Lymphocytes sense antibodies through human FCRL proteins: Emerging roles in mucosal immunity.

Members of the Fc receptor-like (FCRL) family modulate B and T cell responses, yet their functional roles remain enigmatic. Nevertheless, FCRL3 promoter polymorphism that alters gene expression has been associated with autoimmune disease risk, indicating physiologic importance. Providing essential functional context, human FCRL3, FCRL4, and FCRL5 have recently been identified as secretory IgA (SIgA), dimeric IgA, and IgG receptors, respectively, revealing novel ways lymphocytes can interact with antibodies. FCRL3 and FCRL4 are able to distinguish the mucosal and systemic origin of IgA-containing immune complexes, respectively, with clear implications in guiding mucosal responses. SIgA can signal mucosal breach through FCRL3, driving the functional plasticity of regulatory T cells toward inflammatory to help control invading pathogens. Conversely, recognition of dimeric IgA by FCRL4 on memory B cells located in mucosa-associated lymphoid tissues could promote tolerance to commensals. Memory B cells that accumulate under conditions of chronic antigen presence frequently express FCRL4 and FCRL5, and antibody ligands could provide functional feedback to the cells. FCRL5 apparently recognizes the age of the IgG molecule, using deamidation as a molecular clock, conceivably playing regulatory roles in chronic antibody responses. A framework of FCRL3, FCRL4, and FCRL5 operating as sensors of antibodies in immune complexes is proposed. Sensing the spatial origin and age of immune complexes can shape lymphocyte functional attributes and inform their participation in mucosal immune responses. The potential contributions of FCRL3 and SIgA to the pathogenesis of autoimmune diseases are discussed.

Fc receptor-like 4 and 5 define human atypical memory B cells.

Atypical memory B cells accumulate in chronic infections and autoimmune conditions, and commonly express FCRL4 and FCRL5, respective IgA and IgG receptors. We characterized memory cells from tonsils on the basis of both FCRL4 and FCRL5 expression, defining three subsets with distinct surface proteins and gene expression. Atypical FCRL4+FCRL5+ memory cells had the most discrete surface protein expression and were enriched in cell adhesion pathways, consistent with functioning as tissue-resident cells. Atypical FCRL4-FCRL5+ memory cells expressed transcription factors and immunoglobulin genes that suggest poised differentiation into plasma cells. Accordingly, the FCRL4-FCRL5+ memory subset was enriched in pathways responding to endoplasmic reticulum stress and IFN-γ. We reconstructed ongoing B-cell responses as lineage trees, providing crucial in vivo developmental context. Each memory subset typically maintained its lineage, denoting mechanisms enforcing their phenotypes. Classical FCRL4-FCRL5- memory cells were infrequently detected in lineage trees, suggesting the majority were in a quiescent state. FCRL4-FCRL5+ cells were the most represented memory subset in lineage trees, indicating robust participation in ongoing responses. Together, these differences suggest FCRL4 and FCRL5 are unlikely to be passive markers but rather active drivers of human memory B-cell development and function.

Human Fc Receptor-like 3 Inhibits Regulatory T Cell Function and Binds Secretory IgA.

Human Fc receptor-like 3 (FCRL3) is an orphan receptor expressed by lymphocytes, including regulatory T cells. FCRL3 is implicated in several autoimmune diseases; however, its function on regulatory T cells is unknown. We discovered that FCRL3 stimulation of regulatory T cells inhibited their suppressive function. Moreover, FCRL3 stimulation induced IL-17, IL-26, and IFNγ production and promoted expression of the Th17-defining transcription factor RORγt without affecting FOXP3 expression. We suggest that FCRL3 engagement mediates a transition of regulatory T cells to a pro-inflammatory Th17-like phenotype. In addition, we identified secretory IgA as a specific FCRL3 ligand. Secretory IgA could serve as an environmental cue for mucosal breaches and locally drive regulatory T cell plasticity to help control infection. Our findings define a mechanism that explains the recognized association of FCRL3 with autoimmune diseases. Targeting FCRL3 to modulate regulatory T cell activity could be exploited to treat both malignancies and autoimmune diseases.

CD21 and FCRL5 form a receptor complex with robust B-cell activating capacity.

