Function and phenotype of MP1-specific T cells. (A) T cells expanded on HyMP1⁺ T-APCs exhibited HLA-restricted MP1-specific cytolytic response. 4-hour CRA using T-APC–elicited MP1-tetramer⁺ effector T cells and ⁵¹Cr-labeled HLA A2⁺ T2 targets were performed to compare the lytic activity against T2 targets loaded in serum-free media with 10 μM of the MP1-derived peptide GILGFVFTL (□) compared with mock-loaded targets (▪). Representative results of mean ± SD specific lysis of triplicate wells having E/T ratios of 50:1 to 1:1 are shown. (B) MP1-specific T cells elicited by HyMP1⁺ T-APCs recognize endogenously processed and presented HLA class 1 MP-1 epitopes and are activated for cytokine secretion. CD8⁺MP1-tetramer⁺ responder T cells cocultured with autologous CD8⁺HyMP1⁺ T-APCs, followed by OKT3-based rapid expansion, were incubated at a 1:1 responder/stimulator ratio with γ-irradiated autologous CD8⁺Hy⁺ or CD8⁺HyMP1⁺ T-APCs. Controls included T-APCs without addition to culture of responders and responders incubated in CM without stimulators. After 48 hours, cell-free supernatants from these cultures were harvested and subjected to CBA analysis for quantifying IFN-γ and TNF-α content. (C) MP1-specific T cells elicited by HyMP1⁺ T-APCs recognize endogenously processed and presented HLA class 2 MP-1 epitopes and are activated for cytokine secretion. CD4⁺ responder T cells cocultured for 21 days on autologous CD4⁺HyMP1⁺ T-APCs, followed by OKT3-based rapid expansion, were incubated at a 1:1 responder/stimulator ratio with γ-irradiated autologous CD4⁺Hy⁺ or CD4⁺HyMP1⁺ T-APCs. The CD4⁺HyMP1⁺ T-APCs can also stimulate MP1-specific secretion of IFN-γ by CD8⁺MP1-tetramer⁺ responder T cells (data not shown). Controls included T-APCs without addition to culture of responders and responders incubated in CM without stimulators. After 48 hours, cell-free supernatants from these cultures were harvested and subjected to CBA analysis for quantifying IFN-γ content. (D) Phenotype of hygromycin-resistant, CD19R/HyTK-pMG–transfected MP1-specific CTLs. Histograms showing binding of mAbs specific for T-cell cell-surface markers (bold lines), relative to isotype control or unstained cells (dotted lines), to HyMP1⁺ T-APC–elicited CD8⁺ MP1-tetramer⁺ T cells, genetically modified with CD19R/HyTK-pMG and expanded by repeated OKT3-based 14-day stimulation cycles in the presence of cytocidal concentrations of hygromycin B. The relative percentage of cells in each gate is indicated.