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A novel Ets protein was isolated by yeast one-hybrid screening of a cDNA library made from lipopolysaccharide-stimulated mouse splenic B cells, using the SP6 kappa promoter kappaY element as a bait. The novel Ets protein was most closely related to PU.1 and Spi-B within the DNA binding Ets domain and was therefore named Spi-C. However, Spi-C may represent a novel subgroup within the Ets protein family, as it differed significantly from Spi-B and PU.1 within helix 1 of the Ets domain. Spi-C was encoded by a single-copy gene that was mapped to chromosome 10, region C. Spi-C interacted with DNA similarly to PU.1 as judged by methylation interference, band-shift and site selection analysis, and activated transcription of a kappaY element reporter gene upon co-transfection of HeLa cells. Spi-C RNA was expressed in mature B lymphocytes and at lower levels in macrophages. Furthermore, pre-B cell and plasma cell lines were Spi-C-negative, suggesting that Spi-C might be a regulatory molecule during a specific phase of B lymphoid development.

A protein interacting with an A-T-rich region that is a positive control element within the SP6 κ promoter was purified and identified as CArG-box binding factor-A. The purified protein was shown to interact specifically with the coding strand of single-stranded DNA and, with lower affinity, with double-stranded DNA. A mutation that inhibited binding of the protein to the A-T-rich region also aborted the transcriptional stimulatory effect of the region. Two Ets proteins, PU.1 and elf-1, that have previously been shown to bind to an adjacent DNA element were shown to physically interact with CArG-box binding factor-A. An antiserum raised against the protein recognized two different forms indicating either that different splice-forms of CArG-box binding factor-A are expressed, or that the protein is subject to post-translational modification.

Promoter region sequences of human and mouse Igk-V genes were aligned and found to be conserved for about 200-300 base pairs (bp) within subgroups/families. No promoter similarity was found between IGKV promoters from different human subgroups. Related mouse Igk-V gene families were conserved in the promoter region but no similarity was evident when promoters from unrelated Igk-V gene families were compared. Most of the human IGKV promoter subgroups were shown to have mouse counterparts with a similarity region that extended about 150 bp upstream of the translational start codon. All promoters contained an octamer sequence element. The consensus octamer/decamer sequence was favored but only seven residues within the octamer element were strictly conserved. Furthermore, there was also sequence conservation immediately 3' of the octamer where either an A or a G residue was conserved. In addition, other DNA elements were also conserved both within the Igk-V subgroups/families and between mouse and human promoters from related subgroups/families. In several of the subgroups/families an E box of the E2A type was conserved 5' of the octamer and a CCCT element was conserved within the IGKV subgroup II and its related mouse Igk-V families. We conclude from this study that conservation of additional sequence elements besides the octamer is a common feature in Igk-V promoters but that distinct elements are conserved only within a given subgroup/family. Thus, the conservation appears to have operated at the level of function rather than at the level of recognition sequence for defined transcription factors.

The SP6 kappa-promoter pentadecamer (pd) element was found to be unable to stimulate transcription when present in one copy as the only promoter element in a minimal promoter but showed weak stimulatory activity when present as a multimer (four copies). One copy of the pd element acted synergistically with an octamer element, but not with a SP1 site, to stimulate transcription. The effect was orientation dependent with regard to the pd element. Gel shift analysis showed that pd-binding proteins were expressed in transformed as well as nontransformed B lymphocytes, irregardless of their differentiation stage, and in HeLa cells. Two major complexes, binding to different sites within the pd element, were observed in gel shifts. A low-molecular-weight form dominated in resting cells, while a higher-molecular-weight form appeared after mitogenic stimulation. Southwestern analysis showed that the low-molecular-weight pd-binding protein had a molecular mass of 35 kDa, which was confirmed by fractionation by denaturating polyacrylamide gel electrophoresis and molecular sieving. The higher-molecular-weight complex was sensitive to detergent treatment, while the low-molecular-weight complex was not. Mutation analysis showed that the two pd-binding complexes interacted with distinct sites within the element and that dual occupancy was required for functional activity. The functional synergy between the pd element and the octamer was more pronounced in plasmacytomas than in B-cell lymphomas.

