Letters to the Editor binding with an increase of phospho-AKT (p-AKT) FcγRIIb-BCR co-ligation inhibits B-cell receptor and/or phospho-ERK (p-ERK) levels, as well as an signaling in chronic lymphocytic leukemia increase of intracellular Ca++ influx (Online Supplementary Figure S1). BCR responsiveness was neither significantly B-cell receptor (BCR) signaling plays a key role in the associated with FcγRIIb expression (P=0.053) nor with pathogenesis of chronic lymphocytic leukemia (CLL) IgM expression (P=0.307). cells, which exhibit higher intrinsic BCR activity than As a subset of CLL patients show high levels of circu- normal B cells.1 In normal B cells, BCR signaling can be lating IC and express poly-reactive BCR, it is reasonable inhibited by the low affinity receptor FcγRIIb (CD32b). to expect CLL cell interactions with a variety of IC, which This receptor exerts its negative effect when coligated could activate FcγRIIb inhibitory signaling. In murine with the BCR by cognate immune complexes (IC) and B cells, recruitment of SHIP through FcγRIIb-BCR coliga- contributes to maintain peripheral tolerance and to shape tion leads to inactivation of anti-apoptotic kinase AKT7,8 the B-cell repertoire. FcγRIIb-BCR coligation counteracts BCR signaling by inducing phosphorylation of the intra- and/or inhibition of ERK.9,10 Nevertheless, the inhibitory cellular immunoreceptor tyrosine-based inhibition motif effect of FcγRIIb on BCR signaling has been only cursorily (ITIM) of FcγRIIb, and the subsequent recruitment of investigated in CLL.11,12 Gamberale et al.11 suggested that (SH2)-containing inositol phosphatases (SHIP), which FcγRIIb may inhibit BCR signaling in CLL cells by reduc- inhibits Ca++ flux and cell proliferation.2,3 Considering the ing ERK phosphorylation. Here, similarly to what we major role of BCR in the pathogenesis of CLL and the observed in normal B cells (Online Supplementary Figure activated nature of the leukemic cells,1 we aimed at S2A-C), the crosslinking of FcγRIIb and BCR in leukemic investigating whether FcγRIIb could regulate BCR signal- B cells from BCR-responsive patients induced a signifi- ing in this malignancy. For that purpose, we compared cant increase in tyrosine phosphorylation of the ITIM molecular and functional effects of BCR ligation and domain of FcγRIIb as well as SHIP phosphorylation (p- BCR-FcγRIIb cross-linking on purified B-CLL cells and SHIP) and translocation to the membrane (Figure 1). In normal B cells after exposure to equimolar concentra- addition, as observed in normal B cells, FcγRIIb-BCR tions of F(ab’)2 anti-human immunoglobulin M (IgM) (10 coligation blocked the BCR-induced increase of p-AKT mg/mL) or whole anti-human IgM (15 mg/mL) for 5 min- and p-ERK levels (Figure 1C). These inhibitory effects of utes. FcγRIIb were reverted when coligation with BCR was In agreement with previous reports,4,5 BCR ligation abrogated by adding the specific anti-FcγRIIb monoclonal induced highly heterogeneous responses on CLL cells. antibody 2B6 to the whole anti-IgM pAb. Interestingly, Twelve of 22 (55%) CLL samples responded to BCR ligation of BCR alone also induced a significant increase A B C Figure 1. Molecular effects of B-cell receptor (BCR) ligation or FcγRIIb-BCR co-ligation in chronic lymphocytic leukemia cells with responsive BCR. (A) Immunoblot for two representative chronic lymphocytic leukemia (CLL) patient’s samples that responded to BCR ligation. Purified B cells from CLL patients were exposed to F(ab’) anti-human immunoglubin M (IgM) (10 mg/mL) and whole anti-human IgM (15 mg/mL) polyclonal antibodies (pAb) or specific anti-human FcγRIIb monoclonal antibody (2B6) (1 mg/mL) for 5 minutes before being lysed and immunoblotted for p-ITIM domain of FcγRIIb, p-SHIP, p-AKT and p-ERK. Equal 2 loading was checked by immunoblotting with GAPDH (n=9). (B) Immunoblot analyses of SHIP in membrane fraction extracts of CLL cells from two representative patient’s