BLNK is an adaptor protein that functions in B-cell receptor signalling, and is vitally necessary at signalling checkpoints of mammalian B-cell development. However, its importance to avian B-cell development remains unclear. To explore the function of BLNK in chickens, shRNA-mediated RNA interference was delivered to a chicken B-cell line in vitro by replication-competent avian retrovirus (RCAS), and effective shRNA were determined. To observe an shRNA phenotype on chicken B-cell development, we simultaneously explored whether RCAS penetrance was correlated between red blood cells (RBC) and bursal B-cells by infecting chicken embryos with RCAS expressing a fluorescent tag. We found that RCAS penetrance was correlated between RBC and B-cells, which provides a system to observe any in vivo effects of BLNK shRNA on B-cell development. Furthermore, this system for observing BLNK function may be complemented by genetically-modified BLNK, particularly variants resistant to RNA interference.

BLNK is an adaptor protein that functions in B-cell receptor signalling, and is vitally necessary at signalling checkpoints of mammalian B-cell development. However, its importance to avian B-cell development remains unclear. To explore the function of BLNK in chickens, shRNA-mediated RNA interference was delivered to a chicken B-cell line in vitro by replication-competent avian retrovirus (RCAS), and effective shRNA were determined. To observe an shRNA phenotype on chicken B-cell development, we simultaneously explored whether RCAS penetrance was correlated between red blood cells (RBC) and bursal B-cells by infecting chicken embryos with RCAS expressing a fluorescent tag. We found that RCAS penetrance was correlated between RBC and B-cells, which provides a system to observe any in vivo effects of BLNK shRNA on B-cell development. Furthermore, this system for observing BLNK function may be complemented by genetically-modified BLNK, particularly variants resistant to RNA interference.

During a T-dependent immune response, activated B cells enter structures called germinal centers (GC) in the follicles of secondary lymphoid organs. GC B cells proliferate and undergo diversification of their Ig through somatic hypermutation and class-switch recombination. These Ig diversifications require the activity of the enzyme activation-induced deaminase (AID). Clonal selection within GCs selects GC B cells with the highest affinities for antigen to mature into plasma cells and memory B cells. GCs are underpinned by stromal cells called follicular dendritic cells (FDC). FDC functions include secretion of B cell chemokines and the retention of Ag complexes that allow GC B cells to test the affinity of their Ig. FDCs require constitutive signaling through lymphotoxin beta receptor (LTβR) from lymphotoxin alpha-beta (LTαβ) on the surface of B cells to maintain their functions. In these studies, I investigated the properties of GCs using two primary experimental models. First, I employed genetic and pharmacological ablation of LTβR signaling to investigate the expression of AID and the function of GCs in the absence of FDCs. I determined that FDCs are not required for GC formation or the expression of AID in lymph nodes, but that FDCs are crucial for affinity maturation. Second, I examined the competition between AID-/- and WT B cells in the GCs of mixed BM chimeras to investigate the role of affinity maturation during clonal selection. I found that AID increases GC B cell apoptosis, likely by inducing DNA damage, and that this is important in regulating GC size. I also found that AID-/- B cells accumulate at the centrocyte stage of the GC reaction and that this is due to a partial block in plasma cell maturation.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Problem: Herpes simplex virus type-2 (HSV-2) is one of the most prevalent sexually transmitted infections in the world, and rates of infection are higher in women compared to men. Furthermore, vaccines developed against HSV-2 have failed at various stages of clinical trials, due to their inability to induce protective mucosal immunity. In animal models, intranasal (IN) immunization with attenuated HSV-2 (TK−) virus has been shown to confer protection against wildtype HSV-2 challenge. Since IN immunization serves as a more practical and less intrusive vaccination strategy, further studies are warranted to characterize optimal immune responses following IN immunization. We have previously demonstrated that estradiol (E2) treatment promotes enhanced protection against HSV-2 through enhanced anti-viral T cells responses. However, the effect of E2 on B cell responses, which were recently