Ivan Van Riet

Evidence that the clonogenic cell in multiple myeloma originates from a pre‐switched but somatically mutated B cell

Summary. There is still much controversy about the precursor cell type in multiple myeloma (MM). Some authors claim that it is a pre‐B cell, others state that it is a memory B cell or plasmablast. We have recently shown that the VDJ region of the MM immunoglobulin heavy chain gene is somatically hypermutated and antigen selected, without intraclonal variation or evolution in time. By using a patient‐specific PCR approach we have now obtained evidence that the premyeloma cell can be situated in the pre‐switched B‐cell compartment and that heavy chain switching can occur without further somatic mutation. Based on the MM immunoglobulin sequences derived from the bone marrow, patient‐specific CDR2 and CDR3 oligonucleotides were designed. B lymphocytes were separated from plasma cells based on the expression of CD19 and HLA class II or surface bound IgM using immunomagnetic beads. The expressed Ig sequences were amplified by RT‐PCR using patient specific CDR2 primers and isotype specific primers (Cμ, Cγ and Cα). Myeloma‐specific Ig sequences were detected by a myeloma‐specific CDR 3 probe and sequenced. In one out of five cases we found in the peripheral blood clonally related IgM and IgA sequences with the same somatic mutations as the MM‐IgG sequence. In another case of an IgG MM we found in the bone marrow clonally related IgA sequences with the same somatic mutations. These findings, together with the fact that myeloma‐Ig genes contain somatic mutations without intraclonal variation, suggest that the clonogenic cell in multiple myeloma can originate from a pre‐switched but somatically mutated B cell.

Generation of large numbers of dendritic cells in a closed system using Cell Factories

There is a growing interest in using dendritic cells (DC) for vaccine approaches in the treatment of cancer and infectious diseases. This requires a reproducible method for the generation of large numbers of DC in a closed culture system suitable for clinical use and conforming to the current guidelines of good manufacturing practices. We designed a system in which the DC were generated in a closed system from adherent monocytes using Cell Factories (DC-CF). Monocytes were enriched from apheresis products by adherence and then cultured in the presence of AB serum or autologous plasma and GM-CSF and IL-4 for 6 days. The DC generated in Cell Factories were extensively compared to research-grade DC generated in conventional tissue culture flasks (DC-TCF). At day 6, the immature DC were harvested and the yield, the viability, the immunophenotype and the functional characteristics of the DC were compared.DC-CF and DC-TCF showed similar viability and purity and scored equally when tested for stability, dextran and latex bead uptake, in MLR and in the activation of influenza-specific memory cells after electroporation with influenza matrix protein 1 (IMP1) mRNA. These data indicated that large numbers of functional clinical-grade DC could be generated from adherent cells in a closed system using Cell Factories.

Therapeutic vaccination with an autologous mRNA electroporated dendritic cell vaccine in patients with advanced melanoma.

The immunostimulatory capacity of dendritic cells is improved by co-electroporation with mRNA encoding CD40 ligand, constitutively active toll-like receptor 4, and CD70 (TriMix-DC). This pilot clinical trial evaluated the feasibility, safety, and immunogenicity of a therapeutic vaccination containing autologous TriMix-DC co-electroporated with mRNA encoding a human leukocyte antigen class II-targeting signal linked to 1 of 4 melanoma-associated antigens (MAGE-A3, MAGE-C2, tyrosinase, and gp100) in patients with advanced melanoma. Thirty-five American Joint Committee on Cancer stage III/IV melanoma patients received autologous TriMix-DC (4 administrations 2 weeks apart). Immune monitoring was performed by evaluating skin biopsies of delayed type IV hypersensitivity (DTH) reactions for presence of vaccinal antigen-specific DTH-infiltrating lymphocytes (DIL). Thereafter, patients could receive interferon-alpha-2b (IFN-&agr;-2b) 5 MU subcutaneously 3 times weekly and additional TriMix-DC every 8 weeks. TriMix-DC-related adverse events comprised grade 2 local injection site reactions (all patients), and grade 2 fever and lethargy (2 patients). Vaccinal antigen-specific DIL were found in 0/6 patients tested at vaccine initiation and in 12/21 (57.1%) assessed after the fourth vaccine. A positive postvaccination DTH test correlated with IL-12p70 secretion capacity of TriMix-DC. No objective responses to TriMix-DC alone were seen according to RECIST. Twenty-nine patients received IFN-&agr;-2b after the fourth vaccine without unexpected adverse events. During TriMix-DC/IFN-&agr;-2b combination therapy, 1 partial response and 5 stable disease (disease control of >6 months with regression of metastases) were observed in 17 patients with evaluable disease at baseline. In conclusion, this study demonstrated that therapeutic vaccination with autologous TriMix-DC is feasible, safe, and immunogenic and can be combined with sequential IFN-&agr;-2b.

