Peptide-specific CTL and cell-mediated cytotoxicity assay

Cytotoxicity assays were performed as previously described [16]. Target cells were stained with 10µg/mL of calcein-AM (Sigma Aldrich) for fifteen minutes, washed, and plated in a 60-well Terasaki plate (2000 cells/10µL RPMI 10% human serum/well). Effector cells (E75-CTL or Epstein-Barr Virus [EBV]-CTL) were resuspended in 10µL RPMI/10% human serum at increasing effector to target dilutions and then added to the target cells. After 4 hours, trypan blue was added as a quenching agent. Fluorescence was measured by a FLx800 Microplate Fluorescence Reader (Bio-Tek Instruments) and percent specific cytotoxicity was determined using the following calculation: (1 − [fluorescence_{target + effector} − fluorescence_media]/[fluorescence_{target alone} − fluorescence_media]) × 100.

Target cells for cytotoxicity assays included T2 cells pulsed with E75 peptide or irrelevant control peptide, MDA-MB-231 cells, or lymphoblastoid cell lines (LCL) maintained for 14 hours +/− NE (10ug/mL; Athens Research and Technology). EBV-transformed LCL were generated as previously described [17]. Briefly, peripheral blood mononuclear cells (PBMC) were plated and treated with 1ug/mL of cyclosporine A (Sandoz), either alone for spontaneous transformation, or with supernatant derived from cultures of B cells transformed with human type 1 EBV.

E75-specific CTL (E75-CTL) were generated using a modified dendritic cell (DC) method by isolation of monocytes through adherence culturing followed by DC immunostimulation as previously described [18]. Briefly, healthy donor HLA-A2⁺ PBMC were allowed to adhere on 6-well plates at 37°C in Macrophage Serum Free Medium (Gibco). Cells remaining in suspension (lymphocytes) were removed after 4 hours, pulsed with E75-peptide (40µg/mL), and stimulated with IL-7 (10ng/mL) and IL-2 (10ng/mL). These cells were then maintained in RPMI 10% FBS for 5 days. Adherent cells were matured into monocyte-derived DCs by addition of GM-CSF (100ng/mL), IL-4 (50ng/mL), and TNF-α (25ng/mL). After 5 days, DCs were detached by incubation with EDTA (10mM). DCs were pulsed with E75-peptide (40µg/mL) for 4-hours at 37°C and then co-cultured collectively with their autologous lymphocyte population in RPMI 10% FBS. Cells underwent re-stimulation with IL-7 (10ng/mL) and IL-2 (25ng/mL) for 7 days to allow for CTL proliferation. On day 12, cells were harvested and used in calcein-AM cytotoxicity assays. T2 cells, pulsed with either E75 or the irrelevant peptide PR1, were used as control target cells for E75-CTL, to confirm CTL specificity.

EBV-specific CTL (EBV-CTL) were generated as previously described [17]. Briefly, healthy donor PBMC were co-cultured with autologous irradiated LCL. After 10 days, PBMC were harvested using Ficoll gradients, subcultured and restimulated with irradiated LCL. Cultures were then stimulated with IL-2 (20 U/mL; Proleukin, Cetus) 3× weekly for 3 weeks, followed by another stimulation with both IL-2 and irradiated LCL.

Cytotoxicity experiments were also performed using the anti-HLA-A2 antibody BB7.2 in order to block the HLA-A2/T cell receptor interactions, and to further confirm specific killing of target cells by E75-CTL. Briefly, target cells were incubated with BB7.2 antibody for 30 minutes at 37°C prior to co-culture with CTL. BB7.2 antibody was obtained from the supernatant of an established hybridoma in our laboratory