samples. The remaining extract containing cytosolic fraction of non-treated cells was used as control. The absence of GAPDH expression indicated no cytosolic contamination of the membrane fraction. Cadherin was used as a membrane fraction marker, as well as a control for protein loading. (C) Bar charts show mean values and standard deviation for the relative expression of p-ITIM, p-SHIP, p-AKT and p-ERK in CLL patients with responsive BCR (n=9). P-values were calculated using two tail paired t-test (*P<0.05, **P<0.01, ***P<0.001). F: F(ab’) anti-human IgM pAb; W: whole anti-human IgM pAb; 2B6: specific anti- human FcγRIIb monoclonal antibody. 2 306 haematologica | 2021; 106(1) Letters to the Editor A B C D Figure 2. Involvement of SHIP in the inhibitory effect of FcγRIIb-B-cell receptor coligation on chronic lymphocytic leukemia cells. (A) Relative SHIP expression in chronic lymphocytic leukemia (CLL) B cells transfected with short intefering RNA (siRNA) SHIP-1 (n=3). (B) Relative AKT and ERK phosphorylation in SHIP-1 siRNA transfected (n=3) and non-transfected (n=9) CLL B cells. (C) Immunoblot for a representative CLL patient’s sample transfected or not transfected with siRNA SHIP-1. (D) Immunoblot for a representative CLL patient’s sample exposed to 10 mM of 3AC or EtOH. F(ab’) fragments of anti-human immunoglobulin M (IgM) (10 mg/mL) or whole anti-human IgM (15 mg/mL) polyclonal antibodies (pAb) were used to ligate the BCR alone or to coligate it with FcγRIIb, respectively. 2 Cells were lysed and immunoblotted for SHIP, p-SHIP, p-AKT and p-ERK. Equal loading was checked by immunoblotting with GAPDH. Mean values, standard devi- ation and statistical significance between data were determined by two-tailed paired t-test (*P<0.05, **P<0.01). F: F(ab’) anti-human IgM pAb; W: whole anti- human IgM pAb; 2B6: specific anti-human FcγRIIb monoclonal antibody. 2 of p-SHIP levels, as reported before13,14 indicating that flux (Figure 3A) and the increase in the proportion of SHIP can also function independently of FcγRIIb. CD69+ cells (Figure 3B) induced by BCR. We also We also studied the potential involvement of SHIP in observed that FcγRIIb-BCR coligation reduced the per- the inhibition of p-AKT and/or p-ERK induced by centage of apoptotic cells induced by BCR activation after FcγRIIb-BCR cocrosslinking. For that purpose, we inhib- 48 hours (h) with or without IL4/CD40L, with no ited SHIP using two different approaches; cell transfec- inhibitory effects being observed in proliferation ratios tion with a specific SHIP-1 short interfering RNA (siRNA) after 72h (Figure 3C-D, Online Supplementary Figure S5). and cell exposure to 3AC (a molecule that inhibits the In normal B cells, the increase of Ca++ flux, CD69+ cells enzymatic activity of SHIP). In both, SHIP-1 siRNA trans- and proliferation induced by BCR ligation was reduced fected (Figure 2A-C) or 3AC-exposed CLL cells (Figure by FcγRIIb-BCR coligation, but no changes were detected 2D); we observed that BCR ligation was incapable of in apoptosis (Online Supplementary Figure S4). inducing an increase of p-AKT levels, suggesting that Collectively, our study shows that FcγRIIb-BCR coliga- SHIP is responsible for the activation of AKT upon BCR tion can reduce BCR signaling in CLL cells responsive to ligation in CLL cells. Since AKT could not be activated by BCR by reducing the downstream activation of AKT and BCR ligation in these cells, the involvement of SHIP in ERK pathways. Moreover, our results indicate that SHIP the reduction of AKT activation after BCR-FcγRIIb coliga- may be involved in the FcγRIIb inhibitory effect by block- tion could not be assessed. In contrast, in both, SHIP-1 ing the ERK pathway. This inhibitory effect compromises siRNA transfected or 3AC-exposed CLL cells, activation BCR-induced leukemic cell activation, since molecular of ERK (successfully achieved upon BCR ligation), could and cellular inhibitory effects were reverted