T-cell incubation with the media secreted by WM cells overexpressing PD-L1 and PD-L2 reduces T-cell proliferation and cell cycle proliferation. MWCL-1 cells were transfected with control EV, PD-L1, or PD-L2 constructs. (A) Histograms represent the flow cytometry analysis of the cells overexpressing either PD-L1 (left) or PD-L2 (right). Western blot analysis shows the overexpression of PD-L1 and PD-L2 on the cell surface and in the condition media of the cells, respectively. (B) T cells were isolated from PBMCs and incubated with cell-free media from MWCL-1 lines transfected with overexpressing PD-L1 or PD-L2 constructs. Cells were left either nonstimulated or stimulated with suboptimal (0.5 μg/mL) or optimal (5 μg/mL) dose of CD3 (0.5 μg/mL) and CD28. EV-transfected cells were used as control. Proliferation assay was performed using [³H]TdR following 72 hours of incubation. (C) Western blot analysis was performed on the T-cell lysates after 72 hours of incubation with the media from MWCL-1 lines. (D) Respiratory capacity of T cells in response to treatment with soluble PD-L1 and PD-L2. T cells were stimulated with CD3/CD28 dynabeads for 3 days, and then incubated with cell free–conditioned medias containing soluble PD-L1 and PD-L2 for 1 day. Respiratory capacity of T cells was measured using seahorse XFp analyzer. The diagram is a representative of 3 independent experiments.