The intact SP6 kappa promoter stimulated transcription 30 times more efficiently than did a control promoter consisting of a TATA motif as the only promoter element. Mutation of the SP6 kappa promoter decamer in two positions reduced the transcriptional stimulation activity by over 90%. Promoters containing the SP6 kappa promoter octamer or a consensus octamer in front of a TATA box were ineffective immunoglobulin promoters and stimulated at the most 15% of maximal transcription. Identical results were obtained after transfection of untransformed mouse splenic B cells stimulated by lipopolysaccharide, that express high levels of Oct2A, or of S194 cells that express negligible levels of Oct2A. Selective mutations in the penta-decamer (pd), kappa Y or early B cell factor (EBF) elements of the promoter reduced transcriptional stimulation by 20-30% in untransformed B cells. In S194 plasmacytoma cells the EBF mutation was functionally silent while the kappa Y and pd mutations reduced transcriptional activation by 60-70% in this cell line. A mutation in a TATA-proximal E-box motif did not alter the functional activity of the promoter in either cell population. It can be concluded that kappa promoter function is highly dependent on complex interactions between individual promoter elements and that the decamer motif is pivotal for these interactions. The relative functional activity of a given promoter varied according to the target cell population used for the functional assay.

The gut immune system protects against mucosal pathogens, maintains a mutualistic relationship with the microbiota, and establishes tolerance against food antigens. This requires a balance between immune effector responses and induction of tolerance. Disturbances of this strictly regulated balance can lead to infections or the development inflammatory diseases and allergies. Production of secretory IgA is a unique effector function at mucosal surfaces, and basal mechanisms regulating IgA production have been the focus of much recent research. These investigations have aimed at understanding how long-term IgA-mediated mucosal immunity can best be achieved by oral or sublingual vaccination, or at analyzing the relationship between IgA production, the composition of the gut microbiota, and protection from allergies and autoimmunity. This research has lead to a better understanding of the IgA system; but at the same time seemingly conflicting data have been generated. Here, we discuss how gut IgA production is controlled, with special focus on how differences between T cell-dependent and T cell-independent IgA production may explain some of these discrepancies.

Because Peyer's patches (PP) are the main inductive sites for gut IgA responses we have focused this review on what we know about the function of PP germinal centers (GC). The vast majority of IgA gene sequences in the gut lamina propria (LP) are heavily mutated arguing for an origin in GC. Because PP GC formation is dependent on the presence of CD4 T cells, we speculate that all IgA responses in the normal gut are directly or indirectly T cell-dependent (TD). We hypothesize that the CD4 T cell involvement in gut IgA responses against the microbiota is different from that in systemic responses since cognate T-B cell interactions appear not to be required. In the absence of cognate interactions the function of CD4 follicular helper T cells (Tfh) in PP GC is unclear. However, production of IL-21 and IL-6 is more pronounced than in peripheral lymph nodes. Importantly, we discuss how multiple PP are involved in generating specific IgA responses to TD antigens given orally. Recently we found that oral immunization with NP-hapten conjugated to cholera toxin (NP-CT) stimulated a strong highly synchronized, oligoclonal and affinity matured IgA response. This was achieved through re-utilization of GC in multiple PP as GC IgA B cells emigrated into already established GC. Clonally related B cells were present in both inductive and effector lymphoid tissues in the gut and clonal trees involving multiple PP could be constructed in individual mice. Through adoptive transfer of B1-8(hi) NP-specific B cells we demonstrated that GL7(+) PP B cells could enter into pre-existing GC in PP, a process that was antigen-dependent but did not to require cognate Tfh interactions. Finally, we discuss the role of PP GC for the generation of memory B cells and long-lived plasma cells in the light of contrasting findings regarding IgA memory development to colonizing commensal bacteria versus that to oral immunization with enteropathogens or TD antigens.

