MaterialA fter exposure to Ag via immunization or infection, B cells have the capacity to generate plasma cells and develop an extrafollicular response or, together with follicular dendritic cells, initiate the germinal center (GC) reaction (1). The GC is a microanatomical structure formed in B cell follicles in secondary lymphoid organs where Ag-specific B cells undergo division, isotype switching, somatic hypermutation, and differentiation into memory B cells or plasma cells. Cognate interaction between B and T cells in the GC is essential for the selection of high-affinity Ag-specific B cells, and access to T cell help is thought to be a limiting factor for the positive selection of GC B cells (2).The PI3K pathway has been implicated in lymphocyte development and activation. It transduces extracellular signals into the production of the second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) by phosphorylation of phosphatidylinositol 4,5-bisphosphate. In lymphocytes, PIP 3 is generated by the class I PI3K catalytic subunits of which there are four isoforms named a, b, d, and g. The a, b, and d enzymes form heterodimers with one of five Src homology 2 domain-containing regulatory subunits termed p85a, p85b, p55g, p50a, and p55a, which mediate recruitment to phosphotyrosine-containing signalosomes (3). The levels of cellular PIP 3 are also regulated by phosphatases: phosphatase and tensin homolog deleted on chromosome 10 (PTEN) directly opposes PI3K by removing the 39 phosphate from PIP 3 ; the SHIP enzymes generate the second messenger, phosphatidylinositol 3,4-bisphosphate.Mice with germline mutations of the p110d catalytic subunit have demonstrated its importance for the GC reaction. However, it remains unclear whether this is a reflection of the requirement for p110d in B cells, T cells, dendritic cells (DCs), or other cell types required for the GC response (4, 5). B cells from p110d-deficient mice display in vitro defects in survival and proliferation that correlate with defective signal transduction following stimulation with Abs via the BCR or CD19 (6-8). Whether these in vitro defects are relevant in vivo is unclear since B cell-specific deletion of PTEN impaired class-switch recombination but not the magnitude of the GC response (9). T cell-specific deletion of the class IA regulatory subunits showed that the GC reaction and Ag-specific Ab titers were reduced, implicating a T cell-intrinsic requirement for class IA PI3K but leaving unresolved the nature of the relevant catalytic subunits (10). Using the p110d D910A mouse model, which carries a point mutation that renders p110d catalytically inactive, adoptive transfer experiments revealed impaired Th1/Th2 cytokine production and a 2-fold reduction in clonal expansion (5, 11). In contrast, T cell-specific deletion of PTEN removed the requirement for CD28 costimulation (12) and allowed enhanced IL-4 production (13). Gaining a better understanding of the cell intrinsic role for p110d in Ab responses should prove helpful in determining the mech...

B-cell activating factor of the TNF family (BAFF) is critical for the survival and maturation of B cells. The molecular mechanisms by which BAFF regulates the survival of developing B cells are becoming better understood. Recent evidence has begun to emerge demonstrating a role for the PI3K/Akt signalling pathway in response to BAFF. However, the importance of the PI3K family for BAFF-signalling and the effects of loss of PI3K function on BAFF responses are still unknown. We therefore investigated the BAFF-mediated responses of B cells deficient for the PI3K catalytic subunit P110d. We find that the loss of P110d impairs the BAFF-mediated survival of cultured B cells demonstrating a direct role for this member of the PI3K family in regulating the survival of B cells in response to BAFF. P110d was required for the growth of B cells in response to BAFF and was critical for the upregulation of the receptor for BAFF following BCR crosslinking. Our findings reveal an important role for p110d in regulating B-cell responses to BAFF.Key words: B-cell activating factor of TNF family . B cells . PI3K . Survival IntroductionSignals generated by the B-cell activating factor of the TNF family (BAFF; also called BLyS, TNFSF13b, THANK, zTNF4 or TALL-1) upon binding to the BAFF receptor (BAFF-R, also known as BR3, TNFRSF13C, CD268) are necessary for the survival of some mature B-cell subsets. Genetic ablation of BAFF or mutational inactivation of BAFF-R leads to a decrease in the number of follicular (FO) and marginal zone (MZ) B cells illustrating the importance of this cytokine and its receptor for B-cell homeostasis [1,2]. By contrast, transgenic mice overexpressing BAFF have an expansion of mature B cells and elevated levels of serum immunoglobulins, anti-DNA antibodies and immune complexes in the kidney [3,4]. In humans, increased serum levels of BAFF have been demonstrated in diseases such as Sjogren's syndrome, rheumatoid arthritis, systemic lupus erythematosus and multiple sclerosis [5][6][7][8]. This suggests that BAFF may play an important role in the development of autoimmune disorders.The molecular mechanisms by which BAFF exerts its prosurvival effects are not fully understood. BAFF regulates the processing of the NF-kB2 precursor protein p100 to its transcriptionally active derivative p52 and induces phosphorylation and degradation of the NF-kB inhibitor IkBa [9][10][11]. Active NF-kB can promote transcription of anti-apoptotic genes of the Bcl2 family and this may represent a component of BAFF-mediated survival. In addition, the nuclear accumulation of proapoptotic protein kinase Cd (PKCd) and the BCR-induced upregulation of pro-apoptotic Bim are both blocked by BAFF [12,13]. BAFF also stimulates the pro-survival activities of the serine/ threonine kinase Pim2 [14].Recent evidence has shown that BAFF enhances the metabolic activity of B cells and induces cellular growth [15,16]. When B cells are isolated and placed in culture in the presence of BAFF they increase their size and protein content over time [15]. The...

