A study of specific stimulation-induced response in B-chronic lymphocytic leukaemia cell line - MEC1 cells

Author: rusudan chankotadze
Keywords: Chronic lymphocytic leukaemia, CD180, Toll like receptors
Annotation:

Chronic Lymphocytic Leukaemia (CLL) is the most common type of leukaemia in Western countries. The disease affects individuals above 50 years old. CLL cells are mature immuno-competent lymphocytes. B-CLL is the only form of leukaemia, which is radiation-, chemicals- or drug-independent. CLL cells have CD5+CD19+CD23+CD40+ immunophenotype, expression of other surface markers is rather variable (for example, IgM, CD79a, CD79b, CD20 or CD38). CLL can be characterised into two major subgroups with unmutated and mutated immunoglobulin (Ig) heavy (H) chain genes (IGHV), which is prognostic of an aggressive or an indolent disease respectively. It has been shown, that a member of Toll-like receptor (TLR) family - CD180/RP105 is expressed heterogeneously on CLL cells, and mainly on those with mutated IGHV genes. Also it has been demonstrated that CD180-mediated signalling uses the same phosphoinositol-3-kinase (PI3K) signalling pathway as B-cell receptor and proved CD180 participation in pro-survival processes in CLL cells. Ligation of CD180 with monoclonal antibodies (mAb) induced activation of approximately 50% of the CD180+ B-CLL clones, subdividing them into Responders (R) and Non-Responders (NR). It should be emphasized, that in CLL patients proliferation takes place only in so called “proliferation centers” in the lymph nodes and bone marrow, while lymphocytes separated from peripheral blood are blocked in the G0 stage of the cell cycle. The abovementioned fact, is the main cause why while working with patients’ CLL cells it appears impossible to get an actively proliferating lymphocytes population in vitro. Subsequently the research appears to be restricted to lymphocytes population, which is blocked in G0. To avoid this restriction for the current studies we have chosen a model system – cell line MEC1, developed from peripheral blood lymphocytes of CLL patient with mutated IGVH genes, which gives us opportunity to investigate the effect of specific stimulation on pro-survival and proliferative processes in actively cycling cells. Our research group previously has investigated the stability of CD180 surface expression on CLL cells during the progression through the cell cycle in relevance to cell activation state. It has been shown, that MEC1 cells activation level increased together with cell cycle stabilisation. Also it has been demonstrated for the first time, that the percentage of CD180 expressing cells in proliferating CLL culture is not stable: an increase in CD180+ cells number can be seen during exponential growth stage, first 72 hours after the cell culture re-seeding. Therefore, to continue further investigation of CD180 expression dependence on the cells progression through the cell cycle, in the frame of the current research we have studied CD180 or/and IgM ligation induced effect in MEC1 cells: cells distribution between cell cycle phases, percentage of apoptotic cells, cells activation level. The research has been structured into 3 tasks: 1. Study of IgM ligation effect on MEC1 cells; 2. Evaluation of IgM signalling pathway activation effect on CD180 expression; 3. Comparison of IgM and/or CD180 ligation influence on pro-survival and proliferative processes. To evaluate the proliferative state of cells in response to stimulation, we looked at cells distribution between cell cycle stages by staining of double-stranded DNA with ethidium bromide. To evaluate pro-survival processes, we measured apoptosis level, looking at both: percentage of cells on late stages of apoptosis (on EB staining-derived picture – hypoploid section) and percentage of cells on early stages of apoptosis (using detection of phosphatidylserine surface expression by Annexin V, combined with propidium iodide staining). To evaluate cells activation we checked surface expression of the activation marker – CD38. In case of all abovementioned assays, sample analyses has been performed on flow cytometer (FACScan, Becton Dickinson). At the first stage of the research we have evaluated IgM-ligation effect after short-time stimulation – in 0.5 h. This time-point has been chosen due to the fact, that B cell receptor (BCR) ligation has been known to induce an immediate effect. We were simultaneously evaluating proliferation, activation and early apoptosis levels. According the obtained results in response to IgM ligation there was no increase in proliferation (% of cells in G2/M: spont. 2±0.6%; stim. 2.25±0.6%; % of cells in S phase: spont. 5±0.7%; stim. 4.7±1.1%), activation (spont.: 30.6±8.1% ; stim: 24.5±1.2% ) or apoptosis (spont.: 13.45±4.1% ; stim. 9.83±3.9%) levels. In other words, we have revealed , that MEC1 cells are absolutely anergic towards IgM ligation. This fact gives us possibility to observe MEC1 cells line as a model of Non-Responder CLL clone. Usage of this model system will help us in our further studies to investigate functional connection between CD180 and IgM signalling pathways in case of anergic clones. To investigate functional connection between BCR and CD180 we studied IgM ligation effect on the level of CD180 surface expression. The effect has been evaluated at 3 different time-points: 0.5, 6 and 24 hours after stimulation. Beside the fact, that no significant change in CD180 expression level has been detected at short time-points (0.5h: spont.-10.4±2.3; stim. -16.3±3.8; 6h: spont.-28.2±8; stim.-27±2), for 24 hours it has been demonstrated a decrease in CD180 expression level in stimulated cultures (0.5h: spont.-10.4±2.3; stim. -16.3±3.8; 6h: spont.-28.2±8; stim.-27±2). The obtained result once more emphasizes the existing functional connection between CD180 and BCR. As this change has been revealed at a later time-point, we decide to check the possibility, that specific stimulation might has a delayed effect in MEC1 cells. In this case we stimulated cultures with anti-IgM and/or anti-CD180 mAbs. According the obtained data CD180 and IgM simultaneous ligation has resulted in anti-apoptosis protective effect (spont.: % subG0=21±1.8; combined stim.: % subG0=12.5±2.8, p<0.05), which means that in case of MEC1 cells CD180 and IgM ligation have a synergic effect on pro-survival pathways. The study of mechanisms leading to survival and proliferation in MEC1 cells will enable a better understanding of the microenvironmental stimuli that lead to the expansion of the leukaemic cells and progression of the disease, in order to develop new therapies for this currently incurable disease.

Lecture files:

B ქრონიკული ლიმფოციტური ლეიკემიის უჯრედული ხაზის MEC1 უჯრედებში სპეციფიური სტიმულაციით გამოწვეული საპასუხო რეაქციის შესწავლა [ka]