Summary OTHER ARTICLES PUBLISHED IN THIS MINI-REVIEW SERIES ON B CELL SUBSETS IN DISEASE B cells in multiple sclerosis: drivers of disease pathogenesis and Trojan horse for Epstein—Barr virus entry to the central nervous system? Clinical and Experimental Immunology 2012, 167: 1–6. Transitional B cells in systemic lupus erythematosus and Sjögren's syndrome: clinical implications and effects of B cell-targeted therapies. Clinical and Experimental Immunology 2012, 167: 7–14. Haematopoietic stem cell transplantation (HSCT) is an immunological treatment that has been used for more than 40 years to cure a variety of diseases. The procedure is associated with serious side effects, due to the severe impairment of the immune system induced by the treatment. After a conditioning regimen with high-dose chemotherapy, sometimes in combination with total body irradiation, haematopoietic stem cells are transferred from a donor, allowing a donor-derived blood system to form. Here, we discuss the current knowledge of humoral problems and B cell development after HSCT, and relate these to the current understanding of human peripheral B cell development. We describe how these studies have aided the identification of subsets of transitional B cells and also a robust memory B cell phenotype.

A screen of cell surface markers differentially expressed during peripheral B cell differentiation identified that the CD45RB epitope detected by the mAb MEM-55 was highly expressed on CD27+ memory B cells and absent on CD27– naïve B cells. IgG+CD27– memory and a previously unacknowledged CD27– population in blood also expressed high levels of CD45RBMEM55. Naïve and memory B cells from tonsils followed the pattern observed in blood, and CD38high B cells had a bimodal expression pattern when analyzed using flow cytometry. No CD38high GC B cells, however, expressed the CD45RBMEM55 epitope when assayed using immunohistochemistry. Rather, CD38highCD45RBMEM55high B cells had a distinct cellular phenotype and were localized outside of GCs. CD45RB epitopes, detected by other antibody clones, were expressed at high levels through B cell differentiation, and no changes in splicing of the CD45RB exon were observed during B cell differentiation. Instead, B cells regulated their expression of the CD45RBMEM55 epitope through site-specific modifications of an O-linked glycochain. CD4+ T cells differentially spliced CD45 but did not vary the glycosylation of the CD45RBMEM55 epitope, and CD8+ cells modified CD45RBMEM55 expression in a similar manner as B cells. Monocytes expressed the CD45RB exon but not the CD45RBMEM55 epitope. As CD45 is a highly expressed tyrosine phosphatase that regulates antigen receptor signaling strength in lymphocytes, we conclude that regulated O-linked glycosylation of CD45RB can be used to follow B cell differentiation and that this regulation may be involved in fine-tuning antigen signaling in the cell.

The ultimate goal for vaccination is to stimulate protective immunological memory. Protection against infectious diseases not only relies on the magnitude of the humoral immune response, but more importantly on the quality and longevity of it. Adjuvants are critical components of most non-living vaccines. Although little attention has been given to qualitative aspects of the choice of vaccine adjuvant, emerging data demonstrate that this function may be central to vaccine efficacy. In this review we describe efforts to understand more about how adjuvants influence qualitative aspects of memory development. We describe recent advances in understanding how vaccines induce long-lived plasma and memory B cells, and focus our presentation on the germinal center reaction. As mucosal vaccination requires powerful adjuvants, we have devoted much attention to the adenosine diphosphate (ADP)-ribosylating cholera toxin and the CTA1-DD adjuvants as examples of how mucosal adjuvants can influence induction of long-term memory.

During the last two decades our understanding of human B cell differentiation has developed considerably. Our understanding of the human B cell compartment has advanced from a point where essentially all assays were based on the presence or not of class-switched antibodies to a level where a substantial diversity is appreciated among the cells involved. Several consecutive transitional stages that newly formed IgM expressing B cells go through after they leave the bone marrow, but before they are fully mature, have been described, and a significant complexity is also acknowledged within the IgM expressing and class-switched memory B cell compartments. It is possible to isolate plasma blasts in blood to follow the formation of plasma cells during immune responses, and the importance and uniqueness of the mucosal IgA system is now much more appreciated. Current data suggest the presence of at least one lineage of human innate-like B cells akin to B1 and/or marginal zone B cells in mice. In addition, regulatory B cells with the ability to produce IL-10 have been identified. Clinically, B cell depletion therapy is used for a broad range of conditions. The ability to define different human B cell subtypes using flow cytometry has therefore started to come into clinical use, but as our understanding of human B cell development further progresses, B cell subtype analysis will be of increasing importance in diagnosis, to measure the effect of immune therapy and to understand the underlying causes for diseases. In this review the diversity of human B cells will be discussed, with special focus on current data regarding their phenotypes and functions.

