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B-cell maturation antigen (BCMA) is a promising therapeutic target for multiple myeloma (MM), but expression is variable, and early reports of BCMA targeting chimeric antigen receptors (CARs) suggest antigen downregulation at relapse. Dual-antigen targeting increases targetable tumor antigens and reduces the risk of antigen-negative disease escape. "A proliferation-inducing ligand" (APRIL) is a natural high-affinity ligand for BCMA and transmembrane activator and calcium-modulator and cyclophilin ligand (TACI). We quantified surface tumor expression of BCMA and TACI on primary MM cells (n = 50). All cases tested expressed BCMA, and 39 (78%) of them also expressed TACI. We engineered a third-generation APRIL-based CAR (ACAR), which killed targets expressing either BCMA or TACI (P < .01 and P < .05, respectively, cf. control, effector-to-target [E:T] ratio 16:1). We confirmed cytolysis at antigen levels similar to those on primary MM, at low E:T ratios (56.2% ± 3.9% killing of MM.1s at 48 h, E:T ratio 1:32; P < .01) and of primary MM cells (72.9% ± 12.2% killing at 3 days, E:T ratio 1:1; P < .05, n = 5). Demonstrating tumor control in the absence of BCMA, we maintained cytolysis of primary tumor expressing both BCMA and TACI in the presence of a BCMA-targeting antibody. Furthermore, using an intramedullary myeloma model, ACAR T cells caused regression of an established tumor within 2 days. Finally, in an in vivo model of tumor escape, there was complete ACAR-mediated tumor clearance of BCMA+TACI- and BCMA-TACI+ cells, and a single-chain variable fragment CAR targeting BCMA alone resulted in outgrowth of a BCMA-negative tumor. These results support the clinical potential of this approach.

Deficiencies in transmembrane activator and CAML interactor (TACI) result in common variable immune deficiency, a syndrome marked by recurrent infections with encapsulated microorganisms, impaired production of antibodies, and lymphoproliferation. How TACI promotes antibody production and inhibits lymphoproliferation is not understood. To answer this question, we studied the generation of immunity to protein antigens in both TACI-deficient and TACI-proficient mice. We show that TACI promotes sustained Blimp-1 expression by B cells responding to antigen, which in turn limits B-cell clonal expansion and facilitates differentiation of long-lived antibody-secreting cells. Short-term IgG secretion occurs independently of TACI as DNA double-strand breaks associated with isotype class switching induce Blimp-1 transiently, independently of TACI. Our results showing that TACI induces and maintains Blimp-1 provide, for the first time, a unified molecular and cellular mechanism explaining the primary features of common variable immune deficiency, exquisite vulnerability to infection with encapsulated organisms, lymphoproliferation, and hypogammaglobulinemia.

Complement receptor 2–negative (CR2/CD21−) B cells have been found enriched in patients with autoimmune diseases and in common variable immunodeficiency (CVID) patients who are prone to autoimmunity. However, the physiology of CD21−/lo B cells remains poorly characterized. We found that some rheumatoid arthritis (RA) patients also display an increased frequency of CD21−/lo B cells in their blood. A majority of CD21−/lo B cells from RA and CVID patients expressed germline autoreactive antibodies, which recognized nuclear and cytoplasmic structures. In addition, these B cells were unable to induce calcium flux, become activated, or proliferate in response to B-cell receptor and/or CD40 triggering, suggesting that these autoreactive B cells may be anergic. Moreover, gene array analyses of CD21−/lo B cells revealed molecules specifically expressed in these B cells and that are likely to induce their unresponsive stage. Thus, CD21−/lo B cells contain mostly autoreactive unresponsive clones, which express a specific set of molecules that may represent new biomarkers to identify anergic B cells in humans.

A proliferation-inducing ligand (APRIL), as well as its receptors transmembrane activator and calcium-modulating cyclophilin ligand (CAML) interactor (TACI) and B-cell maturation antigen (BCMA), has been shown to be important in B-cell biology, and overexpression of APRIL in mice results in development of lymphoma. Limited data are available on APRIL-specific signaling responses, but knockout models suggest that signaling through TACI is critical to B-cell homeostasis. To better understand the mechanism by which APRIL exerts its effects and how it may contribute to lymphomagenesis, we sought to characterize the outcome of APRIL-TACI interactions. In support of murine studies, we find that APRIL induces proliferation of human patient follicular lymphoma (FL) B cells in a TACI-dependent manner. This study also shows that APRIL is expressed within the tumor microenvironment and that, upon engagement with TACI, APRIL mediates activation of the phosphatidylinositol 3-kinase (PI3K) pathway. Activation of PI3K via APRIL results in phosphorylation of Akt and mammalian target of rapamycin (mTOR) and the mTOR-specific substrates p70S6 kinase and 4E-binding protein 1 in a TACI-dependent manner. APRIL-mediated signaling also results in phosphorylation of Rb and up-regulation of cyclin D1. These studies are the first to characterize APRIL-TACI–specific signaling and suggest a role for this ligand-receptor pair in FL B-cell growth.

