Expression of activation‐induced cytidine deaminase enhances the clearance of pneumococcal pneumonia: evidence of a subpopulation of protective anti‐pneumococcal B1a cells

We describe a protective early acquired immune response to pneumococcal pneumonia that is mediated by a subset of B1a cells. Mice deficient in B1 cells (xid), or activation‐induced cytidine deaminase (AID^?/?), or invariant natural killer T (iNKT) cells (Jα18^?/?), or interleukin‐13 (IL‐13^?/?) had impaired early clearance of pneumococci in the lung, compared with wild‐type mice. In contrast, AID^?/? mice adoptively transferred with AID^+/+ B1a cells, significantly cleared bacteria from the lungs as early as 3 days post infection. We show that this early bacterial clearance corresponds to an allergic contact sensitivity‐like cutaneous response, probably due to a subpopulation of initiating B1a cells. In the pneumonia model, these B1a cells were found to secrete higher affinity antigen‐specific IgM. In addition, as in contact sensitivity, iNKT cells were required for the anti‐pneumococcal B1a cell initiating response, probably through early production of IL‐13, given that IL‐13^?/? mice also failed to clear infection. Our study is the first to demonstrate the importance of AID in generating an appropriate B1a cell response to pathogenic bacteria. Given the antibody affinity and pneumonia resistance data, natural IgM produced by conventional B1a cells are not responsible for pneumonia clearance compared with the AID‐dependent subset.     

Non‐eosinophilic Airway Hyper‐reactivity in Mice,Induced by IFN‐γ Producing CD4+ and CD8+ Lung T cells,is Responsive to Steroid Treatment

Non‐eosinophilic asthma is characterized by infiltration of neutrophils into the lung and variable responsiveness to glucocorticoids. The pathophysiological mechanisms have not been characterized in detail. Here, we present an experimental asthma model in mice associated with non‐eosinophilic airway inflammation and airway hyper‐responsiveness (AHR). For this, BALB/c mice were sensitized by biolistic DNA immunization with a plasmid encoding the model antigen β‐galactosidase (pFascin‐βGal mice). For comparison, eosinophilic airway inflammation was induced by subcutaneous injection of βGal protein (βGal mice). Intranasal challenge of mice in both groups induced AHR to a comparable extent as well as recruitment of inflammatory cells into the airways. In contrast to βGal mice, which exhibited extensive eosinophilic infiltration in the lung, goblet cell hyperplasia and polarization of CD4⁺ T cells into Th2 and Th17 cells, pFascin‐βGal mice showed considerable neutrophilia, but no goblet cell hyperplasia and a predominance of Th1 and Tc1 cells in the airways. Depletion studies in pFascin‐βGal mice revealed that CD4⁺ and CD8⁺ cells cooperated to induce maximum inflammation, but that neutrophilic infiltration was not a prerequisite for AHR induction. Treatment of pFascin‐βGal mice with dexamethasone before intranasal challenge did not affect neutrophilic infiltration, but significantly reduced AHR, infiltration of monocytes and lymphocytes as well as content of IFN‐γ in the bronchoalveolar fluid. Our results suggest that non‐eosinophilic asthma associated predominantly with Th1/Tc1 cells is susceptible to glucocorticoid treatment. pFascin‐βGal mice might represent a mouse model to study pathophysiological mechanisms proceeding in the subgroup of asthmatics with non‐eosinophilic asthma that respond to inhaled steroids.     