The B-cell response to antigen is critically regulated by co-receptors. CD21 (complement receptor 2) amplifies the response to antigen linked to its ligands, specific C3 fragments. In contrast, human Fc receptor-like 5 (FCRL5), a novel IgG receptor, was reported to inhibit B-cell receptor (BCR) signaling. Here, we show that CD21 and FCRL5 physically associate, suggesting that immune complexes containing both C3 fragment and IgG could simultaneously engage the pre-assembled receptors. We found that activating signaling molecules such as CD19, active PLCγ2 and BTK were rapidly recruited to FCRL5 upon engagement, suggesting a novel activating function for FCRL5. We confirmed that FCRL5 through its ITIMs (immunoreceptor tyrosine-based inhibitory motif) inhibited BCR signaling in the absence of CD21 stimulation. In contrast, triple engagement of FCRL5, CD21 and the BCR led to a superior calcium response compared to CD21 and BCR co-stimulation, in both cell lines and tonsil B cells. Furthermore, the novel activating function was independent of established FCRL5 signaling motifs. While human peripheral B cells express either FCRL5 or CD21, we identified a sizable subset of tonsil B cells which co-express the two receptors. We propose that FCRL5 has dual signaling capacity, while CD21 co-engagement serves as molecular switch, converting FCRL5 from a negative to a positive co-receptor. In tissues, B cells that co-express FCRL5 and CD21 could robustly respond to IgG immune complexes loaded with C3 fragments.

Human Fc receptor-like 5 distinguishes IgG2 disulfide isoforms and deamidated charge variants.

Human Fc receptor-like 5 (FCRL5) is a novel IgG receptor. We reported that IgG2 samples display a thousand-fold range affinity for FCRL5, indicating that attributes beyond the isotype affect binding. We hypothesized that the complex interaction could be exploited to identify distinct changes in the IgG2 molecule. We investigated using surface plasmon resonance two factors that might affect the interaction between IgG2 and FCRL5; heterogeneity related to disulfide isoforms and charge variants. We found that panitumumab and denosumab samples enriched for the more flexible A disulfide isoform bound FCRL5 with two-fold and 82-fold higher apparent affinity, respectively, than the B isoform. We next assessed whether FCRL5 binding can distinguish panitumumab charge variants which increase during storage, using two approaches. First, samples were stored at 40°C to promote acidic variants. Heat stressed panitumumab had up to four-fold higher apparent affinity for FCRL5. Next, we used conditions that promoted deamidation, a common cause of acidic variants. We found that deamidated panitumumab had up to 14-fold higher apparent affinity for FCRL5, indicating that deamidation promotes the interaction. Statistical analyses of kinetic parameters and similarity scores obtained from sensogram comparisons indicated that IgG2 disulfide isoforms, heat stressed and deamidated samples each bind FCRL5 differently. We conclude that based on FCRL5 binding, we can discern distinct changes in the IgG2 molecule, including the disulfide isoform structure and charge variants related to deamidation. Since both IgG2 deamidation and conversion of disulfide isoforms occur in vivo, these findings elucidate the biological FCRL5 ligand.

Fc Receptor-like 5 Expression Distinguishes Two Distinct Subsets of Human Circulating Tissue-like Memory B Cells.

Fc receptor-like (FCRL) 5 is a novel IgG binding protein expressed on B cells, with the capacity to regulate Ag receptor signaling. We assessed FCRL5 expression on circulating B cells from healthy donors and found that FCRL5(+) cells are most enriched among atypical CD21(-/lo)/CD27(-) tissue-like memory (TLM) B cells, which are abnormally expanded in several autoimmune and infectious diseases. Using multicolor flow cytometry, FCRL5(+) TLM cells were found to express more CD11c and several inhibitory receptors than did the FCRL5(-) TLM subset. The homing receptor profiles of the two TLM subsets shared features consistent with migration away from lymphoid tissues, but they also displayed distinct differences. Analysis of IgH V regions in single cells indicated that although both subsets are diverse, the FCRL5(+) subset accumulated significantly more somatic mutations. Furthermore, the FCRL5(+) subset had more switched isotype expression and more extensive proliferative history. Microarray analysis and quantitative RT-PCR demonstrated that the two TLM subsets possess distinct gene expression profiles, characterized by markedly different CD11c, SOX5, T-bet, and RTN4R expression, as well as differences in expression of inhibitory receptors. Functional analysis revealed that the FCRL5(+) TLM subset responds poorly to multiple stimuli compared with the FCRL5(-) subset, as reflected by reduced calcium mobilization and blunted cell proliferation. We propose that the FCRL5(+) TLM subset, but not the FCRL5(-) TLM subset, underwent Ag-driven development and is severely dysfunctional. The present study elucidates the heterogeneity of TLM B cells and provides the basis to dissect their roles in the pathogenesis of inflammatory and infectious diseases.

