Cyclin-dependent kinases regulate Ig class switching by controlling access of AID to the switch region.

Ig class switching requires cell proliferation and is division linked, but the detailed mechanism is unknown. By analyzing the first switching cells early in the kinetics, our analysis suggested that proliferating B cells had a very short G1 phase (<3.5 h), a total cell cycle time of ∼ 11 h, and that Ig class switching preferentially occurred in the late G1 or early S phase. Inhibition of cyclin-dependent kinases (CDKs) caused dramatic reduction of switching rate within 6 h. This was associated with less targeting of activation-induced cytidine deaminase (AID) to the Igh locus. Interestingly, ectopically expressed nuclear AID in HeLa cells was preferentially found in the early S phase. Furthermore, in CDK2 hypomorphic cells there was reduced nuclear AID accumulation. Thus, our data are compatible with the idea that division-linked Ig class switching is in part due to CDK2-regulated AID nuclear access at the G1/S border.

The Rho GTPase Cdc42 Is Essential for the Activation and Function of Mature B Cells.

The Rho GTPase Cdc42 coordinates regulation of the actin and the microtubule cytoskeleton by binding and activating the Wiskott-Aldrich syndrome protein. We sought to define the role of intrinsic expression of Cdc42 by mature B cells in their activation and function. Mice with inducible deletion of Cdc42 in mature B cells formed smaller germinal centers and had a reduced Ab response, mostly of low affinity to T cell-dependent Ag, compared with wild-type (WT) controls. Spreading formation of long protrusions that contain F-actin, microtubules, and Cdc42-interacting protein 4, and assumption of a dendritic cell morphology in response to anti-CD40 plus IL-4 were impaired in Cdc42-deficient B cells compared with WT B cells. Cdc42-deficient B cells had an intact migratory response to chemokine in vitro, but their homing to the B cell follicles in the spleen in vivo was significantly impaired. Cdc42-deficient B cells induced a skewed cytokine response in CD4(+) T cells, compared with WT B cells. Our results demonstrate a critical role for Cdc42 in the motility of mature B cells, their cognate interaction with T cells, and their differentiation into Ab-producing cells.

A novel mouse model for the hyper-IgM syndrome: a spontaneous activation-induced cytidine deaminase mutation leading to complete loss of Ig class switching and reduced somatic hypermutation.

We describe a spontaneously derived mouse line that completely failed to induce Ig class switching in vitro and in vivo. The mice inherited abolished IgG serum titers in a recessive manner caused by a spontaneous G → A transition mutation in codon 112 of the aicda gene, leading to an arginine to histidine replacement (AID(R112H)). Ig class switching was completely reconstituted by expressing wild-type AID. Mice homozygous for AID(R112H) had peripheral B cell hyperplasia and large germinal centers in the absence of Ag challenge. Immunization with SRBCs elicited an Ag-specific IgG1 response in wild-type mice, whereas AID(R112H) mice failed to produce IgG1 and had reduced somatic hypermutation. The phenotype recapitulates the human hyper-IgM (HIGM) syndrome that is caused by point mutations in the orthologous gene in humans, and the AID(R112H) mutation is frequently found in HIGM patients. The AID(R112H) mouse model for HIGM provides a powerful and more precise tool than conventional knockout strategies.

STAT6 signaling pathway activated by the cytokines IL-4 and IL-13 induces expression of the Epstein-Barr virus-encoded protein LMP-1 in absence of EBNA-2: implications for the type II EBV latent gene expression in Hodgkin lymphoma.

In line with the B-lymphotropic nature of Epstein-Barr virus (EBV), the virus is present in several types of B-cell lymphomas. EBV expresses a different set of latent genes in the associated tumors, such as EBV nuclear antigen 1 (EBNA-1) and latent membrane proteins (LMPs; type II latency) in classical Hodgkin lymphomas (HLs). We previously reported that exposure of in vitro EBV-converted, HL-derived cell line KMH2-EBV to CD40-ligand and interleukin-4 (IL-4) induced the expression of LMP-1. Here, we show that exposure to IL-4 or IL-13 alone induced LMP-1 in the absence of EBNA-2. Induction of LMP-1 by IL-4 and IL-13 was mediated by the signal transducer signal transducer and activator of transcription 6 (STAT6) and a newly defined high-affinity STAT6-binding site in the LMP-1 promoter. IL-4 induced LMP-1 also in Burkitt lymphoma-derived lines and in tonsillar B cells infected with the EBNA-2-deficient EBV strain P3HR-1. Furthermore, coculture of EBV-carrying Burkitt lymphoma cells with activated CD4(+) T cells resulted in the induction of LMP-1 in the absence of EBNA-2. Because Hodgkin/Reed-Sternberg cells are known to secrete IL-13, to have constitutively activated STAT6, and to be closely surrounded by CD4(+) T cells, these mechanisms may be involved in the expression of LMP-1 in EBV-positive chronic HLs.

