In vivo anti-PD-L1 treatment restores key T-cell effector functions. Fresh splenocytes of isotype (n = 10), aPD-L1–treated (n = 15), and hWT (n = 6) mice were cultured for 6 hours with or without phorbol myristate acetate (PMA)/ionomycin (P/I) in the presence of brefeldin A/monensin during the last 5 hours. (A) After surface staining for CD3, CD4, CD8, and CD44 and permeabilization/fixation, cells were stained for intracytoplasmic IL-2, IL-4, and IFN-γ and analyzed by flow cytometry. Unstimulated cells (unstim.) and FMO controls were included in the analysis, and percentages of IL-2, IL-4, and IFN-γ-positive cells of CD44⁺CD3⁺CD4⁺ viable, single MNCs were calculated. (B) Effector cell cytotoxicity was assessed by CD107a localization to the cell surface upon mitogenic stimulation with P/I. CD107a antibody was added from the beginning of the 6-hour in vitro culture period. Naïve and Ag-exp. CD3⁺CD8⁺ cytotoxic effector T-cell subsets were discriminated by expression of CD44. Unstimulated cells were used as controls. Left panel: representative flow cytometry dot plots. Center panel: quantification of CD107a-expressing cells within CD44^– naïve (light gray) and CD44⁺ Ag-exp. cells (dark gray). Right panel: because of different relative frequencies of CD44⁺ cells between experimental groups, T-cell function was compared by calculating ratios of CD44⁺CD107a⁺:CD44⁺CD107a^– cells of all CD3⁺CD8⁺ T cells to describe enrichment (increased ratio) or loss (decreased ratio) of effector cells within the CD44⁺ population. (C) Splenic T cells taken from aPD-L1, isotype, and hWT mice were mixed with 7-amino-4-chloromethylcoumarin-labeled, superantigen-pulsed healthy syngeneic B cells as antigen-presenting cells at a 1:1 ratio, centrifuged onto poly-lysine-coated microscope slides, and F-actin was stained with rhodamine-phalloidin. Immune synapse formation between T and B cells was quantified by confocal laser-scanning microscopy using AxioVision image analysis software. The synapse area is depicted as mean area of T-cell F-actin immune synapses (μm²) value. (D) To assess ex vivo proliferation of unstimulated cytotoxic T cells, intranuclear Ki-67 staining was analyzed by flow cytometry based on FMO controls and is given as a ratio among CD44⁺ cells as described in (B). (E) In vivo proliferation of CD8⁺ and CD4⁺ T cells in spleen was assessed after injection with 100 µg per gram body weight EdU 20 hours before being euthanized as a percentage of EdU⁺ cells after gating on single CD5⁺ T cells. All graphs depict mean ± SD. *P < .05; **P < .001; ***P < .0001.