Function of MP1- and CD19-bispecific T cells. (A) MP1-tetramer⁺CD19R⁺ T cells lyse MP1⁺ or CD19⁺ target cells. ⁵¹Cr-labeled targets (top) CD19⁺ Daudi cells or (bottom) autologous HyMP1⁺ HLA A2⁺ T-APCs were incubated with anti-CD19 CAR-redirected (▴), HLA-A2–restricted MP1-specific (▪), or MP1×CD19–bispecific (▿) effector T cells. Representative results of mean ± SD specific lysis of triplicate wells having E/T ratios of 50:1 to 1:1 are shown. (B) MP1-tetramer⁺CD19R⁺ T cells lyse primary B-lineage ALL blasts. ⁵¹Cr-labeled thawed ALL blasts, CD19⁺ Daudi cells, and CD19^–K562 cells were incubated with MP1- and CD19-bispecific effector T cells. After a 4-hour incubation period, supernatants were harvested, and CPM was quantified. Mean ± SD specific lysis was calculated from triplicate wells. The ALL blasts (CD19⁺CD10⁺CD45^–) represented 56% of the total population and 78% of the lymphoid-gated population of these specimens. (C) MP1-tetramer⁺CD19R⁺ T cells can be activated by MP1⁺ or CD19⁺ stimulator cells for T_c1 cytokine secretion. (top) IFN-γ and (bottom) TNF-α secreted by HLA A2⁺ MP1- and CD19-bispecific T cells incubated at 37°C with CD19^–K562 cells, autologous Hy⁺ T-APCs, autologous HyMP1⁺ T-APCs, or CD19⁺ Daudi cells. The relative ratio of responder T cells to γ-irradiated stimulator cells is shown. After 48 hours, cytokine concentration was determined by CBA. (D) MP1-tetramer⁺CD19R⁺ T cells can lyse CD19⁺ target cells after previous exposure to MP1⁺ or CD19⁺ target cells, or both. HLA A2⁺ MP1- and CD19-bispecific effector T cells were incubated at 37°C in media (□) or at a 1:1 ratio with γ-irradiated autologous Hy⁺ T-APCs (▴), MP1⁺ T-APCs (▾), CD19⁺ Daudi cells (♦), or 1:1 mixture of MP1⁺ T-APCs and CD19⁺ Daudi cells (•). After 5 days, ⁵¹Cr-labeled Daudi cells were added, and mean ± SD specific lysis was calculated after 4 hours. Lysis of CD19^–K562 cells under these conditions at an E/T ratio of 25:1 was 6% to 13% (data not shown).