B cell receptor induced Fc receptor-like 5 expression is mediated by multiple signaling pathways converging on NF-κB and NFAT.

Fc receptor-like (FCRL) proteins are novel regulators of the B cell response to antigen. Human FCRL5 binds intact IgG and modifies the strength of antigen receptor (BCR) signaling. Altering FCRL5 expression could therefore regulate the B cell response to antigen. In this study, we found that FCRL5 expression is induced specifically upon BCR stimulation and dissected the molecular mechanism. FCRL5 mRNA and cell surface protein expression required prolonged BCR stimulation and de novo protein synthesis. Using chemical inhibitors and activators, we identified roles for several signaling pathways, indicating a complex mechanism. Specifically, the PI3K/AKT, JNK, PKC and IKK2-dependent classical NF-κB pathways were involved in induced FCRL5 expression. Furthermore, induced FCRL5 expression required elevation of intracellular Ca(++) and was partially blocked by cyclosporine A, a calcineurin inhibitor. The importance of the transcription factors NF-κB, NFAT and CREB-binding protein was revealed based on sensitivity to inhibitors. Using reporter gene assays, we showed that the core FCRL5 promoter was sufficient to drive induced gene expression. Mutations of two predicted NF-κB sites or an NFAT site in the core promoter abrogated induced gene expression, suggesting direct regulation of the FCRL5 gene by NF-κB and NFAT. In support, we detected binding of NF-κB and NFAT family proteins to oligonucleotides corresponding to the predicted sites. We propose that the identified intricate mechanism serves to ensure that FCRL5 is expressed on B cells at a precise time following antigen encounter, with potential implications regarding regulation of the B cell response.

U.S. Food and drug administration approval: obinutuzumab in combination with chlorambucil for the treatment of previously untreated chronic lymphocytic leukemia.

On November 1, 2013, the U.S. Food and Drug Administration (FDA) approved obinutuzumab (GAZYVA; Genentech, Inc.), a CD20-directed cytolytic antibody, for use in combination with chlorambucil for the treatment of patients with previously untreated chronic lymphocytic leukemia (CLL). In stage 1 of the trial supporting approval, patients with previously untreated CD20-positive CLL were randomly allocated (2:2:1) to obinutuzumab + chlorambucil (GClb, n = 238), rituximab + chlorambucil (RClb, n = 233), or chlorambucil alone (Clb, n = 118). The primary endpoint was progression-free survival (PFS), and secondary endpoints included overall response rate (ORR). Only the comparison of GClb to Clb was relevant to this approval and is described herein. A clinically meaningful and statistically significant improvement in PFS with medians of 23.0 and 11.1 months was observed in the GClb and Clb arms, respectively (HR, 0.16; 95% CI, 0.11-0.24; P < 0.0001, log-rank test). The ORRs were 75.9% and 32.1% in the GClb and Clb arms, respectively, and the complete response rates were 27.8% and 0.9% in the GClb and Clb arms, respectively. The most common adverse reactions (≥10%) reported in the GClb arm were infusion reactions, neutropenia, thrombocytopenia, anemia, pyrexia, cough, and musculoskeletal disorders. Obinutuzumab was the first Breakthrough Therapy-designated drug to receive FDA approval.

Human Fc receptor-like 5 binds intact IgG via mechanisms distinct from those of Fc receptors.

