Characterization of B cell response alterations resulting from ablation of IκBNS expression

The immune system is a sophisticated organization of cells and tissues that cooperate in safeguarding the integrity of our body by eliminating internal and external threats. Vaccines are the most impactful medical intervention and have contributed tremendously to lowering mortality rates of infectious diseases. The protective immunity induced by vaccination is mediated by eliciting neutralizing antibodies that are sustained for years or even for a lifetime. Antibodies are produced by plasma cells, which are generated from activated and terminally differentiated B cells. Many processes regulating B cell development and function are under the regulation of the NF-κB signaling pathway. Defects in NF-κB signaling have been shown, using mouse models, to be detrimental to the development of distinct B cell subsets as well as their ability to participate in humoral responses. In humans, mutations of components in the NF-κB pathway are increasingly associated with common variable immunodeficiencies. In this thesis, the role of IκBNS, a nuclear regulator of the NF-κB pathway, was investigated to gain a more comprehensive understanding of B cell development and function. In paper I, we addressed the absence of B-1a cells in IκBNS-deficient bumble mice. We identified a precursor population in wildtype mice of IgM+CD93+CD19+CD5+ transitional B1a cells (TrB-1a), which exhibited other indicators of B-1 lineage commitment, such as natural IgM secretion and increased Igλ expression. We did not detect TrB-1a cells in bumble mice whereas the neonatal B-1 progenitor cell (B-1p) population was intact. These results indicate that IκBNS is required for the transition from B-1p to the TrB-1a stage. In paper II, we investigated potential reasons for the impaired T cell-independent (TI) responses in bumble mice. We found impaired expression of the surface receptor TACI, which is essential in responses to TI antigens, and reduced responsiveness to its ligands in bumble mice. In addition, bumble B cells did not fully silence Pax5 expression and exhibited rapid upregulation of Blimp1 during early division cycles. Thus, these results suggest that IκBNS is involved in TACI expression and function as well as in transcriptional regulation of PC differentiation. In paper III, we used nfkbidB- mice in which IκBNS was selectively depleted from B cells to distinguish between B cell intrinsic and extrinsic defects in T cell-independent (TD) responses. NfkbidB- mice exhibited normal GC formation, but antigen-specific antibodies of the IgG2c and IgG3 isotype were reduced. Hence, IκBNS appears to be important for class switching during TD responses. In paper IV, we developed a protocol for evaluation of plasma cell differentiation from human primary B cells. Cells that exhibited a phenotype resembling plasma cells were identified as CD38+IRF4hiPax5lo and CD38+IRF4intPax5lo. Both populations were generated when starting from fresh or cryopreserved samples, or isolated memory and naïve B cells. Application of the methods developed in this paper to patient samples could provide more insight into mechanisms underlying immune disorders. Collectively, the results of this thesis contribute to our understanding of processes that are fundamental to establishing adequate humoral responses and are of direct relevance to immunodeficiency disorders

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

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

Image_3_B-1a Cell Development in Splenectomized Neonatal Mice.tiff

<p>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.</p

Image_2_B-1a Cell Development in Splenectomized Neonatal Mice.tif

<p>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.</p

Image_1_B-1a Cell Development in Splenectomized Neonatal Mice.tiff

<p>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.</p