IL-4 Exerts Opposing Effects on Surface-IgM and CXCR4 Mediated Signalling in Chronic Lymphocytic Leukaemia
Authors: Aguilar Maria Montserrat , Blunt Matthew D , Strefford Jonathan C , Forconi Francesco , Cragg Mark , Stevenson Freda K , Packham Graham , Steele Andrew J 
Published: October 2019

Abstract
The B cell receptor (BCR) is critical for survival and proliferation of chronic lymphocytic leukemia (CLL) cells and is activated following antigen/autoantigen engagement. BCR engagement in vivo triggers a partial receptor down modulation, phosphorylation of tyrosine residues in CD79A and CD79B, signalosome formation and subsequent calcium mobilization and ERK phosphorylation (ERK-P). Interestingly CLL cells incubated in vitro recover surface(s)IgM receptor expression and subsequently their ability to signal (calcium flux and ERK-P). During this recovery of sIgM, expression of a fully glycosylated “mature” form of sIgM increases. Interestingly, sIgD is not downregulated in vivo and expression does not increase in vitro. Downregulation by antigen engagement therefore appears only to affect sIgM. IL-4 appears to be present in CLL tissue sites and has a known role in protecting CLL cells from apoptosis however its effect on BCR expression and subsequent downstream signalling has not previously been demonstrated. Therefore we investigated the effects of IL-4 on BCR signalling and on the expression of surface receptors including chemokine receptors CXCR4 and MHC-class II (MHCII).
CLL cells were pretreated with and without IL-4 for 24h and sIgM and sIgD expression measured by flow cytometry. sIgM naturally recovers with time and this is significantly enhanced further by IL-4 (p=0.006, n=33), whereas sIgD does not (p=0.48, n=30). The same CLL samples pre-treated with IL-4 for 24 hours followed by treatment with anti-IgM (n=22) for 15 minutes showed enhanced calcium flux in 77% (p=0.0005) of samples compared to the control. The increase in sIgM expression correlated strongly with the ability to induce calcium mobilization in IL-4 treated samples (p=0.0007, r=0.7). The fold increase in sIgM expression following pre-treatment with IL-4 was significantly greater in U-CLL compared with M-CLL (p=0.012) and in ZAP70+ve samples (p=0.02). However, analysis of IL-4R expression did not show a significant difference between M-CLL and U-CLL samples (p=0.18, n=20). The increase in sIgM expression by IL-4 coincided with an increase in CD79b protein expression (p<0.0001, n=26). Futhermore IL-4 pretreatment significantly augmented expression of the fully glycosylated µ-chain of sIgM on the surface of the CLL cells (p=0.005), as confirmed by resistance to EndoH cleavage, indicating a role of IL-4 in IgM assembly and transport to the cytoplasmic membrane. These IL-4-specific effects were blocked with either a JAK3 or STAT6 inhibitor (Tofacitinib or Axon 1992, respectively) which specifically inhibit IL-4 signalling.
We next identified whether the effects observed with IL-4 was limited to sIgM. Interestingly, in contrast to the spontaneous recovery of CXCR4 expression which occurs in vitro and parallels the recovery of sIgM expression, IL-4 significantly inhibited CXCR4 recovery in vitro (p<0.0001, n=21) and prevented migration towards CXCL12 in transwell migration assays (p=0.03, n=6). This indicates that IL-4 is not simply amplifying the natural process of recovery of these receptors but is having a differential effect. For other molecules, a significant increase in MHCII expression in IL-4 pre-treated samples (p=0.0011, n=12) was observed mimicking the changes with sIgM, whilst other negative or positive BCR regulator such as CD5 (p=0.47, n=25) and CD19 (p=0.93, n=20) respectively, remained unchanged. Indicating the effect of IL-4 on receptor expression was receptor dependent.
These data suggest a role for IL-4 in augmenting sIgM expression and subsequent downstream signalling, increasing MHCII expression, whilst down modulating chemokine receptors to retain CLL cells within the lymph node. Thus IL-4 may play an important biological role when CLL cells encounter antigen/autoantigen and therefore inhibition of this pathway with JAK3 inhibitors may be a potential clinical target for the treatment of CLL.

Disclosures
No relevant conflicts of interest to declare.