IgG2c Production Research Articles

E6020, a TLR4 Agonist Adjuvant, Enhances Both Antibody Titers and Isotype Switching in Response to Immunization with Hapten-Protein Antigens and Is Diminished in Mice with TLR4 Signaling Insufficiency.

The mechanisms by which TLR4-based adjuvants enhance immunogenicity are not fully understood. We have taken advantage of a novel knock-in mouse strain that homozygously expresses two single-nucleotide polymorphisms (SNPs) that are homologous to human TLR4 (rs4986790 and rs4986791) and have been associated with LPS hyporesponsiveness in vivo and in vitro. TLR4-SNP (coexpressing mutations D298G/N397I in TLR4) mice that recapitulate the human phenotype were compared with wild-type (WT) mice for their hapten-specific Ab responses after immunization with hapten 4-hydroxy-3-nitrophenyl acetyl (NP) NP-Ficoll or NP-OVA in the absence or presence of a water-soluble TLR4 analog adjuvant, E6020. IgM and IgG anti-NP responses were comparable in WT and TLR4-SNP mice after immunization with either NP-Ficoll or NP-OVA only. E6020 significantly yet transiently improved the IgM and IgG anti-NP responses of both WT and TLR4-SNP mice to NP-Ficoll (T-independent), with modestly enhanced Ab production in WT mice. In contrast, T-dependent (NP-OVA), adjuvant-enhanced responses showed sustained elevation of NP-specific Ab titers in WT mice, intermediate responses in TLR4-SNP mice, and negligible enhancement in TLR4-/- mice. E6020-enhanced early humoral responses in WT and TLR4-SNP mice to NP-OVA favored an IgG1 response. After a second immunization, however, the immune responses of TLR4-SNP mice remained IgG1 dominant, whereas WT mice reimmunized with NP-OVA and E6020 exhibited increased anti-NP IgG2c titers and a sustained increase in the IgG1 and IgG2c production by splenocytes. These findings indicate that E6020 increases and sustains Ab titers and promotes isotype class switching, as evidenced by reduced titers and IgG1-dominant immune responses in mice with TLR4 insufficiency.

Single-cell transcriptome mapping identifies a local, innate B cell population driving chronic rejection after lung transplantation.

Bronchiolitis obliterans syndrome (BOS) is the main reason for poor outcomes after lung transplantation (LTx). We and others have recently identified B cells as major contributors to BOS after LTx. The extent of B cell heterogeneity and the relative contributions of B cell subpopulations to BOS, however, remain unclear. Here, we provide a comprehensive analysis of cell population changes and their gene expression patterns during chronic rejection after orthotopic LTx in mice. Of 11 major cell types, Mzb1-expressing plasma cells (PCs) were the most prominently increased population in BOS lungs. These findings were validated in 2 different cohorts of human BOS after LTx. A Bhlhe41, Cxcr3, and Itgb1 triple-positive B cell subset, also expressing classical markers of the innate-like B-1 B cell population, served as the progenitor pool for Mzb1+ PCs. This subset accounted for the increase in IgG2c production within BOS lung grafts. A genetic lack of Igs decreased BOS severity after LTx. In summary, we provide a detailed analysis of cell population changes during BOS. IgG+ PCs and their progenitors - an innate B cell subpopulation - are the major source of local Ab production and a significant contributor to BOS after LTx.

Multidomain peptide hydrogel adjuvants elicit strong bias towards humoral immunity.

Adjuvants play a critical role in enhancing vaccine efficacy; however, there is a need to develop new immunomodulatory compounds to address emerging pathogens and to expand the use of immunotherapies. Multidomain peptides (MDPs) are materials composed of canonical amino acids that form injectable supramolecular hydrogels under physiological salt and pH conditions. MDP hydrogels are rapidly infiltrated by immune cells in vivo and have previously been shown to influence cytokine production. Therefore, we hypothesized that these immunostimulatory characteristics would allow MDPs to function as vaccine adjuvants. Herein, we demonstrate that loading antigen into MDP hydrogels does not interfere with their rheological properties and that positively charged MDPs can act as antigen depots, as demonstrated by their ability to release ovalbumin (OVA) over a period of 7-9 days in vivo. Mice vaccinated with MDP-adjuvanted antigen generated significantly higher IgG titers than mice treated with the unadjuvanted control, suggesting that these hydrogels potentiate humoral immunity. Interestingly, MDP hydrogels did not elicit a robust cellular immune response, as indicated by the lower production of IgG2c and smaller populations of tetramer-positive CD8+ T splenocytes compared to mice vaccinated alum-adjuvanted OVA. Together, the data suggest that MDP hydrogel adjuvants strongly bias the immune response towards humoral immunity while evoking a very limited cellular immune response. As a result, MDPs may have the potential to serve as adjuvants for applications that benefit exclusively from humoral immunity.

