Figure 4. Blocking of programmed death receptor ligand 1 (PD-L1) in co-cultures.

(A) PD-L1 expression on the CML cell lines K562, BV-173 and CML-T1 was evaluated by flow cytometry. The thick line represents staining with specific antibody and the filled histograms represent the staining with isotype control antibody. (B) Proliferative response of healthy donor PBMCs to CML cell lines with or without addition of PD-L1 blocking antibody. The bars show the proliferative response in different wells after subtraction of proliferation seen when cells lines were cultured alone. The experiment was repeated twice with four donors. Proliferation was measured by thymidine H³ incorporation on triplicate samples. (C) T cell-depleted leukocytes from CML patients (HR n = 7, LR n = 4)) and HCs (n = 15) were co-cultured with PBMCs from a control subject (responder) with addition of an irrelevant antibody (iso) or an antibody blocking PD-L1 (αPD-L1). The percentage of proliferating T cells in the co-culture is shown. (D) The concentration of IL-2 in co-culture supernatants from HCs (n = 15), LR (n = 4) and HR (n = 7) was assessed by ELISA. The increase/decrease in IL-2 concentration for the individual control subjects and CML patients are shown in the figure.