Subjects with common variable immune deficiency may have mutations in transmembrane activator calcium modulator and cyclophilin ligand interactor (TACI). Unlike the murine gene, human TACI undergoes alternative messenger (m)RNA splicing to produce isoforms with 1 or 2 ligand-binding domains. Because both isoforms are found in human B cells, we compared their functions in transduced murine B and human pre-B cells. Although murine cells and pre-B cells transduced with the long TACI isoform retained surface CD19 and immunoglobulin G, cells transduced with the short TACI isoform completely lost these B-cell characteristics. Expression of the short TACI isoform produced intense nuclear factor κB activation, nuclear p65 translocation, and colocalization with myeloid differentiation factor 88 and calcium-modulating cyclophilin ligand. The short TACI-transduced cells became larger and CD138 positive, demonstrated upregulated BLIMP1 and XBP1 mRNA, and acquired the morphology of plasma cells. In contrast, cells bearing the long isoform had significantly less BLIMP1 and XBP1 mRNA and, for human pre-B cells, remained CD138 negative. Although human B cells express both isoforms, the short isoform predominates in CD27(+) B cells, toll-like receptor 9-activated peripheral B cells, and splenic marginal zone B cells. Although the transcriptional controls for alternative splicing of isoforms remain unknown, differential signals via isoforms may control plasma-cell generation in humans.

B-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.

Chronic lymphocytic leukemia (CLL) B cells express BR3, the specific receptor for the B cell–activating factor of tumor necrosis factor family (BAFF). CLL cells also express 2 other receptors for BAFF, namely B-cell maturation antigen (BCMA) and the transmembrane activator and calcium modulator and cyclophilin ligand-interactor (TACI), which also bind a proliferation-inducing ligand (APRIL). We found that signaling through BR3, but not BCMA or TACI, activated the alternative nuclear factor of κ B (NF-κB) pathway in CLL cells, whereas signaling through BCMA/TACI induced activation of the canonical NF-κB pathway. Blocking BR3 did not inhibit the capacity of BAFF to support CLL cell survival in vitro. On the other hand, specifically blocking the canonical NF-κB pathway with UTC, an inhibitor of IκB kinase β (IKKβ), or transfection of CLL cells with the IκBα super-repressor, blocked the capacity of BAFF and APRIL to promote CLL cell survival in vitro. This contrasts what is found with normal blood B cells, which apparently depend on activation of the alternative NF-κB pathway for BAFF-enhanced survival. These findings suggest that inhibitors of protein kinase IKKβ, which is required for activation of the canonical NF-κB pathway, might have a therapeutic role in this disease.

Hodgkin lymphoma (HL) originates from the clonal expansion of malignant Hodgkin and Reed-Sternberg (HRS) cells. These B-cell–derived elements constitute less than 10% of the tumoral mass. The remaining tissue is comprised of an inflammatory infiltrate that includes myeloid cells. Myeloid cells activate B cells by producing BAFF and APRIL, which engage TACI, BCMA, and BAFF-R receptors on the B cells. Here, we studied the role of BAFF and APRIL in HL. Inflammatory and HRS cells from HL tumors expressed BAFF and APRIL. Unlike their putative germinal center B-cell precursors, HRS cells lacked BAFF-R, but expressed TACI and BCMA, a phenotype similar to that of plasmacytoid B cells. BAFF and APRIL enhanced HRS cell survival and proliferation by delivering nonredundant signals via TACI and BCMA receptors through both autocrine and paracrine pathways. These signals caused NF-κB activation; Bcl-2, Bcl-xL, and c-Myc up-regulation; and Bax down-regulation, and were amplified by APRIL-binding proteoglycans on HRS cells. Interruption of BAFF and APRIL signaling by TACI-Ig decoy receptor, which binds to and neutralizes BAFF and APRIL, or by small-interfering RNAs targeting BAFF, APRIL, TACI, and BCMA inhibited HRS cell accumulation in vitro and might attenuate HL expansion in vivo.