IL‐10 production in murine IgM+CD138hi cells is driven by Blimp‐1 and downregulated in class‐switched cells

In contrast to antibody‐induced inflammatory responses, some B‐cell subpopulations suppress inflammation through the production of interleukin (IL)‐10. However, the mechanisms underlying Il10 gene expression during B‐cell development is elusive. Here, we identify IgM⁺B220^loCD138^hi cells responsible for marked IL‐10 production in the bone marrow and spleen of mice. These murine IL‐10‐producing cells predominantly secrete IgM and have unique characteristics of long‐lived plasma cells in spite of high expression of surface IgM. We found that IL‐10 production is strongly correlated with the expression level of Prdm1 (encoding the Blimp‐1 protein), an essential regulator of plasma cell development. Furthermore, overexpression of Prdm1 induces Il10 expression in naïve B cells. Immunoglobulin class‐switching recombination events resulted in the downregulation of both Il10 and Prdm1 expression in differentiating B cells. Thus, the prolonged elevation of Blimp‐1 expression during the formation of IgM⁺CD138^hi cells without class‐switching elicits IL‐10 production. Adoptive transfer of Il10‐deficient B cells into B‐cell‐deficient mice demonstrated that IgM⁺CD138^hi cell‐derived IL‐10 supports the survival of class‐switched plasma cells and their antibody production in response to antigen challenge. These findings reveal an important role for IL‐10 secretion by IgM⁺CD138^hi cells in the complete and efficient humoral response.     

Interferon-γ and pulmonary macrophages contribute to the mechanisms underlying prolonged airway hyperresponsiveness

Background Airway hyperresponsiveness (AHR) in asthmatics includes a variable component that persists following an allergen challenge. This may be dissociated from inflammatory cell recruitment, implying a role for resident pulmonary cells in regulating the response. Objective Using improved methods of assessing AHR in a mouse model of allergic airway disease, to investigate the basis of the development of prolonged AHR. Method BALB/c mice were systemically sensitized and then challenged with aerosolized ovalbumin (OVA). Airway and tissue responsiveness were measured at baseline and at 1 day, and 1, 2 and 3 weeks after the last OVA challenge. Inflammatory cell numbers in BALF and levels of mRNA for eotaxin‐1 and ‐2, IFN‐γ, IL‐5 and ‐13 in the lung were measured at each time‐point. In further experiments, the roles of IFN‐γ and of CCR3⁺ and CD4⁺ cells in the development of prolonged AHR were assessed by blockade or depletion with monoclonal antibodies. The role of pulmonary macrophages was assessed by selective chemical depletion of these cells. Results Airway responsiveness was increased above baseline at 1 day after the last OVA challenge, and this was sustained for 1 week. In contrast, tissue‐specific responsiveness was only significantly increased above baseline at 1 day. Development of prolonged AHR was inhibited by neutralization of IFN‐γ or by depletion of pulmonary macrophages, but not by depletion of either CD4⁺ T cells or CCR3⁺ eosinophils. Conclusion An interaction between IFN‐γ and pulmonary macrophages contributed to the prolongation of airway hyperresponsiveness. In contrast, T cells and eosinophils did not contribute to prolongation of AHR. These findings emphasize the importance of the innate host response in the development of manifestations of asthma, as well as its potential relevance as a target for therapeutic intervention. Cite this as: M. Yang, R. K. Kumar and P. S. Foster, Clinical & Experimental Allergy, 2010 (40) 163–173.     

Airway remodelling and inflammation in asthma are dependent on the extracellular matrix protein fibulin‐1c

