Ocular surface complications of local anticancer drugs for treatment of ocular tumors.

Local chemotherapy is increasingly used, either in combination with surgery or as monotherapy, for management of ocular tumors. Yet many of the local chemotherapeutic agents used for ocular tumors are cytotoxic drugs that are frequently associated with toxicities in normal ocular tissues. Understanding and managing these side effects are important because they affect treatment tolerability, outcome and quality of vision. Herein, we review local anticancer drugs administered for the treatment of ocular tumors, with an emphasis on their toxicities to the ocular surface, adnexa and lacrimal drainage system. We provide the underlying mechanisms and management strategies for the ocular side effects. Recent innovations in anticancer immunotherapy and ocular drug delivery systems also are discussed as new potential therapeutic modalities for alleviation of side effects.

Myeloid-Derived Suppressor Cells Mediate Inflammation Resolution in Humans and Mice with Autoimmune Uveoretinitis.

Resolution of inflammation is an active process that leads to tissue homeostasis and involves multiple cellular and molecular mechanisms. Myeloid-derived suppressor cells (MDSCs) have recently emerged as important cellular components in the resolution of inflammation because of their activities to suppress T cell activation. In this article, we show that HLA-DR-CD11b+CD33+CD14+ human MDSCs and CD11b+Ly6G-Ly6C+ mouse MDSCs markedly increased in patients and mice during and before the resolution phase of autoimmune uveoretinitis. CD11b+Ly6C+ monocytes isolated from autoimmune uveoretinitis mice were able to suppress T cell proliferation in culture, and adoptive transfer of the cells accelerated the remission of autoimmune uveoretinitis in mice. Alternatively, depletion of CD11b+Ly6C+ monocytes at the resolution phase, but not CD11b+Ly6G+ granulocytes, exacerbated the disease. These findings collectively indicate that monocytic MDSCs serve as regulatory cells mediating the resolution of autoimmune uveoretinitis.

Mesenchymal stromal cells promote B-cell lymphoma in lacrimal glands by inducing immunosuppressive microenvironment.

Mesenchymal stromal cells (MSCs) have therapeutic potential for various diseases because of their anti-inflammatory and immunosuppressive properties. However, the immunosuppressive microenvironment allows tumor cells to evade immune surveillance, whereas maintenance of inflammation is required for tumor development and progression. Hence, MSCs may promote or suppress tumors in a context-dependent manner. We here investigated the effects of bone marrow-derived MSCs in a murine model of lacrimal gland B-cell lymphoma. Co-injection of MSCs with B lymphoma cells enhanced tumor growth in lacrimal glands without long-term engraftment. Of note, MSCs induced greater infiltration of immune and immune-regulatory cells near tumor: CD4+ cells, CD11b+ cells, CD4+Foxp3+ regulatory T cells and CD11b+Ly6C+Ly6G- myeloid-derived suppressor cells. Concurrently, there was up-regulation of immune-related molecules including TNF-α, IL-1ß, TGF-ß1, and arginase in glands treated with MSCs. Apoptosis in tumor was less severe in mice treated with MSCs compared to those without MSCs; however, MSCs did not directly inhibit apoptosis of B lymphoma cells in an in vitro co-culture. Together, data demonstrate that MSCs create immunosuppressive milieu by recruiting regulatory immune cells and promote B-cell lymphoma growth in lacrimal glands.

Rapamycin regulates macrophage activation by inhibiting NLRP3 inflammasome-p38 MAPK-NFκB pathways in autophagy- and p62-dependent manners.

Excessive and prolonged activation of macrophages underlies many inflammatory and autoimmune diseases. To regulate activation and maintain homeostasis, macrophages have multiple intrinsic mechanisms, one of which is modulation through autophagy. Here we demonstrate that autophagy induction by rapamycin suppressed the production of IL-1ß and IL-18 in lipopolysaccharide- and adenosine triphosphate-activated macrophages at the post-transcriptional level by eliminating mitochondrial ROS (mtROS) and pro-IL1ß in a p62/SQSTM1-dependent manner. In addition, rapamycin activated Nrf2 through up-regulation of p62/SQSTM1, which further contributed to the reduction of mtROS. Reduced IL-1ß subsequently diminished the activation of p38 MAPK-NFκB pathways, leading to transcriptional down-regulation of IL-6, IL-8, MCP-1, and IκBα in rapamycin-treated macrophages. Therefore, our results suggest that rapamycin negatively regulates macrophage activation by restricting a feedback loop of NLRP3 inflammasome-p38 MAPK-NFκB pathways in autophagy- and p62/SQSTM1-dependent manners.