Fc receptor-like (FCRL) 5 regulates B cell Ag receptor signaling and has been reported to bind aggregated IgG. Using surface plasmon resonance, we analyzed the interaction of native IgG samples with FCRL5, revealing a complex binding mechanism, where isotype is just one factor. FCRL5 bound IgG1 and IgG4 with ~1 µM KD, whereas the interaction with IgG3 was a magnitude weaker. However, IgG2 samples displayed a wide range of affinities, indicating that additional factors affect binding. We used a panel of 19 anti-FCRL5 mAbs with defined reactivity to identify domains involved in ligand binding. Six mAbs blocked IgG binding, indicating critical roles of FCRL5 domains 1 and 3, as well as epitopes at the domain 1/2 and domain 2/3 boundaries. We found that only glycosylated IgG containing both Fab arms and the Fc region bound with high affinity. Furthermore, the presence of sialic acid in the IgG carbohydrate altered FCRL5 binding. The interaction of IgG and FCRL5 consisted of two kinetic components, suggesting a complex binding mechanism. We established that the IgG-Fc and IgG-F(ab')2 fragments bind FCRL5 independently but with low affinity, revealing the mechanism behind the two-step binding of whole IgG. This complex binding mechanism is distinct from that of Fc receptors, which bind through the Fc. We propose that FCRL5 is a new type of receptor that recognizes intact IgG, possibly enabling B cells to sense Ig quality. Recognition of undamaged IgG molecules by FCRL5 could allow B cells to engage recently produced Abs.

Fc receptor-like 5 promotes B cell proliferation and drives the development of cells displaying switched isotypes.

The biological roles of B cell membrane proteins in the FCRL family are enigmatic. FCRL proteins, including FCRL5, were shown to modulate early BCR signaling, although the subsequent, functional consequences of receptor engagement are poorly understood. We found that FCRL5 surface protein itself was induced temporarily upon BCR stimulation of human, naive B cells, indicating precise control over timing of FCRL5 engagement. Cross-linking of FCRL5 on cells induced to express FCRL5 enhanced B cell proliferation significantly. This enhancement required costimulation of the BCR and TLR9, two signals required for optimal proliferation of naive B cells, whereas T cell help in the form of anti-CD40 and IL-2 was dispensable. In addition, we found that FCRL5 stimulation generated a high proportion of cells displaying surface IgG and IgA. Optimal development of cells expressing switched isotypes required T cell help, in addition to stimuli found necessary for enhanced proliferation. Surprisingly, cells that developed upon FCRL5 stimulation simultaneously displayed surface IgM, IgG, and IgA. Cells expressing multiple Ig isotypes were described in hairy cell leukemia, a disease in which FCRL5 is overexpressed. Enhanced proliferation and downstream isotype expression upon FCRL5 stimulation could reflect a physiological role for FCRL5 in the expansion and development of antigen-primed B cells. In addition, FCRL5 may promote growth of malignant cells in hairy cell leukemia and other FCRL5-expressing tumors.

Considerations for the development of therapeutic monoclonal antibodies.

An increasing number of Investigational New Drug (IND) applications for therapeutic monoclonal antibodies (mAbs) have been submitted to US FDA over the past several years. Monoclonal antibodies and related products are under development for a wide range of indications. In addition, the diversity of antibody-related products is increasing including IgG2/IgG4 subclasses and engineered Fc regions to enhance or reduce antibody effector functionality. Recent findings highlight the need to more fully characterize these products and their activity. Advances in product characterization tools, immunogenicity assessments, and other bioanalytical assays can be used to better understand product performance and facilitate development.

Elf-1 binds to GGAA elements on the FcRgamma promoter and represses its expression.

The Fc receptor (FcR) gamma-chain has been shown to be up-regulated in T cells when the TCR zeta-chain is decreased. We demonstrate that Elf-1, but not other Ets family transcription factors, bind to a cluster of GGAA sites located within the 200 bp upstream from the transcription initiation site of the FcRgamma promoter. Forced expression of Elf-1 results in the suppression of FcRgamma expression, whereas silencing its expression with small interfering RNA Elf-1 results in increased FcRgamma expression. Elf-1 represents the first transcription factor identified to be involved in the transcriptional regulation of FcRgamma, and cells that fail to express Elf-1, as is the case with human systemic lupus erythematosus T cells, will express FcRgamma-chain.

Epstein-Barr virus nuclear antigen 2 induces FcRH5 expression through CBF1.