Type I interferons directly down-regulate BCL-6 in primary and transformed germinal center B cells: differential regulation in B cell lines derived from endemic or sporadic Burkitt's lymphoma.

Type I interferons (IFN) exert multiple effects on both the innate and adaptive immune system in addition to their antiviral and antiproliferative activities. Little is known, however about the direct effects of type I IFNs on germinal center (GC) B cells, the central components of adaptive B cell responses. We used Burkitt's lymphoma (BL) lines, as a model system of normal human GC B cells, to examine the effect of type I IFNs on the expression of BCL-6, the major regulator of the GC reaction. We show that type I IFNs, but not IFNγ, IL-2 and TNFα rapidly down-regulate BCL-6 protein and mRNA expression, in cell lines derived from endemic, but not from sporadic BL. IFNα-induced down-regulation is specific for BCL-6, independent of Epstein-Barr virus and is not accompanied by IRF-4 up-regulation. IFNα-induced BCL-6 mRNA down-regulation does not require de novo protein synthesis and is specifically inhibited by piceatannol. The proteasome inhibitor MG132 non-specifically prevents, while inhibitors of alternate type I IFN signaling pathways do not inhibit IFNα-induced BCL-6 protein downregulation. We validate our results with showing that IFNα rapidly down-regulates BCL-6 mRNA in purified mouse normal GC B cells. Our results identify type I IFNs as the first group of cytokines that can down-regulate BCL-6 expression directly in GC B cells.

Methods to Study the Role of Cdc42, Rac1, and Rac2 in B-Cell Cytoskeletal Responses.

B-cell migration and adhesion are critical to form a germinal center response, the site for B-cell production of high-affinity antibodies. Here, we describe two assays that can be used to examine B-cell cytoskeletal responses needed during the germinal center response: B-cell spreading and homotypic adhesion. Spreading of B cells is dependent on Cdc42, while Rac1 and Rac2 are necessary for homotypic adhesion. These in vitro assays can be used to examine functional responses of B cells mediated by the cell cytoskeleton, for example when comparing B cells from different gene knockout animals.

Deletion of Dock10 in B Cells Results in Normal Development but a Mild Deficiency upon In Vivo and In Vitro Stimulations.

We sought to identify genes necessary to induce cytoskeletal change in B cells. Using gene expression microarray, we compared B cells stimulated with interleukin-4 (IL-4) and anti-CD40 antibodies that induce B cell spreading, cell motility, tight aggregates, and extensive microvilli with B cells stimulated with lipopolysaccharide that lack these cytoskeletal changes. We identified 84 genes with 10-fold or greater expression in anti-CD40 + IL-4 stimulated B cells, one of these encoded the guanine nucleotide exchange factor (GEF) dedicator of cytokinesis 10 (Dock10). IL-4 selectively induced Dock10 expression in B cells. Using lacZ expression to monitor Dock10 promoter activity, we found that Dock10 was expressed at all stages during B cell development. However, specific deletion of Dock10 in B cells was associated with a mild phenotype with normal B cell development and normal B cell spreading, polarization, motility, chemotaxis, aggregation, and Ig class switching. Dock10-deficient B cells showed lower proliferation in response to anti-CD40 and IL-4 stimulation. Moreover, the IgG response to soluble antigen in vivo was lower when Dock10 was specifically deleted in B cells. Together, we found that most B cell responses were intact in the absence of Dock10. However, specific deletion of Dock10 in B cells was associated with a mild reduction in B cell activation in vitro and in vivo.

The Small Rho GTPases Rac1 and Rac2 Are Important for T-Cell Independent Antigen Responses and for Suppressing Switching to IgG2b in Mice.