BMP-7 induces apoptosis in human germinal center B cells and is influenced by TGF-ß receptor type I ALK5.

Selection and maturation of B cells into plasma cells producing high-affinity antibodies occur in germinal centers (GC). GCs form transiently in secondary lymphoid organs upon antigen challenge, and the GC reaction is a highly regulated process. TGF-ß is a potent negative regulator, but the influence of other family members including bone morphogenetic proteins (BMPs) is less known. Studies of human peripheral blood B lymphocytes showed that BMP-6 suppressed plasmablast differentiation, whereas BMP-7 induced apoptosis. Here, we show that human naïve and GC B cells had a strikingly different receptor expression pattern. GC B cells expressed high levels of BMP type I receptor but low levels of type II receptors, whereas naïve B cells had the opposite pattern. Furthermore, GC B cells had elevated levels of downstream signaling components SMAD1 and SMAD5, but reduced levels of the inhibitory SMAD7. Functional assays of GC B cells revealed that BMP-7 suppressed the viability-promoting effect of CD40L and IL-21, but had no effect on CD40L- and IL-21-induced differentiation into plasmablasts. BMP-7-induced apoptosis was counteracted by a selective TGF-ß type I receptor (ALK4/5/7) inhibitor, but not by a selective BMP receptor type I inhibitor. Furthermore, overexpression of truncated ALK5 in a B-cell line counteracted BMP-7-induced apoptosis, whereas overexpression of truncated ALK4 had no effect. BMP-7 mRNA and protein was readily detected in tonsillar B cells, indicating a physiological relevance of the study. Altogether, we identified BMP-7 as a negative regulator of GC B-cell survival. The effect was counteracted by truncated ALK5, suggesting greater complexity in regulating BMP-7 signaling than previously believed.

Tumor associated macrophages provide the survival resistance of tumor cells to hypoxic microenvironmental condition through IL-6 receptor-mediated signals.

Hypoxia and infiltration of tumor-associated macrophages (TAM) are intrinsic features of the tumor microenvironment. Tumor cells that remain viable in hypoxic conditions often possess an increased survival potential and tend to grow aggressively. TAM also respond to a variety of signals in the hypoxic tumor microenvironment and express a more M2-like phenotype. In this study, the established mouse tumor tissues showed a dense infiltration of CD206+ macrophages at the junctions between the normoxic and hypoxic regions and an increased IL-6 receptor (IL-6R) expression of tumor cells in the areas of CD206+ TAM accumulation, which indicates a role of M2 phenotype TAM in survival adaptation of tumor cells preparing for an impending hypoxic injury before changes in oxygen availability. Cocultured mouse FM3A or human MCF-7 tumor cells with tumor infiltrating macrophages isolated from mouse tumor tissues and M2-polarized macrophages generated from human THP-1 cells, respectively, showed significantly decreased rate of cell death in cultures exposed to hypoxia. The acquisition of survival resistance was attributed to increased IL-6 production by M2 TAM and increased expression of IL-6R in tumor cells in the coculture system. MCF-7 cells cocultured with M2 TAM showed activated JAK1/STAT3 and Raf/MEK/JNK pathways contributing to tyrosine and serine phophorylation of STAT3, respectively. However, only tyrosine phosphorylated STAT3 was detected in the nucleus, which induced upregulation of Bcl-2 and downregulation of Bax and Bak. Finally, knockdown of IL-6R by small interfering RNA significantly counteracted coculture-induced signals and completely abolished the survival resistance to hypoxic injury. Thus, we present evidence for the role of M2 phenotype TAM in IL-6 receptor-mediated signals, particularly tyrosine phosphorylation of STAT3, responsible for the prosurvival adaptation of tumor cells to hypoxia.

Mesenchymal stem/stromal cells precondition lung monocytes/macrophages to produce tolerance against allo- and autoimmunity in the eye.