shown to be critical in protecting the host following IN immunization, remain poorly understood. Therefore, in this study we aimed to examine if following IN immunization, E2 enhances the memory B cell (MBC) and antibody-secreting plasma cell populations within the secondary lymphoid tissues and nasal effector sites, and whether this enhancement leads to an overall better protection against intravaginal IVAG WT-HSV-2 challenge. Methodology: Ovariectomized (OVX) mouse model of HSV-2 were pre-treated with E2 or placebo pellets. Subsequently, both groups were immunized intranasally with TK- HSV-2. Four weeks later nasal associated lymphoid tissues, nasal mucosa, cervical and iliac lymph nodes, spleen and vaginal tract were collected and processed and MBC and antibody-secreting plasma cells were characterized by flow cytometric analysis. HSV-2 specific IgM and IgG antibody responses in serum and vaginal secretions were measured by ELISA. In parallel experiments, animals were IVAG challenged with WT-HSV-2 and the B cell subsets were characterized as above. Results: The formation of MBC subsets, as seen by the presence of CD19+ IgD- cells and the heterogenous expression of CD73, CD80, and PD-L2, were observed four-weeks post immunization within the cervical and iliac lymph nodes and spleen, which were further enhanced in the presence of E2. Additionally, E2-treated mice had increased number of B220- CD138+ IgG2c+ plasma cells within the nasal mucosa following immunization. These enhancements translated into increased levels of HSV-2 specific IgG2b and IgG2c antibodies within the serum and vaginal secretions of E2-treated mice at four-weeks post IN immunization. Upon IVAG challenge, E2-treated mice, but not control mice, were protected. Since the antibody isotypes that were enhanced in E2 treated mice are correlated with Th17 responses, E2 mediated antibody enhancement was tested in IL-17 knockout mice. E2 treatment in IL-17-knockout mice failed to induce similar responses observed in WT mice, indicating that the enhancement of B cells and antibodies seen following E2 treatment was mediated in an IL-17 dependent manner. Conclusion: This study highlights the importance of sex-dependent differences in vaccine-induced immunity. Specifically, the findings from this study will provide valuable information for the design of a potentially efficacious mucosal vaccine strategy, whereby immunization in the context of E2 could significantly enhance antigen-specific antibody responses in the genital tract. / Thesis / Master of Science (MSc)

<p>Several HIV-1 neutralizing antibodies (e.g. 2F5, 4E10) have been shown to react with self-antigens, suggesting that effective humoral responses to HIV-1 may be constrained by the tolerization of HIV-reactive B cells that also recognize self-antigens. I have tracked the development of 2F5-like HIV-1 gp41 membrane proximal external region (MPER)-reactive B cells throughout ontogeny using B-cell tetramer reagents. In BL/6 mice, MPER-binding populations are lost during normal B-cell development and immunization with HIV-1 MPER antigen does not elicit robust humoral responses. I have identified Kynureninase as a self-antigen that is recognized by 2F5 antibody and, therefore, is a molecule that could mediate the developmental loss of B cells reactive to an epitope shared by HIV gp41 and Kynureninase. To recover these MPER-reactive cells, I describe and characterize a stromal-cell independent culture system that efficiently supports pro-B cell to IgM+ B-cell development with near normal levels of IgH and Igkappa diversity. B-cell development in vitro closely follows the patterns of development in vivo with culture derived (CD) B cells demonstrating characteristic patterns of surface antigen expression and gene activation. Immature and transitional B-cell compartments are reduced, due to the induction of tolerance, in the bone marrow of 3H9 IgH knockin mice ; however, cultures of 3H9 IgH knockin pro-B cells yields high frequencies of "forbidden", autoreactive IgM+ B cells. Furthermore, serum IgG autoantibody exceeded that present in autoimmune, C4-/- animals following the reconstitution of RAG-1-/- mice with IgM+ CD cells derived from BL/6 mice. I show that HIV-1 MPER-reactive B cells are recovered from both BL/6 and 2F5 IgH knockin bone marrow using this in vitro culture system. RAG-1-/- mice reconstituted with these culture-derived B and T cells generate strong germinal center and antibody responses to HIV-1 MPER antigens. These data demonstrate that the humoral immune response to this HIV-1 gp41 MPER antigen can be restored in mice when the constraints of B-cell tolerance have been relaxed.</p> / Dissertation