Asthma is a chronic inflammatory disease of the airways. It is characterized by allergic airway inflammation, airway remodelling, and airway hyperresponsiveness (AHR). Asthma patients, in particular those with chronic or severe asthma, have airway remodelling that is associated with the accumulation of extracellular matrix (ECM) proteins, such as collagens. Fibulin‐1 (Fbln1) is an important ECM protein that stabilizes collagen and other ECM proteins. The level of Fbln1c, one of the four Fbln1 variants, which predominates in both humans and mice, is increased in the serum and airways fluids in asthma but its function is unclear. We show that the level of Fbln1c was increased in the lungs of mice with house dust mite (HDM)‐induced chronic allergic airway disease (AAD). Genetic deletion of Fbln1c and therapeutic inhibition of Fbln1c in mice with chronic AAD reduced airway collagen deposition, and protected against AHR. Fbln1c‐deficient (Fbln1c^–/–) mice had reduced mucin (MUC) 5 AC levels, but not MUC5B levels, in the airways as compared with wild‐type (WT) mice. Fbln1c interacted with fibronectin and periostin that was linked to collagen deposition around the small airways. Fbln1c^–/– mice with AAD also had reduced numbers of α‐smooth muscle actin‐positive cells around the airways and reduced airway contractility as compared with WT mice. After HDM challenge, these mice also had fewer airway inflammatory cells, reduced interleukin (IL)‐5, IL‐13, IL‐33, tumour necrosis factor (TNF) and CXCL1 levels in the lungs, and reduced IL‐5, IL‐33 and TNF levels in lung‐draining lymph nodes. Therapeutic targeting of Fbln1c reduced the numbers of GATA3‐positive Th2 cells in the lymph nodes and lungs after chronic HDM challenge. Treatment also reduced the secretion of IL‐5 and IL‐13 from co‐cultured dendritic cells and T cells restimulated with HDM extract. Human epithelial cells cultured with Fbln1c peptide produced more CXCL1 mRNA than medium‐treated controls. Our data show that Fbln1c may be a therapeutic target in chronic asthma. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Tim1 and Tim3 are not essential for experimental allergic asthma

Background Initial studies suggested that polymorphisms in Tim1 and Tim3 contribute to the development of airway hyperreactivity (AHR) in an acute mouse model of asthma. This was also mirrored in human genetic studies where polymorphisms in Tim1 and Tim3 have been associated with atopic populations. Objective Further studies using anti‐Tim1 or ‐Tim3 antibodies, or Tim fusion proteins, have also suggested that these molecules may function as regulators of type‐1 and type‐2 immunity. However, their role in the development of AHR and airway inflammation remains unclear. Given the proposed roles for Tim1 and Tim3 in type‐1 and type‐2 responses, we sought to determine whether these molecules were important in regulating antigen‐driven lung allergy and inflammation. Method We used Tim1‐ and Tim3‐deficient mice and determined how the development of allergic lung inflammation was affected. Results AHR was induced normally in the absence of both Tim1 and Tim3, although Tim1‐deficient mice did show a small but significant decrease in cell infiltration in the lung and blood eosinophilia. Although Tim3 was expressed on CD4⁺ T cells in the allergic lung, Tim1 expression was restricted to CD86⁺ B cells. Conclusions and clinical relevance Thus, Tim1 and Tim3 are not essential for the induction of the type‐2 response in lung allergy. This is contrary to what was proposed in a number of other studies using neutralizing and activating antibodies and questions the clinical relevance of Tim1 and Tim3 for novel allergy therapies. Cite this as: J. L. Barlow, S. H. Wong, S. J. Ballantyne, H. E. Jolin and A. N. J. McKenzie, Clinical & Experimental Allergy, 2011 (41) 1012–1021.     

Surface immunoglobulin phenotype of complement receptor-positive and complement receptor-negative B cells of normal and xid immunodeficient mice

Single- and dual-parameter fluorescence-activated cell sorter analyses of complement receptor-positive and -negative (CR⁺ and CR^?) splenic B cells of normal and xid immunodeficient mice were performed to determine the frequencies of surface (s) IgM⁺ and sIgD⁺ cells, as well as the quantities of sIgM and sIgD on these cells. Single parameter analysis revealed identical frequencies of sIgM⁺ and sIgD⁺ cells in the CR⁺ B cell population of normal mice. In contrast, the percentage of sIgM⁺ cells exceeded that of sIgD⁺ cells in the CR^? normal and CR⁺ xid B cell population to a similar extent. CR^? xid B cells showed an even greater discrepancy between sIgM⁺ and sIgD⁺ cells. Dual-parameter analysis of CR⁺ normal B cells showed the typical sIg phenotype of mature B cells, i.e. an intermediate density of sIgM and a high density of sIgD. The CR⁺ xid B cell population displayed an increase in cells with high sIgM and no-to-low sIgD and a decrease in cells with intermediate sIgM and high sIgD when compared to CR⁺ normal cells. CR^? B cells from normal and xid immunodeficient mice were similar in that both consisted largely of a cell population with low sIgM and no-to-low sIgD. However, the CR^? normal B cell population also contained cells with intermediate sIgM and high sIgD, which were lacking in the CR^? xid cell population. Similar results were obtained when CR⁺ and CR^? B cells from normal and xid mice were purified from a small resting B cell population, isolated by elutriation. This indicates that the sIg phenotype of CR^? cells is not due to the presence of large cells which have lost sIgD during activation.     