Monophosphoryl Lipid A and Poly I:C Combination Adjuvant Promoted Ovalbumin-Specific Cell Mediated Immunity in Mice Model.

Simple SummaryMany research groups have investigated and developed new adjuvant candidates to promote vaccine efficacy, but only few of them were licensed. A combination of toll-like receptor (TLR) agonists can be a promising vaccine adjuvant candidate by stimulating innate immune cells and inducing antigen-specific cell-mediated immunity (CMI). In this study, a monophosphoryl lipid A (MPL) and Poly I:C combination, in low doses with ovalbumin (OVA) protein, elicited strong OVA-specific antibody production and more effective memory T cell responses compared with OVA only, OVA+MPL, OVA+Poly I:C groups at the site of immunization, as well as innate immune cell recruitment and activation. This study suggests MPL+Poly I:C as a potential CMI-inducing vaccine adjuvant candidate.Induction of antigen-specific cell-mediated immunity (CMI), as well as humoral immunity, is critical for successful vaccination against various type of pathogens. Toll-like receptor (TLR) agonists have been developed as adjuvants to promote vaccine efficacy and induce appropriate immune responses. Monophosphoryl lipid A (MPL); a TLR4 agonist, and Poly I:C; a TLR3 agonist, are known as a strong immuno-stimulator which induce Th1 response. Many studies proved and compared the efficacy of each adjuvant, but no study has investigated the combination of them. Using ovalbumin protein antigen, MPL+Poly I:C combination induced more effective antigen-specific CMI response than single adjuvants. Production of inflammatory cytokines, recruitment of innate immune cells and antigen-specific CD4/CD8 memory T cell at the immunized site had been significantly enhanced by MPL+Poly I:C combination. Moreover, MPL+Poly I:C combination enhanced ovalbumin-specific serum IgG, IgG1, and IgG2c production and proliferative function of CD4 and CD8 T cells after in vitro ovalbumin peptide stimulation. Taken together, these data suggest that the combination of MPL and Poly I:C has a potency as a CMI-inducing vaccine adjuvant with synergistically increased effects.

T-bet+ B cells promote adipose tissue inflammation and metabolic disorder during obesity.

Abstract Obesity is a public health crisis affecting a large and ever-growing portion of the global population. Obesity is associated with an increased risk of death and comorbidities, resulting in lower quality of life for obese patients and increased economic costs on healthcare systems. Obesity is linked with a low-grade and chronic inflammation in the adipose tissue, which further exacerbates metabolic dysfunctions. Understanding the mechanisms and immune cell types promoting this inflammation would allow for the development of new therapies to restore immune regulation in obese patients. Our lab has identified a subset of B lymphocytes which is expanded in the adipose tissue of humans and mice during obesity. This subset is characterized by the co-expression of T-bet and CD11c, production of IgG2c, and its expansion is dependent on the presence of iNKT cells. In vivo studies with mice fed a high fat diet reveal that these Tbet+ B cells promote inflammation. Serum transfer experiments and flow cytometry analysis revealed that this effect is mediated through the production of CXCL10 and antibodies. The absence of T-bet+ B cells does not appear to affect adipose CD4+ T cells or NK cells, but it reduces macrophage polarization towards a more proinflammatory phenotype. Transgenic mice engineered to lack T-bet+ B cells not only have reduced adipose tissue inflammation, but present improved metabolic function as well, gaining less weight on a high-fat diet than littermate controls and presenting improved glucose tolerance. Based on these results, T-bet+ B cells represent an interesting potential target to reduce inflammation in obese patients and improve their metabolic functions.

B cell-intrinsic MyD88 signaling controls IFN-γ-mediated early IgG2c class switching in mice in response to a particulate adjuvant.

