HBZ_157-176 is a candidate for the development of an anti-HTLV-1 peptide vaccine. (A-B) Mouse splenocytes and MM558 PBMCs were prestimulated with (A) HBZ-PA or (B) HBZ-PB. Thereafter, mouse (upper panels) and monkey (lower panels) cells were restimulated with single peptide–pulsed DCs (for mice) or single peptide–pulsed auto-PBMCs (for monkeys). IFN-γ and TNF-α production in CD8⁺ T cells were measured. Cells stimulated with nonpulsed APCs were used as a reference. (C-D) Cytotoxicity assay using B-lymphoblastoid cell lines (B-LCL) overexpressing HBZ. (C) B-LCLs from MM558 were transfected with HBZ, labeled with tracer dye, and used as target cells. (D) Target cells were cocultured with HBZ_109-128- or HBZ_157-176-stimulated MM558 PBMCs, and cytotoxicity was evaluated by counting living target cells. Relative index = E:T_(x:1)/E:T_(0:1) in GFP-positive cells (%). (E-G) Cytokine production by HBZ-specific CTLs. (E) HLA-A2 and HLA-A24 expression in PBMCs from a healthy donor. (F) Production of IFN-γ and TNF-α. CTLs were induced by using HBZ_109-128 or HBZ_157-176-pulsed DCs from an HLA-A2– and HLA-A24–positive healthy donor. The cells were cocultured with auto-B-LCLs pulsed with the matched peptide, and production of IFN-γ and TNF-α was analyzed. (G) HBZ-specific CTLs generated by HBZ_157-176-pulsed DCs produced cytokines in a concentration-dependent manner. (H) Peptide reconstitution assay of HBZ_109-128 and HBZ_157-176 peptide on HLA-positive HTLV-1–infected and ATL cell lines. Binding of HLA and the peptides was measured by using HTLV-1–infected cell lines TL-Om1 and MT-2. OVA_323-339 peptide was used as negative control. Peptide-HLA binding was measured as a recovered mean fluorescence intensity (MFI) of HLA-A2 and HLA-A24 by using flow cytometry. The bars represent the mean ± SD of triplicate experiments.