B-BiTE and specific activation of human T cells by a mAb/B-BiTE complex. (A) B-BiTE was purified and then detected using anti-His mAb. (B) K562, K562/CD20, K562/CD38, Raji, Jurkat, and Jurkat 76 cells were stained with each mAb/B-BiTE complex (blue), containing 10 μg/mL isotype, rituximab, or daratumumab bound to 3.3 μg/mL B-BiTE. K562/CD20 and K562/CD38 cells were established by transduction with the CD20 or CD38 gene, respectively. The gray histogram shows the staining with PE-anti-His mAb alone. (C-E) Freshly isolated human peripheral blood CD3+ T cells derived from 6 different donors were cocultured with K562 (red), K562/CD20 (blue), or K562/CD38 (green) cells in the presence of isotype/B-BiTE, rituximab/B-BiTE, or daratumumab/B-BiTE (mAb, 7.5 μg/mL; B-BiTE, 2.5 μg/mL). Multiple cytokine production by CD8+ T cells and CD4+ T cells was evaluated by intracellular staining assay. The gating strategy and representative cytokine plots of CD8+ T cells and CD4+ T cells against K562/CD38 cells are shown (C), and the results for K562/CD20 cells (D) and K562/CD38 cells (E) are summarized. Each dot represents a donor, and an isotype/B-BiTE complex was prepared as a negative control. (F) Freshly isolated human peripheral blood CD3+ T cells derived from 5 different donors were labeled with CFSE, and stimulated with irradiated K562/CD20 and K562/CD38 cells at an E:T ratio of 10:1 in the presence of rituximab/B-BiTE, daratumumab/B-BiTE, or isotype/B-BiTE complex (mAb, 7.5 μg/mL; B-BiTE, 2.5 μg/mL). After 5 days of incubation, the mean fluorescence intensity (MFI) of CFSE–labeled CD4+ T cells and CD8+ T cells was examined. Representative stainings of CD4+ T cells and CD8+ T cells derived from the same donor are depicted (left), and their proliferation (%) against indicated target cells is summarized (right). The percentage was calculated as: (control MFI – experimental MFI)/(control MFI) × 100 (%). (G) CD45RA+FoxP3+ and CD45RA–FoxP3++ populations among CD4+CD25+ T cells after stimulation, similar to panel F, were measured. Representative stainings are shown (left), and the frequency of the indicated population from each donor is depicted (right). All paired data were evaluated statistically by a paired t test (2-sided). Error bars show the SD. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. CFSE, carboxyfluorescein diacetate succinimidyl ester; E:T, effector to target; MFI, mean fluorescence intensity; n.s., not significant; PE, phycoerythrin; SD, standard deviation.

B-BiTE and specific activation of human T cells by a mAb/B-BiTE complex. (A) B-BiTE was purified and then detected using anti-His mAb. (B) K562, K562/CD20, K562/CD38, Raji, Jurkat, and Jurkat 76 cells were stained with each mAb/B-BiTE complex (blue), containing 10 μg/mL isotype, rituximab, or daratumumab bound to 3.3 μg/mL B-BiTE. K562/CD20 and K562/CD38 cells were established by transduction with the CD20 or CD38 gene, respectively. The gray histogram shows the staining with PE-anti-His mAb alone. (C-E) Freshly isolated human peripheral blood CD3+ T cells derived from 6 different donors were cocultured with K562 (red), K562/CD20 (blue), or K562/CD38 (green) cells in the presence of isotype/B-BiTE, rituximab/B-BiTE, or daratumumab/B-BiTE (mAb, 7.5 μg/mL; B-BiTE, 2.5 μg/mL). Multiple cytokine production by CD8+ T cells and CD4+ T cells was evaluated by intracellular staining assay. The gating strategy and representative cytokine plots of CD8+ T cells and CD4+ T cells against K562/CD38 cells are shown (C), and the results for K562/CD20 cells (D) and K562/CD38 cells (E) are summarized. Each dot represents a donor, and an isotype/B-BiTE complex was prepared as a negative control. (F) Freshly isolated human peripheral blood CD3+ T cells derived from 5 different donors were labeled with CFSE, and stimulated with irradiated K562/CD20 and K562/CD38 cells at an E:T ratio of 10:1 in the presence of rituximab/B-BiTE, daratumumab/B-BiTE, or isotype/B-BiTE complex (mAb, 7.5 μg/mL; B-BiTE, 2.5 μg/mL). After 5 days of incubation, the mean fluorescence intensity (MFI) of CFSE–labeled CD4+ T cells and CD8+ T cells was examined. Representative stainings of CD4+ T cells and CD8+ T cells derived from the same donor are depicted (left), and their proliferation (%) against indicated target cells is summarized (right). The percentage was calculated as: (control MFI – experimental MFI)/(control MFI) × 100 (%). (G) CD45RA+FoxP3+ and CD45RA–FoxP3++ populations among CD4+CD25+ T cells after stimulation, similar to panel F, were measured. Representative stainings are shown (left), and the frequency of the indicated population from each donor is depicted (right). All paired data were evaluated statistically by a paired t test (2-sided). Error bars show the SD. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. CFSE, carboxyfluorescein diacetate succinimidyl ester; E:T, effector to target; MFI, mean fluorescence intensity; n.s., not significant; PE, phycoerythrin; SD, standard deviation.

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