Adjuvants improve the potency of vaccines, but the modes of action (MOAs) of most adjuvants are largely unknown. TLR-dependent and -independent innate immune signaling through the adaptor molecule MyD88 has been shown to be pivotal to the effects of most adjuvants; however, MyD88's involvement in the TLR-independent MOAs of adjuvants is poorly understood. Here, using the T-dependent antigen NIPOVA and a unique particulate adjuvant called synthetic hemozoin (sHZ), we show that MyD88 is required for early GC formation and enhanced antibody class-switch recombination (CSR) in mice. Using cell-type-specific MyD88 KO mice, we found that IgG2c class switching, but not IgG1 class switching, was controlled by Bcell-intrinsic MyD88 signaling. Notably, IFN-γ produced by various cells including Tcells, NK cells, and dendritic cells was the primary cytokine for IgG2c CSR and B-cell intrinsic MyD88 is required for IFN-γ production. Moreover, IFN-γ receptor (IFNγR) deficiency abolished sHZ-induced IgG2c production, while recombinant IFN-γ administration successfully rescued IgG2c CSR impairment in mice lacking B-cell intrinsic MyD88. Together, our results show that Bcell-intrinsic MyD88 signaling is involved in the MOA of certain particulate adjuvants and this may enhance our specific understanding of how adjuvants and vaccines work.

DAMP-Inducing Adjuvant and PAMP Adjuvants Parallelly Enhance Protective Type-2 and Type-1 Immune Responses to Influenza Split Vaccination.

Recently, it was reported that 2-hydroxypropyl-β-cyclodextrin (HP-β-CyD), a common pharmaceutical additive, can act as a vaccine adjuvant to enhance protective type-2 immunogenicity to co-administered seasonal influenza split vaccine by inducing host-derived damage-associated molecular patterns (DAMPs). However, like most other DAMP-inducing adjuvants such as aluminum hydroxide (Alum), HP-β-CyD may not be sufficient for the induction of protective type-1 (cellular) immune responses, thereby leaving room for improvement. Here, we demonstrate that a combination of HP-β-CyD with a humanized TLR9 agonist, K3 CpG-ODN, a potent pathogen-associated molecular pattern (PAMP), enhanced the protective efficacy of the co-administered influenza split vaccine by inducing antigen-specific type-2 and type-1 immune responses, respectively. Moreover, substantial antigen-specific IgE induction by HP-β-CyD, which can cause an allergic response to immunized antigen was completely suppressed by the addition of K3 CpG-ODN. Furthermore, HP-β-CyD- and K3 CpG-ODN-adjuvanted influenza split vaccination protected the mice against lethal challenge with high doses of heterologous influenza virus, which could not be protected against by single adjuvant vaccines. Further experiments using gene deficient mice revealed the unique immunological mechanism of action in vivo, where type-2 and type-1 immune responses enhanced by the combined adjuvants were dependent on TBK1 and TLR9, respectively, indicating their parallel signaling pathways. Finally, the analysis of immune responses in the draining lymph node suggested that HP-β-CyD promotes the uptake of K3 CpG-ODN by plasmacytoid dendritic cells and B cells, which may contributes to the activation of these cells and enhanced production of IgG2c. Taken together, the results above may offer potential clinical applications for the combination of DAMP-inducing adjuvant and PAMP adjuvant to improve vaccine immunogenicity and efficacy by enhancing both type-2 and type-1 immune responses in a parallel manner.