Role of CCR1 and CCR5 in homing and growth of multiple myeloma and in the development of osteolytic lesions: a study in the 5TMM model

Multiple myeloma (MM) is a plasma cell malignancy, characterized by the localization of the MM cells in the bone marrow (BM), where they proliferate and induce osteolysis. The MM cells first need to home or migrate to the BM to receive necessary survival signals. In this work, we studied the role of CCR1 and CCR5, two known chemokine receptors, in both chemotaxis and osteolysis in the experimental 5TMM mouse model. A CCR1–specific (BX471) and a CCR5–specific (TAK779) antagonist were used to identify the function of both receptors. We could detect by RT-PCR and flow cytometric analyses the expression of both CCR1 and CCR5 on the cells and their major ligand, macrophage inflammatory protein 1α (MIP1α) could be detected by ELISA. In vitro migration assays showed that MIP1α induced a 2-fold increase in migration of 5TMM cells, which could only be blocked by TAK779. In vivo homing kinetics showed a 30% inhibition in BM homing when 5TMM cells were pre-treated with TAK779. We found, in vitro, that both inhibitors were able to reduce osteoclastogenesis and osteoclastic resorption. In vivo end-term treatment of 5T2MM mice with BX471 resulted in a reduction of the osteolytic lesions by 40%; while TAK779 treatment led to a 20% decrease in lesions. Furthermore, assessment of the microvessel density demonstrated a role for both receptors in MM induced angiogenesis. These data demonstrate the differential role of CCR1 and CCR5 in MM chemotaxis and MM associated osteolysis and angiogenesis.

Expression of cytoadhesion molecules (CD56, CD54, CD18 and CD29) by myeloma plasma cells

Recently we reported the expression of the human natural killer cell associated antigen CD56 (Leu 19/NKH1) in plasma cells of a majority of multiple myeloma (MM) patients. CD56 is known to be an isoform of the human neural adhesion molecule N‐CAM which is involved in homotypic adhesive interactions. By immunophenotyping using four CD56 specific monoclonal antibodies and immunoprecipitation analysis we here confirm that the Leu 19 antigen expressed by myeloma plasma cells is identical to N‐CAM and corresponds to the 145 kDa isoform. Because of the possible biological role of adhesion molecules on myeloma cells, we compared the expression of N‐CAM with the intercellular adhesion molecule 1 (ICAM‐1) and the β1 and β2 integrins. By immunogold‐silver staining of cytospin preparations of mononuclear cell suspensions, bone marrow plasma cells of 17 MM patients were analysed. Plasma cells expressed N‐CAM (CD56) in 14 patients, ICAM‐1 (CD54) in 16 patients, and β2 intergrins (CD18) in eight patients. β1 integrins (CD29) were expressed in all patients. The expression of β2 integrins was always very weak while N‐CAM, ICAM‐1 and the β1 integrins showed a moderate to strong positivity. The plasma cells of five haematological normal individuals lacked significant N‐CAM expression but were positive for ICAM‐1 and both integrin subgroups. One plasma cell leukaemia patient and two out of four end‐stage MM patients showed no expression of N‐CAM or β2 integrins on their circulating plasma cells. Among 11 previously established myeloma cell lines, surface expression of ICAM‐1 and the integrins was detected in most cases, while N‐CAM was present in only four lines. Most cell lines showed coexpression of the fibronectin receptors (VLA‐4 and VLA‐5) and the laminin receptor (VLA‐6). The collagen receptor (VLA‐2) was not expressed. The N‐CAM negative cell lines included four cell lines that were derived from plasma cell leukaemia patients. These results indicate that the expression of adhesion molecules is an intrinsic part of the biology of multiple myeloma.