CD23 has long been appreciated to be a natural, negative regulator of IgE synthesis. This understanding is due in part to animal models in which CD23 deficient or CD23 transgenic animals display exacerbated or reduced IgE levels respectively. Interestingly, CD23 is susceptible to proteolytic cleavage from the cell surface. When this occurs, CD23 loses its regulatory capability and the solubilized form can lead to pro-inflammatory events through its cytokinergic activity on macrophages. Thus, targeting this specific cleavage would be beneficial to the control of allergic disease by stabilizing CD23 at the cell surface. Inhibitor studies performed by our group as well as others indicate that the enzyme responsible for CD23 ectodomain shedding is a hydroxamate-sensitive metalloproteinase. Through collaboration with the Blobel group, we analyzed various ADAM KO mouse embryonic fibroblasts (MEFs) and found no involvement of ADAMs 8,9,12,15,17,19, and 33 in CD23 shedding, however we did find a role for ADAM10. Using ADAM10 KO MEFs and ADAM10 specific inhibitors, we discovered that ADAM10 is indeed the CD23 cleaving enzyme or “sheddase”. Thus, developing strategies that would target ADAM10 could have an effect on sCD23 release and IgE production. In the CNS, signaling through the kainate receptor (KAR) by glutamate causes an increase in ADAM10 expression. Human B cells were found to express a GluK2 containing kainate receptor and its activation increased ADAM10 expression which is in agreement with KAR activation in the CNS. Although glutamate is considered a neurotransmitter, it signals in the periphery and elevated levels are associated with certain immune disorders. A significant corresponding increase in sCD23 release is observed as well. Remarkably, this activation induced a strong increase in B cell proliferation, IgG, and IgE production and these events can be reversed through the use of NS102, a specific KAR antagonist. Thus, we report for the first time the unique presence on B cells of a neurotransmitter receptor and that activation of this receptor could serve as a novel mechanism for enhancing B cell activation and Ig production. This enhancement and control thereof has implications for allergic and autoimmune diseases. Lastly, the CD23-ADAM10 axis was examined in a non-allergic disease state, B cell chronic lymphocytic leukemia (BCLL). BCLL is characterized by a large accumulation of CD23+ cells and high levels of soluble CD23 in the sera. After further analysis, we show that ADAM10 is indeed over-expressed in BCLL and could account for the high levels seen in this patient population. Furthermore, specifically targeting ADAM10 resulted in reduced soluble CD23 release, reduced proliferation, and enhanced apoptosis induction. Taken together the novel finding that ADAM10 is involved in CD23 shedding allows for targeted therapeutic intervention of both atopic and non-atopic disease states.

Thesis advisor: Welkin Johnson / Thesis advisor: Thomas Chiles / B cell activation is an energetically demanding process during which B lymphocytes undergo reprogramming and shift from a resting state to a highly proliferative, metabolically active state. Little is known about the metabolic reprogramming process or the role extracellular nutrients play in the activation response. Here we demonstrate that there are distinct requirements for the nutrients L-glutamine and glucose during activation. We show that cells activated in glucose-depleted conditions are still able to undergo growth and signaling events. In contrast, we show that extracellular L-glutamine is essential for all but the earliest activation events, and cells cultured in L-glutamine-deprived conditions are unable to enter the cell cycle. Consistently, we show that extracellular supplementation of the cell-permeable derivative of α-ketoglutarate (α-KG), a glutaminolytic product, is able to rescue cell activation in the absence of glutamine. We also show the induction of the high affinity amino acid transporter ASCT2 is required for glutamine uptake following B cell receptor (BCR) crosslinking. Specifically, we found that halting glutamine uptake or processing by inhibiting ASCT2 or the glutaminolytic enzyme glutaminase causes activation defects that parallel those observed in glutamine deprived conditions, indicating a requirement for glutaminolysis during the very early stages of activation. We found that -KG does not contribute to epigenetic remodeling, but is necessary for mammalian target of rapamycin complex 1 (mTORC1) activation. In turn, mTORC1 activity is required for upregulation of the glucose transporter Glut1 during the