B cell responses to apoptotic cells in MFG-E8-/- mice

Defective clearance of apoptotic cells in MFG-E8 deficient mice results in lupus-like disease in the mixed B6x129, but not pure B6 background. The lack of overt autoimmunity in MFG-E8-/- B6 mice suggests that accumulation of apoptotic cells is not sufficient to break central tolerance. However, the delayed clearance of apoptotic cells in the follicles of MFG-E8-/- B6 mice provides an excellent opportunity to investigate how B cells respond to excessive apoptotic cells in the periphery under relatively non-inflammatory conditions. In MFG-E8-/- B6 mice, we found increased IgG2c production against apoptotic cells and oxidized LDL. Apoptotic cell induced antibody responses depended on MyD88 signal and T cell help. In addition, MFG-E8-/- B6 mice had enlarged MZ B cell compartments as well as an enhanced antibody response to NP-Ficoll. Moreover, a significant percentage of MZ B cells in aged MFG-E8-/- B6 mice migrated into follicles. Injecting apoptotic cells or oxidized LDL into wild type mice as well as physiological accumulation of LDL in ApoE-/- mice recapitulated the translocation of MZ B cells. To determine how MFG-E8 deficiency affects the functions of autoreactive B cells specific for nucleic acids in the periphery under non-inflammatory conditions, we utilized BCR transgenic mice to bypass central selection and compared the differentiation of TLR9 dependent anti-dsDNA 56R B cells and TLR7 dependent anti-ssRNA H564 B cells in MFG-E8-/- mice. In MFG-E8-/- 56R mice, anti-dsDNA specific 56R/Vκ38c B cells differentiated into MZ B cells but not AFCs. On the contrary, in MFG-E8-/-H564 mice, anti-ssRNA specific H564 B cells further differentiated into GC B cells and AFCs. Adoptive transfer of activated autoreactive B cells confirmed that H564 B cells were more sensitive to apoptotic cell antigens than 56R B cells. Our observations provide new insights about the MZ B cell translocation in lupus patients as well as the dichotomy of TLR9 and TLR7 signals in the pathogenesis of lupus.

Altered Humoral Immune Responses and IgG Subtypes in NOX2-Deficient Mice and Patients: A Key Role for NOX2 in Antigen-Presenting Cells.

Chronic granulomatous disease (CGD) is a primary immunodeficiency resulting from loss of function mutations in the reactive oxygen species generating phagocyte NADPH oxidase (NOX2). CGD patients are prone to infection, but also have an increased susceptibility to autoimmune diseases. The aim of this study was to investigate the role of NOX2 in the regulation of specific immunity. In both CGD patients and NOX2-deficient mice, we observed an alteration in the basal proportions of IgG subtypes. Upon immunization with curdlan—a dectin 1 agonist—NOX2-deficient mice showed increased production of IgG2c compared to controls, and restimulation of lymph node-derived cells led to increased production of IFNγ, but not IL-5, indicative hallmark of an enhanced Th1 response. T cell activation was increased in NOX2-deficient mice and a similar trend was observed in vitro when T cells were co-cultured with NOX2-deficient bone marrow-derived cells. In contrast, no difference in T cell activation was observed when NOX2-deficient T cells were co-cultured with wild-type BMDC. Following stimulation of NOX2-deficient dendritic cells (DCs), no difference in costimulatory molecules was observed, while there was an increase in the release of Th1-driving cytokines. In summary, both CGD patients and CGD mice have an altered IgG subtype distribution, which is associated with an increased IFNγ production. Thus, NOX2 within DCs appears to be an important regulator at the interface of innate and specific immunity, especially after activation of the dectin 1 pathway, limiting immune activation and the development of autoimmunity.

Atherogenic dyslipidemia promotes autoimmune follicular helper T cell responses via IL-27.

The incidence of atherosclerosis is higher among patients with systemic lupus erythematosus (SLE); however, the mechanism by which an atherogenic environment affects autoimmunity remains unclear. We found that reconstitution of atherosclerosis-prone Apoe-/- and Ldlr-/- mice with bone marrow from lupus-prone BXD2 mice resulted in increased autoantibody production and glomerulonephritis. This enhanced disease was associated with an increase in CXCR3+ follicular helper T cells (TFH cells). TFH cells isolated from Apoe-/- mice had higher expression of genes associated with inflammatory responses and SLE and were more potent in inducing production of the immunoglobulin IgG2c. Mechanistically, the atherogenic environment induced the cytokine IL-27 from dendritic cells in a Toll-like receptor 4 (TLR4)-dependent manner, which in turn triggered the differentiation of CXCR3+ TFH cells while inhibiting the differentiation of follicular regulatory T cells. Blockade of IL-27 signals diminished the increased TFH cell responses in atherogenic mice. Thus, atherogenic dyslipidemia augments autoimmune TFH cell responses and subsequent IgG2c production in a TLR4- and IL-27-dependent manner.

Disruption of Pathogenic Cellular Networks by IL-21 Blockade Leads to Disease Amelioration in Murine Lupus.

