IDX combined with cis enhances T cells expansion with cytolytic potential in killing tumor cells
(A) To capture and visualize all mononuclear cell subpopulations in a single two-dimensional (2D) map, representation of t-distributed Stochastic Neighbor Embedding (t-SNE) analysis of concatenated mononuclear cells (3,000 cells/sample) from PBMCs collected from cocultures was applied. T-SNE represent phenotypic markers defining specific cells and are assigned specific colors (CD19 B cells in purple; CD56 natural killer cells in pink; CD14 monocyte in green; CD4 T helper cells in orange; CD8 cytotoxic T cells in blue). (B) PBMCs cocultured with tumor cells following treatments as followed: IDX at 1 µM and 2 µM (IDX1 in orange and IDX2 in green) or cis at 1.2 µM (cis1.2 in pink), combinatorial treatment with IDX1 µM and cis 1.2 µM (IDX1+cis1.2 in purple) or IDX 2 µM and cis 1.2 µM (IDX2+cis1.2 in dark green), or left untreated with DMSO (blue). Differential expression patterns are shown. (C) Flow cytometry analysis of the frequencies of CD4+and CD8+ cells in the cocultures. (D) CD8/CD4 ratio was calculated according to the cellular percentage changes between different treatments. (E) Representative flow cytometry plots of NPC cell line C17 (target cell; T) stained with CFSE before getting in contact with PBMCs (effector cell; E) collected from cocultures at indicated effector:target ratio (E:T) for 3 days. Cells were then collected and stained with fixable viability dye (FVD) and the dead cells (%) were measured by flow cytometry. CFSE+ cells are target cells and FVD+ cells are dead cells, such that dead target cells are represented by the CFSE + FVD +. (F) Quantitative data of % lysis (in terms of CFSE+FVD+ cells) was obtained from three independent experiments. Statistical significance was analyzed using 2-way ANOVA, *p < 0.05; when compared with control (DMSO).