Functional Characterization In vitro

Workpackage 8

Objectives

To compare the growth and differentiation potential of blood circulating myeloma memory B cells and normal memory B cells into plasmablasts. To evaluate the ability of genes overexpressed in myeloma cells compared to normal plasma cells to promote survival and differentiation of normal plasmablasts.

Description of work 1

Partner 1 has developed a method which can quantitate the number of low frequent cells in a phenotypically defined population expressing a gene of interest by combining RT-PCR method working on whole single cells with sorting and limiting dilution. Recently, our collaborators have succeeded in selecting specific single chain FV bearing phages from libraries specifically directed against the immunoglobulin idiotype expressed by myeloma tumour cells. Panning of semi-synthetic phage library against purified myeloma paraproteins select clones reactive with monoclonal membrane Ig B cells confirmed by flow cytometry. Such a technique has been described to detect rare idiotype positive cells in blood, which e.g. can be sorted as CD19+/CD38- memory B cells or CD19+/CD38+ plasmablasts. We intend to develop an autologous in vitro culture system to study patients' idiotypic B cells differentiation into plasma cells in the presence of stromal cell lines transfected by CD40L and recombinant human cytokines as described for normal memory B cells by partner 7 (Tarte, et al , Blood, 2002). The differentiation and proliferative response will be followed by real-time RT-PCR against myeloma specific gene products. For individual studies interphase multicolor FISH and laser scanning cytometry will document functional plasma cells if present.

Description of work 2

Partner 7 has developed a reproducible methodology making it possible to get a virtually pure population of plasmablasts (Tarte, et al , Blood, 2002). Peripheral blood memory B cells are cultured for 4 days with a CD40 ligand transfectant and cytokines yielding expansion of activated B cells. These activated B cells are then induced to differentiate into plasmablasts by removal of CD40 ligand and change in cytokine combinations. Only memory B cells can generate plasmablasts. These plasmablasts are still proliferating, but apoptose within 4 days, and are unable to complete a full differentiation into mature plasma cells. Plasmablasts expressed no more B cell markers, express the plasma cell transcription factors Blimp1 and XBP-1, but did not express syndecan-1, a hallmark of mature plasma cells. Comparisons of gene expression profiles between memory B cells and plasmablasts and bone marrow plasma cells and plasmablasts demonstrates a down regulation of genes coding for anti-apoptotic proteins (A1, bcl-2, Mcl-1) and up regulation of apoptotic genes (bik) in plasmablasts. Several interesting genes that could introduced are those that we found already poorly expressed in normal plasmablastic cells compared to normal bone marrow plasma cells as well as some putative oncogenes in MMC. However, this list of genes will be improved according to data obtained. We will use biscistronic lentiviral vectors making it possible to have expression of a green fluorescent protein (GFP) together with the protein of interest. Transduced GFP+ plasmablasts will be FACS sorted after 2 day transduction to avoid the variability of transduction experiments.

We will monitor the survival of plasmablasts, the cell cycle, the ability to differentiate in to mature plasma cells as evidenced by induction of syndecan-1, increased immunoglobulin production, induction of intercellular communication proteins. The survival of transduced plasmablasts will be studied in the presence of various growth factors (in particular the heparan sulfate binding members of the EGF family), osteoclast or MSC (Mahtouk, et al 2005).

Deliverables and cooperations

• Analysis of proliferation and differentiation potential of patients' idiotypic memory B cells.
• Contribution of myeloma genes to promote survival, proliferation and differentiation of normal plasmablasts.
• Creation of an in vitro model of multiple myeloma.

Milestones and expected result

• Purification of memory B cells from 20 newly diagnosed patients. Month 1-24
• Obtaining anti-idiotypic antibodies and purification of idiotypic B cells. Months 6-24.
• Preparation of 10 lentiviral vectors with myeloma genes coding for proteins involved in the survival, proliferation or differentiation of myeloma cells. Months 1-12.
• Biological effect of expression of myeloma genes in normal plasmablasts transduced with lentiviral vectors. Months 6-36.