Page 142 - Haematologica3

E.M. Coulter et al.
reduced ability to activate ERK following BCR stimulation
(Figure 3H; P=0.024, n=12). Discussion
In the study herein, we investigated the capacity of nor- mal and CLL B cells to internalize ligands that bind to the BCR. Using two complementary techniques, we showed that BCR internalization and transit to acidified endo-
somes occurs in both normal and CLL B cells. When cor- rected for the level of sIgM expression, we found that BCR internalization and accumulation in endosomes is three times more efficient in CLL than normal B cells, and is highest of all in cases with anergic BCRs. Using agonistic antibodies to sIgM and sCD79B we also showed that internalization of sIgM is not accompanied by internaliza- tion of CD79B and vice versa. A comparison of LN with PB CLL cells and PB CXCR4dimCD5bright cells, representing
AB
CD
EF
GH
502
Figure 3. In vivo relationship between BCR signaling and internalization in CLL cells. BCR expression and uptake index were investigated firstly in subsets of periph- eral blood (PB) cells (CXCR4brightCD5dim and CXCR4dimCD5bright expressing CLL cells (A-B), and secondly in CD19+CD5+ cells from the matched lymph node (LN) and PB samples from seven unmutated CLL patients (C-D). Thirdly, sIgM expression was measured in CD19+CD5+ PB cells from CLL patients before, one month and at least 12 months after commencing BTKi treatment (ibrutinib, n=6 or acalabrutinib, n=1; presented as a percentage change in surface (s)IgM expression (E-F). Uptake index (G) and the capacity to induce pERK activation following BCR stimulation (H: pERK levels normalized to PMA/positive control) was also examined and compared in CLL B cells, pre- and during treatment. Statistical analysis was performed via Wilcoxon matched pairs test. MFI: mean fluorescent intensity; MESF: molecules of equivalent soluble fluorochrome.
haematologica | 2018; 103(3)