when coliga- not be inhibited by BCR-FcγRIIb coligation, demonstrat- tion was abrogating by using a specific anti-human ing that SHIP is involved in the inhibitory effect of FcγRIIb monoclonal antibody. These data are in agree- FcγRIIb through inhibition of ERK signaling in CLL cells. ment with a recent report published by Lemm et al.12 In normal B cells, the ability of FcγRIIb-BCR coligation to inhibit p-ERK was not altered by 3AC exposure or which suggests that SHIP is a potential regulator of BCR SHIP-1 siRNA transfection (Online Supplementary Figure signaling in CLL cells and may explain our previous find- S3). ings suggesting a correlation between high expression of We further explored whether BCR-FcγRIIb coligation FcγRIIb and better prognosis in early stage CLL.15 could inhibit the BCR-induced activation of CLL cells. Rosa Bosch,1,2,3* Alba Mora,1,2,3,4* Carolina Cuellar,1,2,3 Similarly to normal B cells (Online Supplementary Figure Gerardo Ferrer,5 Sergey Gorlatov,5 Josep Nomdedéu,7 S4A-B), the crosslinking of FcγRIIb and BCR with whole Emili Montserrat,8 Jorge Sierra,1,2,3 Kanti R. Rai,5 anti-IgM pAb significantly inhibited the increase in Ca++ Nicholas Chiorazzi5 and Carol Moreno1,2,3,4 haematologica | 2021; 106(1) 307 Letters to the Editor A B C D Figure 3. Activation, apoptosis and proliferation of chronic lymphocytic leukemia cells upon B-cell receptor (BCR) ligation or FcγRIIb-BCR coligation. (A) Calcium kinetics for a representative chronic lymphocytic leukemia (CLL) patient’s sample in which purified leukemic cells were loaded with Indo1 ratio and acquired for 60 seconds prior to the addition of F(ab’) anti-immunoglobulin M (anti-IgM), whole anti-IgM or anti-FcγRIIb. (B) Box plots represents the median mean fluores- cence intensity ratio of Indo1 (4 mM) for each stimuli in CLL patient’s samples (n=10). Purified B cells from CLL patients with responsive BCR were exposed to 2 the indicated stimuli for 48 hours (h) or 72h and then analyzed by flow cytometry for CD69 expression on viable cells (B), Annexin V and TO-PRO®-3 (Annexin V+ cells were considered apoptotic cells) (C) or EdU incorporation using the Click-iTTM EdU Alexa FluorTM 488 Flow Cytometry Assay Kit (D) (n=10). Box plots encom- pass the data points within 1.5-times the interquartile range from the first or third quartile. P-values were calculated using two tail paired t-test (*P<0.05, **P<0.01). F: F(ab’) anti-human IgM ployclonal antibodies (pAb); W: whole anti-human IgM pAb; 2B6: specific anti-human FcγRIIb monoclonal antibody. 2 1 Laboratory of Oncology/Hematology and Transplantation, Institute paper; CC, SG, JN, JS and KR supplied essential data, samples of Biomedical Research, IIB Sant Pau, Barcelona, Spain; 2Department or reagent; RB, AM, NC and CM designed the study. All authors of Hematology, Hospital de la Santa Creu i Sant Pau, Autonomous have reviewed and approved the final version of the manuscript University of Barcelona, Barcelona, Spain; 3Biomedical Research Funding: this work was supported in part by the Spanish Ministry Institute (IIB-Sant Pau) and José Carreras Leukemia Research of Science through grants from Red Temática de Investigación Institute, Barcelona, Spain; 4Department of Medicine, Autonomous Cooperativa en Cáncer (RD12/0036/0071 to JS) Instituto de Salud University of Barcelona, Barcelona, Spain; 5Karches Center for Carlos III (FIS PI11/01740 and FIS PI19/00753 to CM), grants Oncology Research, The Feinstein Institute for Medical Research, from Catalan Government AGAUR (2014 SGR 1281), Cellex Manhasset, NY, USA; 6MacroGenics, Inc., Rockville, MD, USA; Foundation Barcelona, Asociación Española contra el Cáncer (AECC), 7 Laboratory of Hematology, Hospital de la Santa Creu i Sant Pau, José Carreras Foundation and from the NIH, National Cancer Institute Barcelona, Spain and 8Department of Hematology, Institute of (RO1 CA0 81554 to NC). 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