Fig. 3.

Multiparameter flow cytometry. Freshly isolated peripheral blood mononuclear cells (PBMCs) and tumor cell suspensions prepared from resected tumor material were stained using a panel of directly labelled mAbs to detect different T-cell and myeloid cell subpopulations. Gating strategies are described in detail in “Material and Methods”. a Pre- and posttreatment analysis of lymphocyte subpopulations, mainly CD3⁺, CD4⁺ and CD8⁺ T-cells, CD3⁻CD56⁺ NK cells and CD4⁺CD25^highCD127^low regulatory T-cells. b Pre- and posttreatment analysis of CD45⁺CD11b⁺ myeloid cell subsets including HLA-DR expression on tumor-associated CD45^++/+++CD14^high myeloid cells (TAMC) and CD45^+/++CD14^low microglia. c Tumor cell suspensions were stimulated with PMA/Ionomycin, and CD4⁺ and CD8⁺ CD45RA⁻ memory T cells were analysed for the expression of INF-γ and IL-4 by intracellular FACS staining. Th1/Th2 ratios were calculated by dividing the proportion of IFN-γ positive T-cells by the proportion of IL-4 positive cells. d Pre- and posttreatment expression of PD-L1 on TAMC and microglia (black line = PD-L1; grey line = isotype control)