CD5+ B Cell-Dependent Regulation of the Murine T-Cell Independent Immune Response Against the Human Blood Group a Antigen

The CD5⁺B lymphocyte (B1a) population is known to be involved in most immune responses to microorganism Tl antigens. Moreover, xid mice deficient for immune responses against Tl-2 antigens are known to lack the B1a population, suggesting a role for B1a cells in Tl-2 immune responses. We previously established that the oligosaccharide human blood group A antigen stimulated murine Tl-2 immune responses. In this work, we show that the frequency of anti-A-secreting hybridomas was higher in mice with larger splenic B1a populations and that in vivo anti-CD5 treatment reduced anti-A immune response without affecting the response against TD RBC antigens. A similar effect was observed by in vitro anti-CD5 treatment of splenocytes. The in vivo anti-CD5 treatment also interfered with the immunization-dependent increase in splenocyte numbers. These results are in agreement with an important role for the B-cell CD5 receptor in the regulation of Tl-2 immune responses possibly mediated by its interaction with the CD72 ligand.     

B and CD4+ T‐cell expression of TLR2 is critical for optimal induction of a T‐cell‐dependent humoral immune response to intact Streptococcus pneumoniae

TLR2^?/? mice immunized with Streptococcus pneumoniae (Pn) elicit normal IgM, but defective CD4⁺ T‐cell‐dependent type 1 IgG isotype production, associated with a largely intact innate immune response. We studied the T‐cell‐dependent phosphorylcholine (PC)‐specific IgG3 versus the T‐cell‐independent IgM response to Pn to determine whether TLR2 signals directly via the adaptive immune system. Pn‐activated TLR2^?/? BMDC have only a modest defect in cytokine secretion, undergo normal maturation, and when transferred into naïve WT mice elicit a normal IgM and IgG3 anti‐PC response, relative to WT BMDC. Pn synergizes with BCR and TCR signaling for DNA synthesis in purified WT B and CD4⁺T cells, respectively, but is defective in cells lacking TLR2. Pn primes TLR2^?/? mice for a normal CD4⁺ T‐cell IFN‐γ recall response. Notably, TLR2^?/? B cells transferred into RAG‐2^?/? mice with WT CD4⁺T cells, or TLR2^?/? CD4⁺T cells transferred into athymic nude mice, each elicit a defective IgG3, in contrast to normal IgM, anti‐PC response relative to WT cells. These data are the first to demonstrate a major role for B‐cell and CD4⁺ T‐cell expression of TLR2 for eliciting an anti‐bacterial humoral immune response.     

MMP induced by Gr‐1+ cells are crucial for recruitment of Th cells into the airways

