Significance A subpopulation of antibody-secreting cells, B-1 cells, provides early protection against several types of pathogens. Both the development and function differ between B-1 cells and the better known B-2 cells, and exclusively B-1 cells are lacking in mice deficient for the nuclear inhibitory κB protein, IκBNS. B-1 cells mature similar to B-2 cells via a transitional stage. We demonstrate here the existence of a phenotypically distinct B-1 transitional B (TrB)-cell population in the neonatal spleen of wild-type mice. This TrB-1a–cell subset was lost in the absence of IκBNS, thus revealing a requirement for intact NF-κB signaling via IκBNS during this stage of the development of B-1 cells. Learning more about the development of B-1 cells may reveal new targets for therapeutic intervention.

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.

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 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 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 mice revealed no alteration in the cellular MZ microenvironment, and analysis of bone marrow chimeras indicated that the MZ B cell development defect in mice was B cell intrinsic. Further, we demonstrate that the B cells that repopulate the MZ in aged 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 mice. Finally, plasma cell differentiation of sorted LPS-stimulated MZ B cells was impaired, and aged 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.

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.

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.

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.

Serological studies are critical for understanding pathogen−specific immune responses and informing public health measures (1,2). By developing highly sensitive and specific trimeric spike (S)−based antibody tests, we report IgM, IgG and IgA responses to SARS−CoV−2 in COVID−19 patients (n=105) representing different categories of disease severity. All patients surveyed were IgG positive against S. Elevated anti−SARS−CoV−2 antibody levels were associated with hospitalization, with IgA titers, increased circulating IL−6 and strong neutralizing responses indicative of intensive care status. Antibody−positive blood donors and pregnant women sampled during the pandemic in Stockholm, Sweden (weeks 14−25), displayed on average lower titers and weaker neutralizing responses compared to patients; however, inter−individual anti−viral IgG titers differed up to 1,000−fold. To provide more accurate estimates of seroprevalence, given the frequency of weak responders and the limitations associated with the dichotomization of a continuous variable (3,4), we used a Bayesian approach to assign likelihood of past infection without setting an assay cut−off. Analysis of blood donors (n=1,000) and pregnant women (n=900) sampled weekly demonstrated SARS-CoV-2-specific IgG in 7.2% (95% Bayesian CI [5.1−9.5]) of individuals two months after the peak of spring 2020 COVID−19 deaths. Seroprevalence in these otherwise healthy cohorts increased steeply before beginning to level−off, following the same trajectory as the Stockholm region deaths over this time period.

No abstract