Figure 2.

Specific IGHV associated point mutations of autonomous active BCR affect cell-autonomous signal. (A) In the upper part sequence alignment of 4 identified germline alleles of IGHV3-48 are shown, differences are marked in blue. Allele 01 and 04 are identical. In the lower part sequence alignment of CLL-patient P6540 derived IGHV is displayed, which is related to IGHV3-48*02 allele (Mutations marked in red). Several single point-mutations were reverted to their germline version, depicted in green. (B-D) IGHV and IGLV sequences obtained from the CLL patient P6540 were cloned into retroviral expression vectors for human μHC and λLC and expressed in the TKO system (see Supplementary Material and Method ). Proper BCR expression on the TKO cells was assessed via flow cytometry (data not shown). Germline IGHV3-48*02 (GL) was included for comparison. IGHVs were co-expressed with autonomously active subset #2 IGLV3-21^R110 LC variant. As control, P6540 IGHV was expressed with IGLV3-21^G110. Indo-1 staining was performed to analyze intracellular calcium release. In particular, cell-autonomous signaling was assessed via calcium mobilization after 4-Hydroxytamoxifen administration, (B) shown in representative dot plots with kinetics and (C) statistically summarized as area under the curve of the kinetic (AUC). (D) As control B-cell receptors were additionally stimulated with anti-LC antibody to mimic ligand-dependent signal. One-Way ANOVA (Bonferroni Correction), significance of mean to mean of P6540 R110 is shown, (**p < 0,01) N = 5.