The catalytic subunit of polymerase zeta is encoded from the Rev3 gene. The enzyme is conserved through eukaryotic evolution and its main function appears to be translesion synthesis (TLS) over damaged bases that stall DNA replication. In non-vertebrate cells, inactivation of polymerase zeta results in a moderate hypersensitivity to DNA damage but no proliferative defect in the absence of exogenous damage. Mouse embryos that lack Rev3 however have a severe growth defect and are aborted at midgestation. This has suggested that polymerase zeta may be involved in vital processes in mammalian cells. Here we describe the establishment of immortalized mouse fibroblast cell lines that lack a functional Rev3 gene. These were established from homozygously Rev3-targeted mouse embryos that were also heterozygously targeted at the p53 locus, but the cell lines lost the wild type p53 allele during transformation. Cell lines in which the Rev3 gene is targeted on both alleles grow more slowly than control lines and the deficiency is also associated with an increased frequency of cells at the G2/M phase of the cell cycle and augmented apoptosis. Targeted cells are hypersensitive to UV irradiation and cisplatin treatment and arrest at the S or G2/M phase of the cell cycle if exposed to these treatments. Thus, although vital for murine embryonic development, polymerase zeta activity is not essential for continuous proliferation of transformed mammalian cells that lack p53. It does, however, appear to play an important role in allowing mammalian cells to tolerate DNA damage.

Recent debate over the ability to generate memory B cells in the gut immune system prompted us to explore a novel model with transgenic NP-hapten-specific GFP+ B cells and oral immunizations with NP-conjugated to cholera toxin (NP-CT). We found that orally immunized mice developed exceptional long term memory B cell populations that resided in B cell follicles in Peyer’s patches, mesenteric lymph nodes and spleen. A majority of the GFP+ memory cells, defined by CD80, CD73 and PD-L2 expression, were IgM+, but also a significant number were IgA+ and in spleen IgG+ memory B cells were frequent. Upon analysis of the NP binding VH186.2 IgA gene sequences between 20-40 % carried high affinity mutations and VH186.2 IgG (bone marrow) and IgA (intestine and bone marrow) gene sequences were clonally related. A challenge immunization after 1.5 years, either orally or i.p, elicited a strong NP-specific gut lamina propria (LP) IgA and serum IgG response, starting from a calculated frequency of 1 NP-memory / total 10.000 B cells in these mice. Gut LP IgA plasma cells increased after challenge from 0.05% long-lived NP-specific IgA plasma cells to >5% NP-specific IgA plasma cells of all LP IgA plasma cells. The boosted IgA plasma cells carried more mutations and higher affinities (>60%) than long-lived memory B cells, demonstrating unexpectedly effective selection and maturational processes in gut IgA memory responses.

Somatic hypermutation and isotype switch recombination occur in germinal center B cells, are linked to transcription, and are similarly affected by deficiency in MutS homologue (MSH)2. Class-switch recombination is abrogated by disruption of genes encoding components of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs)/Ku complex and likely involves nonhomologous end joining (NHEJ). That somatic hypermutation might also be associated with end joining is suggested by its association with the creation of deletions, duplications, and sites accessible to terminal transferase. However, a requirement for NHEJ in the mutation process has not been demonstrated. Here we show that somatic mutation in mice deficient in NHEJ can be tested by introduction of rearranged immunoglobulin and T cell receptor transgenes: the transgene combination not only permits reconstitution of peripheral lymphoid compartments but also allows formation of germinal centers, despite the wholly monoclonal nature of the lymphocyte antigen receptors in these animals. Using this strategy, we confirm that somatic hypermutation like class-switching can occur in the absence of recombination-activating gene (RAG)1 but show that the two processes differ in that hypermutation can proceed essentially unaffected by deficiency in DNA-PKcs activity.