The Rho GTPases Cdc42, Rac1, and Rac2 coordinate receptor signaling to cell adhesion, migration, and proliferation. Deletion of Rac1 and Rac2 early during B cell development leads to failure in B cell entry into the splenic white pulp. Here, we sought to understand the role of Rac1 and Rac2 in B cell functionality and during the humoral antibody response. To circumvent the migratory deficiency of B cells lacking both Rac1 and Rac2, we took the approach to inducibly delete Rac1 in Rac2-/- B cells in the spleen (Rac1BRac2-/- B cells). Rac1BRac2-/- mice had normal differentiation of splenic B cell populations, except for a reduction in marginal zone B cells. Rac1BRac2-/- B cells showed normal spreading response on antibody-coated layers, while both Rac2-/- and Rac1BRac2-/- B cells had reduced homotypic adhesion and decreased proliferative response when compared to wild-type B cells. Upon challenge with the T-cell-independent antigen TNP-conjugated lipopolysaccharide, Rac1BRac2-/- mice showed reduced antibody response. In contrast, in response to the T-cell-dependent antigen sheep red blood cells, Rac1BRac2-/- mice had increased serum titers of IgG1 and IgG2b. During in vitro Ig class switching, Rac1BRac2-/- B cells had elevated germline γ2b transcripts leading to increased Ig class switching to IgG2b. Our data suggest that Rac1 and Rac2 serve an important role in regulation of the B cell humoral immune response and in suppressing Ig class switching to IgG2b.

CEACAM1 is a potent regulator of B cell receptor complex-induced activation.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1, CD66a) is a member of the immunoglobulin (Ig) superfamily, previously characterized as an adhesion and signaling molecule in epithelial, endothelial, and hematopoietic cells. Here, we show that the CEACAM1 isoform expression pattern is different in nonactivated and activated primary mouse B lymphocytes and that CEACAM1 influences B cell receptor complex-mediated activation. A CEACAM1-specific monoclonal antibody strongly triggered proliferation of mouse B cells when combined with surface IgM cross-linking. However, anti-CEACAM1 was not mitogenic when added alone. The proliferation was more pronounced and lasted longer as compared with other activators of B cells, such as anti-IgM in the presence of interleukin-4 or lipopolysaccharide. A similar, costimulatory effect was exerted by CEACAM1-expressing fibroblasts, indicating that homophilic CEACAM1-CEACAM1 cell-mediated binding is the physiological stimulus for CEACAM1-triggered B cell signaling. The anti-CEACAM1/anti-IgM-activated cells aggregated in a lymphocyte function-associated antigen-1-dependent manner. Furthermore, cells that were activated by anti-CEACAM1/anti-IgM secreted Ig but did not go through Ig class-switching. Anti-CEACAM1 induced phosphorylation of c-Jun N-terminal kinase (stress-activated protein kinase) but did not activate the extracellular signal-regulated kinase or p38 mitogen-activated protein kinases.

Binding of Ikaros to germline Ig heavy chain gamma1 and epsilon promoters.

Immunoglobulin (Ig) class switching occurs in activated B cells and results in production of antigen-specific IgA, IgE or IgG. It involves a DNA recombination event and is partly regulated by germline (GL) immunoglobulin heavy chain promoters. Ikaros is an abundant nuclear protein expressed in hematopoietic cells. Many different functions have been ascribed to Ikaros, such as transcriptional activation or repression, cell cycle control and tumor suppression. A typical feature of Ikaros is its expression in large clusters in the nucleus of activated lymphocytes. We give evidence that Ikaros can bind to several sites in the germline gamma1 and epsilon immunoglobulin heavy chain promoters, in a cooperative manner. Using a promoter reporter assay, we found evidence that Ikaros can suppress germline gamma1 and epsilon promoter activity in a B cell line. When a mutated non-DNA-binding form of Ikaros was introduced into primary activated B cells by retrovirus transduction, the endogenous Ikaros clusters were disrupted. In spite of this, there was no effect on transcription or Ig class switching. The data are discussed in relation to the different hypotheses for the function of Ikaros.

Regulation of epsilon germline transcription and switch region mutations by IgH locus 3' enhancers in transgenic mice.

Germline (GL) transcription is regulated by specific promoters and immunoglobulin heavy chain (IgH) 3' locus enhancers and is necessary for Ig class-switch recombination (CSR). We have generated different transgenic lines containing the GL epsilon promoter, switch (S) epsilon region, and constant (C) epsilon region with or without the DNase I-sensitive regions (HS) 3A-HS1,2 or HS3B-HS4 3' IgH enhancer pairs. The enhancerless construct was expressed in B cells activated by interleukin (IL)-4 and CD40, thus resembling regulation of the endogenous gene. Both enhancer-containing transgenes efficiently increased expression in B cells and were strongly up-regulated by stimuli. In addition, Sepsilon regions of the transgene containing HS3B-HS4 were mutated in activated, sorted B cells. Such mutations are known to precede CSR and are dependent on activation-induced cytidine deaminase (AID). Our findings show that all elements necessary for recruitment of the recombination machinery are present in the transgene containing HS3 and HS4. These enhancers probably provide something more specific than mere increased accessibility of switch regions. We propose that transcription factors binding the enhancers help to target the recombination machinery to the switch regions.

FcgammaRIIb controls bone marrow plasma cell persistence and apoptosis.

