Generation and Activation of Epitope-Specific CTL

E183-91 epitope-specific human CTL, C18-91, or E183-91 epitope-specific TCR-transduced human T cells were made as described^{40 (link),41 (link)}. The E183 CTL have not been authenticated and not been tested for mycoplasma. PBMC were freshly isolated by Ficoll-Paque PLUS (GE Healthcare Life Sciences) according to the manufacturer’s protocol and irradiated at 30 Gy. PBMC were resuspended in Aim-V medium with 2% human serum (Sigma-Aldrich) at a concentration of 2 × 10⁶ per ml. Human CTL were resuspended in the same medium at 10⁶ per ml. Totally, 500 μl of PBMC and 500 μl of human CTL were pooled per well in a 24-well plate. Final concentrations of 20 U ml⁻¹ human recombinant IL-2, 10 ng ml⁻¹ of IL-7, 10 ng ml⁻¹ of IL-15 (R&D Systems) were added to the human CTL culture. Lectin from Phaseolus vulgaris (Sigma-Aldrich) was added to the human CTL culture at a concentration of 1.5 μg ml⁻¹. PBMC were collected from healthy volunteers under the protocol approved by NUS IRB. Informed consent was obtained from all donors. GAG-A2-specific TCR-transduced T cells were made as described^{27 (link)}.