Th2 lymphocytes deliver essential signals for induction of asthmatic airway inflammation. We previously found that airway antigen challenge induces recruitment of Gr‐1⁺ neutrophils prior to the recruitment of Th2 cells. We examined, therefore, whether Gr‐1⁺ cells contribute to the development of Th2‐dependent airway inflammation. Systemic depletion of Gr‐1⁺ cells using the RB6‐8C5 monoclonal antibody reduced Th2 cell recruitment following i.n. antigen challenge. The levels of both MMP‐9 and the tissue inhibitor of matrix metalloproteinases‐1 mRNA were up‐regulated in the lungs of mice 12 h after i.n. antigen challenge. Up‐regulation of tissue inhibitor of matrix metalloproteinases‐1 was independent of Gr‐1⁺ cells, whereas up‐regulation of MMP‐9 RNA and total gelatinolytic activity was dramatically reduced in mice depleted of Gr‐1⁺ cells. At 24 h after challenge, total lung collagenolytic activity was also up‐regulated, in a Gr‐1⁺ cell‐dependent fashion. Systemic inhibition of MMP‐8 and MMP‐9 reduced the airway recruitment of Th cells, resulting in significantly reduced eosinophilic inflammation. These data suggest that antigen challenge via the airway activates Gr‐1⁺ cells and consequently MMP to facilitate the recruitment of Th cells in the airway inflammatory response.     

PB76: a novel surface glycoprotein preferentially expressed on mouse pre-B cells and plasma cells detected by the monoclonal antibody G-52

A monoclonal antibody (mAb) G-5-2 was isolated which binds to transformed as well as normal cells of the B lineage but not to cells of the T cell, myeloid lineages nor to fibroblasts. mAb G-5-2 reacts with pre-B and plasma cell-transformed lines, and it preferentially recognizes normal pre-B cells from fetal liver and bone marrow as well as plasma cells from spleen of mice. G-5-2⁺ fetal liver cells isolated by cell sorter express mRNA for μ heavy chain Ig gene and generate in vitro antibody-producing cells when co-cultured with lipopolysaccharide and rat thymocyte filler cells. During development the frequency and staining intensity of G-5-2⁺ cells in fetal liver from normal mice increases from 1% G-5-2⁺ cells at day 14 to～7% positive cells at day 18 of gestation. Several strains or normal mice contain comparable numbers of G-5-2⁺ cells as well as B-220⁺ and BP-1⁺ B cell precursors in the fetal liver. Mice carrying the xid mutation have 3-4-fold less G-5-2⁺ as well as B-220⁺ and BP-1⁺ cells in the fetal liver, suggesting that the effects of the xid mutation may be manifested from early stages of B cell development. Fetal liver cells from mice carrying the scid mutation were found to contain normal numbers of G-5-2⁺ as well as B-220⁺ and BP-1⁺ pre-B cells. These results indicate that differentiation from progenitors to pre-B cells in scid mice may occur normally; the scid mutation would thus appear to affect the process of rearrangement and expression of the Ig genes in the developing pre-B cells. mAb G-5-2 precipitates a 76-kDa glycoprotein from surface-radiolabeled pre-B cells and plasma cells. Taken together, these results indicate that G-5-2 mAb recognizes a novel B cell lineage-specific surface molecule called PB76 which is preferentially expressed by pre-B cells and plasma cells.     

Ligation of TLR7 on CD19+CD1dhi B cells suppresses allergic lung inflammation via regulatory T cells

B cells have been described as having the capacity to regulate cellular immune responses and suppress inflammatory processes. One such regulatory B‐cell population is defined as IL‐10‐producing CD19⁺CD1d^hi cells. Previous work has identified an expansion of these cells in mice infected with the helminth, Schistosoma mansoni. Here, microarray analysis of CD19⁺CD1d^hi B cells from mice infected with S. mansoni demonstrated significantly increased Tlr7 expression, while CD19⁺CD1d^hi B cells from uninfected mice also demonstrated elevated Tlr7 expression. Using IL‐10 reporter, Il10^?/? and Tlr7^?/‐ mice, we formally demonstrate that TLR7 ligation of CD19⁺CD1d^hi B cells increases their capacity to produce IL‐10. In a mouse model of allergic lung inflammation, the adoptive transfer of TLR7‐elicited CD19⁺CD1d^hi B cells reduced airway inflammation and associated airway hyperresponsiveness. Using DEREG mice to deplete FoxP3⁺ T regulatory cells in allergen‐sensitized mice, we show that that TLR7‐elicited CD19⁺CD1d^hi B cells suppress airway hyperresponsiveness via a T regulatory cell dependent mechanism. These studies identify that TLR7 stimulation leads to the expansion of IL‐10‐producing CD19⁺CD1d^hi B cells, which can suppress allergic lung inflammation via T regulatory cells.     

