Functional interaction between CD180 Toll-like receptor (TLR) and B cell receptor (BCR) in the biology of Chronic Lymphocytic Leukaemia (CLL)

Chronic Lymphocytic Leukaemia (CLL) is the most common leukaemia in the western world and remains incurable. It is driven by as yet unknown (auto)antigens via the B cell receptor (BCR) and growth, survival and expansion signals it receives from the microenvironment through a range of cytokines and receptors, including Toll-like receptors (TLR). The role of the microenvironment in the development and progression of CLL is currently of major interest. Pathogen- and damage-associated molecular patterns (PAMPs and DAMPs, respectively) represent exogenous and endogenous microenvironmental factors acting via a range of receptors, including TLR. CD180/RP105 is a membrane-associated orphan receptor of the TLR family which is expressed on various cells of the immune system including macrophages, peripheral blood monocytes, dendritic cells, naive and mature B cells and marginal zone/mantle zone B cells driving normal B-cell activation and proliferation. However, it is not expressed on germinal centre (GC) B cells. CD180 is expressed heterogeneously on CLL cells, and predominantly on CLL with mutated IGVH genes (M-CLL). Although approximately 60% of CLL clones expressed CD180, only half responded to ligation with anti-CD180 monoclonal antibody (mAb) by activation, cycling, and reduced basal apoptosis. They were termed responders (R) and CD180+ CLL samples that failed to respond to anti-CD180 mAb despite expressing a high density of CD180 receptors, were termed non-responders (NR).

Our group has previously demonstrated that CD180 ligation significantly induced phosphorylation of ZAP70/Syk, ERK, p38MAPK, and AKT in R-CLL cells whilst CD180-mediated signalling in NR CLL cells did not progress downstream from ZAP70/Syk phosphorylation indicating a block in activation of downstream protein kinases, and possible anergy.

However, the responses were quite heterogeneous and to further understand the CD180-mediated signalling pathways in CLL; downstream signal transduction was studied by defining CLL samples into R and NR through their proximal ability to activate AKT. R-CLL cells could be divided into two categories as AKT signallers (AKT-S) and AKT non-signallers (AKT-NS) based on the ability to increase level of phosphorylation of AKT (Ser 473) compared with the basal levels. AKT-NS showed a significant increase in p38MAPK-P and this group was, therefore, re-categorised as p38MAPK signallers (p38MAPK-S). A small cohort of patients showed phosphorylation of both AKT and p38MAPK and they were categorised as double signallers (DS) whereas the remaining CLL samples which showed a decrease in percentages of both AKT-P and p38MAPK-P expressing cells and were categorised as non-signallers (NS). Ligation of CD180 with mAb on CLL cells can activate two alternative signalling pathways, one being pro-survival and operating through activation of protein kinases (PK) BTK-AKT (AKT-signallers); and the second - predominantly pro-apoptotic, operating through activation of p38MAPK (p38MAPK-signalers) but not through BTK. This may have implications for CLL therapy where BTK inhibitors are being used.

To assess cross-talk between CD180 and BCR (sIgM) signalling pathways in CLL, the effect of pre-engagement of CD180 on sIgM-mediated signalling was investigated. While sIgM ligation with goat anti-human IgM F(ab)2 alone led to a significant activation of pro-survival BTK-AKT pathway, pre-treatment with anti-CD180 mAb redirected pro-survival signalling mediated through BCR towards pro-apoptotic p38MAPK pathway. Application of specific inhibitors of AKT and p38MAPK signalling pathways confirmed that, in many of the CLL samples, activation of AKT and p38MAPK pathways is exclusive. This dichotomy appears to be a feature of CLL cells, and not of normal B cells that responded to CD180 ligation as double AKT/p38MAPK signallers.

It is becoming apparent that the clinical outcome of patients with CLL is significantly affected by intraclonal diversity. CLL clones which can respond more vigorously to external stimuli may gain selective advantage for growth and survival and perhaps promote clonal evolution. That CD180 expression modulates in some categories of CD180+ CLL cells suggests a possible link between dynamic expression of CD180 and CD180-mediated intracellular signalling, survival or apoptosis. Finding on rewiring of signalling pathways from BTK/AKT pro-survival circuit to p38MAPK pro-apoptotic pathway in AKT-S CLL cells may suggest a possible cross-talk between CD180 and BCR in AKT-S is consistent with that CD180+sIgM+ CLL cells may receive simultaneous signals through both receptors in vivo and microenvironment plays a major role in the survival of CLL cells. Hence, this study helped defineify mechanisms leading to the expansion of the leukaemic cells and thus contribute to development of novel therapies of CLL.