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Fig. 1 | Journal of Hematology & Oncology

Fig. 1

From: Polymorphonuclear myeloid-derived suppressor cells impair the anti-tumor efficacy of GD2.CAR T-cells in patients with neuroblastoma

Fig. 1

Presence of PMN-MDSC in PB of NB patients and their effect on GD2.CAR T-cells. A, B Mononuclear cells isolated from the PB of NB patients were analyzed by flow-cytometry for the expression of specific markers allowing the identification of PMN-MDSC subsets. A Percentages of PMN-MDSC on CD45+cells (CD66b+CD14cells) in healthy donors (HD, n = 10) and in NB patients (NB pts, n = 9). B A representative gating strategy for PMN-MDSC identification by flow-cytometry is shown. C, D Second- and third-generation GD2.CAR T-cells were cultured either in the absence (w/o, blue bars) or in the presence (cond, red bars) of PMN-MDSC collected from stem cell donors given G-CSF and undergoing to leukapheresis. After 48h, GD2.CAR T-cells and non-transduced (NT) cells (used as control) were collected, purified and co-cultured at the effector:target ratio 1:1 with the SH-SY5Y-eGFP+ NB cell line. Percentages of SH-SY5Y-eGFP+ NB residual live cells at day 5 of co-culture with (C) second- (n = 10) and D third- (n = 10) generation GD2.CAR T-cells are reported. Patient derived GD2.CAR T-cells expressing CD28.4-1BBζ (the same used in our clinical trial) were cultured in the presence of PB-derived (n = 8) or BM-derived (n = 3) PMN-MDSC or PB-derived neutrophils (n = 3) collected from NB patients. After 48h, GD2.CAR T-cells were collected, purified from PMN-MDSC/neutrophils as described in supplementary data (Additional file 6), and co-cultured at an Effector:Target (E/T) ratio 1:1 with SH-SY5Y-eGFP+ NB cell line for 3 days. Percentages of SH-SY5Y-eGFP+ NB residual live cells were analyzed. E One representative co-culture experiment of GD2.CAR T-cells and PMN-MDSC is shown. F Percentages of residual live NB tumor cells in different culture conditions is reported. G Correlation between PMN-MDSC and GD2.CAR T-cells in CD45+ cells in NB patients (n = 55, different time point after GD2.CAR T-cell infusion), r2 = 0.07 and p value is indicated. Mononuclear cells present in the PB of NB patients were analyzed by flow-cytometry. Graphs represent percentages of PMN-MDSC and the corresponding absolute number/µl of GD2.CAR T-cells in NB patients. H Graphs represent GD2.CAR T-cells/µl (blue squares) and the percentages of PMN-MDSC (red dots) in CD45+ cells in responder (n = 5) and non-responder (n = 6) patients (multiple comparison with mixed-effect analysis). *p < 0.05, **p < 0.01, ***p < 0.005. Where not indicated, data were not statistically significant. A Mann–Whitney and C, D, F Wilcoxon Student’s t test. Data are shown as mean ± standard error of the mean (SEM)

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