Systemic lupus erythematosus (lupus) is characterized by autoantibody-mediated organ injury. Follicular Th (Tfh) cells orchestrate physiological germinal center (GC) B cell responses, whereas in lupus they promote aberrant GC responses with autoreactive memory B cell development and plasma cell-derived autoantibody production. IL-21, a Tfh cell-derived cytokine, provides instructional cues for GC B cell maturation, with disruption of IL-21 signaling representing a potential therapeutic strategy for autoantibody-driven diseases such as systemic lupus erythematosus. We used blockade of IL-21 to dissect the mechanisms by which this cytokine promotes autoimmunity in murine lupus. Treatment of lupus-prone B6.Sle1.Yaa mice with an anti-IL-21 blocking Ab reduced titers of autoantibodies, delayed progression of glomerulonephritis and diminished renal-infiltrating Tfh and Th1 cells, and improved overall survival. Therapy inhibited excessive accumulation of Tfh cells coexpressing IL-21 and IFN-γ, and suppressed their production of the latter cytokine, albeit while not affecting their frequency. Anti-IL-21 treatment also led to a reduction in GC B cells, CD138hi plasmablasts, IFN-γ-dependent IgG2c production, and autoantibodies, indicating that Tfh cell-derived IL-21 is critical for pathological B cell cues in lupus. Normalization of GC responses was, in part, caused by uncoupling of Tfh-B cell interactions, as evidenced by reduced expression of CD40L on Tfh cells and reduced B cell proliferation in treated mice. Our work provides mechanistic insight into the contribution of IL-21 to the pathogenesis of murine lupus, while revealing the importance of T-B cellular cross-talk in mediating autoimmunity, demonstrating that its interruption impacts both cell types leading to disease amelioration.

MiR-17-92 Is Required for the Pathogenicity of T and B Cells in Chronic Gvhd

Chronic graft-versus-host disease (cGVHD) remains to be a major cause of mortality and morbidity after allogeneic hematopoietic cell transplantation (allo-HCT). cGVHD is characterized as autoimmune-like fibrosis and antibody production, mediated by pathogenic T and B cells. Through producing pro-inflammatory cytokines, CD4 T cells are the driving force of cGVHD. Donor B cells augment the pathogenesis of cGVHD not only by acting as antigen-presenting cells (APCs) and promoting CD4 T-cell expansion and survival, but also by producing autoantibodies. microRNA (miR)-17-92 has been shown to regulate T-cell immunity including allogeneic, anti-viral, and anti-tumor responses. Recently, miR-17-92 was found to act together with Bcl-6 to promote the differentiation of Follicular help T (Tfh) cells. Furthermore, B-cell deficiency of miR-17-92 impairs IgG2c production. Since Tfh differentiation and antibody production are required for the development of cGVHD, we hypothesize that miR-17-92 contributes to the pathogenesis of cGVHD by promoting pathogenic T- and B-cell responses.By using Cre-loxp system, we generated B6 mice with conditional deficiency of miR-17-92 in T cells (CD4cre), B cells (CD19cre), or both (CD4CD19cre). aGVHD to cGVHD transition model (B6 to BALB/c) was utilized to test the effects of individual and combinational deficiency of miR-17-92 in T and/or B cells in the development of cGVHD. BALB/c mice were lethally irradiated and transferred with splenocytes plus BM cells derived from CD4cre, CD19cre or CD4CD19cre miR-17-92flox/flox B6 mice. WT B6 (Cre- miR-17-92flox/flox) mice were used as control donors. A significantly reduction of GVHD mortality was observed only in the recipients with CD4CD19cre grafts, but not with CD4cre or CD19cre grafts. Deficiency of miR-17-92 in donor T or B cells indeed improved the clinical manifestation of cGVHD, but the deficiency in both T and B cells showed further improvement, indicating the additive role of miR-17-92 in T and B cells in the pathogenesis of cGVHD. Mechanistically, deficiency of miR-17-92 in T cells resulted in the reduction of Tfh generation (Fig. A), germinal center (GC) B-cell and plasma cell differentiation, and the expression of MHC-II and CD86 on donor B cells in recipient spleens. Furthermore, deficiency of miR-17-92 in B cells significantly reduced the levels of total IgG and IgG2c in recipient serum (Fig. A). These data suggest that miR-17-92 contributes to both T- and B-cell differentiation and function, which is required for the development of cGVHD.To extend our findings, we used a bronchiolitis obliterans cGVHD model (B6 to B10.BR). Recipient mice were pre-conditioned and received either BM alone from WT or CD19cre B6 mice, or BM plus purified T cells from WT or CD4cre B6 mice. Deficiency of miR-17-92 in T cells or BM-derived B cells resulted in significant improvement in pulmonary functions in recipient mice, as demonstrated by a decrease in resistance and elastance and an increase in compliance (Fig. B). Consistently, we found that miR-17-92 promoted Tfh and GC B-cell differentiation (Fig. B), while inhibiting differentiation of T follicular regulatory cells in recipient spleens 60 days after allo-HCT.For translational purpose, we tested whether inhibition of miR-17-92 could ameliorate cGVHD using locked nucleic acid (LNA) antagomirs specific for miR-17 or miR-19, key members in this microRNA cluster. In a SLE cGVHD model (DBA2 to BALB/c), administration of anti-miR-17, but not anti-miR-19, significantly suppressed the incidence of proteinuria and the severity of clinical manifestation by inhibiting donor splenocyte expansion, expression of costimulatory molecules on donor B cells, and differentiation of GC B cells and plasma cells (Fig. C). In addition, systemic delivery of anti-miR-17 significantly improved skin cGVHD by restraining IL-17 producing CD4 T-cell infiltration in skin-draining lymph nodes in a scleroderma-cGVHD model (B10.D2 to BALB/c).Taken together, the current work reveals that miR-17-92 is required for T- and B-cell differentiation and function, and thus for the development of cGVHD. Furthermore, pharmacological inhibition of miR-17 represents a potential therapeutic strategy for the control of cGVHD after allo-HCT. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

