The aim of this in vitro study was to evaluate if osteoclasts (OCs) and dendritic cells (DCs), both of monocyte origin, can support the survival of normal human plasma cells (PCs). PCs differentiate ...from plasmablasts (PBs) arising from activated B cells, essentially memory B cells. To study the survival of both PBs (CD20lowCD38highCD138neg) and PCs (CD20negCD38brightCD138bright), we generated pre-PBs (CD20lowCD38posCD138neg) from CD40-activated B cells (CD20highCD38negCD138neg) and cultured them on DCs or OCs in the presence of added IL-6. By quantitative and qualitative study, we showed that DCs support the survival of PBs and early PCs, but not that of PCs. In contrast, OCs support the survival of PBs, early PCs and PCs. PCs surviving on OCs 12 days after pre-PB input display phenotypic features of bone marrow PCs, CD138brightCD38brightHLA-DRlowCD45dim. The ability for OCs to support the survival of PCs was fully dependent on cell–cell contact and not inhibited by BCMA-Fc suggesting that secreted BAFF and APRIL were not involved.
Plasma cells (PCs) have a heterogeneous phenotype in humans. While bone marrow PCs are CD20-CD138+, tonsil PCs are CD20+CD138+/- and peripheral plasmablasts (PBs) are CD20-CD138-. In vitro, PCs are ...mainly generated by the activation of CD27+ memory B-cells through transient stimulation of CD40, and their phenotype appears similar to that of bone marrow PCs. While CD20 expression is lost at the plasmablastic stage, CD138 expression appears only at the PC stage. Thus, the CD20+CD138± phenotype of tonsil PCs does not represent an intermediate stage in the differentiation of memory B-cells into PCs. Because it has been previously shown that TLR9 activation was more able than CD40 stimulation to induce the differentiation of IgM+ CD27+ B-cells, we wondered whether TLR9 or CD40 stimulation would induce the same phenotype of PCs. Thus, we compared the differentiation of CD27+ B-cells isolated from either the tonsils or peripheral blood and stimulated with either CD40L-expressing fibroblasts or a TLR9 ligand, CpG oligodeoxynucleotide (CpG ODN). We observed that CpG ODN mainly induced CD27+ B-cell differentiation into CD20+CD38+CD138- PBs and CD20+CD38+CD138± PCs, which appear similar to tonsil PCs. Removal of CpG ODN during differentiation induced a decrease in the CD20+ plasmablastic population, and, conversely, stimulation of CD40L-induced pre-plasmablasts with CpG ODN increased the population of CD20+CD38+ PBs. Analysis of Ig secretion showed that CpG ODN induced increased IgM secretion compared to CD40L. PCs from patients with multiple myeloma, the malignant counterpart of bone marrow PCs, rarely express CD20. We show that CpG ODN did not induce or increase CD20 in nine IgG or IgA myeloma cell lines. These data strongly suggest that CpG ODN mainly targets CD27+ IgM+ B-cells.
We investigated the generation of myeloma-specific CTLs from normal donors HLA mismatched with the myeloma cell line SBN. The T-cell line obtained was cloned and each CTL was assessed against SBN and ...SBN-EBV (a B-EBV cell line obtained by EBV infection of B cells from SBN patient) simultaneously. Among >270 clones evaluated, 2 CTLs (Vbeta13.1 and Vbeta17) killed SBN but spared SBN-EBV cells. Antibodies against HLA-I, but not HLA-A2, molecules abrogated their recognition of SBN. Moreover, SBN recognition was abrogated by anti-HLA-Cw6 antiserum. Both clones recognized two other HLA-Cw*0602 myeloma cell lines. Neither of them recognized HLA-Cw*0602 B-EBV cell lines, the PBMCs of HLA-Cw*0602-unrelated donors or HLA-Cw*0602 melanoma cell lines. We showed that HLA-Cw6 molecules were more expressed at the cell surface of B-EBV cells as compared with myeloma cells, suggesting that the lack of reactivity against B-EBV cells was not related to a low level of HLA expression. Since CTL clones did not express any KIR or NKG2D, we excluded the fact that NK cell receptors could be involved in myeloma-specific recognition through KIR–HLA-I or NKG2D–MICA,B interactions. Cold target competition and acid elution experiments confirmed that myeloma cell recognition was peptide dependent.
