Figure 3.

Visualization of target cell killing by co-culture of T cells from tumor-draining LN with tumor cells. (A) FACS analysis of CD169⁺CD8⁺ T cells on fresh lymphocytes isolated from peri-LN or distant-LN tissues of CRC patients (upper). Lymphocytes were gated as CD45⁺CD3⁺CD14⁻ cells. T cells (effector cell, E) from tumor-draining LN were assessed for non-specific cytotoxicity against the HCT116 colorectal cancer cell line (target cells, T) in vitro by a CFSE-based assay (E:T = 5:1). PI staining was used to discriminate viable and intact cells from dead subpopulations. Cells were co-cultured and harvested for analysis after 5 h of incubation. (B) The histograms depict the viable PI positive target dead cell subpopulations. Results are representative of at least three different CRC patients. Paired t-test (*p < 0.05; **p < 0.01; ***p < 0.001). (C) T cells isolated from distant-LN stained with Cell Tracker™ Red were mixed with T cells from peri-LN and then co-cultured with target cells (HCT116) in vitro. T cell-mediated killing of target cells was monitored by time-lapse epifluorescence and bright field microscopy. The combination of bright field and fluorescence microscopy allowed the detection of interactions between T cells and target cells.