Figure 4.

Functional analysis of KWV3.1‐transfected T cells. (a) Flow cytometric analysis of KWV3.1‐transfected T cells. The mRNA encoding KWV3.1 α‐ and β‐chains or buffer alone (control) were electroporated in T cells activated by CD3/CD28 beads. The electroporated T cells were stained with anti‐mouse T‐cell receptor‐β chain (mTRBC) antibody or AFP_2–11/HLA‐A24 tetramer and analyzed on a flow cytometer. (b) Specific activation of KWV3.1‐transfected T cells against T2‐A24 cells pulsed with various concentrations of AFP_2–11. Interferon‐γ (IFN‐γ) release was determined by elispot assay. The irrelevant peptide‐pulsed T2‐A24 and the unpulsed T2‐A24 were used as negative controls. (c) Specific activation of KWV3.1‐transfected T cells co‐cultured with tumor cell lines. KWV3.1‐transfected T cells were specifically activated by HepG2 (AFP⁺ HLA‐A24⁺) and SNU398‐AFP (AFP overexpressed, HLA‐A24⁺), but not by SNU398 (AFP⁻ HLA‐A24⁺). IFN‐γ release was determined by elispot assay. A6 was used as a negative TCR control. (d) Cytotoxicity of KWV3.1‐transfected T cells against T2‐A24 loaded with varied concentrations of AFP_2–11 peptide. Lactate dehydrogenase (LDH) assay was implemented at an effector : target ratio of 5 : 1. The A6‐transfected T cells incubated with T2‐A24 loaded with 10⁻⁶ m AFP_2–11 were used as negative control. (e) The cytotoxic activity of KWV3.1‐transfected T cells against AFP‐expressing tumor cell lines at effector : target ratios of 1 : 1, 3 : 1, 5 : 1 and 10 : 1. A6 was used as a negative TCR control. Error bars indicate standard deviation of triplicate measurements