BANK1 Regulates IgG Production in a Lupus Model by Controlling TLR7-Dependent STAT1 Activation.

The purpose of our study was to investigate the effects of the adaptor Bank1 in TLR7 signaling using the B6.Sle1.yaa mouse, a lupus model that develops disease through exacerbated TLR7 expression. Crosses of B6.Sle1.yaa with Bank1-/- mice maintained several B and myeloid cell phenotypes close to normal wild-type levels. Most striking was the reduction in total serum IgG antibodies, but not of IgM, and reduced serum levels of autoantibodies, IL-6, and BAFF. Bank1 deficiency did modify numbers of MZ B cells and total B cell numbers, as well as expression of CXCR4 by follicular helper T cells. Other T cell changes were not observed. Bank1 deficiency did not modify numbers of germinal center B cells or plasma cells or clinical disease outcomes. Purified B cells from Bank1 deficient mice had strongly reduced Ifnb, Ifna4, Irf7, Aicda and Stat1 gene expression following TLR7 agonist stimulation. Interestingly, phosphorylation of Tyr701, but not of Ser727 of STAT1, was impaired in splenic B cells from B6.Sle1.yaa.Bank1-/- mice, as was the nuclear translocation of IRF7 in response to TLR7 agonist stimulation. Further, Bank1 deficiency in B6.Sle1.yaa mice reduced the production of IgG2c after in vitro TLR7 agonist stimulation. Our results demonstrate that Bank1 controls TLR7-mediated type I interferon production. Combined with the control of the nuclear translocation of IRF7, the modulation of STAT1 transcription and phosphorylation, Bank1 contributes to IgG production during development of autoimmune disease.

BANK1 Controls IgG Production Through TLR7-Dependent STAT1 Activation in a Lupus Model

Abstract The purpose of our study was to investigate the effects of the adaptor Bank1 in TLR7 signaling using the B6.Sle1.yaa mouse, a lupus model that develops disease through exacerbated TLR7 expression. Crosses of B6.Sle1.yaa with Bank1−/−mice maintained several B and myeloid cell phenotypes close to normal levels in wild-type mice. Most striking was the reduction in total serum IgG antibodies, but not of IgM, and reduced serum levels of autoantibodies, IL-6, and BAFF. Bank1 deficiency did modify frequencies of MZ B cells and splenic B cells, as well as expression of CXCR4 by follicular helper T cells. Bank1 deficiency did not modify frequencies of germinal center B cells or plasma cells or clinical disease outcomes. Purified B cells from B6.Sle1.yaa.Bank1−/−mice had lost strong induction of Ifnb, Ifna4, Irf1, 7, 9, Aicda and Stat1 gene expression following TLR7 agonist stimulation, compared with B6.Sle1.yaa mice. Interestingly, phosphorylation of Tyr701, but not of Ser727 of STAT1, was impaired in splenic B cells from B6.Sle1.yaa.Bank1−/− mice, as was the nuclear translocation of IRF7 in response to TLR7 agonist stimulation. Further, Bank1 deficiency in B6.Sle1.yaa mice reduced the production of IgG2c after in vitro TLR7 agonist stimulation. Our results demonstrate that Bank1 controls TLR7-mediated type I interferon production, leading to positive feedback loop on IFNAR and IFN I-mediated gene expression, including Tlr7 gene. Combined with the control of the nuclear translocation of IRF7, and the modulation of transcription and STAT1 phosphorylation, Bank1 contributes to IgG production during development of autoimmune disease.