Fc-receptor homolog 5 (FcRH5) is a recently identified B-cell membrane protein of unknown function. In Burkitt lymphoma cell lines with chromosome 1q21 abnormalities, FcRH5 expression is deregulated, implicating FcRH5 in lymphomagenesis. Epstein-Barr virus infects and immortalizes B cells, and is implicated in the etiology of several tumors of B-cell origin. Overexpression of genes located on 1q21-25 has been proposed as a surrogate for Epstein-Barr virus in Burkitt lymphoma. We now report that Epstein-Barr virus nuclear antigen 2 (EBNA2) markedly induces the expression of the FcRH5 gene, encoded on chromosome 1q21. Induction occurred in the absence of other viral proteins and did not require de novo protein synthesis. EBNA2 lacks a DNA-binding domain and can target responsive genes through the host DNA binding protein CBF1. We show that induction of FcRH5 by EBNA2 is strictly CBF1 dependent, as it was abolished in CBF1-deficient cells. Accordingly, EBNA2 targeted CBF1 binding sites present in the FcRH5 promoter in vivo, as detected by chromatin immunoprecipitation. These results identify FcRH5 as a novel, direct target of EBNA2 that may contribute to the development of Epstein-Barr virus-associated tumors.

The cyclic AMP response element modulator regulates transcription of the TCR zeta-chain.

Systemic lupus erythematosus T cells display decreased amounts of TCR zeta mRNA that results in part from limited binding of the transcriptional enhancer Elf-1 to the TCR zeta promoter. We have identified a new cis-binding site for the cAMP response element (CRE) modulator (CREM) on the TCR zeta promoter, centered on the -390 nucleotide. Transfection of T cells with an antisense CREM alpha plasmid reduced the binding of CREM to the TCR zeta promoter, as shown by chromatin and reporter chromatin immunoprecipitation assays, and enhanced the production of TCR zeta mRNA and protein. Mutagenesis of the -390 CRE site prevented the binding of CREM to the TCR zeta promoter. The mechanism of CREM-mediated repression appears to be chromatin dependent, because antisense CREM promotes the acetylation of histones on the TCR zeta promoter. Finally, we established an enhanced binding of CREM to the TCR zeta-chain promoter in systemic lupus erythematosus cells compared with control T cells. Our studies demonstrate that CREM alpha binds to the TCR zeta promoter and repress its activity.

Protein kinase A regulatory subunit type II beta directly interacts with and suppresses CREB transcriptional activity in activated T cells.

Levels of the type IIbeta regulatory subunit (RIIbeta) of protein kinase A are abnormally high in the nuclei of T cells of some subjects with the autoimmune disorder systemic lupus erythematosus (SLE). However, the role of nuclear RIIbeta in the regulation of T cell function is unknown. Based on previous studies demonstrating that nuclear protein kinase A-RII subunits can modify cAMP response element (CRE)-dependent transcription, we tested the hypothesis that nuclear RIIbeta can alter CRE-directed gene expression in T cells through interaction with the nuclear transcription factor CRE-binding protein CREB. To test this hypothesis, we used the RIIbeta-deficient S49 and the Jurkat T cell lines. In both cell lines, transient transfection of RIIbeta resulted in nuclear localization of a portion of the ectopically expressed RIIbeta. In vitro and in vivo analyses revealed a novel, specific interaction between RIIbeta and CREB that mapped to the N-terminal 135 aa of RIIbeta. In functional studies, RIIbeta inhibited the transcriptional activity of a GAL4-CREB fusion protein by 67% in Jurkat T cells following activation with anti-CD3 and anti-CD28 mAbs. Importantly, deletion of the CREB-binding region of RIIbeta completely abrogated inhibition. Additionally, RIIbeta suppressed CRE-directed reporter gene expression and substantially reduced induction of promoter activity and endogenous protein levels of the CREB-dependent gene, c-fos, in activated T cells. We conclude that nuclear RIIbeta can act as a repressor of CREB transcriptional activity in T cells, providing a potential functional significance for aberrant levels of nuclear RIIbeta in systemic lupus erythematosus T cells.

The cyclic adenosine 5'-monophosphate response element modulator suppresses IL-2 production in stimulated T cells by a chromatin-dependent mechanism.

