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BCR internalization and signaling in CLL
significantly higher on cells derived from the LN com- pared to PB (Figure 3C; P=0.03, n=7; Online Supplementary Figure S5). Again, there was great variability in pHrodo- αIgM uptake efficiency with higher levels in the LN than PB in five out of seven patients, but no significant differ- ence overall (Figure 3D; P=0.218, n=7).
BTKi-treated CLL B-cells display features of anergy
We next investigated the effect of BCR pathway block- ade on BCR expression and function. PB samples were col-
lected over time from BTKi-treated patients (ibrutinib and acalabrutinib) with a prolonged lymphocytosis for a min- imum of 12 months (Online Supplementary Table S2). On comparison of CD19+CD5+ CLL B-cells before and after one month of treatment, most cases showed an initial increase in sIgM expression (Figure 3E; P=0.02, n=7) fol- lowed by a decrease in sIgM levels after at least 12 months of treatment (Figure 3F; P=0.008, n=7). After 12 months or more of BTKi therapy, CLL B cells exhibited more efficient BCR internalization (Figure 3G; P=0.023, n=7) and had
A
B
Figure 2. Dissociation of BCR signaling and internalization in CLL cells. (A) A correlation was detected between the
CD expression levels of surface (s)IgM and sCD79B on CD19+5+ cells derived from 21 CLL patients (both anergic and sig- naling competent B cells, Pearson’s correlation). Values are expressed as molecules of equivalent soluble fluo- rochrome (MESF) and derived from mean fluorescence intensity (MFI) val- ues. (B) To examine the relationship within an individual patient, 15 subsets of CD19+5+ cells were created, as defined by increasing sIgM expression, and mean sCD79B MFI values were recorded within the same gate. (C) A representative patient demonstrating a correlation between sIgM and sCD79B. (D) The percentage of sCD79B receptor EF expression was measured on CLL and normal B cells following agonistic αIgM ligation; a gradual reduction and inter- nalization of sCD79B on normal CD19+ B cells was detected after 60 min incu- bation (n=6; P=0.03*; Wilcoxon matched pairs test). In addition, CD79B internalization (E) and sIgM receptor expression (F) was measured upon αCD79B ligation, and compared between anergic, signaling competent and normal B cells. CD79B internaliza- tion occurred more rapidly (2 min time point) in anergic B cells compared to signaling competent and normal B cells (P=0.01 and P=0.001, respectively*; Mann-Whitney test), however, the per- GH centage of sIgM receptor expression remained unchanged in all CLL cases. Finally, signaling competent CLL B cells were pre-incubated with agonistic αCD79B for 10mins at 37°C prior to αIgM stimulation to determine the effect of CD79B internalization on αIgM-induced pERK activation (G: n=9; pERK levels were normalized to the positive control), as well as pHrodo- αIgM uptake (H: n=9). Statistical analy- sis was performed via Wilcoxon matched pairs test. CLL: chronic lym- phocytic leukemia; SAV: streptavidin-
APC.
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