Enhancement of anti-OVA IgG2c production in vivo by enalapril.

Angiotensin-converting enzyme (ACE) inhibitors have non-hemodynamic, pleiotropic effects on the immune response. The effects of ACE inhibitors on the production of cytokines and T-cell functions are well established. However, little is known on the effects of these medicines on humoral response to foreign antigens. In this study, we investigated the effect of enalapril treatment on ovalbumin (OVA)-specific IgG1 and IgG2c production in mice determined by ELISA. Two groups of 8-week-old C57BL/6 females mice (3–4/group) were subcutaneously immunized with OVA (10 μg/animal) in presence of Alhydrogel (1 mg/mouse) and boosted at day 21. The mice were treated with enalapril (5 mg/kg daily, po) or were left without treatment for one month. The animals were bled from the orbital plexus on days 0, 7, 14, 21, and 28 after the first immunization and the sera were stored at –20°C until usage. OVA-specific serum IgG1 and IgG2c were determined by ELISA using serum from each individual animal. The results showed that enalapril significantly increased anti-OVA serum IgG2c in the secondary response without affecting IgG1 synthesis. These data expand our understanding on the properties of enalapril on the immune response, including antibody production.

T follicular helper, but not Th1, cell differentiation in the absence of conventional dendritic cells.

Development of long-lived humoral immunity is dependent on CXCR5-expressing T follicular helper (Tfh) cells, which develop concomitantly to effector Th cells that support cellular immunity. Conventional dendritic cells (cDCs) are critical APCs for initial priming of naive CD4(+) T cells but, importantly, also provide accessory signals that govern effector Th cell commitment. To define the accessory role of cDCs during the concurrent development of Tfh and effector Th1 cells, we performed high-dose Ag immunization in conjunction with the Th1-biased adjuvant polyinosinic:polycytidylic acid (pI:C). In the absence of cDCs, pI:C failed to induce Th1 cell commitment and IgG2c production. However, cDC depletion did not impair Tfh cell differentiation or germinal center formation, and long-lived IgG1 responses of unaltered affinity developed in mice lacking cDCs at the time point for immunization. Thus, cDCs are required for the pI:C-driven Th1 cell fate commitment but have no crucial accessory function in relation to Tfh cell differentiation.

Mir-17-92 regulates bone marrow homing of plasma cells and production of immunoglobulin G2c.

The polycistronic mir-17-92 cluster, also known as oncomir-1, was previously shown to be essential for early B lymphopoiesis. However, its role in late-stage B-cell differentiation and function remains unexplored. Here we ablate mir-17-92 in mature B cells and demonstrate that mir-17-92 is dispensable for conventional B-cell development in the periphery. Interestingly, mir-17-92-deficiency in B cells leads to enhanced homing of plasma cells to the bone marrow during T-cell-dependent immune response and selectively impairs IgG2c production. Mechanistically, mir-17-92 directly represses the expression of Sphingosine 1-phosphate receptor 1 and transcription factor IKAROS, which are, respectively, important for plasma cell homing and IgG2c production. We further show that deletion of mir-17-92 could reduce IgG2c anti-DNA autoantibody production and hence mitigate immune complex glomerulonephritis in Shp1-deficient mice prone to autoimmunity. Our results identify important roles for mir-17-92 in the regulation of peripheral B-cell function.

Slc15a4 function is required for intact class switch recombination to IgG2c in response to TLR9 stimulation.

