B-cell receptor (BCR) signaling plays a key role in the pathogenesis of chronic lymphocytic leukemia (CLL) cells, which exhibit higher intrinsic BCR activity than normal B cells.1 In normal B cells, BCR signaling can be inhibited by the low affinity receptor FcγRIIb (CD32b). This receptor exerts its negative effect when coligated with the BCR by cognate immune complexes (IC) and contributes to maintain peripheral tolerance and to shape the B-cell repertoire. FcγRIIb-BCR coligation counteracts BCR signaling by inducing phosphorylation of the intracellular immunoreceptor tyrosine-based inhibition motif (ITIM) of FcγRIIb, and the subsequent recruitment of (SH2)-containing inositol phosphatases (SHIP), which inhibits Ca++ flux and cell proliferation.2,3 Considering the major role of BCR in the pathogenesis of CLL and the activated nature of the leukemic cells,1 we aimed at investigating whether FcγRIIb could regulate BCR signaling in this malignancy. For that purpose, we compared molecular and functional effects of BCR ligation and BCR-FcγRIIb cross-linking on purified B-CLL cells and normal B cells after exposure to equimolar concentrations of F(ab’)2 anti-human immunoglobulin M (IgM) (10 mg/mL) or whole anti-human IgM (15 mg/mL) for 5 minutes.
In agreement with previous reports,4,5 BCR ligation induced highly heterogeneous responses on CLL cells. Twelve of 22 (55%) CLL samples responded to BCR binding with an increase of phospho-AKT (p-AKT) and/or phospho-ERK (p-ERK) levels, as well as an increase of intracellular Ca++ influx (Online Supplementary Figure S1). BCR responsiveness was neither significantly associated with FcγRIIb expression (P=0.053) nor with IgM expression (P=0.307).
As a subset of CLL patients show high levels of circulating IC and express poly-reactive BCR, it is reasonable to expect CLL cell interactions with a variety of IC, which could activate FcγRIIb inhibitory signaling. In murine B cells, recruitment of SHIP through FcγRIIb-BCR coligation leads to inactivation of anti-apoptotic kinase AKT7,8 and/or inhibition of ERK.9,10 Nevertheless, the inhibitory effect of FcγRIIb on BCR signaling has been only cursorily investigated in CLL.11,12 Gamberale et al.11 suggested that FcγRIIb may inhibit BCR signaling in CLL cells by reducing ERK phosphorylation. Here, similarly to what we observed in normal B cells (Online Supplementary Figure S2A-C), the crosslinking of FcγRIIb and BCR in leukemic B cells from BCR-responsive patients induced a significant increase in tyrosine phosphorylation of the ITIM domain of FcγRIIb as well as SHIP phosphorylation (p- SHIP) and translocation to the membrane (Figure 1). In addition, as observed in normal B cells, FcγRIIb-BCR coligation blocked the BCR-induced increase of p-AKT and p-ERK levels (Figure 1C). These inhibitory effects of FcγRIIb were reverted when coligation with BCR was abrogated by adding the specific anti-FcγRIIb monoclonal antibody 2B6 to the whole anti-IgM pAb. Interestingly, ligation of BCR alone also induced a significant increase of p-SHIP levels, as reported before13,14 indicating that SHIP can also function independently of FcγRIIb.
We also studied the potential involvement of SHIP in the inhibition of p-AKT and/or p-ERK induced by FcγRIIb-BCR cocrosslinking. For that purpose, we inhibited SHIP using two different approaches; cell transfection with a specific SHIP-1 short interfering RNA (siRNA) and cell exposure to 3AC (a molecule that inhibits the enzymatic activity of SHIP). In both, SHIP-1 siRNA transfected (Figure 2A-C) or 3AC-exposed CLL cells (Figure 2D); we observed that BCR ligation was incapable of inducing an increase of p-AKT levels, suggesting that SHIP is responsible for the activation of AKT upon BCR ligation in CLL cells. Since AKT could not be activated by BCR ligation in these cells, the involvement of SHIP in the reduction of AKT activation after BCR-FcγRIIb coligation could not be assessed. In contrast, in both, SHIP-1 siRNA transfected or 3AC-exposed CLL cells, activation of ERK (successfully achieved upon BCR ligation), could not be inhibited by BCR-FcγRIIb coligation, demonstrating that SHIP is involved in the inhibitory effect of FcγRIIb through inhibition of ERK signaling in CLL cells. In normal B cells, the ability of FcγRIIb-BCR coligation to inhibit p-ERK was not altered by 3AC exposure or SHIP-1 siRNA transfection (Online Supplementary Figure S3).
We further explored whether BCR-FcγRIIb coligation could inhibit the BCR-induced activation of CLL cells. Similarly to normal B cells (Online Supplementary Figure S4A-B), the crosslinking of FcγRIIb and BCR with whole anti-IgM pAb significantly inhibited the increase in Ca++ flux (Figure 3A) and the increase in the proportion of CD69+ cells (Figure 3B) induced by BCR. We also observed that FcγRIIb-BCR coligation reduced the percentage of apoptotic cells induced by BCR activation after 48 hours (h) with or without IL4/CD40L, with no inhibitory effects being observed in proliferation ratios after 72h (Figure 3C-D, Online Supplementary Figure S5). In normal B cells, the increase of Ca++ flux, CD69+ cells and proliferation induced by BCR ligation was reduced by FcγRIIb-BCR coligation, but no changes were detected in apoptosis (Online Supplementary Figure S4).
Collectively, our study shows that FcγRIIb-BCR coligation can reduce BCR signaling in CLL cells responsive to BCR by reducing the downstream activation of AKT and ERK pathways. Moreover, our results indicate that SHIP may be involved in the FcγRIIb inhibitory effect by blocking the ERK pathway. This inhibitory effect compromises BCR-induced leukemic cell activation, since molecular and cellular inhibitory effects were reverted when coligation was abrogating by using a specific anti-human FcγRIIb monoclonal antibody. These data are in agreement with a recent report published by Lemm et al.12 which suggests that SHIP is a potential regulator of BCR signaling in CLL cells and may explain our previous findings suggesting a correlation between high expression of FcγRIIb and better prognosis in early stage CLL.15
this work was supported in part by the Spanish Ministry of Science through grants from Red Temática de Investigación Cooperativa en Cáncer (RD12/0036/0071 to JS) Instituto de Salud Carlos III (FIS PI11/01740 and FIS PI19/00753 to CM), grants from Catalan Government AGAUR (2014 SGR 1281), Cellex Foundation Barcelona, Asociación Española contra el Cáncer (AECC), José Carreras Foundation and from the NIH, National Cancer Institute (RO1 CA0 81554 to NC).
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