Multiple DNA polymerases exist in eukaryotes. Polymerases α , δ and e are mainly responsible for chromosomal DNA replication in the nucleus and are required for proliferation. In contrast, the repair polymerases β and η are not essential for cellular proliferation in yeast or mice, but a lack of either polymerase can lead, respectively, to defects in base excision repair or the ability to replicate past lesions induced by ultraviolet (UV) radiation [1–3]. Here, we have focused on polymerase ζ . This was first described as a non-essential product of the yeast REV3 / REV7 genes involved in UV-induced mutagenesis, and was later implicated in trans-lesion synthesis [4,5]. Unlike in yeast, the mouse homologue ( mRev3 ) was found to be essential for life. Homozygous mutant mice died in utero . Mutant embryos were considerably reduced in size at day 10.5 of development and usually aborted around day 12.5. It is likely that this block reflects a need for mRev3 in proliferative clonal expansion (rather than in the production of a particular cell type) as mutant blastocysts showed greatly diminished expansion of the inner cell mass in culture. Thus, mRev3 could be required to repair a form of externally induced DNA damage that otherwise accumulates during clonal expansion or, consistent with the high homology shared between its Rev7 partner and the mitotic checkpoint gene product Mad2 [6], mRev3 might play a role in cell proliferation and genomic stability even in the absence of environmentally induced damage.

The physiological mechanism for producing antigen-specific antibodies is based on a two-phase neo-Darwinian process: the first phase consists of diversity generation (formation of the repertoire), and the second phase is antigen-mediated selection. In this article, we consider how the natural immunoglobulin gene-diversification processes can be exploited both in vivo and in vitro in order to allow the generation of novel antibody (and heterologous protein) repertoires.

Immunoglobulin kappa promoters show sequence divergence but conserved function between different subgroups. Here we show that three separate 5' elements are required for synergistic stimulation of transcription with the decamer in a kappa promoter. These sites are a 5' E-box, a 3' AT-rich region in the pentadecamer (pd) element, and the kappa-Y element. Elf-1 is a novel kappa-Y element ligand induced upon mitogenic stimulation of resting B lymphocytes. Furthermore, the 5' E2A-like E-box in the pd element could be substituted by an upstream stimulatory factor motif with conservation of function. Thus, the synergistic activation requirements of kappa transcription is strictly dependent on the quantitative presence of transcription factor-binding motifs 5' of the decamer, but these differ qualitatively in that they may bind an array of proteins with conserved function.

Whereas recent studies have pointed to multi-centered and diverse gut IgA responses to the microbiota, little information is available on IgA responses following oral immunization with T cell-dependent antigens. Here we have addressed how gut IgA responses develop using a novel approach where NP-hapten was conjugated to cholera toxin (CT), which allowed us to follow, at the molecular level, the site of initiation, expansion, differentiation and distribution of a specific IgA B cell response. Clonal relationships and affinity maturation of specific IgA cells at gut inductive and effector sites were investigated. Unexpectedly, we found gut IgA B cell responses to be oligoclonal and dominated by high affinity maturation. Extensive lineage trees of gut NP-specific IgA cells were generated, revealing strong clonal relationships throughout the entire gut mucosal immune system. Thus, clonally related IgA cells were found in Peyer’s patches, mesenteric lymph nodes and the small and large intestine, suggesting an effective expansion and selection process. This was achieved through synchronization of multiple PP hosting the same high affinity B cell clones. Adoptive transfer experiments showed that this was possible through re-utilization of already existing germinal centers (GC) in multiple PP by previously activated GC GL7+ B cells. The re-utilization required that oral antigen was given prior to cell transfer.