Chromatin remodeling agent trichostatin A: a key-factor in the hepatic differentiation of human mesenchymal stem cells derived of adult bone marrow

BackgroundThe capability of human mesenchymal stem cells (hMSC) derived of adult bone marrow to undergo in vitro hepatic differentiation was investigated.ResultsExposure of hMSC to a cocktail of hepatogenic factors [(fibroblast growth factor-4 (FGF-4), hepatocyte growth factor (HGF), insulin-transferrin-sodium-selenite (ITS) and dexamethasone)] failed to induce hepatic differentiation. Sequential exposure to these factors (FGF-4, followed by HGF, followed by HGF+ITS+dexamethasone), however, resembling the order of secretion during liver embryogenesis, induced both glycogen-storage and cytokeratin (CK)18 expression. Additional exposure of the cells to trichostatin A (TSA) considerably improved endodermal differentiation, as evidenced by acquisition of an epithelial morphology, chronological expression of hepatic proteins, including hepatocyte-nuclear factor (HNF)-3β, alpha-fetoprotein (AFP), CK18, albumin (ALB), HNF1α, multidrug resistance-associated protein (MRP)2 and CCAAT-enhancer binding protein (C/EBP)α, and functional maturation, i.e. upregulated ALB secretion, urea production and inducible cytochrome P450 (CYP)-dependent activity.ConclusionhMSC are able to undergo mesenchymal-to-epithelial transition. TSA is hereby essential to promote differentiation of hMSC towards functional hepatocyte-like cells.

Extravasation and homing mechanisms in multiple myeloma

Multiple myeloma (MM) is a malignant B-cell disorder characterized by a monoclonal expansion of plasma cells (PC) in the bone marrow (BM). During the main course of disease evolution, MM cells depend on the BM microenvironment for their growth and survival. Reciprocal interactions between MM cells and the BM mediate not only MM cell growth, but also protect them against apoptosis and cause bone disease and angiogenesis. A striking feature of MM represents the predominant localization and retention of MM cells in the BM. Although BM PC indeed represent the main neoplastic cell type, small numbers of MM cells can also be detected in the peripheral blood circulation. It can be assumed that these circulating cells represent the tumour-spreading component of the disease. This implicates that MM cells have the capacity to (re)circulate, to extravasate and to migrate to the BM (homing). In analogy to the migration and homing of normal leucocytes, the BM homing of MM cells is mediated by a multistep process of extravasation with adhesion to the endothelium, invasion of the subendothelial basement membrane, followed by further migration within the stroma, mediated by chemotactic factors. At the end stage of disease, MM cells are thought to develop autocrine growth supporting loops that enable them to survive and proliferate in the absence of the BM microenvironment and to become stroma-independent. In this stage, the number of circulating cells increases and growth at extramedullary sites can occur, associated with alteration in adhesion molecule and chemokine receptor expression. This review summarizes the recent progress in the study of the extravasation and homing mechanisms of MM cells.

