T cell stimulation by FC preparations.

<p>(A) T cells (1×10⁶/mL) (n = 3) were stimulated with four types of FC preparations (Mock/FCs, TGF-β/FCs, E-Mock/FCs, and E-TGF-β/FCs) in the absence of rhIL-2 for 2 days and IFN-γ-production was immediately analyzed by ELISA. The background cytokine levels in conditioned medium were subtracted from each sample. (B) T cells (n = 3) were stimulated with the same number of fused cells that coexpressed both MUC1 and HLA-DR in all four types of FC preparations in the same set of experiments. Stimulated T cells were incubated with PKH-26-labeled PANC-1 cells at the indicated E:T ratios (60∶1, 20∶1, 6∶1) for cytotoxicity assays. Percentage of cytotoxicity (mean ± SD) was determined by flow cytometry CTL assays. (C) T cells (HLA-A2⁺) (n = 3) stimulated by each types of FCs (HLA-A2⁺) were purified through nylon wool and stained with FITC-conjugated mAbs against CD8 and PE-conjugated pentamer against HLA-A2/MUC1 or irrelevant IgG (left panel). T cell populations were gated based on their forward- <i>vs.</i> side-scatter profile. The percentage of CD8⁺ T cells reactive to MUC1 among the whole CD8⁺ T cell population was shown as a percentage of the double-positive population (pentamer⁺CD8⁺) in the total CD8⁺ T cells (right panel). The results are expressed as the mean ± SD. ***<i>P</i><0.001; **<i>P</i><0.01; *<i>P</i><0.05.