The survival of long-lived plasma cells, which produce most serum immunoglobulin, is central to humoral immunity. We found here that the inhibitory Fc receptor FcgammaRIIb was expressed on plasma cells and controlled their persistence in the bone marrow. Crosslinking FcgammaRIIb induced apoptosis of plasma cells, which we propose contributes to the control of their homeostasis and suggests a method for therapeutic deletion. Plasma cells from mice prone to systemic lupus erythematosus did not express FcgammaRIIb and were protected from apoptosis. Human plasmablasts expressed FcgammaRIIb and were killed by crosslinking, as were FcgammaRIIb-expressing myeloma cells. Our results suggest that FcgammaRIIb controls bone marrow plasma cell persistence and that defects in it may contribute to autoantibody production.

Wiskott-Aldrich syndrome protein deficiency leads to reduced B-cell adhesion, migration, and homing, and a delayed humoral immune response.

The Wiskott-Aldrich syndrome protein (WASp) is mutated in the severe immunodeficiency disease Wiskott-Aldrich syndrome (WAS). The function of B cells and the physiologic alterations in WAS remain unclear. We show that B cells from WAS patients exhibited decreased motility and had reduced capacity to migrate, adhere homotypically, and form long protrusions after in vitro culture. WASp-deficient murine B cells also migrated less well to chemokines. Upon antigen challenge, WASp-deficient mice mounted a reduced and delayed humoral immune response to both T-cell-dependent and -independent antigens. This was at least in part due to deficient migration and homing of B cells. In addition, the germinal center reaction was reduced in WASp-deficient mice. Thus, WASp is crucial for optimal B-cell responses and plays a pivotal role in the primary humoral immune response.

Efficient antigen presentation of soluble, but not particulate, antigen in the absence of Wiskott-Aldrich syndrome protein.

B cells and dendritic cells, lacking functional Wiskott-Aldrich syndrome protein (WASP), have aberrant formation of membrane protrusions. We hypothesized that protrusions may play a role in antigen presentation, and consequently, that impaired antigen presentation may be an underlying factor of the immune deficiency in patients with Wiskott-Aldrich syndrome. In this paper, we investigated the antigen presentation capacity of B cells and dendritic cells from WASP knockout mice, using soluble and particulate antigen, to CD4+ T cells from T-cell receptor transgenic DO11.10 mice. As antigen we used soluble ovalbumin (OVA), a peptide thereof (amino acids 323-339) or bacteria expressing OVA. We found that WASP-deficient B cells and dendritic cells efficiently processed and presented soluble OVA protein as well as its peptide in vitro, inducing proliferation and cytokine production from CD4+ T cells. Antigen presentation of soluble protein was efficient also in vivo, because immunization of WASP-deficient mice with OVA elicited proliferation of transferred, fluorescent-labelled, CD4+ T cells. Although we could detect uptake of bacteria in dendritic cells, processing and presentation of bacterial-expressed OVA was impaired in WASP-deficient dendritic cells. In conclusion, our data suggest that WASP is not needed for processing and presentation of soluble antigen, but that efficient presentation of particulate antigen require WASP.

Microvilli structures on B lymphocytes: inducible functional domains?

Interactive contact between B lymphocytes and T cells is necessary for their expansion during an immune response. It has been shown that B lymphocytes receive signals from T cells, such as IL-4 and cross-linking of CD40, which are crucial for their differentiation. We previously found that these factors induce formation of microvilli on B cells and that this was correlated with increased homotypic adhesion of B lymphocytes. In this study we have investigated if IL-4 induce segregation of proteins to microvilli and lipid rafts. Using immuno-electron microscopy we analyzed cell-surface distribution of molecules involved in B-T cell co-activation. Recruitment to detergent-resistant membrane fractions was analyzed using sucrose gradient centrifugation. We found that microvilli were enriched in ICAM-1 and MHC class II molecules. In contrast, LFA-1 and CD40 were more abundant on the smooth cell surfaces, while B7-2 (CD86) was randomly distributed. We also discovered that depletion of cholesterol, using beta-methyl-cyclodextrin, lowered the number of microvilli, indicating that intact lipid rafts are required for their expression. Moreover, activation of B lymphocytes by lipopolysaccharide (LPS) induced increased expression of GM(1), a marker for lipid rafts. However, although both surface and total levels of GM(1) were similar in B lymphocytes stimulated with either LPS or LPS plus IL-4, GM(1) was mainly expressed on microvilli in LPS plus IL-4-stimulated cells. Taken together, our results indicate that microvilli represent distinct inducible membrane domains that can regulate direct cell-cell interactions via grouping and three-dimensional presentation of cell-surface receptors.