A PRoliferation-Inducing TNF Ligand (APRIL) costimulates B-cell activation. When overexpressed in mice, APRIL induces B-cell neoplasia, reminiscent of human B-cell chronic lymphoid leukemia (B-CLL). We analyzed APRIL expression in situ in human non-Hodgkin lymphomas. APRIL up-regulation was only observed in high-grade B-cell lymphomas, diffuse large B-cell lymphoma (DLBCL), and Burkitt lymphoma (BL). Up-regulation was seen in 46% and 20% of DLBCL and BL, respectively. In DLBCL, neutrophils, constitutively producing APRIL and infiltrating the tumor tissue, were the main cellular source of APRIL. Rare DLBCL cases showed a predominance of histiocytes or mesenchymal cells as APRIL source. APRIL secreted by neutrophils accumulated on tumor cells via proteoglycan binding. In addition to proteoglycans, DLBCL tumor cells expressed the APRIL signaling receptor, TACI and/or BCMA, indicating that these tumor cells are fully equipped to respond to APRIL. A retrospective clinical analysis revealed a significant correlation between high expression of APRIL in tumor lesions and decreased overall patient survival rate. Hence, APRIL produced by inflammatory cells infiltrating lymphoma lesions may increase tumor aggressiveness and affect disease outcome.

B-lymphocyte homeostasis and function are regulated by complementary actions of the TNFR family members TACI, BCMA, and BAFF-R, which are expressed by mature B cells. How these receptors are differentially activated is not entirely understood, because the primary ligand BAFF binds to all three. We searched for alternative ligands for TACI using recombinant TACI-Fc fusion protein as a probe and identified syndecan-2 as a new binding partner. TACI binding appears to require heparan sulfate posttranslational modifications of syndecan-2, because free heparin or pretreatment with heparitinase blocked the interaction. Syndecan-2 bound TACI but bound neither BAFF-R nor BCMA. Transfected cells expressing syndecan-2 activated signaling through TACI, as indicated by an NFAT-specific reporter. Syndecan-1 and syndecan-4 were also able to induce TACI signaling in a similar manner. This is the first identification of ligands that selectively activate TACI without simultaneously triggering BCMA or BAFF-R. This finding may help explain the alternative outcomes of signaling from this family of receptors in B cells.

B-cell chronic lymphocytic leukemia (CLL) is the most common human leukemia. 13q14 deletions are most common chromosomal alterations in CLL. We previously reported that miR-15/16 is a target of 13q14 deletions and plays a tumor suppressor role by targeting BCL2. Because DLEU7 is located near miR-15/16 and is also positioned within a minimal deleted region, we investigated whether DLEU7 could also play a tumor suppressor role. Recent studies of transgenic mouse models demonstrated the importance of the nuclear factor-κB (NF-κB) pathway in CLL. To examine the possible role of DLEU7 in CLL, we investigated the effect of DLEU7 expression on NF-κB and nuclear factor of activated T cells (NFAT) activity. We found that DLEU7 functions as a potent NF-κB and NFAT inhibitor by physically interacting and inhibiting TACI and BCMA, members of the tumor necrosis factor (TNF) receptor family involved in B-CLL. In addition, DLEU7 expression in A549 lung cancer cells resulted in a decrease in S phase and increased apoptosis. The results suggest that loss of DLEU7 may cooperate with the loss of miR-15/16 in the pathogenesis of CLL.

TNFRSF13B, which encodes TACI (transmembrane activator and calcium-modulator and cyclophilin ligand interactor), is mutated in 10% of patients with common variable immune deficiency (CVID). One of the 2 most common TACI mutations in CVID, A181E, introduces a negative charge into the transmembrane domain. To define the consequence of the A181E mutation on TACI function, we studied the effect of its murine equivalent, mTACI A144E, on TACI signaling in transfected cells and on TACI function in transgenic mice. The mTACI A144E mutant, like its human TACI A181E counterpart, was expressed on the surface of 293T transfectants and was able to bind ligand, but exhibited impaired constitutive and ligand-induced NFκB signaling. In addition, constitutive and ligand-induced clustering of the intracellular domain was deficient for A144E as measured by fluorescence resonance energy transfer. Transgenic mice expressing the A144E mutant on TACI−/− background had low serum IgA levels and significantly impaired antibody responses to the type II T-independent antigen TNP-Ficoll. B cells from A144E transgenic mice were impaired in their capacity to proliferate and secrete IgG1 and IgA in response to TACI ligation. These results suggest that mTACI A144E mutation and its human counterpart, A181E, disrupt TACI signaling and impair TACI-dependent B-cell functions.