Bonnie K. Arendt

Regulated Expression of BAFF-Binding Receptors during Human B Cell Differentiation

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.

Inhibition of survivin expression suppresses the growth of aggressive non-Hodgkin's lymphoma

Survivin is a member of the inhibitor of apoptosis protein (IAP) family and functions both as an apoptosis inhibitor and a regulator of cell division. Survivin overexpression is common in many human tumors and correlates with survival in large cell non-Hodgkins lymphoma. To evaluate this molecule as a potential therapeutic target in large-cell lymphoma, we evaluated the effect of survivin inhibition both in vitro and in vivo. Using an antisense oligonucleotide (ASO) approach, cell growth was significantly inhibited in the DoHH2, RL and HT lymphoma cell lines. In a lymphoma xenograft model, the development of tumors as well as the growth of established tumors was inhibited in the survivin ASO-treated mice compared to controls. To assess the efficacy of the survivin ASO in combination with other biological agents, we combined the survivin ASO with an anti-CD20 monoclonal antibody, rituximab. The effect of survivin ASO and rituximab in combination was additive in vitro. In vivo, however, suppression of tumor growth with the combination was not significantly superior to controls. We conclude that inhibition of survivin expression is an attractive therapeutic strategy in aggressive non-Hodgkins lymphomas, and that combining survivin ASO with rituximab may enhance the efficacy of this approach.

Divergent Effects of BAFF on Human Memory B Cell Differentiation into Ig-Secreting Cells

B cell-activating factor belonging to the TNF family (BAFF) plays a critical role in B cell maturation, yet its precise role in B cell differentiation into Ig-secreting cells (ISCs) remains unclear. In this study, we find that upon isolation human naive and memory B (MB) cells have prebound BAFF on their surface, whereas germinal center (GC) B cells lack detectable levels of prebound BAFF. We attribute their lack of prebound BAFF to cell activation, because we demonstrate that stimulation of naive and MB cells results in the loss of prebound BAFF. Furthermore, the absence of prebound BAFF on GC B cells is not related to a lack of BAFF-binding receptors or an inability to bind exogenous BAFF. Instead, our data suggest that accessibility to soluble BAFF is limited within GCs, perhaps to prevent skewing of the conventional B cell differentiation program. In support of this concept, whereas BAFF significantly enhances ISC differentiation in response to T cell-dependent activation, we report for the first time the ability of BAFF to considerably attenuate ISC differentiation of MB cells in response to CpG stimulation, a form of T cell-independent activation. Our data suggest that BAFF may be providing regulatory signals during specific T cell-independent events, which protect the balance between MB cells and ISCs outside GCs. Taken together, these data define a complex role for BAFF in humoral immune responses and show for the first time that BAFF can also play an inhibitory role in B cell differentiation.

Biologic and genetic characterization of the novel amyloidogenic lambda light chain–secreting human cell lines, ALMC-1 and ALMC-2

Primary systemic amyloidosis (AL) is a rare monoclonal plasma cell (PC) disorder characterized by the deposition of misfolded immunoglobulin (Ig) light chains (LC) in vital organs throughout the body. To our knowledge, no cell lines have ever been established from AL patients. Here we describe the establishment of the ALMC-1 and ALMC-2 cell lines from an AL patient. Both cell lines exhibit a PC phenotype and display cytokine-dependent growth. Using a comprehensive genetic approach, we established the genetic relationship between the cell lines and the primary patient cells, and we were also able to identify new genetic changes accompanying tumor progression that may explain the natural history of this patients disease. Importantly, we demonstrate that free lambda LC secreted by both cell lines contained a beta structure and formed amyloid fibrils. Despite absolute Ig LC variable gene sequence identity, the proteins show differences in amyloid formation kinetics that are abolished by the presence of Na(2)SO(4). The formation of amyloid fibrils from these naturally secreting human LC cell lines is unprecedented. Moreover, these cell lines will provide an invaluable tool to better understand AL, from the combined perspectives of amyloidogenic protein structure and amyloid formation, genetics, and cell biology.

Differential human multiple myeloma cell line responsiveness to interferon-alpha. Analysis of transcription factor activation and interleukin 6 receptor expression.