TLR2 and TLR4 signaling shapes specific antibody responses to Salmonella typhi antigens

TLR directly induce innate immune responses by sensing a variety of microbial components and are critical for the fine‐tuning of subsequent adaptive immune responses. However, their impact and mechanism of action on antibody responses against bacterial antigens are not yet fully understood. Salmonella enterica serovar Typhi (S. typhi) porins have been characterized as inducers of long‐lasting specific antibody responses in mice. In this report, we show that immunization of TLR4‐deficient (TLR4^?/?), myeloid differentiating gene 88‐deficient and Toll/IL‐R domain‐containing adaptor‐inducing IFN‐β‐deficient mice with S. typhi porins led to significantly reduced B‐cell responses. TLR2^?/? mice, as well, showed reduced IgG titers with a more pronounced impairment in the production of IgG3 anti‐porins antibodies. Adoptive transfer of TLR2^?/?‐ or TLR4^?/?‐B cells into B‐cell‐deficient mice revealed a direct effect of TLR4 on B cells for the primary IgM response, whereas stimulation of B cells via TLR2 was important for IgG production. Furthermore, S. typhi porins were found to efficiently elicit maturation of CD11c⁺ conventional DC. Taken together, S. typhi porins represent not only a suitable B‐cell antigen for vaccination, but exhibit potent TLR‐dependent stimulatory functions on B cells and DC, which help to further enhance and shape the antibody response.     

Effects of furosemide on allergic asthmatic responses in mice

Background The syndrome of allergic asthma features reversible bronchoconstriction, airway inflammation and hyperresponsiveness as well as airway remodelling, including goblet cell hyperplasia. Managing severe asthma is still a clinical challenge. Numerous studies report that furosemide, an inhibitor of Na⁺–K⁺–Cl^? cotransporter (NKCC) reduces airway hyperresponsiveness (AHR) in asthmatic patients. However, the mechanism by which furosemide exerts anti‐asthmatic action remains unclear. Objective This study sought to investigate the cellular profile of NKCC1 expression in the lung and examine the effects of furosemide on several outcome measurements in a mouse model of allergic asthma. Methods Mice were sensitized and challenged with ovalbumin (OVA). Before challenge, the OVA‐sensitized mice were treated with furosemide (4.0 mg/kg/day, via daily intraperitoneal injection for 5 days). Outcome measurements in naïve, OVA‐exposure, furosemide‐treated naïve and furosemide‐treated OVA‐exposed mice included the slope of the relationship between inhaled methacholine (MCh) concentration and respiratory system resistance (Slope·R_RS), bronchoalveolar lavage (BAL) cell counts and immunohistochemical and immunoblotting assays of lung tissues. Results NKCC1 immunoreactivity was observed in airway epithelial cells (AECs) and alveolar type II (ATII) cells of the control mice. OVA exposure enhanced the expression of NKCC1 in AECs and ATII cells, and increased the infiltration of NKCC1‐expressing T lymphocytes in the lung. NKCC1 immunoreactivity was not detected in the airway smooth muscle (ASM) cells. Furosemide treatment reduced the Slope·R_RS in both naïve and OVA‐exposed mice by about 50%. Furosemide treatment also increased T lymphocyte infiltration to the lung in OVA‐exposed mice by approximately 53%, but had no effect on pulmonary goblet cell hyperplasia. Conclusions and Clinical Relevance Furosemide decreases basal airway responsiveness, thereby reducing the extent of allergen‐induced AHR. However, the same treatment also increases T lymphocytes infiltration in the course of allergic asthma. Further studies are necessary to address the usefulness of furosemide in the clinical treatment of asthma. Cite this as: S. Wang, Y.‐Y. Xiang, R. Ellis, J. Wattie, M. Feng, M. D. Inman and W.‐Y. Lu, Clinical & Experimental Allergy, 2011 (41) 1456–1467.     

