FcμR Triggering Activates BCR Signaling Pathways and Increases Survival of CLL Cells
Authors: Gobessi Stefania , Sasi Binu K , Pozzato Gabriele , Innocenti Idanna , Laurenti Luca , Efremov Dimitar G 
Published: October 2019

Abstract
Soluble IgM was recently shown to activate B cell receptor (BCR) signaling pathways in chronic lymphocytic leukemia (CLL) B cells and protect them from spontaneous and CpG DNA-induced apoptosis (Wagner M et al, Blood 2016, 127:436-48; Gobessi S et al, Blood 2015, 126:4131, abstract 4131). The exact mechanism behind these effects is still unknown, but interaction of the leukemic BCRs with internal epitopes in the framework regions of soluble IgM has been considered as a possible explanation. An alternative explanation for these effects is that they are mediated by binding of soluble IgM to the Fcμ receptor (FcμR), which is highly overexpressed in CLL compared to normal B cells (Li FJ et al, Blood. 2011; 118:4902-9) and was recently shown to physically interact with the BCR in normal murine B cells (Ouchida R et al, J Immunol. 2015; 194:3096-101).
To evaluate the possibility that triggering of the FcμR is responsible for the activation of BCR signaling pathways and for the increased apoptosis resistance of CLL cells, we first investigated the viability of CLL cells cultured for 72 hours with or without soluble human IgM or Fcμ fragment. Both soluble human IgM and Fcμ significantly increased the viability of CLL cells with respect to unstimulated control, suggesting that triggering of the FcμR is sufficient to increase CLL cell survival (n=28, % viable IgM-stimulated: 52.6±18.3, % viable Fcμ-stimulated: 55.0±14.8, % viable unstimulated: 43.7±17.0, p<0.001 for IgM- and Fcμ-stimulated vs unstimulated, p=n.s. for IgM-stimulated vs Fcμ-stimulated). We next investigated whether triggering of FcμR can activate BCR signaling pathways. CLL cells were stimulated for 10 min with Fcμ or anti-IgM and the levels of phospho-SYK, phospho-AKT, phospho-ERK (n=7) and intracellular Ca2+ (n=5) were analyzed by immunoblotting and flow cytometry, respectively. Increased phosphorylation of SYK, AKT and ERK and increased Ca2+ flux were detected in 5 and 4 of the investigated samples, respectively. The effects of Fcμ stimulation appeared greater in samples that showed a weaker response to anti-IgM stimulation, suggesting that FcμR and BCR signaling are reciprocally regulated.
Because IL-4 was recently shown to increase surface IgM expression on CLL cells (Aguilar-Hernandez MM et al, Blood. 2016, 127:3015-25; Guo B et al, Blood 2016, 128:553-562), we next investigated whether it will have an opposite effect on FcμR expression. Stimulation of CLL cells (n=7) for 48 hours with IL-4 resulted in a mean 2.4 fold reduction in surface FcμR expression and a 3.9 fold increase in surface IgM expression compared to unstimulated cells (P<0.001 and P=0.016, respectively). Since IL-4 is produced by T cells, which typically interact with CLL cells in lymph nodes (LN), we next compared surface FcμR and IgM expression in two paired LN and peripheral blood (PB) CLL samples. Interestingly, in both cases the levels of surface FcμR were lower on LN than PB CLL cells, whereas no difference was detected in the expression of surface IgM.
We next compared signaling in CLL cells cultured in the presence or absence of IL-4. As previously reported, CLL cells cultured in the presence of IL-4 showed a considerably greater increase in anti-IgM induced phosphorylation of SYK, PLCγ2 and AKT compared to controls. Interestingly, greater activation of SYK, PLCγ2 and AKT was also observed when FcμR was downregulated by RNA interference in one primary CLL sample, suggesting that FcμR expression could be in part responsible for the reduced capacity of PB CLL cells to respond to anti-IgM stimulation.
In summary, this study shows that stimulation of FcμR by soluble IgM increases CLL cell survival and activates BCR signaling pathways, presumably because the two receptors are physically associated on the cellular membrane. Expression of FcμR is higher on PB than LN CLL cells and is negatively regulated by IL-4, which has an opposite effect on surface IgM expression. Preliminary data suggest that FcμR expression may reduce the capacity of the BCR to respond to external ligand. The purpose of such a mechanism is currently unclear, but one possibility could be to prevent inappropriate activation of PB CLL cells by (auto)antigens in the absence of co-stimulatory signals.

Disclosures
Efremov: Gilead: Honoraria.