -KO EVs; PBS was used as control. Data shows 1 representative experiment of 3, with 5 replicates. (E) Flow cell cytometer detection of PD-L1 in shCTRL and shTP53 hMB cells, treated with mafosfamide (CTX) or vehicle for 24 hours. Left panel shows half offset histogram representation of 1 representative experiment. Right panel displays PD-L1 protein expression percentage from 6 independent experiments. (F) Anti-CD20 (18B12)-mediated ADCP of shCTRL and shTP53 hMB cells pretreated or not with mafosfamide and cocultured with peritoneal macrophages exposed to anti-PD1 antibody (GS-696882) from 4 hours prior to the assay. Data shows 1 representative experiment of 3, with 5 replicates. (G) Survival curve of NSG mice IV injected with shTP53 hMB tumor cells treated IP with cyclophosphamide and alemtuzumab (CA) or CA and anti-PD1 antibody (GS-696882). PBS was used as control treatment (n = 12-18). (H) Alemtuzumab-mediated ADCP of different shTP53/PD-L1–WT and -KO clones coculture with J774A.1 macrophages (n = 3). (I) Number of mafosfamide (CTX) pretreated GFP+ hMB cells (shTP53/PD-L1–KO, shTP53/RAB27A-KO, and the corresponding shTP53 empty vector) remaining in the coculture with J774A.1 macrophages after ADCP assay performed in combination or not with alemtuzumab and normalized to the amount of hMB cells in a hMB single culture. Data shows 1 representative experiment, with 5 replicates. (J) Survival curve of NSG mice IV injected with shTP53/PD-L1-WT or shTP53/PD-L1-KO hMB tumor cells and IP treated after 10 days with cyclophosphamide and alemtuzumab (CA) or PBS as control treatment (n = 3-6). *P < .05, **P ≤  .01, ***P ≤  .001, ****P ≤ .0001. IP, intraperitoneal.

Identification of EVs and PD-L1 expression roles on the resistance to chemotherapy in TP53-deficeint B-cell lymphomas. (A) Flow cytometer analysis of shCTRL− and shTP53-EVs bound to polystyrene microspheres and stained for the indicated proteins or the correspondent isotype control (1 representative experiment of 3). (B) Alemtuzumab-mediated ADCP assay with normal hMB cells and J774A.1 macrophages in the presence of EVs derived from shCTRL and shTP53 hMB cells that were incubated (10 minutes) with anti–PD-L1 antibody (atezolizumab) prior to their addition to the coculture. The same concentration of anti–PD-L1 antibody diluted in PBS was used as control. Data shows 1 representative experiment of 3, with 5 replicates. (C) Immunoblotting detection of p53 and PD-L1 protein in shTP53/PD-L1–WT and -KO hMB cells. The corresponding total protein staining of the samples as a protein-loading control is shown. (D) Alemtuzumab-mediated ADCP assay with normal hMB cells with J774A.1 macrophages in the presence of shTP53/PD-L1–WT and -KO EVs; PBS was used as control. Data shows 1 representative experiment of 3, with 5 replicates. (E) Flow cell cytometer detection of PD-L1 in shCTRL and shTP53 hMB cells, treated with mafosfamide (CTX) or vehicle for 24 hours. Left panel shows half offset histogram representation of 1 representative experiment. Right panel displays PD-L1 protein expression percentage from 6 independent experiments. (F) Anti-CD20 (18B12)-mediated ADCP of shCTRL and shTP53 hMB cells pretreated or not with mafosfamide and cocultured with peritoneal macrophages exposed to anti-PD1 antibody (GS-696882) from 4 hours prior to the assay. Data shows 1 representative experiment of 3, with 5 replicates. (G) Survival curve of NSG mice IV injected with shTP53 hMB tumor cells treated IP with cyclophosphamide and alemtuzumab (CA) or CA and anti-PD1 antibody (GS-696882). PBS was used as control treatment (n = 12-18). (H) Alemtuzumab-mediated ADCP of different shTP53/PD-L1–WT and -KO clones coculture with J774A.1 macrophages (n = 3). (I) Number of mafosfamide (CTX) pretreated GFP+ hMB cells (shTP53/PD-L1–KO, shTP53/RAB27A-KO, and the corresponding shTP53 empty vector) remaining in the coculture with J774A.1 macrophages after ADCP assay performed in combination or not with alemtuzumab and normalized to the amount of hMB cells in a hMB single culture. Data shows 1 representative experiment, with 5 replicates. (J) Survival curve of NSG mice IV injected with shTP53/PD-L1-WT or shTP53/PD-L1-KO hMB tumor cells and IP treated after 10 days with cyclophosphamide and alemtuzumab (CA) or PBS as control treatment (n = 3-6). *P < .05, **P ≤  .01, ***P ≤  .001, ****P ≤ .0001. IP, intraperitoneal.

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