Darce, Jaime

Abstract One of the major mechanisms by which the immune system combats unpredictable foreign pathogens is to produce highly specific and effective antibodies by B cells. Somatic hypermutation (SHM) and class switch recombination (CSR) are two key events required for the production of highly specific and effective antibodies. Both SHM and CSR occur in the germinal center (GC), and are mediated by a common enzyme, activation-induced cytidine deaminase (AID) that specifically mutates the variable regions (IgV) and switch regions (IgS) of immunoglobulin genes by converting cytidines to uridines. It has been shown that in addition to the central deaminase domain, an N-terminal domain is essential to target SHM to the IgV region whereas a C-terminal domain is necessary for Ig CSR. Given its mutagenic nature, it has been postulated that AID may play causative roles in B cell lymphomagenesis. Consistent with this notion, AID is expressed and alternatively spliced in B cell tumors such as B-cell chronic lymphocytic leukemia (B-CLL) and non-Hodgkin’s lymphoma (NHL). However, it remains unclear whether post-GC malignant B cell AID expression and alternative splicing is a consequence of B cell transformation and/or is required to maintain growth and/or malignant B-cell survival, or whether post-GC AID expression and alternative splicing may also occur in normal B cells. To discriminate between these two possibilities, we analyzed AID expression and splicing in normal human B cells. Our data demonstrate that human tonsillar B cells prominently express the full-length AID transcript, and of interest, also express all four alternative transcripts that have been previously described in B-CLL and NHL B cells. To determine if all normal B cells express all of the AID transcripts, we performed limiting dilution of tonsillar B cells and used RT-PCR to detect AID expression. We surprisingly discovered that AID splicing variants are singly expressed by individual B-cells. To address the functional significance of each splicing variant, we considered the possibility that B cells expressing the alternative transcripts may be by-products of the normal GC reaction and are eliminated prior to GC exit. Conversely those B cells may contribute to the memory B cell pool if those AID variants are functional. Therefore, we examined AID expression and splicing in memory B cells using a more sensitive nested PCR approach. Our data show that AID is indeed expressed in peripheral blood memory cells but not in naïve B cells. Moreover, at least one splicing variant was expressed at levels comparable to the full-length AID transcript. Using limiting dilution, we again found that the full-length and alternatively spliced AID transcripts are singly expressed by individual memory B cells. Our results suggest that AID expression beyond the GC stage may play a role in sustaining selection pressure. Since the AID domains specific for SHM and CSR are encoded by different exons, we hypothesize that those variants may function differentially in SHM and CSR and this concept is under current study. Since each B cell only expressed one AID transcript, our data strongly suggest that SHM and CSR are coordinated in a stepwise process by generation of alternative AID splice variants that exhibit selective activity thereby allowing fine-tuning of humoral immunity.

Abstract B cell-activating factor (BAFF) is known to regulate normal B cell development and homeostasis primarily by signaling through the high affinity receptor, BAFF-R, one of three BAFF binding receptors (BBRs). BAFF also binds two other receptors, BCMA and TACI with lesser affinity. We have recently shown that normal peripheral blood (PB) B cells express high levels of prebound soluble BAFF, which is lost upon B cell activation. Because of BAFF’s activity on normal B cells, we have been interested in the roles of BAFF and BBRs in B cell chronic lymphocytic leukemia (B-CLL). We and others have demonstrated that BAFF promotes primary CLL B cell survival and that serum BAFF levels are elevated in some patients. Although CLL B cells are known to express BBRs, a comprehensive and quantitative analysis of BBR levels and CLL B cell capacity to bind BAFF has not yet been done. We began this study by characterizing the level of soluble BAFF bound to freshly isolated CLL B cells, measured by both western blot analysis and flow cytometry. To assess receptor occupancy, cells were incubated with or without exogenous BAFF before assessing anti-BAFF reactivity and changes in median fluorescence intensity (ΔMFI; defined by dividing the MFI of the anti-BAFF antibody by the MFI of the isotype matched control antibody) were calculated. Normal B cells have higher detectable levels of bound BAFF with a ΔMFI ranging from 16 to 35 (mean=22.2). Upon addition of exogenous BAFF, the ΔMFI range increased to 27–96.6 (mean=49.1; n=8). Thus, despite evidence of prebound BAFF, clearly not all BBRs were occupied on normal PB B cells. By contrast, the levels of prebound BAFF on CLL B cells were significantly lower with a ΔMFI ranging from 1 to 13.1 (mean=2.7; n=36). Of note, 10/36 patients did not exhibit increased anti-BAFF reactivity upon incubation with exogenous BAFF (mean fold induction=0.8) whereas 26/36 patients displayed a mean fold induction of anti-BAFF reactivity of 3.5. These observations prompted us to next quantitate CLL B cell BBR expression. All patient CLL B cells expressed BAFF-R but at significantly lower levels than observed in normal B cells (p=0.0009). When CLL patients were categorized into IGHV mutated (M; n=22) and unmutated (UM; n=24), UM patients were observed to express higher levels of BAFF-R (ΔMFI =8.9) than M patients (ΔMFI =5.24). Regarding TACI, we previously demonstrated that normal memory B cells uniformly express TACI (ΔMFI =12.7; n=10) and there is a small population of activated naïve B cells that express TACI at lower levels (ΔMFI =8.3; n=10). In our CLL cohort, 14/22 M patients were TACI+ (ΔMFI =7.0) and 19/24 UM patients were TACI+ (ΔMFI =4.7). Finally, whereas normal PB B cells completely lack BCMA expression, 7/22 M and 4/22 UM patients expressed BCMA. Thus, using the BBR profile and analysis of expression levels relative to normal PB B cells, the following subgroups of B-CLL can be defined: BAFF-R+; BAFF-R/TACI+; BAFF-R/BCMA+; BAFF-R/TACI/BCMA+. It remains to be determined if these BBR profiles correlate with aspects of clinical disease. In addition, given the putative importance of BAFF in this disease, it is interesting to note that in general, CLL B cells display overall lower levels of prebound BAFF. Current studies are focused on determining whether this reflects CLL B cell activation status, increased competition for BAFF, and/or reduced levels of BBR expression.

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 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.

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.

Abstract 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 undefined. Because of the knowledge that B-lymphocyte stimulator (BLyS), a tumor necrosis factor (TNF) family member shown to be critical for maintenance of normal B-cell development and homeostasis, promotes the survival of malignant B cells, we began a coordinated study of BLyS and its receptors in MM. All MM cells studied expressed one or more of 3 known receptors (B-cell maturation antigen [BCMA], transmembrane activator and CAML interactor [TACI], and B-cell activating factor receptor [BAFF-R]) for BLyS; however, the pattern of expression was variable. Additionally, we provide evidence that BLyS can modulate the proliferative capacity and survival of MM cells. Finally, we provide evidence that BLyS is expressed by MM cells and is present in the bone marrow of patients with MM. Expression of BCMA, TACI, and BAFF-R by MM taken together with the ability of BLyS to support MM cell growth and survival has exciting implications because they may be potential therapeutic targets.