Intravenously administered mesenchymal stem/stromal cells (MSCs) engraft only transiently in recipients, but confer long-term therapeutic benefits in patients with immune disorders. This suggests that MSCs induce immune tolerance by long-lasting effects on the recipient immune regulatory system. Here, we demonstrate that i.v. infusion of MSCs preconditioned lung monocytes/macrophages toward an immune regulatory phenotype in a TNF-α-stimulated gene/protein (TSG)-6-dependent manner. As a result, mice were protected against subsequent immune challenge in two models of allo- and autoimmune ocular inflammation: corneal allotransplantation and experimental autoimmune uveitis (EAU). The monocytes/macrophages primed by MSCs expressed high levels of MHC class II, B220, CD11b, and IL-10, and exhibited T-cell-suppressive activities independently of FoxP3(+) regulatory T cells. Adoptive transfer of MSC-induced B220(+)CD11b(+) monocytes/macrophages prevented corneal allograft rejection and EAU. Deletion of monocytes/macrophages abrogated the MSC-induced tolerance. However, MSCs with TSG-6 knockdown did not induce MHC II(+)B220(+)CD11b(+) cells, and failed to attenuate EAU. Therefore, the results demonstrate a mechanism of the MSC-mediated immune modulation through induction of innate immune tolerance that involves monocytes/macrophages.

Mesenchymal stem/stromal cells protect against autoimmunity via CCL2-dependent recruitment of myeloid-derived suppressor cells.

Exogenously administered mesenchymal stem/stromal cells (MSCs) suppress autoimmunity despite transient engraftment. However, the mechanism is unclear. In this study, we report a novel mechanism by which MSCs modulate the immune system by recruiting myeloid-derived suppressor cells in a mouse model of experimental autoimmune uveitis (EAU). Intravenous infusion of MSCs blocked EAU development and reduced Th1 and Th17 responses. Time course analysis revealed an increase of MHC class II(lo)Ly6G(-)Ly6C(hi)CD11b(+) cells in draining lymph nodes by MSCs. These Ly6C(hi)CD11b(+) cells suppressed CD4(+) cell proliferation and Th1/Th17 differentiation and induced CD4(+) cell apoptosis. Adoptive transfer of Ly6C(hi)CD11b(+) cells ameliorated EAU, whereas depletion of Ly6C(hi)CD11b(+) cells abrogated the effects of MSCs. 1.8% of MSCs were present in draining lymph nodes 1 d after infusion, and MSCs with CCL2 knockdown did not increase MHC class II(lo)Ly6G(-)Ly6C(hi)CD11b(+) cells and failed to attenuate EAU. Therefore, our findings demonstrate that MSCs suppress autoimmunity by recruiting myeloid-derived suppressor cells into sites of inflammation in a CCL2-dependent manner.

Gamma secretase inhibitors enhance vincristine-induced apoptosis in T-ALL in a NOTCH-independent manner.

Activating mutations in the NOTCH1 gene are found in over 50 % of T-ALL cases. Since Notch signaling contributes to the leukemia cell survival and growth, targeting Notch signaling using γ-secretase inhibitors (GSI) has been proposed as a molecularly targeted therapy for the treatment of T-ALL. However, not all T-ALL with NOTCH1 activating mutations respond to GSI treatment. We examined whether GSI could enhance the cytotoxic effect of anti-leukemic agents in the GSI-resistant T-ALL cells although GSI does not have anti-tumor effect as a single agent. GSI significantly increased cell death induced by Vincristine (VCR) but not other anti-leukemic drugs (Methotrexate, Asparaginase, and Cytarabine). The GSI effect in enhancing VCR efficacy was not the result of inhibition of Notch signaling. GSI augmented VCR-induced mitotic arrest, followed by apoptosis. GSI accelerated VCR-triggered loss of mitochondrial membrane potential and caspase-mediated apoptosis. Our finding suggests that GSI has other functions besides inhibiting Notch signaling in T-ALL and incorporating GSI into the conventional regimen containing VCR may offer therapeutic advantage by potentiating VCR treatment in leukemia patients.

CD9 may contribute to the survival of human germinal center B cells by facilitating the interaction with follicular dendritic cells.

