Combinations of TLR Ligands: A Promising Approach in Cancer Immunotherapy
Figure 4
Coculture experiments. Tumor cells were cocultured with PBLs of three different healthy donors in the presence of (a) single TLR ligands (10 μM) or in the presence of (b) TLR ligand combinations (0.01 μM) for 72 h. Thereafter, numbers of viable tumor cells were quantified by flow cytometry using microsphere beads as calibrator. Tumor cells without TLR ligand were set as 1 and all other data were given as X-fold increase. (b) Results for single substance treatment (0.01 μM) are additionally shown. ((a), (b)) For each approach, cells treated with the particular TLR ligand but without PBL are shown. (c) Representative dot plot illustrating the gating strategy for quantification of tumor cells. Shown are FACS data of HROC69 cells treated with PBL alone (control, upper panel) and treated with PBL + R848 (10 μM, middle panel) for 72 h. Dead cells present in the gated tumor cells (Gate1) were excluded by staining with propidium iodide (PI+ cells; upper left quadrant in the right upper and middle blots). To illustrate the reliability of the gating-based separation of tumor cells and lymphocytes after coculture, FACS data of PBL alone and HROC69 cells alone are shown in addition (lower panel).