Our previous studies have shown that overexpression of bovine FcRn (bFcRn) in transgenic (Tg) mice leads to an increase in the humoral immune response, characterized by larger numbers of Ag-specific B cells and other immune cells in secondary lymphoid organs and higher levels of circulating Ag-specific antibodies (Abs). To gain additional insights into the mechanisms underlying this increase in humoral immune response, we further characterized the bFcRn Tg mice. Our Western blot analysis showed strong expression of the bFcRn transgene in peritoneal macrophages and bone marrow derived dendritic cells; and a quantitative PCR analysis demonstrated that the expression ratios of the bFcRn to mFcRn were 2.6- and 10-fold in these cells, respectively. We also found that overexpression of bFcRn enhances the phagocytosis of Ag-IgG immune complexes (ICs) by both macrophages and dendritic cells and significantly improves Ag presentation by dendritic cells. Finally, we determined that immunized bFcRn mice produce a much greater diversity of Ag-specific IgM, whereas only the levels, but not the diversity, of IgG is increased by overexpression of bFcRn. We suggest that the increase in diversity of IgG in Tg mice is prevented by a selective bias towards immunodominant epitopes of ovalbumin, which was used in this study as a model antigen. These results are also in line with our previous reports describing a substantial increase in the levels of Ag-specific IgG in FcRn Tg mice immunized with Ags that are weakly immunogenic and, therefore, not affected by immunodominance.

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Mouse strains with specific deficiency of given hematopoietic lineages provide invaluable tools for understanding blood cell function in health and disease. Whereas neutrophils are dominant leukocytes in humans and mice, there are no widely useful genetic models of neutrophil deficiency in mice. In this study, we show that myeloid-specific deletion of the Mcl-1 antiapoptotic protein in Lyz2Cre/CreMcl1flox/flox (Mcl1ΔMyelo) mice leads to dramatic reduction of circulating and tissue neutrophil counts without affecting circulating lymphocyte, monocyte, or eosinophil numbers. Surprisingly, Mcl1ΔMyelo mice appeared normally, and their survival was mostly normal both under specific pathogen-free and conventional housing conditions. Mcl1ΔMyelo mice were also able to breed in homozygous form, making them highly useful for in vivo experimental studies. The functional relevance of neutropenia was confirmed by the complete protection of Mcl1ΔMyelo mice from arthritis development in the K/B×N serum-transfer model and from skin inflammation in an autoantibody-induced mouse model of epidermolysis bullosa acquisita. Mcl1ΔMyelo mice were also highly susceptible to systemic Staphylococcus aureus or Candida albicans infection, due to defective clearance of the invading pathogens. Although neutrophil-specific deletion of Mcl-1 in MRP8-CreMcl1flox/flox (Mcl1ΔPMN) mice also led to severe neutropenia, those mice showed an overt wasting phenotype and strongly reduced survival and breeding, limiting their use as an experimental model of neutrophil deficiency. Taken together, our results with the Mcl1ΔMyelo mice indicate that severe neutropenia does not abrogate the viability and fertility of mice, and they provide a useful genetic mouse model for the analysis of the role of neutrophils in health and disease.

