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. 2022 Jul 21;10(7):e004695. doi: 10.1136/jitc-2022-004695

Figure 4.

Figure 4

Induction of T-cell activation by PD-1/PD-L1 inhibition using co-culture experiments. (A) Experimental workflow. Tumor cells were obtained from surgical resections of melanoma (Mel), bone metastases of breast (BBM) and lung cancer (LBM). The tumor cells were stimulated with IFN-γ for 18 hours prior to co-culture to enhance the PD-L1 expression. The PBMC and tumor cells were co-cultured and treated with anti-CD28 (blue) or treated with anti-CD28 plus PD-L1 inhibitors (small-molecule inhibitor 69 (yellow) and anti-PD-L1 (red)) for 72 hours. The PD-1/PD-L1 inhibition and T-cell activation were assessed using flow cytometry. (B) Cell-surface PD-L1 levels were determined by flow cytometry. Data indicate mean fluorescence intensity (MFI) of anti-PD-L1-BV711 minus MFI of isotype control. (C) Cell-surface PD-1 levels were determined by flow cytometry. (D) Representative flow cytometry plots for T cell reactivity after 72 hours of co-culture with autologous tumor cells. The plots indicate the percentage of IFN-γ and CD107a on CD8+ T cells. Quantification of tumor cells-induced IFN-γ (E) production and CD107a (F) cell-surface expression of CD45+CD3+CD8+ T cells was obtained after 72 hours of co-culture. Data are presented as mean±SD, N=1, n=3 or 2, from one independent experiment performed in triplicate or duplicate (limited amounts of tumor or blood available). Statistical analysis: one-way analysis of variance and Tukey’s post test. FACS, fluorescence-activated cell sorting; IFN, interferon; IL, interleukin; PBMC, peripheral blood mononuclear cells; PD-1, programmed cell death protein 1; PD-L1, PD-ligand 1.