The production of IL-2 is tightly controlled by several transcription factors that bind to the IL-2 promoter. The cAMP response element modulator (CREM) is known to form complexes with CREB and bind to the -180 site of the IL-2 promoter in anergic and in systemic lupus erythematosus T cells. In this study we show that CREM is transcriptionally induced in T cells following stimulation through CD3 and CD28, binds to the IL-2 promoter in vivo, and suppresses IL-2 production. Transfection of an antisense CREM plasmid into T cells blocked the expression and binding of CREM to the IL-2 promoter and the decrease of IL-2 production, which follows the early increase after T cell stimulation with CD3 and CD28. In addition, as assessed by chromatin immunoprecipitation experiments, antisense CREM prevented the binding of protein 300 and cAMP response element binding protein and promoted the acetylation of histones. Antisense CREM also enhanced the accessibility of the IL-2 promoter to endonucleases and prevented the condensation of chromatin in vivo. Our data suggest that upon T cell activation, CREM gradually replaces phosphorylated CREB at the -180 site of the IL-2 promoter. CREM, in turn, binds protein 300 and cAMP response element binding protein, but CREM is unable to activate its histone acetyltransferase activity, which results in condensation of chromatin and down-regulation of IL-2 production.

NF-kappaB regulates the expression of the human complement receptor 2 gene.

CR2 is a key regulator of the B cell response to Ag. Here we show that NF-kappaB enhances the expression of the human CR2 gene. Promoter truncation, deletion, and mutagenesis studies indicated a functional role for a consensus NF-kappaB promoter element, as well as a heterogeneous nuclear ribonucleoprotein D element and an overlapping X box/E box. By supershift analysis, the first two elements bound NF-kappaB p50 and p65 and heterogeneous nuclear ribonucleoprotein RNP D, respectively. The X box/E box bound regulatory factor X5 and, surprisingly, NF-kappaB p50 and p65. Overexpression of NF-kappaB p50 enhanced the activity of the CR2 promoter in B cell lines and primary B cells, suggesting a direct role for NF-kappaB in regulating promoter activity. Importantly, mutation of the NF-kappaB element or the X box/E box rendered the promoter unresponsive to NF-kappaB p50. Using chromatin immunoprecipitation in live B cell lines and primary B cells, we found that NF-kappaB proteins p50, p65, and c-Rel bound to the genomic promoter at two locations that overlap with the consensus NF-kappaB element or the X box/E box. Finally, stimuli that activate NF-kappaB enhanced the activity of the CR2 promoter, and LPS rapidly increased the number of CR2 proteins on the surface of primary B cells. We propose that the NF-kappaB signaling pathway enhances the expression of the CR2 gene, as a result of NF-kappaB proteins binding to two CR2 promoter elements. Thus, at the onset of an infection, LPS could sensitize the B cell to Ag by enhancing the level of CR2-costimulatory molecules on the cell surface.

Protein kinase A enhances, whereas glycogen synthase kinase-3 beta inhibits, the activity of the exon 2-encoded transactivator domain of heterogeneous nuclear ribonucleoprotein D in a hierarchical fashion.

Heterogeneous nuclear ribonucleoprotein D (hnRNP D) is implicated in transcriptional regulation. Alternative splicing of exons 2 and 7 generates four isoforms of the protein. We report here that only isoforms that contain the product of exon 2 (amino acids 79-97) were able to transactivate. Moreover, the exon 2-encoded protein domain alone was sufficient to drive transcription. TATA-binding protein and p300 interacted with a synthetic peptide corresponding to exon 2, and both proteins co-precipitated with hnRNP D. Stimulation of protein kinase A (PKA) and protein kinase C (PKC) synergistically induced the transactivating ability of hnRNP D, and the exon 2-encoded domain was sufficient for this inducibility. In kinase assays PKA phosphorylated Ser-87 of hnRNP D, whereas glycogen synthase kinase-3 beta (GSK-3 beta) phosphorylated Ser-83, but only if Ser-87 had been pre-phosphorylated by PKA. Phosphorylation of Ser-87 enhanced, whereas phosphorylation of Ser-83 repressed, transactivation. Overexpression of GSK-3 beta inhibited transactivation by hnRNP D, but stimulation of PKC negated the inhibitory effect of GSK-3 beta. We suggest that a hierarchical phosphorylation pathway regulates the transactivating ability of hnRNP D: PKA activates hnRNP D, but at the same time renders it sensitive to inhibition by GSK-3 beta; the latter inhibition can be suspended by inactivating GSK-3 beta with PKC.