Detection of monoclonal B lymphocytes in bone marrow and peripheral blood of multiple myeloma patients by immunoglobulin gene rearrangement studies

To investigate whether B lymphocytes are involved in the malignant cell clone of multiple myeloma (MM), we performed immunoglobulin gene rearrangement analysis of mononuclear cells and separated B lymphocytes, isolated from bone marrow and peripheral blood of MM patients. The B lymphocytes were separated by immunomagnetic beads, coated with an HLA class II specific antibody. Southern blot analysis with a JH probe revealed in the bone marrow of three out of seven patients identical immunoglobulin gene rearrangements in the B lymphocytes when compared to the plasma cells. Out of 10 patients, two patients with a high tumour burden were found to have monoclonal B lymphocytes in the peripheral blood. These results suggest that B lymphocytes in the bone marrow are part of the myeloma clone and that they can circulate in the peripheral blood. Although previous studies indicated that the ratio of K to λ bearing lymphocytes in the peripheral blood can provide evidence for B cell monoclonality, we did not find a correlation between the results of K/λ analysis and immunoglobulin gene rearrangement.

Homing and migration of mesenchymal stromal cells: How to improve the efficacy of cell therapy?

Mesenchymal stromal cells (MSCs) are currently being investigated for use in a wide variety of clinical applications. For most of these applications, systemic delivery of the cells is preferred. However, this requires the homing and migration of MSCs to a target tissue. Although MSC homing has been described, this process does not appear to be highly efficacious because only a few cells reach the target tissue and remain there after systemic administration. This has been ascribed to low expression levels of homing molecules, the loss of expression of such molecules during expansion, and the heterogeneity of MSCs in cultures and MSC culture protocols. To overcome these limitations, different methods to improve the homing capacity of MSCs have been examined. Here, we review the current understanding of MSC homing, with a particular focus on homing to bone marrow. In addition, we summarize the strategies that have been developed to improve this process. A better understanding of MSC biology, MSC migration and homing mechanisms will allow us to prepare MSCs with optimal homing capacities. The efficacy of therapeutic applications is dependent on efficient delivery of the cells and can, therefore, only benefit from better insights into the homing mechanisms.

Bone Marrow-Derived Mesenchymal Stromal Cells Are Attracted by Multiple Myeloma Cell-Produced Chemokine CCL25 and Favor Myeloma Cell Growth In Vitro and In Vivo

Multiple myeloma (MM) is a malignancy of terminally differentiated plasma cells that are predominantly localized in the bone marrow (BM). Mesenchymal stromal cells (MSCs) give rise to most BM stromal cells that interact with MM cells. However, the direct involvement of MSCs in the pathophysiology of MM has not been well addressed. In this study, in vitro and in vivo migration assays revealed that MSCs have tropism toward MM cells, and CCL25 was identified as a major MM cell‐produced chemoattractant for MSCs. By coculture experiments, we found that MSCs favor the proliferation of stroma‐dependent MM cells through soluble factors and cell to cell contact, which was confirmed by intrafemoral coengraftment experiments. We also demonstrated that MSCs protected MM cells against spontaneous and Bortezomib‐induced apoptosis. The tumor‐promoting effect of MSCs correlated with their capacity to enhance AKT and ERK activities in MM cells, accompanied with increased expression of CyclinD2, CDK4, and Bcl‐XL and decreased cleaved caspase‐3 and poly(ADP‐ribose) polymerase expression. In turn, MM cells upregulated interleukin‐6 (IL‐6), IL‐10, insulin growth factor‐1, vascular endothelial growth factor, and dickkopf homolog 1 expression in MSCs. Finally, infusion of in vitro‐expanded murine MSCs in 5T33MM mice resulted in a significantly shorter survival. MSC infusion is a promising way to support hematopoietic recovery and to control graft versus host disease in patients after allogeneic hematopoietic stem cell transplantation. However, our data suggest that MSC‐based cytotherapy has a potential risk for MM disease progression or relapse and should be considered with caution in MM patients. STEM CELLS 2012; 30:266–279.