Chang, Sook Kyung

Abstract Multiple myeloma (MM) is a malignant disease of plasma cells that accumulate in the bone marrow. The survival mechanisms of myeloma cells are still not fully understood. There are several cytokines that are known to support the survival and growth of myeloma cells, such as IL-6, IGF-I, TNF-a, and IL-1β. Recent evidence has also shown that the TNF family member, B lymphocyte stimulator (BLyS), is critical for normal B cell development and homeostasis, as well as for survival of malignant B cells, including MM. However, it should be noted that the precise mechanisms by which BLyS promotes the survival of MM cells remain uncertain. Our group has already reported that MM cell lines and primary myeloma cells can express BLyS and its receptors. Thus, exogenous BLyS augmented the survival of MM cells, including the IL-6 dependent MM cell line, KAS-6/1. To better study the mechanism underlying BLyS-mediated survival of myeloma cells, we sought to establish a KAS-6/1 cell line variant which was solely dependent on exogenous BLyS for growth and survival. We have previously reported that KAS-6/1 cells do not appear to express autocrine IL-6, and therefore are highly dependent on exogenous IL-6 for survival. To establish a BLyS-dependent KAS-6/1 variant, cells were cultured long-term in RPMI containing 0.5% BSA or 1% FCS in the absence of exogenous IL-6, and in the presence or absence of exogenous BLyS. KAS-6/1 cells cultured in the absence of cytokines were non-viable after two weeks of culture, whereas KAS-6/1 cells cultured with BLyS alone remained viable and proliferated, albeit at a lower rate than parental KAS-6/1 cells maintained with IL-6. We next focused on identifying the mechanisms of BLyS-dependent survival of the KAS-6/1/BLyS (KAS-6/B) variant cell line. Of interest, KAS-6/B cells placed in short term culture exhibited cytokine independent survival and proliferation and this was completely inhibited using a neutralizing IL-6 antibody. These results therefore suggested that the mechanism underlying BLyS-mediated survival and proliferation of the KAS-6/B cells is induction of an autocrine IL-6 pathway. This notion is further supported by the ability of BLyS to induce IL-6 expression at the transcriptional level, and at the protein level as revealed by an IL-6 specific ELISA. Consistent with the concept that BLyS induced autocrine IL-6 expression in the KAS-6/B cells, we also observed that STAT-3 was constitutively activated in these cells. In addition, we tested the ability of BLyS-conditioned media obtained from KAS-6/B cells to stimulate the proliferation of a different IL-6 dependent MM cell line, JMW. Whereas BLyS alone failed to stimulate JMW cell proliferation, BLyS-conditioned media obtained from the KAS-6/B cells stimulated proliferation, and in a manner that was inhibited by a neutralizing IL-6 antibody. Finally, BLyS stimulation of primary patient MM cells similarly resulted in increased IL-6 expression. IL-6 is known to play a major role in the malignant progression of MM by regulating the growth and survival of tumor cells. IL-6 levels often increase during disease progression, and it has been demonstrated that IL-6 may derive from the tumor cells themselves or from other cell types in the bone marrow. Because of this, it is important to fully understand what signals are capable of inducing IL-6 expression in this disease. In summary, our results demonstrate for the first time the ability of BLyS to maintain myeloma cell survival via the induction of an autocrine IL-6 pathway. Our results underscore the importance of IL-6 in this disease, and further demonstrate the complexity of the deregulated cytokine network in this disease.

Abstract 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.

Abstract B lymphocyte stimulator (BLyS) is a well-known direct costimulator of adaptive immune cells, particularly B lineage cells. However, we have reported recently that BLyS is also able to activate monocytes. Other innate immune cells, such as dendritic cells (DCs), play a key role in the initiation of adaptive immune responses and the purpose of the current study was to assess whether there is a direct role for BLyS in modulating human DC functions. In this study, we show that BLyS induces DC activation and maturation. Thus, BLyS strongly induced up-regulation of surface costimulatory molecule expression and secretion of specific cytokines and chemokines in DCs. BLyS-stimulated DCs (BLyS-DCs) were also able to augment allogeneic CD4 T cell proliferation to a greater extent than control DCs. BLyS-DCs secreted elevated levels of the major Th1-polarizing cytokine, IL-12p70, and they promoted naive CD4 T cell differentiation into Th1 T cells. Regarding BLyS receptor expression, DCs primarily express cytoplasmic transmembrane activator and CAML interactor; however, low levels of cell surface transmembrane activator and CAML interactor are expressed as well. Collectively, our data suggest that BLyS may modulate adaptive immune cells indirectly by inducing DC maturation.

Abstract The mutagenic enzyme activation-induced cytidine deaminase (AID) is required for immunoglobulin class switch recombination (CSR) and somatic hypermutation (SHM) in germinal center (GC) B cells. Deregulated expression of AID is associated with various B-cell malignancies and, currently, it remains unclear how AID activity is extinguished to avoid illegitimate mutations. AID has also been shown to be alternatively spliced in malignant B cells, and there is limited evidence that this also occurs in normal blood B cells. The functional significance of these splice variants remains unknown. Here we show that normal GC human B cells and blood memory B cells similarly express AID splice variants and show for the first time that AID splicing variants are singly expressed in individual normal B cells as well as malignant B cells from chronic lymphocytic leukemia patients. We further demonstrate that the alternative AID splice variants display different activities ranging from inactivation of CSR to inactivation or heightened SHM activity. Our data therefore suggest that CSR and SHM are differentially switched off by varying the expression of splicing products of AID at the individual cell level. Most importantly, our findings suggest a novel tumor suppression mechanism by which unnecessary AID mutagenic activities are promptly contained for GC B cells.

AbstractB-lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor (TNF) ligand superfamily. Although BLyS costimulates adaptive immune cells, the ability of BLyS to stimulate innate immune cells has not been described. Here, we show that BLyS strongly induces human monocyte survival, and activation as measured by proinflammatory cytokine secretion and up-regulation of costimulatory molecule expression. In addition, monocytes cultured with BLyS differentiated into macrophage-like cells. Regarding BLyS receptor(s) expression, freshly isolated monocytes bound low levels of exogenous BLyS and expressed primarily intracellular TACI, and cell surface TACI levels increased following monocyte activation. Of interest, bone marrow monocytes from some multiple myeloma patients expressed significant levels of cell surface TACI at isolation. Our findings indicate that BLyS plays a role in activating innate immune cells. Moreover, this study may explain more clearly why high BLyS production is often correlated with certain inflammatory autoimmune diseases and B-lymphocyte malignancies.