Enhanced B Cell Receptor Signaling Partially Compensates for Impaired Toll-like Receptor 4 Responses in LPS-Stimulated IκBNS-Deficient B Cells.

Lipopolysaccharide (LPS) stimulates dual receptor signaling by bridging the B cell receptor and Toll-like receptor 4 (BCR/TLR4). B cells from IκBNS-deficient bumble mice treated with LPS display reduced proliferative capacity and impaired plasma cell differentiation. To improve our understanding of the regulatory role of IκBNS in B cell activation and differentiation, we investigated the BCR and TLR4 signaling pathways separately by using dimeric anti-IgM Fab (F(ab')2) or lipid A, respectively. IκBNS-deficient B cells exhibited reduced survival and defective proliferative capacity in response to lipid A compared to B cells from wildtype (wt) control mice. In contrast, anti-IgM stimulation of bumble B cells resulted in enhanced viability and increased differentiation into CD138+ cells compared to control B cells. Anti-IgM-stimulated IκBNS-deficient B cells also showed enhanced cycle progression with increased levels of c-Myc and cyclin D2, and augmented levels of pCD79a, pSyk, and pERK compared to control B cells. These results suggest that IκBNS acts as a negative regulator of BCR signaling and a positive regulator of TLR4 signaling in mouse B cells.

IκBNS expression in B cells is dispensable for IgG responses to T cell-dependent antigens.

Mice lacking the atypical inhibitory kappa B (IκB) protein, IκBNS, a regulator of the NF-κB pathway encoded by the nfkbid gene, display impaired antibody responses to both T cell-independent (TI) and T cell-dependent (TD) antigens. To better understand the basis of these defects, we crossed mice carrying floxed nfkbid alleles with mice expressing Cre under the transcriptional control of the Cd79a gene to create mice that lacked IκBNS expression only in B cells. Analyses of these conditional knock-out mice revealed intact CD4+ and CD8+ T cell populations, including preserved frequencies of FoxP3+ regulatory T cells, which are known to be reduced in IκBNS knock-out mice. Like IκBNS knock-out mice, mice with conditional IκBNS ablation in B cells displayed defective IgM responses to TI antigens and a severe reduction in peritoneal B-1a cells. However, in contrast to mice lacking IκBNS altogether, the conditional IκBNS knock-out mice responded well to TD antigens compared to the control mice, with potent IgG responses following immunization with the viral antigen, rSFV-ßGal or the widely used hapten-protein model antigen, NP-CGG. Furthermore, B cell intrinsic IκBNS expression was dispensable for germinal center (GC) formation and T follicular helper cell responses to NP-CGG immunization. The results presented here suggest that the defect in antibody responses to TD antigens observed in IκBNS knock-out mice results from a B cell extrinsic defect.

Impaired plasma cell differentiation associates with increased oxidative metabolism in IκBNS-deficient B cells.

Mutations causing loss of the NF-κB regulator IκBNS, result in impaired development of innate-like B cells and defective plasma cell (PC) differentiation. Since productive PC differentiation requires B cell metabolic reprogramming, we sought to investigate processes important for this transition using the bumble mouse strain, deficient for IκBNS. We report that LPS-activated bumble B cells exhibited elevated mTOR activation levels, mitochondrial accumulation, increased OXPHOS and mROS production, along with a reduced capacity for autophagy, compared to wildtype B cells. Overall, our results demonstrate that PC differentiation in the absence of IκBNS is characterized by excessive activation during early rounds of B cell division, increased mitochondrial metabolism and decreased autophagic capacity, thus improving our understanding of the role of IκBNS in PC differentiation.

Flow Cytometry-Based Protocols for the Analysis of Human Plasma Cell Differentiation.

