Page 138 - Haematologica3

498
E.M. Coulter et al.
patients are left with low level residual disease, which
regrows on discontinuation of drug or when resistance mutations develop.11,12 This persistent disease also suggests that, within individual patients, the tumor may not behave in a homogeneous manner.13
Despite the central importance of BCR signaling in CLL and the efficacy of drugs that block this pathway, there is relatively little known about BCR dynamics in leukemic B cells. Surface levels of IgM and other BCR components are generally lower in CLL compared to normal B cells, and it has been suggested that this might be due to a failure to properly assemble the sIg α/β subunits CD79A and CD79B.14 Recent studies have shown that total IgM and CD79A levels are near normal in CLL but that CD79B expression, which is required for the transport of BCR to the cell surface,15 is reduced, thus trapping IgM within the cell.16 Exposure to interleukin 4 (IL4) increases CD79B expression and allows sIgM levels to increase and BCR signaling capacity to improve.16,17 CLL cell surface BCRs have an immature pattern of glycosylation that matures following ex vivo incubation18 or exposure to IL4,17 in keep- ing with accelerated BCR turnover induced by chronic activation. It has also been reported that, within the peripheral blood (PB) of individual patients with CLL, leukemic cells with the lowest sIgM expression show bio- chemical features of recent activation and proliferation, presumably because they have recently been released from lymphoid tissues where BCR stimulation and activa- tion are thought to occur.19,20 Taken together, these previ- ous data suggest that the reduced sIgM levels observed in CLL are due to a combination of increased turnover con- sequent to chronic activation coupled with defective transport to the cell surface resulting from a deficiency of CD79B. The ability of CLL BCRs to become internalized also has implications for how the tumor interacts with other cells, such as T cells. We, and others, have previous- ly shown that, as in normal lymph nodes (LNs), activated CD4+ T cells colocalize with proliferating tumor cells and, in vitro, can supply signals that cause tumor proliferation.21- 24 This process normally involves endocytosis and the pro- cessing of antigen bound to BCR, however it is not known whether CLL B cells are capable of providing this function. Herein we investigated whether, like normal B cells, CLL cells can internalize their BCR and to what extent this is linked to downstream signaling in both untreated patients and those receiving therapy with the BTKis ibrutinib and acalabrutinib. Our results shed new light on BCR function in CLL and have implications for understanding the mode of action of this important new class of drugs.
Methods
Patients and samples
PB samples were obtained from 19 healthy volunteers, 40 untreated patients with confirmed CLL (Online Supplementary Table S1), and an additional 15 patients receiving BTKi therapy (Online Supplementary Table S2). LN fine needle aspirate (FNA) and paired matched PB samples were derived from seven untreated CLL patients (Online Supplementary Table S3). Ethical approval was obtained from the National Research Ethics Service (08/H0906/94); all patients provided written informed consent. Patients were classified as having unmutated IGHV genes if homology with germline was >98%25 and as CD38+ if expression levels were 7% or higher (Online Supplementary Tables S1-S3).26
Flow cytometry
Cells were stained according to the manufacturer’s recommen- dations using fluorochrome-coupled antibodies (Online Supplementary Table S4). Viable CD19+CD5+ CLL and CD19+ nor- mal B cells were acquired on a FACS Canto II flow cytometer (BD Biosciences) and analyzed using FlowJo software (TreeStar).
Surface (s)IgM and IgD expression was assessed using QuantumTM fluorescein isothiocyanate molecules of equivalent soluble fluorochrome (FITC MESF) microsphere kits and the QuickCal v. 2.3 program, according to the manufacturer’s recom- mendations. Surface CD79B was quantified using R-phycoery- thrin (R-PE) MESF microsphere kits.
BCR signaling competence was determined using the ratiomet- ric Ca2+ detector Indo1-AM (Life Technologies) to measure intra- cellular calcium (Ca2+) levels and Ca2+ influx following αIgM stim- ulation, as previously described.4,9
ERK1/2 phosphorylation activation was analyzed in both treat- ment-naïve and BTKi-treated (collected before and during BTKi therapy) CLL patient B cells. Cells were incubated with or without αIgM, or with phorbol 12-myristate 13-acetate (PMA; served as positive control) for ten minutes at 37°C prior to intracellular and surface staining.
B-cell receptor internalization in normal and CLL B cells
BCR internalization was assessed in two ways. The uptake and retention of ligand/receptor complexes in acidified endosomes was measured using the pH sensitive fluorescent sensor, pHrodoTM Red avidin (Life Technologies) linked to agonistic αIgM or IgD (pHrodo-αIgM or D). Target cells were incubated with either pHrodo-αIgM or D for 30 minutes at 4°C and then 37°C for 1h. Results are expressed as the pHrodo mean fluorescent intensi- ty (MFI) after subtraction of the background signal (MFI of unla- beled anti-IgM). BCR internalization in normal and CLL B cells was also directly assessed by measuring the rate of disappearance of sIgM following ligation by agonistic αIgM. Full methodology is provided in the Online Supplementary Information.
Immunofluorescence Staining
CLL B cells were isolated using a human B-cell negative selec- tion kit without CD43 depletion (StemCell Technologies) accord- ing to the manufacturer’s instructions, and purity was confirmed by flow cytometry. Cells were labeled with pHrodo-avidin or pHrodo-αIgM, deposited onto poly-l-lysine coated glass slides by cytospin and stained with CytoPainter Phalloidin-iFluor 488 reagent. Images were acquired on a Nikon Eclipse Ti-E inverted microscope equipped with the Nikon A1R Si confocal imaging system. Image analysis was with Nikon Elements v4.2 software (see Online Supplementary Information).
Statistical analysis
Statistical analyses were performed using GraphPad Prism soft- ware version 5 (GraphPad Software, La Jolla, CA, USA). The Shapiro–Wilk test, t-test, Mann–Whitney and/or Wilcoxon’s test were used where indicated. P-values of <0.05 were considered sig- nificant.
Results
Internalization of normal and CLL BCRs
The uptake and retention of pHrodo-αIgM labeled BCRs in acidified endosomes was similar in normal and CLL B cells (MFI ± standard deviation: normal control: 147.5±16.8, n=19, P=0.825, CLL: 151.3±12.0, n=40; Figure
haematologica | 2018; 103(3)