Gut-derived antigens trigger immunoglobulin A (IgA) immune responses that are initiated by cognate B cells in Peyer's patches (PPs). These cells colonize the subepithelial domes (SEDs) of the PPs and subsequently infiltrate pre-existing germinal centers (GCs). Here we defined the pre-GC events and the micro-anatomical site at which affinity-based B cell selection occurred in PPs. Using whole-organ imaging, we showed that the affinity of the B cell antigen receptor (BCR) regulated the infiltration of antigen-specific B cells into GCs but not clonal competition in the SED. Follicular helper-like T cells resided in the SED and promoted its B cell colonization, independently of the magnitude of BCR affinity. Imaging and immunoglobulin sequencing indicated that selective clonal expansion ensued during infiltration into GCs. Thus, in contrast to the events in draining lymph nodes and spleen, in PPs, T cells promoted mainly the population expansion of B cells without clonal selection during pre-GC events. These findings have major implications for the design of oral vaccines.

The germinal center (GC) reaction in Peyer′s patches (PP) requires continuous access to antigens, but how this is achieved is not known. Here we show that activated antigen-specific CCR6+CCR1+GL7− B cells make close contact with M cells in the subepithelial dome (SED). Using in situ photoactivation analysis of antigen-specific SED B cells, we find migration of cells towards the GC. Following antigen injection into ligated intestinal loops containing PPs, 40% of antigen-specific SED B cells bind antigen within 2 h, whereas unspecifc cells do not, indicating B cell-receptor involvment. Antigen-loading is not observed in M cell-deficient mice, but is unperturbed in mice depleted of classical dendritic cells (DC). Thus, we report a M cell-B cell antigen-specific transporting pathway in PP that is independent of DC. We propose that this antigen transporting pathway has a critical role in gut IgA responses, and should be taken into account when developing mucosal vaccines., Gut lumen antigens must be continuously sampled by the immune system to maintain proper immune homeostasis. Here the authors show that activated CCR6+CCR1+GL7- gut B cells retrieve lumen antigens from specialized M cells and transfer them across the subepithelial dome in the Peyer’s patch to contribute to the maintenance of gut humoral immunity.

Summary Antibodies secreted within the intestinal tract provide protection from the invasion of microbes into the host tissues. Germinal center (GC) formation in lymph nodes and spleen strictly requires SLAM-associated protein (SAP)-mediated T cell functions; however, it is not known whether this mechanism plays a similar role in mucosal-associated lymphoid tissues. Here, we find that in Peyer’s patches (PPs), SAP-mediated T cell help is required for promoting B cell selection in GCs, but not for clonal diversification. PPs of SAP-deficient mice host chronic GCs that are absent in T cell-deficient mice. GC B cells in SAP-deficient mice express AID and Bcl6 and generate plasma cells in proportion to the GC size. Single-cell IgA sequencing analysis reveals that these mice host few diversified clones that were subjected to mild selection forces. These findings demonstrate that T cell-derived help to B cells in PPs includes SAP-dependent and SAP-independent functions., Graphical Abstract, Highlights • Chronic germinal centers in Peyer’s patches are formed in SAP-deficient mice • SAP-independent germinal centers arise in response to influenza infection • Few highly diversified clones dominate the SAP-independent germinal centers • Germinal center B cells in SAP-deficient mice are subjected to mild selection forces, SAP is required for proper T cell help in germinal centers (GCs). Biram et al. show that SAP-independent GCs are formed within Peyer’s patches. These GCs host highly diversified clones that are subjected to mild selection forces, demonstrating that clonal diversification can be uncoupled from clonal selection in chronic GCs.

B cell responses are critical for antiviral immunity. However, a comprehensive picture of antigen-specific B cell differentiation, clonal proliferation, and dynamics in different organs after infection is lacking. Here, by combining single-cell RNA and B cell receptor (BCR) sequencing of antigen-specific cells in lymph nodes, spleen, and lungs after influenza infection in mice, we identify several germinal center (GC) B cell subpopulations and organ-specific differences that persist over the course of the response. We discover transcriptional differences between memory cells in lungs and lymphoid organs and organ-restricted clonal expansion. Remarkably, we find significant clonal overlap between GC-derived memory and plasma cells. By combining BCR-mutational analyses with monoclonal antibody (mAb) expression and affinity measurements, we find that memory B cells are highly diverse and can be selected from both low- and high-affinity precursors. By linking antigen recognition with transcriptional programming, clonal proliferation, and differentiation, these finding provide important advances in our understanding of antiviral immunity.