We evaluated the ability of 2 human mAbs directed against TRAILR1 (HGS-ETR1) and TRAILR2 (HGS-ETR2) to kill human myeloma cells. HGS-ETR1 and HGS-ETR2 mAbs killed 15 and 9 human myeloma cell lines ...(HMCLs; n = 22), respectively. IL-6, the major survival and growth factor for these HMCLs, did not prevent their killing. Killing induced by either HGS-ETR1 or HGS-ETR2 was correlated with the cleavage of Mcl-1L, a major molecule for myeloma survival. Mcl-1L cleavage and anti-TRAILR HMCL killing were dependent on caspase activation. Kinetic studies showed that Mcl-1L cleavage occurred very early (less than 1 hour) and became drastic once caspase 3 was activated. Our data showed that both the extrinsic (caspase 8, Bid) and the intrinsic (caspase 9) pathways are activated by anti–TRAIL mAb. Finally, we showed that the HGS-ETR1 and, to a lesser extent, the HGS-ETR2 mAbs were able to induce the killing of primary myeloma cells. Of note, HGS-ETR1 mAb was able to induce the death of medullary and extramedullary myeloma cells collected from patients at relapse. Taken together, our data clearly encourage clinical trials of anti–TRAILR1 mAb in multiple myeloma, especially for patients whose disease is in relapse, at the time of drug resistance.
Long-lived plasma cells (PCs) located within the bone marrow are the source of protective antibody for extended periods of time. Recent studies pointed out that PC survival depends on the ability of ...PCs to find a suitable niche. Bone marrow environment of PCs is constituted by cells of either mesenchymal (stromal cells and osteoblasts) or hematopoietic origin (macrophages, dendritic cells, osteoclasts (OCs)). In this work, we evaluated the ability of these five types of cells to support the long-term survival of human PCs in vitro.
Stromal cells were derived from normal bone marrow mononuclear cells and osteoblasts were SAOS2 cells (malignant osteoblastic cells). Macrophages, dendritic cells and OCs were derived from adherent circulating mononuclear cells with M-CSF, IL4 and GM-CSF or RANKL and M-CSF, respectively. Morphology, phenotype and functional features were used to characterize each cell type. On the other hand, plasmablasts (PBs) were generated from tonsil CD27+ B cells activated by CD40L expressing fibroblasts and then induced to differentiate by removal of fibroblasts in the continuous presence of IL2 and IL10. PBs were purified (by CD20 depletion) and cocultured with the five types of cells previously described.
To assess PC differentiation and survival in cocultures, we monitored the frequency of PCs by flow cytometry using an anti-CD38 and anti-CD138 double staining, along with Ig accumulation. Among cells of either mesenchymal (stromal cells and osteoblasts) or hematopoietic origin (macrophages, dendritic cells, OCs), only OCs allowed PBs to survive and differentiate into PCs that remained alive more than 14 days. Indeed, after a 5-day coculture with OCs, 100 000 PCs were alive (400 000 PBs were seeded at day 0) as compared to less than 10 000 PCs in the other culture conditions. Moreover, the total frequency of PCs after 14 days in culture with OCs was approximately 5% of the original input at day 0. PCs fully maintained their antibody secretory capacity since the level of secreted antibodies increased. Cellular interactions were required since PBs cultured with either OC conditioned medium or with OCs in transwell did not survive nor differentiate. Identification of membrane and soluble molecules supporting PC survival is now under investigation.
Our data indicate that the interaction with OCs is not limited to malignant PCs i.e., myeloma cells, and pointed out that OC is an essential partner of normal PC.
We investigated TRAILR expression and sensitivity of myeloma cells in vitro. This study was done using a panel of 20 myeloma cell lines that are representative of primary myeloma cells (14q ...chromosomal translocation, IL-6 dependency, phenotype, oncogenes mutation). TRAILR were stimulated with agonistic human antibodies directed against either TRAIL-R1/DR4 (HGS-ETR1, mapatumumab) or TRAIL-R2/DR5 (HGS-ETR2), provided by Human Genome Sciences, Rockville, MD. This approach allowed us to analyze the contribution of each receptor separately. We show that a wide majority of cell lines, 16 of 20 were killed upon either TRAIL-R1 or R2 stimulation in the presence or absence of IL-6. However, 4 cell lines were resistant to HGS-ETR1 and 6 to HGS-ETR2 and 3 to both. Activation of both caspase 8 and Bid has been extensively described as being associated with TRAIL response. Indeed, we observed an activation of both caspase 8 and Bid. Cleaved molecules were detected 6 to 18h after antibody addition but after detection of cellular apoptosis. However, we show that Mcl-1L, a key molecule for myeloma survival, was downregulated and cleaved as soon as 3h after Ab addition. The cleaved form of Mcl-1 has been shown to behave like a proapoptotic molecule. Since caspase 3 has been reported to cleave Mcl-1, we looked at caspase 3 activation. Indeed, we observed that caspase 3 cleavage occured early and concomitantly to the one of Mcl-1. Our data show that in a wide majority of myeloma cell lines (80%) TRAILR triggering induces massive apoptosis that was not prevented by IL-6, the major myeloma cell growth and survival factor. Moreover, apoptosis induced upon TRAILR triggering was fully correlated to an early cleavage of both caspase 3 and Mcl-1 and to a delayed one of both caspase 8 and Bid.
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