IntroductionBacterial lipopolysaccharide (LPS) is a potent B-cell mitogen both in vivo 1 and in vitro. 2 Activation of B cells by LPS triggers immunoglobulin (Ig) secretion and Ig class switching. 3 LPS also promotes the ability of B cells to function as antigen-presenting cells by increasing expression of major histocompatibility complex type II (MHC-II) and costimulatory molecules such as CD86. 4,5 Collectively, these responses of B cells contribute to the neutralization of Gram-negative bacteria.B cells express 2 receptors that function as recognition and signal-transducing receptors for LPS. Toll-like receptor 4 (TLR4) is a leucine-rich repeat protein with a large cytoplasmic domain that contains a signal transducing Toll/IL-1 (interleukin 1) receptor (TIR) homology domain. 6 CD180, previously called RP105, similarly contains a leucine-rich extracellular domain but bears an 11-amino acid cytoplasmic domain with no homology to TLR4 or other known proteins. 7 The extracellular domains of TLR4 and CD180 associate with small molecules called MD2 and MD1, respectively. These molecules contribute to recognition of LPS as well as the surface expression and intracellular distribution of TLR4 and CD180. 8,9 Although B cells express relatively little TLR4 when compared with macrophages, analysis of mutant mice demonstrate TLR4 is essential for B-cell responses to LPS. Engineered mutations of CD180 and MD1 have revealed roles for each of these in LPS-mediated activation of B cells and in LPS-induced antibody responses. 9,10 While mutation of TLR4 yields a more severe defect in humoral LPS responses than that of CD180 or MD1, the genetic experiments indicate a nonredundant requirement for both classes of LPS receptor for the optimal responses of B cells.TLR4 is ubiquitously expressed, and the signal transduction pathways it activates have been well studied. The cytoplasmic domain of TLR4 recruits adapter proteins MyD88 (myeloid differentiation factor 88), TIRAP (TIR-containing adaptor protein), and TRIF (TIR-containing adaptor protein inducing interferon ␤), which, in turn, lead to the activation of ERK (extracellular receptor-activated kinase), JNK (c-JUN n-terminal kinase), and p38 MAP (mitogen-activated protein) kinases and subsequently to the activation of transcription factors, including NF-B (nuclear factor B) and IRF3 (interferon response factor 3) (reviewed by Sabroe et al 11 ). By contrast, expression of CD180 is restricted to mature B lymphocytes. However, the mechanisms by which CD180 signals are less well studied. CD180 crosslinking by antibody leads to activation of ERK, JNK, and p38. 12,13 B cells deficient in MyD88 proliferate normally following CD180 crosslinking, 14 while B cells deficient in PKC␤ (protein kinase C ␤) or from xid (X-linked immunodeficient) mice lacking normal BTK (Bruton tyrosine kinase) function, respond poorly to CD180 crosslinking. 13 Recently, it has been reported that the B-cell coreceptor CD19 is required for optimal proliferative responses to CD180 ligation. 14 Furthermore, it wa...

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The neonatal Fc receptor (FcRn) plays key roles in IgG and albumin homeostasis, maternal IgG transport, and antigen presentation of IgG-opsonized antigens. Previously, we reported that transgenic (Tg) mice that overexpress the bovine FcRn (bFcRn) have augmented T-dependent humoral immune response with increased IgG protection, higher level of antigen-specific antibodies, greater number of antigen-specific B cells, and effective immune response even against weakly immunogenic epitopes. In the current study, we analyzed the localization of the bFcRn in secondary lymphoid organs, and focused to demonstrate the in vivo impact of its overexpression in the spleen on the course of antibody production. bFcRn was highly expressed by red pulp macrophages and marginal zone macrophages in the spleen and by subcapsular sinus macrophages and macrophage-like cells in the interfollicular areas in the lymph node cortex. We also demonstrated that splenic dendritic cells of Tg mice express bFcRn and intraperitoneal immunization of these mice with T-dependent antigens led to more than threefold increase in the number of antigen-specific activated T helper cells with increased size and numbers of germinal centers compared to wild-type controls. bFcRn expression in splenic B cells was also detected and that may also contribute to the enhanced B cell activation. Finally, we demonstrated that these Tg mice developed efficient immune response against very low dose of antigen, reflecting another important practical benefit of these Tg mice.