Signalling through Toll-like receptors (TLRs) by endogenous components of viruses or bacteria can promote antibody (Ab) isotype switching to IgG2a/c. Multiple cell types are capable of responding to TLR stimulation in vivo and the processes underlying TLR-induced Ab isotype switching are not fully defined. Here, we used feeble mice, which are deficient in the peptide/histidine transporter solute carrier family 15 member 4 (Slc15a4), and fail to produce cytokines including interferon alpha (IFNα) in response to TLR9 stimulation, to study Ab isotype switching to IgG2c in response to vaccination. We demonstrate that the production of IgG2c in response to CpGA-adjuvanted vaccines was severely reduced in feeble mice, while a more subtle defect was observed for CpGB. The reduced IgG2c production in feeble could not be ascribed to defective plasmacytoid dendritic cell (pDC) responses alone as we found that splenic cDCs and B cells from feeble mice were also defective in response to TLR9 ligation ex vivo. We conclude that Slc15a4 is required for intact function of TLR9-expressing cells and for effective Ab isotype switching to IgG2c in response to CpG-adjuvanted vaccines.

Adjuvanticity and Toxicity of Cobalt Oxide Nanoparticles as An Alternative Vaccine Adjuvant

There are very few adjuvants licensed for use in human vaccination, and alum-based adjuvants are the most widely used. Alum adjuvants predominantly boost Th2 immune responses and there is a need for new adjuvants that also stimulate Th1 immunity. We recently reported that cobalt oxide nanoparticles (Co(3)O(4)NPs) stimulate Th1-type immune responses in vivo. Here, we exploited this property to examine whether Co(3)O(4)NP could act as an adjuvant using the model antigen ovalbumin. Female C57BL/6 mice were immunized subcutaneously twice with ovalbumin plus adjuvant (Co(3)O(4)NPs or Imject® Alum) followed by intraperitoneal stimulation with soluble ovalbumin. Co(3)O(4)NPs induced a more balanced Th1- and Th2-type response, triggering higher specific Th1-dependent IgG2c production in addition to Th2-dependent IgG1 and less 'allergic' IgE production, and induced less inflammation at both the subcutaneous and intraperitoneal injection sites. Co(3)O(4)NPs could be a very useful adjuvant where both Th1 and Th2 responses are needed to clear pathogens.

Abstract 293: Id3 Regulates High-Fat-Diet--Induced IgG2c Production

Background: High-fat diet (HFD)-induced adipose tissue inflammation leads to insulin resistance and glucose intolerance, increasing the risk of cardiovascular disease. Recently, B cells and IgG antibodies have been shown to promote the metabolic consequences of obesity. HFD induces IgG2c production - an IgG isotype that promotes inflammatory disorders. Mice null for Id3, a helix-loop-helix protein known to mediate IgG2c responses in vivo, develop significantly attenuated HFD-induced obesity; raising the interesting hypothesis that Id3 promotes HFD-induced obesity by mediating HFD-induced IgG2c production. Methods and results: Western Blot analysis revealed that primary splenic B cells stimulated with IFN + CD40L (an IgG2c-inducing cocktail) expressed significantly greater amounts of Id3 protein compared to B cells treated with CD40L alone ((1.26+/-0.05 vs 0.88+/-0.03; n=3; p=0.003). ELISA determination of immunoglobulin levels from whole adipose tissue of fed C57Bl/6J (WT) mice fed a HFD (60% kcal fat) revealed a 4 fold increase in IgG2c (3810+/-789 ng vs 993+/-422 ng; n=5; p=0.01) - but not IgM, IgG1, or IgG2b - compared to chow-fed controls. No differences in serum IgG2c were observed. In contrast, HFD-induced adipose tissue IgG2c levels were significantly attenuated in Id3 -/- mice (1643+/-493 vs 889+/-272 ng; n=6-7; p=0.19), despite the fact that Id3 -/- mice had significantly more adipose tissue B cells per (g) fat compared to WT (1.6E5+/-0.2E5 vs 0.5E5+/-0.09E5; n=5; p=0.001). Conclusions: Id3 promotes HFD-induced local adipose tissue IgG2c production; an effect not dependent on B cell number. Studies in animals with B cell-specific loss of Id3 are ongoing to identify the mechanisms whereby Id3 mediates HFD-induced IgG2c production and downstream metabolic effects.