W6 32 or l243 monoclonal antibody

A standard 4-hour LDH release cytotoxicity assay (LDH Release Assay Kit, Promega, USA) was performed to evaluate the cytotoxic activity of the SP17-stimulated T cells. Cytotoxicity against autologous breast cancer primary cells was determined at various effector:target cell ratios. For the measurement of CTL-mediated lysis of autologous lymphoblastoid cells (LCL) pulsed with SP17 or HPV16-E6 antigen, cytotoxicity assay was performed with 20:1 effector:target ratio. LCL were generated from autologous PBMCs as described previously [63 (link)]. To determine HLA restricted response, a cytotoxicity assay was performed with or without 25 μg/mL HLA-I or HLA-II (W6/32 or L243 monoclonal antibody, respectively, BioLegend 11080 Roselle Street, San Diego, CA) blocking antibodies (effector:target ratio 20:1).

A standard 4-hour LDH release cytotoxicity assay (LDH Release Assay Kit, Promega, USA) was performed to evaluate the cytotoxic activity of the SP17-stimulated T cells. Cytotoxicity against autologous breast cancer primary cells was determined at various effector:target cell ratios. For the measurement of CTL-mediated lysis of autologous lymphoblastoid cells (LCL) pulsed with SP17 or HPV16-E6 antigen, cytotoxicity assay was performed with 20:1 effector:target ratio. LCL were generated from autologous PBMCs as described previously [26 (link)]. To determine HLA restricted response, a cytotoxicity assay was performed with or without 25 μg/mL HLA-I or HLA-II (W6/32 or L243 monoclonal antibody, respectively, BioLegend 11080 Roselle Street, San Diego, CA) blocking antibodies (effector:target ratio 20:1). To block the perforin-mediated cell lysis, 5 μg/mL concanamycin A (CMA, Sigma-Aldrich, USA) was added to the co-cultures [27 (link)].