Humoral immunity is established after differentiation of antigen-specific B cells into plasma cells (PCs) that produce antibodies of relevant specificities. Defects in the development, activation, or differentiation of B cells severely compromises the immune response. Primary immunodeficiencies are often characterized by hypogammaglobulinemia and the inability to mount effective antigen-specific antibody responses, resulting in increased susceptibility to infections. After IgA deficiency, which is most often asymptomatic, common variable immunodeficiency (CVID) is the most prevalent symptomatic primary immunodeficiency, but in most cases the underlying genetic causes are unknown or their roles in disease pathogenesis are poorly understood. In this study, we developed a protocol for in vitro stimulation of primary human B cells for subsequent analyses of PC differentiation and antibody production. With this approach, we were able to detect a population of CD38+ IRF4+ Blimp-1+ cells committed to PC fate and IgG production, including when starting from cryopreserved samples. The application of functional assays to characterize PC differentiation and possible defects therein in B cells from patients suffering from primary antibody deficiencies with late B cell defects could increase our understanding of the disease pathophysiology and underlying mechanisms.

TACI expression and plasma cell differentiation are impaired in the absence of functional IκBNS.

Impaired classical NF-κB pathway signaling causes reduced antibody responses to T-independent (TI) antigens. We investigated the potential reasons for defective TI responses in mice lacking the atypical inhibitory kappa B (IκB) protein of the NF-κB pathway, IκBNS. Analyses of the plasma cell compartment in vitro and in vivo after challenge with lipopolysaccharide (LPS) showed significant decreases in the frequencies of plasma cells in the absence of IκBNS. In vitro activation of B cells via the B cell receptor or via Toll-like receptor 4 revealed that early activation events were unaffected in IκBNS-deficient B cells, while proliferation was reduced compared to in similarly stimulated wildtype (wt) B cells. IκBNS-deficient B cells also displayed impaired upregulation of the transmembrane activator and calcium modulator cyclophilin ligand interactor (TACI), which is essential for TI responses, and decreased sensitivity to TACI ligands upon stimulation. Furthermore, IκBNS-deficient B cells, in contrast to wt B cells, displayed altered expression of IRF4, Blimp-1 and Pax5 upon LPS-induced differentiation, indicating impaired transcriptional regulation of plasma cell generation.

B-1a Cell Development in Splenectomized Neonatal Mice.

B-1a cells are mainly generated from fetal liver progenitor cells, peri- and neonatally. The developmental steps and anatomical sites required for these cells to become mature B-1a cells remain elusive. We recently described a phenotypically distinct transitional B cell subset in the spleen of neonatal mice that generated B-1a cells when adoptively transferred. This, in combination with findings demonstrating that B-1a cells are lacking in congenitally asplenic mice, led us to hypothesize that the neonatal spleen is required for B-1a cell development. In accordance with previous reports, we found that B-1a cell numbers were reduced in adult mice that had undergone splenectomy compared to after sham surgery. In contrast, neonatal splenectomy led to peritoneal B-1a cell frequencies comparable to those observed in sham-operated mice until 6 weeks after surgery, suggesting that an intact spleen is required for B-1a cell maintenance rather than development. To study the role of the prenatal spleen in generating B-1a cells, we transferred fetal liver cells from pre-splenic embryos [embryonic age 11 (E11) days] into splenectomized recipient mice. B-1a cells were generated in the absence of the spleen, albeit at slightly reduced frequencies, and populated the peritoneal cavity and bone marrow. Lower bone marrow B-1a cell frequencies were also observed both after neonatal and adult splenectomy. These results demonstrated that B-1a cells could be generated in the complete absence of an intact spleen, but that asplenia led to a decline in these cells, suggesting a role of the spleen for maintaining the B-1a compartment.

Altered Marginal Zone B Cell Selection in the Absence of IκBNS.

