CD19/CD3 bsAb’s induce CLL cell death and expansion of autologous T cells in vitro. PBMCs from treatment-naïve CLL patients were cultured with either CD19/CD3-scFv-Fc, blinatumomab, HER2/CD3-scFv-Fc, or medium alone at 6.6 nM (n = 12). Response to treatment was analyzed after 7 and 12 days by flow cytometry. (A) Representative histograms after 12 days of culture. Percent dead CLL cells (LIVE/DEAD positive) is indicated. (B) Percent CLL specific killing by treatment condition after 7 and 12 days in culture, calculated as: (%Untreated CLL Viability – % Treated CLL Viability)/(% Untreated CLL Viability) × 100. Untreated samples had average (range) CLL viability of 62% (35% to 82%) on day 7 and 64% (40% to 79%) on day 12. (C) Representative flow cytometry plots showing CD8 and CD4 staining in live cells after 12 days in culture. Frequencies of CD8⁺, CD4⁺, and CD8⁻/CD4⁻ (composed mostly of CLL cells) populations are shown. (D) CD8 and CD4 cell counts after 7 and 12 days in culture. (E) Correlation of CLL specific killing by treatment condition with either CD8 or CD4 T-cell count after 7 days in culture by Spearman’s rank coefficient. Each dot represents 1 sample and treatment condition; color and shape indicate treatment. Color of data points in panels B and D represent patient-matched samples and correspond with colors in supplemental Table 1; median and IQRs are shown. Asterisks indicate statistical significance using Dunn’s multiple comparisons test. *P < .05; **P < .01; ***P < .001; ****P < .0001. Blin, blinatumomab; CD19/CD3, CD19/CD3-scFv-Fc; H2/CD3, HER2/CD3-scFv-Fc; No Ab, medium alone.