initial 24 hours of activation, as well as increased glucose uptake. These findings indicate a distinct metabolic profile that begins with glutamine uptake, and acts through mTORC1 signaling to later promote glucose uptake. Finally, we show that nutrients contribute to functional differentiation events during B cell activation. Glucose is required to support biogenesis of the endoplasmic reticulum as well as differentiation into plasma-like cells, while glutamine is required to support differentiation into IL-10 secreting regulatory B cell subsets. The requirement for glutamine for in vitro B10 cell differentiation is the first reported link between nutrient signaling and regulatory B cell development, and is a novel finding in the field. / Thesis (PhD) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Renal transplantation is the optimum treatment for end-stage renal failure. B cells have been identified in chronic allograft damage (CAD) and are associated with the development of tertiary lymphoid tissue within the human renal allograft. To investigate this pathology we utilized a mouse model of renal transplantation. A mouse model of kidney transplantation was first described in 1973. Although the mouse model is technically difficult it is attractive for several reasons: the mouse genome has been characterized and in many aspects is similar to man and there is a greater diversity of experimental reagents and techniques available for mouse studies than other experimental models. We reviewed the literature on all studies of mouse kidney transplantation to report the donor and recipient strain combinations that have been investigated and the resultant survival and histological outcomes. Some models of kidney transplantation have used the transplanted kidney as a life-supporting organ, however in many studies the recipient mouse’s native kidney has been left in situ. Several different combinations of inbred mouse strains have been reported, with varying degrees of injury, survival, or tolerance due to haplotype differences. Both cellular and humoral rejection processes have been observed. This model has been exceptionally useful as an investigational tool to understand multiple aspects of transplantation including acute rejection, cellular and humoral rejection mechanisms and their treatment. Furthermore this model has been used to investigate disease mechanisms beyond transplant rejection including intrinsic renal disease and infection-associated pathology. We performed renal transplantation in mice to model CAD and identified B cells forming tertiary lymphoid tissue with germinal centres. Intra-allograft B220+ B cells comprised of IgMhigh CD23- marginal zone, IgMlo CD23+ follicular zone and IgMlo CD23- transitional-type B cells similar to spleen, and these compartments had elevated expression of CD86. Depletion of B cells with anti-CD20 was associated with an improvement in CAD but only when administered after transplantation and not before. Isolated intra-allograft B cells were cultured and shown to synthesise multiple cytokines, the most abundant of these being GRO-α (CXCL1), RANTES (CCL5), IL-6 and MCP-1 (CCL2). Tubular loss was associated with T cell mediated injury and interstitial fibrosis, whilst type III collagen deposition driven by F4/80+ macrophages and PDGFR-β+ and transgelin+ fibroblasts, all of which were reduced by B cell depletion. In this report we show that intra-allograft B cells are key mediators of chronic damage to the transplant allograft kidney by cytokine orchestration of T cell, macrophage infiltration and fibroblast activation.

Piperine, the major alkaloid derived from black pepper corns, has played an important role in traditional medicine worldwide. Current research has demonstrated piperine to have several anti-inflammatory properties, however, little is known concerning the effect of piperine on B cells. Spleen-derived murine B cells were cultured in the presence or absence of piperine during T-dependent or T-independent activation. Piperine reversibly inhibited B cell proliferation in a dose-dependent manner. This was due to a G0/1-phase cell cycle arrest, and was associated with a reduction in phospho-ERK, phospho-AKT, and Cyclin D1, D2, and D3. Piperine also inhibited antibody and cytokine production. Furthermore, piperine treatment diminished B cell-mediated antigen presentation determined by measuring OT-II transgenic T cell proliferation in response to OVA, which was attributed to the decreased MHC-II ad co-stimulatory molecule expression observed. This in vitro study shows that piperine has potent immuno-suppressive effects on B cell activation and effector function.