The germinal center (GC) is a dynamic microenvironment where antigen (Ag)-activated B cells rapidly expand and differentiate, generating plasma cells (PC) that produce high-affinity antibodies. Precise regulation of survival and proliferation of Ag-activated B cells within the GC is crucial for humoral immune responses. The follicular dendritic cells (FDC) are the specialized stromal cells in the GC that prevent apoptosis of GC-B cells. Recently, we reported that human GC-B cells consist of CD9+ and CD9- populations and that it is the CD9+ cells that are committed to the PC lineage. In this study, we investigated the functional role of CD9 on GC-B cells. Tonsillar tissue section staining revealed that in vivo CD9+ GC-B cells localized in the light zone FDC area. Consistent this, in vitro CD9+ GC-B cells survived better than CD9- GC-B cells in the presence of HK cells, an FDC line, in a cell-cell contact-dependent manner. The frozen tonsillar tissue section binding assay showed that CD9+ GC-B cells bound to the GC area of tonsillar tissues significantly more than the CD9- GC-B cells did and that the binding was significantly inhibited by neutralizing anti-integrin ß1 antibody. Furthermore, CD9+ cells bound to soluble VCAM-1 more than CD9- cells did, resulting in activation and stabilization of the active epitope of integrin ß1. All together, our data suggest that CD9 on GC-B cells contributes to survival by strengthening their binding to FDC through the VLA4/VCAM-1 axis.

GSI promotes vincristine-induced apoptosis by enhancing multi-polar spindle formation.

Gamma secretase inhibitors (GSI), cell-permeable small-molecule inhibitors of gamma secretase activity, had been originally developed for the treatment of Alzheimer disease. In recent years, it has been exploited in cancer research to inhibit Notch signaling that is aberrantly activated in various cancers. We previously found that GSI could synergize with anti-microtubule agent, vincristine (VCR) in a Notch-independent manner. Here, we delineate the underlying cell cycle-related mechanism using HeLa cells, which have strong mitotic checkpoints. GSI enhanced VCR-induced cell death, although GSI alone did not affect cell viability at all. GSI augmented VCR-induced mitotic arrest in a dose-dependent manner, which was preceded by apoptotic cell death, as shown by an increase in Annexin V-positive and caspase-positive cell population. Furthermore, GSI amplified multi-polar spindle formation triggered by VCR. Altogether, we show the evidence that GSI enhances VCR-induced apoptosis in HeLa cells via multi-polar mitotic spindle formation, independent of Notch signaling. These data suggest that one or more GS substrates, yet to be identified, in a post-GS processed form, may play a role in maintaining functional centrosomes/mitotic spindles. More significantly, the synergistic effect of GSI in combination with VCR could be exploited in clinical setting to improve the efficacy of VCR.

CD9 is a novel marker for plasma cell precursors in human germinal centers.

The germinal center (GC) is the dynamic microenvironment where Ag-activated B cells rapidly expand and differentiate, generating plasma cells (PC) that produce high affinity antibodies. B cells within the GC have great heterogeneity, containing B cells at different stages of activation and differentiation. However, there are few surface markers that allow subsets of GC-B cells to be distinguished. In the present study, we show that GC-B cells in human tonsils contain two distinct populations regarding CD9 expression; CD9- and CD9+ cells. CD9+ GC-B cells are functionally more differentiated towards PC based upon the following evidence; (1) CD9+ cells express higher levels of PC transcription factor, Blimp-1 while lower levels of B cell transcription factors, Bcl-6 and Pax-5, compared to CD9- cells, (2) CD9+ cells differentiate into plasmablasts faster than CD9- cells in the presence of cytokines that generate PC, and (3) CD9 expression was induced in CD9- GC-B cells under PC generating condition and gradually increased in the course of PC differentiation. Taken together, our data suggest that CD9 is a novel marker for a human GC-B cell subset that is committed to PC lineage.

Expression of mesenchyme-specific gene signatures by follicular dendritic cells: insights from the meta-analysis of microarray data from multiple mouse cell populations.

Follicular dendritic cells (FDC) are an important subset of stromal cells within the germinal centres of lymphoid tissues. They are specialized to trap and retain antigen-containing immune complexes on their surfaces to promote B-cell maturation and immunoglobulin isotype class-switching. However, little is known of the cell types from which FDC originate. To address fundamental questions associated with the relationships between FDC and other cell populations, we took advantage of the growing body of publicly available data for transcriptome analysis. We obtained a large number of gene expression data files from a range of different primary mouse cells and cell lines and subjected these data to network-based cluster analysis using BiolayoutExpress(3D) . Genes with related function clustered together in distinct regions of the graph and enabled the identification of transcriptional networks that underpin the functional activity of distinct cell populations. Several gene clusters were identified that were selectively expressed by cells of mesenchymal lineage and contained classic mesenchymal cell markers and extracellular matrix genes including various collagens, Acta2, Bgn, Fbn1 and Twist1. Our analysis showed that FDC also express highly many of these mesenchyme-associated genes. Promoter analysis of the genes comprising the mesenchymal clusters identified several regulatory motifs that are binding sites for candidate transcription factors previously known to be candidate regulators of mesenchyme-specific genes. Together, these data suggest FDC are a specialized mesenchymal cell population within the germinal centres of lymphoid tissues.

