CD180/RP105 Toll-like Receptor (TLR) mediated signalling in Chronic Lymphocytic Leukaemia

The role of the microenvironment in the development and progression of chronic lymphocytic leukaemia (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 Toll-like receptors (TLR). CD180/RP105 is a membrane-associated orphan receptor that belongs to the TLR family, is expressed by professional antigen-presenting cells, and drives normal B-cell activation and proliferation.

We have previously shown that approximately 60% of CLL samples expressed surface CD180 but only half responded to ligation with anti-CD180 monoclonal antibody (mAb) resulting in activation, cycling, and reduced basal apoptosis and were termed responders (R). In contrast, CD180+CLL samples that failed to respond to anti-CD180 mAb, despite expressing a high density of CD180 receptors, were termed non-responders (NR). We further demonstrated that in R-CLL cells, CD180 ligation significantly induced phosphorylation of ZAP70/Syk, ERK, p38MAPK, and AKT. In contrast, 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.

To further clarify the CD180-mediated signalling pathways in CLL, here we studied signal transduction downstream from ZAP70/Syk by delineating CLL samples into R and NR through their proximal ability to activate AKT. We have studied major signalling protein kinases associated with the BCR signalling pathway: PI3K, Btk, ERK, p38MAPK and AKT.

Segregation of CLL samples responding to CD180 ligation by signalling via pAKT, rather than by CD86 upregulation, revealed that CD180 ligation on CLL cells can activate two alternative signalling pathways: pro-survival that operates via PI3K-Btk-AKT protein kinases, or mostly pro-apoptotic, that operates via p38MAPK but not through Btk. This may have implications for CLL therapy where Btk inhibitors are being used.

Here we demonstrate that albeit ligation of sIgM alone also activates pro-survival PI3K-Btk-AKT pathway pre-engagement of CD180 redirected BCR-mediated signalling towards the potentially pro-apoptotic p38MAPK pathway that opens new horizons for immunotherapy.

Since the tissue microenvironment plays a crucial role in generation and survival of the CLL clones, studies pertaining to CD180 expression in the lymphoid tissues were undertaken. Our pilot data suggests that in normal tonsils CD180 is expressed by the mantle zone (MZ) B cells and not the germinal centre (GC) B cells. However in CLL lymph nodes complete obliteration of the normal tissue architecture and a weak expression of CD180 has been detected, whilst expression of CD180 on bone marrow CLL cells was heterogeneous. Since CLL cells migrate to and from the solid tissues into the peripheral circulation in any CLL clone, there is always an intra-clonal kinetic heterogeneity through a suggested continuum between the 'proliferative' or CXCR4dimCD5bright, and 'resting' CXCR4 brightCD5dim fractions. Here we report that the 'resting' compartment was enriched for CD180+ cells compared to the 'proliferating' subset. In contrast, sIgM+ cells were more frequent in the proliferating fraction. Since the “resting” subset of CLL cells is also considered as the one “returning” to the solid tissues supported by the increased expression of CXCR4, our data might suggest possible attraction of the CD180+ cells towards the putative ligand in the lymphoid tissues.

It is becoming apparent that intraclonal diversity plays an important role in the clinical outcome of patients with CLL. Subsets of the CLL clone that respond more robustly to external stimuli may well gain a growth and survival advantage and possibly promote clonal evolution. Identification of these CLL subpopulations was therefore of prime importance, as these cells may be preferred targets for future therapeutics. We have established that CD180 expression on CLL cells helps identifying different subsets and delineating their physiological status. Our findings on modulation of signalling pathways through CD180 and sIgM and the temporal effects of their ligation is consistent with multiple ligands in the, in vivo, microenvironment playing an important role in the survival of CLL cells. Since TLR can shuttle between inhibition and promotion of leukemic growth they may play a key role in immune evasion impacting on clinically relevant tumour-host microenvironment interactions. The identification of distinct CD180-mediated signalling pathways that promote tumour cell proliferation and survival will allow specific targeting of key players in the pathways with immunotherapy and chemotherapy.