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BCR internalization and signaling in CLL
1A) and correlated with sIgM expression (r2=0.405, P=0.0001, n=40; Figure 1B). Confocal immunofluores- cence microscopy confirmed that the labeled BCRs accu- mulate in an intracellular compartment (Online Supplementary Figure S1A) and pre-incubation with excess unlabeled anti-IgM, sodium azide and cytochalasin D showed the process to be specific and dependent on ener- gy and the cytoskeleton (Online Supplementary Figures S1B- S1D). Repeated measurements confirmed the repro- ducibility of the assay within individual CLL cases (r2=0.874, P<0.0001, n=30; Online Supplementary Figure
S1E). It has previously been reported that sIgM expression is lower in CLL compared to normal B cells, and this was also the case in our patients (Figure 1C). Since BCR uptake by normal and CLL B cells is similar and dependent on sIgM expression, this suggested that the process might be more efficient in CLL compared to normal B cells. Correction of the pHrodo-αIgM uptake value for the num- ber of surface IgM molecules (uptake index) confirmed this to be the case (Figure 1D). After correcting for the level of surface IgM expression, the pHrodo-αIgM signal, which reflects accumulation in acidified endosomes, was
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CD
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Figure 1. B-cell receptor expression and internalization in CLL patients and healthy controls. (A) PBMCs from CLL patients (n=40) and healthy controls (n=19) were incubated with pHrodo-αIgM and the MFI of cells internalizing pHrodo-labeled avidin was measured within the CD19+5+ (CLL) and CD19+ (normal) B-cell population by flow cytometry. (B) B-cell receptor (BCR) internalization correlates with sIgM expression (Pearson’s correlation); correlation plot comparing the relationship between pHrodo-αIgM uptake and surface IgM (sIgM) expression in CD19+5+ cells derived from 40 CLL patients. (C) Although the level of pHrodo-αIgM uptake was comparable between CLL patients and healthy controls, marked differences in surface expression (sIgM) were observed (P=0.0001). (D) After correcting the level of uptake per molecule of sIgM, the uptake index was measured in both CLL patients and healthy controls. Within the CLL patient subsets, CLL B cells from CD38 negative, anergic and mutated patients were identified as having a greater uptake index than their counterparts, although mutational status was not significant (P=0.05, P=0.05 and P=0.436, respectively; unpaired t-test). (E) To assess the rate of BCR internalization more directly, the disappearance of sIgM following ligation of agonistic αIgM was measured. Accelerated BCR endocytosis was detected (2 min time point) in anergic B cells (n=6) compared to signaling competent (n=6) and normal (n=6) B cells (P=0.02 and P=0.01, respectively*; Mann-Whitney test). MFI: mean fluorescent intensity; MESF: molecules of equivalent soluble fluorochrome; CLL: chronic lymphocytic leukemia; SAV: streptavidin-APC.
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