Marginal zone (MZ) B cells reside in the splenic MZ and play important roles in T cell-independent humoral immune responses against blood-borne pathogens. IκBNS-deficient bumble mice exhibit a severe reduction in the MZ B compartment but regain an MZ B population with age and, thus, represent a valuable model to examine the biology of MZ B cells. In this article, we characterized the MZ B cell defect in further detail and investigated the nature of the B cells that appear in the MZ of aged bumble mice. Flow cytometry analysis of the splenic transitional B cell subsets demonstrated that MZ B cell development was blocked at the transitional-1 to transitional-2-MZ precursor stage in the absence of functional IκBNS. Immunohistochemical analysis of spleen sections from wild-type and bumble mice revealed no alteration in the cellular MZ microenvironment, and analysis of bone marrow chimeras indicated that the MZ B cell development defect in bumble mice was B cell intrinsic. Further, we demonstrate that the B cells that repopulate the MZ in aged bumble mice were distinct from age-matched wild-type MZ B cells. Specifically, the expression of surface markers characteristic for MZ B cells was altered and the L chain Igλ+ repertoire was reduced in bumble mice. Finally, plasma cell differentiation of sorted LPS-stimulated MZ B cells was impaired, and aged bumble mice were unable to respond to NP-Ficoll immunization. These results demonstrate that IκBNS is required for an intact MZ B cell compartment in C57BL/6 mice.

Heterozygous Mutation in IκBNS Leads to Reduced Levels of Natural IgM Antibodies and Impaired Responses to T-Independent Type 2 Antigens.

Mice deficient in central components of classical NF-κB signaling have low levels of circulating natural IgM antibodies and fail to respond to immunization with T-independent type 2 (TI-2) antigens. A plausible explanation for these defects is the severely reduced numbers of B-1 and marginal zone B (MZB) cells in such mice. By using an ethyl-N-nitrosourea mutagenesis screen, we identified a role for the atypical IκB protein IκBNS in humoral immunity. IκBNS-deficient mice lack B-1 cells and have severely reduced numbers of MZB cells, and thus resemble several other strains with defects in classical NF-κB signaling. We analyzed mice heterozygous for the identified IκBNS mutation and demonstrate that these mice have an intermediary phenotype in terms of levels of circulating IgM antibodies and responses to TI-2 antigens. However, in contrast to mice that are homozygous for the IκBNS mutation, the heterozygous mice had normal frequencies of B-1 and MZB cells. These results suggest that there is a requirement for IκBNS expression from two functional alleles for maintaining normal levels of circulating natural IgM antibodies and responses to TI-2 antigens.

B-1a transitional cells are phenotypically distinct and are lacking in mice deficient in IκBNS.

B-1 cells mediate early protection against infection by responding to T cell-independent (TI) antigens found on the surface of various pathogens. Mice with impaired expression of the atypical IκB protein IκBNS have markedly reduced frequencies of B-1 cells. We used a mouse strain with dysfunctional IκBNS derived from an N-ethyl-N-nitrosourea (ENU) screen, named bumble, to investigate the point in the development of B-1 cells where IκBNS is required. The presence of wild-type (wt) peritoneal cells in mixed wt/bumble chimeras did not rescue the development of bumble B-1 cells, but wt peritoneal cells transferred to bumble mice restored natural IgM levels and response to TI antigens. The bumble and wt mice displayed similar levels of fetal liver B-1 progenitors and splenic neonatal transitional B (TrB) cells, both of which were previously shown to give rise to B-1 cells. Interestingly, we found that a subset of wt neonatal TrB cells expressed common B-1a markers (TrB-1a) and that this cell population was absent in the bumble neonatal spleen. Sorted TrB-1a (CD93(+)IgM(+)CD5(+)) cells exclusively generated B-1a cells when adoptively transferred, whereas sorted CD93(+)IgM(+)CD5(-) cells gave rise to B-2 cells and, to a lesser extent, B-1b and B-1a cells. This study identifies a phenotypically distinct splenic population of TrB-1a cells and establishes that the development of B-1a cells is blocked before this stage in the absence of IκBNS.