CD19/CD3 bsAb promotes Th1 polarization independent of BTKi therapy. PBMCs from BTKi-naïve (BN) and BTKi-treated (BTKi) patients were cultured with either CD19/CD3 bsAb, or medium only. Th1 and Th2 polarization of CD4⁺ T cells was assessed based on CCR6 and CXCR3 expression. Th1/Th2 ratio was calculated from the percentage of Th1 and Th2 subsets within CD4⁺ T cells. (A) Comparison of Th1/Th2 ratio (log2 transformed) at baseline, before in vitro culture between BN (n = 16, gray triangles), acalabrutinib-treated (Aca, n = 11, purple circles), and ibrutinib-treated (Ibr, n = 19, green diamonds) patient samples. (B) Th1/Th2 ratio (log2 transformed) in BN (n = 14), Aca (n = 11), and Ibr (n = 19) patient samples, after (B) 3 days, and (C) 5 days of culture with CD19/CD3 bsAb (CD19/CD3, red bars) or medium only (NoAb, blue bars). Each symbol represents 1 patient sample. Results are displayed as median and 95% confidence interval (CI). (D) Cytokine levels in media from untreated (No Ab) and treated (CD19/3) conditions after 5 days were measured by Luminex assay. The heatmap depicts the median cytokine concentration for each group of samples (BN, n = 7; Ibr, n = 8; and Aca, n = 8). Asterisks indicate statistical significance using Wilcoxon matched-pair signed-rank test for comparison of different treatments applied to individual patient samples and Mann-Whitney test for comparison of different patient groups. *P < .05; **P < .01; ****P < .0001.