Autonomous signaling activity of BCR of DLBCL cell lines. (A) Calcium flux assay of TKO cell transduced with combinations of BCR heavy (HC) and light chains (LC) of ABC-DLBCL cell lines TMD8 (CARD11^WT/WT) and OCI-Ly3 (CARD11^L251P/L251P). Black triangles indicate administration of 4-OHT. Open triangles indicate anti-Ig heavy chain crosslinking. (B) Histograms of fluorescence intensity of APC-labeled CD79A phosphorylated on tyrosine (Y) 182 and BLNK phosphorylated at Y84 as measured by phospho-flow cytometry of 4-OHT–treated TKO cells transduced with the BCR of DLBCL cell lines TMD8 and Karpas 422 (K422). Light gray: anti-Ig heavy chain (anti-Ig) crosslinking. Dark gray: no BCR crosslinking. (C) Phospho-flow cytometry of 4-OHT–treated TKO cells transduced with the BCR of DLBCL cell lines TMD8 and Karpas 422. Triangles represent individual measurements. Bars represent MFI of AF647/APC after normalization to the MFI induced by BCR crosslinking with anti-Ig. MFI of cells without crosslinking were compared by unpaired t test. (D) Cell viability assay after 96 h of culture with increasing concentrations of the BTK-inhibitor acalabrutinib. Values were normalized to medium control. Error bars show standard deviation of duplicate measurements. Dotted line: IC50.