Although IFN-alpha is commonly used as maintenance treatment for multiple myeloma patients, its effectiveness is varied. In this study, we have used a panel of IL-6 responsive myeloma cell lines that vary remarkably in responsiveness to IFN-alpha. Three cell lines were growth arrested by IFN-alpha; however, IFN-alpha significantly stimulated growth of the fourth cell line, KAS-6/1. Our studies have focused on elucidating the mechanism of differential IFN-alpha responsiveness. First, we have shown that IFN-alpha-stimulated growth of the KAS-6/1 cells did not result from induction of autocrine IL-6 expression. Second, analysis of Stats 1, 2, and 3 and IFN regulatory factor-1 (IRF-1) and IRF-2 activation failed to reveal differences between the IFN-alpha growth-arrested or growth-stimulated cells. Third, although IFN-alpha treatment of the IFN-alpha growth-inhibited cell lines reduced IL-6 receptor (IL-6R) expression, IFN-alpha also reduced KAS-6/1 IL-6R expression. Finally, although IFN-alpha treatment reduced IL-6R numbers on each cell line, analysis of Stat protein activation revealed that the receptors were still functional. We conclude that myeloma cell responsiveness to IFN-alpha is heterogeneous and that mechanisms of IFN-alpha-mediated growth inhibition other than IL-6R downregulation must exist in myeloma. Identification of these mechanisms may allow development of agents that are more universally effective than IFN-alpha.

Interleukin 6 induces monocyte chemoattractant protein-1 expression in myeloma cells

Interleukin 6 (IL-6) is known to play an important role in the biology of the malignant plasma cells in multiple myeloma. In an effort to better understand IL-6 stimulated myeloma cell growth, we have performed gene expression profiling to identify IL-6 early response genes. Using the KAS-6/1 IL-6-dependent human myeloma cell line, IL-6 stimulation dramatically induced expression of monocyte chemoattractant protein-1 (MCP-1) mRNA. To verify this result, we used reverse transcriptase PCR and RNAse protection assays and demonstrated using both assays that MCP-1 is indeed an IL-6 responsive gene in a variety of IL-6-responsive myeloma cell lines. Moreover, we also demonstrated IL-6 stimulated MCP-1 secretion by the myeloma cell lines as well as by fresh patient tumor cells. Lastly, we present evidence that fresh patient tumor cells express mRNA for the MCP-1 receptor, CCR2, as do myeloma cell lines along with a second MCP-1 receptor, CCR11. Although MM cell chemotaxis in response to MCP-1 was only minimal, we were able to demonstrate that MCP-1 stimulated activation of MAPK. Because of the important role that this chemokine plays in both angiogenesis and bone homeostasis, and the ability of MCP-1 to activate myeloma cells, these results suggest a new mechanism by which IL-6 may contribute to disease pathogenesis.

CD147 regulates the expression of MCT1 and lactate export in multiple myeloma cells.

Increased use of the glycolytic pathway, even in the presence of oxygen, has recently been recognized as a key characteristic of malignant cells. However, the glycolytic phenotype results in increased lactic acid production and, in order to prevent cellular acidosis, tumor cells must increase proton efflux via upregulation of pH regulators such as proton-pumps, sodium-proton exchangers, and/or monocarboxylate transporters (MCT) (e.g., MCT1, MCT4). Interestingly, expression of MCT1 and MCT4 has been previously shown to be dependent upon expression of the transmembrane glycoprotein CD147. Recently, we demonstrated that primary patient multiple myeloma (MM) cells and human MM cell lines (HMCLs) overexpress CD147. Therefore, the goal of the current study was to specifically determine if MCT1 and MCT4 were also overexpressed in MM cells. RT-PCR analysis demonstrated both primary patient MM cells and HMCLs overexpress MCT1 and MCT4 mRNA. Notably, primary MM cells or HMCLs were found to express variable levels of MCT1 and/or MCT4 at the protein level despite CD147 expression. In those HMCLs positive for MCT1 and/or MCT4 protein expression, MCT1 and/or MCT4 were found to be associated with CD147. Specific siRNA-mediated downregulation of MCT1 but not MCT4 resulted in decreased HMCL proliferation, decreased lactate export, and increased cellular media pH. However, western blot analysis revealed that downregulation of MCT1 also downregulated CD147 and vice versa despite no effect on mRNA levels. Taken together, these data demonstrate the association between MCT1 and CD147 proteins in MM cells and importance of their association for lactate export and proliferation in MM cells.

