Multiple myeloma (MM) is a progressive disease that is thought to result from multiple genetic insults to the precursor plasma cell that ultimately affords the tumor cell with proliferative potential despite its differentiated phenotype and resistance to undergoing apoptosis. Altered expression of antiapoptotic factors as well as growth factors have been described in a significant number of patients. However, the key regulatory elements that control myeloma development and progression remain largely unde-

BAFF plays a central role in B-lineage cell biology; however, the regulation of BAFF-binding receptor (BBR) expression during B cell activation and differentiation is not completely understood. In this study, we provide a comprehensive ex vivo analysis of BBRs in human B-lineage cells at various stages of maturation, as well as describe the events that drive and regulate receptor expression. Our data reveal that B-lineage cells ranging from naive to plasma cells (PCs), excluding bone marrow PCs, express BAFF-R uniformly. In contrast, only tonsillar memory B cells (MB) and PCs, from both tonsil and bone marrow tissues, express BCMA. Furthermore, we show that TACI is expressed by MB cells and PCs, as well as a subpopulation of activated CD27neg B cells. In this regard, we demonstrate that TACI is inducible early upon B cell activation and this is independent of B cell turnover. In addition, we found that TACI expression requires activation of the ERK1/2 pathway, since its expression was blocked by ERK1/2-specific inhibitors. Expression of BAFF-R and B cell maturation Ag (BCMA) is also highly regulated and we demonstrate that BCMA expression is only acquired in MB cells and in a manner accompanied by loss of BAFF-R expression. This inverse expression coincides with MB cell differentiation into Ig-secreting cells (ISC), since blocking differentiation inhibited both induction of BCMA expression and loss of BAFF-R. Collectively, our data suggest that the BBR profile may serve as a footprint of the activation history and stage of differentiation of normal human B cells.

IntroductionMonocytes are produced in the bone marrow; they circulate in blood for 1 to 3 days before entering tissues or undergo apoptosis if they do not encounter specific survival signals. [1][2][3] The mechanisms regulating monocyte apoptosis are not fully understood. It is known, however, that lipopolysaccharide (LPS), granulocytemacrophage colony stimulating factor (GM-CSF), macrophage CSF (M-CSF), TNF-␣, and interleukin-1␤ (IL-1␤) all inhibit monocyte apoptosis and induce prolonged monocyte survival. [3][4][5] Monocytes play an important role in initiating activation of the innate immune system, for example, phagocytosis further activates monocytes to secrete proinflammatory cytokines and chemokines in inflamed tissues. 6 Monocytes can differentiate into either macrophages, 7 which display enhanced ability to phagocytose, or dendritic cells (DCs), 8 which function as antigen-presenting cells that play a central role in activating the adaptive immune system. These cells provide a first-line host defense against viral and bacterial infection.B-lymphocyte stimulator (BLyS) is a member of the TNF family (also named BAFF, zTNF4, THANK, and Tall-1), which is expressed as a full-length 285-amino acid transmembrane molecule, and cleaved from cells as a 152-amino acid soluble ligand following processing by a furinlike protease. 9,10 Soluble BLyS exists as a trimer or oligomer and is thought to be the primary effector of in vivo function. However, cell-associated BLyS also induced proliferation of anti-IgM-stimulated B lymphocytes, 10 and more recently, T cells were shown to respond only to immobilized BLyS. 11,12 BLyS is produced by myeloid lineage cells, malignant B cells, activated T cells, and bone marrow stromal cells. [13][14][15][16][17] Monocyte stimulation with IFN-␥, TNF-␣, or IL-10 increases BLyS production. 14 Bacterial components such as LPS and peptidoglycan can also up-regulate BLyS secretion by macrophages, dendritic cells, and monocytes. 14,18 BLyS has 3 receptors: BCMA (B-cell maturation antigen), TACI (transmembrane activator and CAML interactor), and BAFF-R (BAFF receptor). [19][20][21][22] These receptors belong to the TNF receptor superfamily; all receptors possess an extracellular domain containing multiple cysteine-rich domains (CRDs) and intracellular sequences containing TNF receptor-associated protein (TRAF) binding sites. These receptors are primarily expressed in B-lineage cells. BCMA is exclusively expressed in B cells. However, a subset of T cells has also been shown to express TACI and BAFF-R. 11,12,23 Because of the expression pattern of BLyS receptors, most studies to date have focused on the effects of BLyS on adaptive immune cells, and these studies show that BLyS costimulates B-cell proliferation and induces cell survival 18,24 and can also function as a T-cell costimulatory molecule. 11,12,23 Whether BLyS has functional effects on other cell lineages has not been reported.BLyS transgenic mice developed a syndrome with similarities to systemic lupus erythematosus (SLE) in humans...

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DNA double strand breaks (DSBs) are deleterious lesions that can lead to chromosomal anomalies, genomic instability and cancer. The histone protein H2AX plays an important role in the DNA damage response (DDR) and the presence of phospho-H2AX (γH2AX) nuclear foci is the hallmark of DSBs. We hypothesize that ongoing DNA damage provides a mechanism by which chromosomal abnormalities and intratumor heterogeneity are acquired in malignant plasma cells (PCs) in patients with multiple myeloma (MM). Therefore, we assessed PCs from patients with the premalignant condition, monoclonal gammopathy of undetermined significance (MGUS) and MM, as well as human MM cell lines (HMCLs) for evidence of DSBs. γH2AX foci were detected in 2/5 MGUS samples, 37/40 MM samples and 6/6 HMCLs. Notably, the DSB response protein 53BP1 colocalized with γH2AX in both MM patient samples and HMCLs. Treatment with wortmannin decreased phosphorylation of H2AX and suggests phosphoinositide (PI) 3-kinases and/or PI3-kinase like family members underlie the presence of γH2AX foci in MM cells. Taken together, these data imply that ongoing DNA damage intensifies across the disease spectrum of MGUS to MM and may provide a mechanism whereby clonal evolution occurs in the monoclonal gammopathies.

Multiple myeloma (MM) is characterized by the clonal expansion of malignant plasma cells within the bone marrow. There is a growing literature that tumor cells release biologically active microvesicles (MVs) that modify both local and distant microenvironments. In this study, our goals were to determine if MM cells release MVs, and if so, begin to characterize their biologic activity. Herein we present clear evidence that not only do both patient MM cells and human MM cell lines (HMCLs) release MVs, but that these MVs stimulate MM cell growth. Of interest, MM-derived MVs were enriched with the biologically active form of CD147, a transmembrane molecule previously shown by us to be crucial for MM cell proliferation. Using MVs isolated from HMCLs stably transfected with a CD147-GFP fusion construct (CD147GFP), we observed binding and internalization of MV-derived CD147 with HMCLs. Cells with greater CD147GFP internalization proliferated at a higher rate than did cells with less CD147GFP association. Lastly, MVs obtained from CD147 downregulated HMCLs were attenuated in their ability to stimulate HMCL proliferation. In summary, this study demonstrates the significance of MV shedding and MV-mediated intercellular communication on malignant plasma cell proliferation, and identifies the role of MV-enriched CD147 in this process.

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