Serum IgM/Fcmr Interactions Inhibit BCR Signaling and Influence the Cinical Course of CLL
Authors: Gobessi Stefania , Laurenti Luca , Porro Fabiola , Martines Claudio , Fazio Rosa , Xerxa Elena , Innocenti Idanna , Zucchetto Antonella , Chen Shih-Shih , Yan Xiao-Jie , Chiorazzi Nicholas , Gattei Valter , Efremov Dimitar G 
Published: October 2019

Abstract
CLL cases display heterogeneous responses to B cell receptor (BCR) engagement which correlate with clinical course and survival. Typically, cases with more aggressive disease show greater activation of downstream signaling molecules, such as the kinases SYK, AKT and ERK, upon BCR engagement. The heterogeneous BCR signaling responses have been attributed to varying levels of anergy occurring as a consequence of chronic antigen engagement in the absence of T cell help. However, an alternative explanation for the anergic phenotype is that it is induced by binding of serum IgM to the high-affinity Fcμ receptor (FCMR), which is highly overexpressed on CLL cells and has been reported to physically interact with the BCR in normal murine B cells. To investigate whether binding of soluble IgM to the FCMR can affect BCR signaling, we investigated activation of downstream BCR signaling molecules in primary CLL B cells after FCMR prestimulation with monoclonal IgM or Fcμ fragment followed by BCR stimulation with polyclonal anti-light chain antibody. For 10 of the 18 cases tested, substantial reduction in the levels of phospho-SYK, phospho-AKT and phospho-ERK was observed in IgM- or Fcμ-prestimulated CLL cells consistent with FCMR stimulation inhibiting BCR signaling. Stimulation with IgM or Fcμ did not cause any appreciable change in surface IgM expression, suggesting that inhibition of BCR signaling is not caused by receptor downmodulation but rather by recruitment of negative regulatory mechanism(s). Investigation of these mechanisms is still ongoing, but preliminary data suggest involvement of the phosphatases SHIP and SHP1.
To investigate the impact of serum IgM/FCMR interactions on the clinical course, we correlated FCMR expression and serum IgM levels with treatment-free survival (TFS) in a large series of CLL patients (Figure 1). Cases with FCMR expression levels below median value (n=67) had a median TFS of 41 months, which was significantly shorter compared to cases with FCMR levels equal to or above median value (n=71, median TFS=132 months, P<0.001). This difference did not correlate with higher expression of FCMR on IGHV-mutated (M-CLL) cases (n=80, mean MFI=123+/-79), considering that no difference was observed with respect to the IGHV-unmutated (U-CLL) cases (n=58, mean MFI=114+/-127, P=n.s). Interestingly, when analyzed independently, only M-CLL cases showed a significant correlation with TFS (FCMRlow cases: n=36, median TFS=75 months; FCMRhigh cases: n=44, median TFS=170 months, P=0.006); this difference was lost in U-CLL cases (FCMRlow cases: n=31, median TFS=21; FCMRhigh cases: n=27, median TFS=31, P=n.s.). In contrast, serum IgM levels below normal range (<40 mg/dl) significantly correlated with shorter TFS in U-CLL (low serum IgM cases: n=28, median TFS=20 months; normal serum IgM cases: n=44, median TFS=51 months, P=0.009) but not in M-CLL (low serum IgM cases: n=34, median TFS=83 months; normal serum IgM cases: n=72, median TFS=125 months, P=n.s.). To further establish whether low serum IgM levels are a cause rather than a consequence of clinically more aggressive disease, we are currently conducting experiments with leukemias derived from the Eμ-TCL1 CLL model that have been adoptively transferred into secretory IgM-deficient or normal mice; the results from these ongoing experiments will be presented at the meeting.
Collectively, these data show that serum IgM/FCMR interactions inhibit BCR signaling, which could at least in part explain the anergic phenotype of peripheral blood CLL cells. Along with our finding that the FCMR is downregulated by IL4 and expressed at lower levels on LN compared to PB CLL cells (Gobessi S et al, Blood 2016 128:2015), these data suggest that serum IgM/FCMR interactions are involved in regulating the intensity of the BCR signal in different compartments and consequently can influence the clinical behavior of the disease.

Disclosures
Chen: Janssen: Research Funding; Pharmacyclics: Research Funding; ArgenX: Research Funding; Beigene: Research Funding; Verastem: Research Funding. Chiorazzi:Janssen, Inc: Consultancy; AR Pharma: Equity Ownership.