Induction of Malignant Plasma Cell Proliferation by Eosinophils

The biology of the malignant plasma cells (PCs) in multiple myeloma (MM) is highly influenced by the bone marrow (BM) microenvironment in which they reside. More specifically, BM stromal cells (SCs) are known to interact with MM cells to promote MM cell survival and proliferation. By contrast, it is unclear if innate immune cells within this same space also actively participate in the pathology of MM. Our study shows for the first time that eosinophils (Eos) can contribute to the biology of MM by enhancing the proliferation of some malignant PCs. We first demonstrate that PCs and Eos can be found in close proximity in the BM. In culture, Eos were found to augment MM cell proliferation that is predominantly mediated through a soluble factor(s). Fractionation of cell-free supernatants and neutralization studies demonstrated that this activity is independent of Eos-derived microparticles and a proliferation-inducing ligand (APRIL), respectively. Using a multicellular in vitro system designed to resemble the native MM niche, SCs and Eos were shown to have non-redundant roles in their support of MM cell growth. Whereas SCs induce MM cell proliferation predominantly through the secretion of IL-6, Eos stimulate growth of these malignant cells via an IL-6-independent mechanism. Taken together, our study demonstrates for the first time a role for Eos in the pathology of MM and suggests that therapeutic strategies targeting these cells may be beneficial.

Atypical expression of ErbB3 in myeloma cells: cross-talk between ErbB3 and the interferon-alpha signaling complex.

We have previously demonstrated that the responsiveness of multiple myeloma (MM) cells to interferon-alpha (IFN-α) stimulation is variable, with an atypical growth response displayed by some cells. Here we report the ability of IFN-α to induce tyrosine phosphorylation of a 180 kDa band in the KAS-6/1 MM cell line, which is growth responsive to IFN-α. Further characterization demonstrated that this band corresponds to ErbB3. To our knowledge, this is the first report of ErbB3 expression in a cell type of the hematopoietic lineage. Although ErbB receptors have been shown to crosscommunicate with various other receptors, our results show for the first time that the IFN-α receptor can crosscommunicate with ErbB3. To address the significance of these observations, we transfected ErbB3-negative DP-6 MM cells with ErbB3 and used siRNA to silence ErbB3 in the KAS-6/1 cell line. Although IFN-α transactivated ErbB3 in the DP-6 transfectants, it did not confer growth responsiveness to IFN-α. Interestingly, silencing ErbB3 expression in the KAS-6/1 cells decreased the overall growth response to IFN-α and to interleukin-6. These results suggest that ErbB3 expression alone does not uniquely confer IFN-α growth responsiveness, but instead may amplify proliferation rates in MM cells that have acquired atypical expression of this receptor.

Differentiation of Chronic Lymphocytic Leukemia B Cells into Immunoglobulin Secreting Cells Decreases LEF-1 Expression

Lymphocyte enhancer binding factor 1 (LEF-1) plays a crucial role in B lineage development and is only expressed in B cell precursors as B cell differentiation into mature B and plasma cells silences its expression. Chronic lymphocytic leukemia (CLL) cells aberrantly express LEF-1 and its expression is required for cellular survival. We hypothesized that modification of the differentiation status of CLL cells would result in loss of LEF-1 expression and eliminate the survival advantage provided by its aberrant expression. In this study, we first established a methodology that induces CLL cells to differentiate into immunoglobulin (Ig) secreting cells (ISC) using the TLR9 agonist, CpG, together with cytokines (CpG/c). CpG/c stimulation resulted in dramatic CLL cell phenotypic and morphologic changes, expression of cytoplasmic Ig, and secretion of light chain restricted Ig. CpG/c stimulation also resulted in decreased CLL cell LEF-1 expression and increased Blimp-1 expression, which is crucial for plasma cell differentiation. Further, Wnt pathway activation and cellular survival were impaired in differentiated CLL cells compared to undifferentiated CLL cells. These data support the notion that CLL can differentiate into ISC and that this triggers decreased leukemic cell survival secondary to the down regulation of LEF-1 and decreased Wnt pathway activation.