Down-regulation of CD9 expression and its correlation to tumor progression in B lymphomas.

Histological transformation, a pivotal event in the natural history of cancers including lymphomas, is typically associated with more aggressive clinical behavior. L3055, a B lymphoma cell line of germinal center (GC) origin, is dependent on follicular dendritic cells (FDCs) for survival and proliferation, similar to GC-B cells. However, L3055 cells become less FDC-dependent after prolonged culture, which is analogous to transformation in vivo. Comparison of two L3055 subclones (i.e., the FDC-dependent indolent clone 12 and the FDC-independent aggressive clone 33) by DNA microarray revealed that CD9 was the most differentially expressed gene (P = 0.05). L3055-12 expresses high levels of CD9 while L3055-33 does not. Reduced levels or loss of CD9 expression is also observed in other CD9-positive B lymphoma cell lines. The resultant CD9-negative cells grow faster than CD9-positive cells due to their greater resistance to apoptosis. Furthermore, CD9-negative cells are less dependent on FDCs for their survival and growth compared with CD9-positive cells. CD9 down-regulation in B lymphomas appears to be controlled epigenetically, mainly through histone modifications. These findings imply that CD9 is inversely correlated with B lymphoma progression, and CD9 inactivation may play an important role in B lymphoma transformation.

IL-21 and IL-10 have redundant roles but differential capacities at different stages of Plasma Cell generation from human Germinal Center B cells.

The GC is the anatomical site where antigen-activated B cells differentiate into PC, producing high-affinity antibodies in physiological and pathological states. PC differentiation is regulated by multiple factors within the GC microenvironment, including cytokines. IL-21, a recently identified type I cytokine produced by GC-Th cells, promotes differentiation of human B cells into ISC. In this study, we investigated in detail the functional role of IL-21 in the course of GC-B cell differentiation into terminally differentiated PC compared with that of IL-10, a well-known PC differentiation factor. IL-21 had a greater capacity to initiate PC differentiation from CD77(+) centroblasts than IL-10 by strongly inducing PC transcription factors through activation of STAT3; however, IL-10 was more potent than IL-21 in generating CD138(+) PC from CD20(-)CD38(++) plasmablasts in the terminal stage of GC-B cell differentiation. This differential effect of IL-21 and IL-10 was reflected in receptor expression on B cell subsets emerging in the course of differentiation. Our studies have revealed that IL-21 is a critical decision-maker for driving initial PC differentiation at the stage of CD77(+) centroblasts, yet IL-10 is more effective in producing IgG by generating terminally differentiated CD138(+) PC at the later stage of PC differentiation in the GC.

Notch ligands expressed by follicular dendritic cells protect germinal center B cells from apoptosis.

The Notch signaling pathway is one of the most conserved mechanisms to regulate cell fate in many tissues during development and postnatal life. In the immune system, Notch signaling regulates T and B cell development and modulates the differentiation of T and B cells. In this study, we investigated the functional roles of Notch signaling in human B cell differentiation within the germinal center (GC). Notch ligands, Delta-like 1 (Dll1) and Jagged 1 (Jg1), are expressed by follicular dendritic cells (FDC) but not by B cells in the GC, while GC-B cells express the Notch receptors, Notch1 and Notch2. The blockade of Notch signaling pathways using a gamma-secretase inhibitor, DAPT (N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester), reduces the survival of GC-B cells in the presence of FDC/HK cells. Jg1 has a dominant effect on GC-B cell survival mediated by Notch signaling. Furthermore, Notch cooperates with another anti-apoptotic factor, BAFF/Blys produced by FDC to support GC-B cell growth. Taken together, our data shows the important role of Notch signaling provided by FDC in the survival of GC-B cells in vitro.

Expression of CD320 in human B cells in addition to follicular dendritic cells.

CD320 has been recently discovered and reported as a follicular dendritic cell (FDC) protein. Although CD320 is known to enhance proliferation of germinal center (GC) B cells, little other information is available. In this study, we investigated its cellular distribution in the GC. Confocal microscopy of human tonsil sections revealed co-localization of CD320 with CD19 and CD38 but not with CD3 indicating that GC B cells expressed CD320 in addition to FDC. In purified GC B cells, CD320 expression was inhibited in the nucleus, membrane and cytoplasm. Reverse transcriptase-polymerase chain reaction confirmed CD320 mRNA expression in B cells. These finding indicate that CD320 is expressed in B cells in addition to FDC, and that its GC activity may be more complicated than previously thought.

Affinity maturation of an anti-hepatitis B virus PreS1 humanized antibody by phage display.

In a previous study we generated an anti-Hepatitis B Virus (HBV) preS1 humanized antibody (HzKR127) that showed in vivo HBV-neutralizing activity in chimpanzees. However, the antigen-binding affinity of the humanized antibody may not be sufficient for clinical use and thus affinity maturation is required for better therapeutic efficacy. In this study, phage display technique was employed to increase the affinity of HzKR127. All six amino acid residues (Glu95-Tyr96-Asp97-Glu98-Ala99-Tyr100) in the heavy (H) chain complementarydetermining region 3 (HCDR3) of HzKR127 were randomized and phage-displayed single chain Fv (scFv) library was constructed. After three rounds of panning, 12 different clones exhibiting higher antigen-binding activity than the wild type ScFv were selected and their antigen-binding specificity for the preS1 confirmed. Subsequently, five ScFv clones were converted to whole IgG and subjected to affinity determination. The results showed that two clones (B3 and A19) exhibited an approximately 6 fold higher affinities than that of HzKR127. The affinity-matured humanized antibodies may be useful in anti-HBV immunotherapy.

Construction, affinity maturation, and biological characterization of an anti-tumor-associated glycoprotein-72 humanized antibody.

The tumor-associated glycoprotein (TAG)-72 is expressed in the majority of human adenocarcinomas but is rarely expressed in most normal tissues, which makes it a potential target for the diagnosis and therapy of a variety of human cancers. Here we describe the construction, affinity maturation, and biological characterization of an anti-TAG-72 humanized antibody with minimum potential immunogenicity. The humanized antibody was constructed by grafting only the specificity-determining residues (SDRs) within the complementarity-determining regions (CDRs) onto homologous human immunoglobulin germ line segments while retaining two mouse heavy chain framework residues that support the conformation of the CDRs. The resulting humanized antibody (AKA) showed only about 2-fold lower affinity compared with the original murine monoclonal antibody CC49 and 27-fold lower reactivity to patient serum compared with the humanized antibody HuCC49 that was constructed by CDR grafting. The affinity of AKA was improved by random mutagenesis of the heavy chain CDR3 (HCDR3). The highest affinity variant (3E8) showed 22-fold higher affinity compared with AKA and retained the original epitope specificity. Mutational analysis of the HCDR3 residues revealed that the replacement of Asn(97) by isoleucine or valine was critical for the affinity maturation. The 3E8 labeled with (125)I or (131)I showed efficient tumor targeting or therapeutic effects, respectively, in athymic mice with human colon carcinoma xenografts, suggesting that 3E8 may be beneficial for the diagnosis and therapy of tumors expressing TAG-72.

BAFF supports human B cell differentiation in the lymphoid follicles through distinct receptors.

B cell-activating factor of the tumor necrosis factor family (BAFF/BLys) plays a critical role in B cell survival and immune responses through its three receptors: BAFF receptor (BAFF-R/BR3), transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) and B cell maturation antigen (BCMA). Using specific antibodies, we have investigated the expression of BAFF-R on human tonsillar B cells and their functional roles in naive and germinal center (GC) B cell differentiation. Our studies show that BAFF-R is the dominant receptor on naive B cells. However, three receptors are differentially modulated during in vitro GC-B cell differentiation. BAFF-R expression increased initially and then decreased with a corresponding induction of TACI and BCMA expression during differentiation to plasma cells (PCs). Consistently, blocking of BAFF-R alone with specific mAb inhibited GC-B cell proliferation and PC generation in the early period of their differentiation, whereas depletion of BAFF with TACI-Ig exhibited consistent inhibition throughout the differentiation. Finally, histological and molecular analyses of human tonsil tissue revealed that follicular dendritic cells produce BAFF. In conclusion, BAFF in the GC plays an important role through more than one receptor, and the three known receptors are differentially modulated as GC-B cells differentiate to PCs.