EGF receptor activation during allergic sensitization affects IL‐6‐induced T‐cell influx to airways in a rat model of asthma

EGF receptor (EGFR) is involved in cell differentiation and proliferation in airways and may trigger cytokine production by T cells. We hypothesized that EGFR inhibition at the time of allergic sensitization may affect subsequent immune reactions. Brown Norway rats were sensitized with OVA, received the EGFR tyrosine kinase inhibitor, AG1478 from days 0 to 7 and OVA challenge on day 14. OVA‐specific IgE in serum and cytokines and chemokines in BAL were measured 24 h after challenge. To evaluate effects on airway hyperresponsiveness (AHR), rats were sensitized, treated with AG1478, intranasally challenged, and then AHR was assessed. Furthermore chemotactic activity of BALF for CD4⁺ T cells was examined. The eosinophils, neutrophils and lymphocytes in BAL were increased by OVA and only the lymphocytes were reduced by AG1478. OVA significantly enhanced IL‐6 concentration in BAL, which was inhibited by AG1478. However AHR, OVA‐specific IgE and IL‐4 mRNA expression in CD4⁺ T cells were not affected by AG1478. BALF from OVA‐sensitized/challenged rats induced CD4⁺ T‐cell migration, which was inhibited by both AG1478 treatment in vivo and neutralization of IL‐6 in vitro. EGFR activation during sensitization may affect the subsequent influx of CD4⁺ T cells to airways, mainly mediated through IL‐6.     

CD4+CD25+ regulatory T cells utilize FasL as a mechanism to restrict DC priming functions in cutaneous immune responses

Recent studies have suggested Fas‐mediated elimination of antigen‐presenting cells as an important mechanism down‐regulating the induction of autoimmune responses. It remains unknown whether this mechanism restricts the magnitude of immune responses to non‐self antigens. We used a mouse model of a cutaneous CD8⁺ T‐cell‐mediated immune response (contact hypersensitivity, CHS) to test if CD4⁺CD25⁺ T cells expressing FasL regulate hapten‐specific effector CD8⁺ T cell expansion through the elimination of Fas‐expressing hapten‐presenting DC. In WT mice, attenuation of CD4⁺CD25⁺ T regulatory cell activity by anti‐CD25 mAb increased hapten‐presenting DC numbers in skin‐draining LN, which led to increased effector CD8⁺ T‐cell priming for CHS responses. In contrast, CD4⁺CD25⁺ T cells did not regulate hapten‐specific CD8⁺ T‐cell priming and CHS responses initiated by Fas‐defective (lpr) DC. Thus, restricting DC priming functions through Fas–FasL interactions is a potent mechanism employed by CD4⁺CD25⁺ regulatory cells to restrict CD8⁺ T‐cell‐mediated allergic immune responses in the skin.     

Fcγ receptor‐mediated antigen uptake by lung DC contributes to allergic airway hyper‐responsiveness and inflammation

During asthma, lung DC capture and process antigens to initiate and maintain allergic Th2 cell responses to inhaled allergens. The aim of the study was to investigate whether allergen‐specific IgG, generated during sensitization, can potentiate the acute airway inflammation through Fcγ receptor (FcγR)‐mediated antigen uptake and enhance antigen presentation resulting in augmented T‐cell proliferation. We examined the impact of antigen presentation and T‐cell stimulation on allergic airway hyperresponsiveness and inflammation using transgenic and gene‐deficient mice. Both airway inflammation and eosinophilia in bronchoalveolar lavage fluid were markedly reduced in sensitized and challenged FcγR‐deficient mice. Lung DC of WT, but not FcγR‐deficient mice, induced increased antigen‐specific CD4⁺ T‐cell proliferation when pulsed with anti‐OVA IgG immune complexes. Intranasal application of anti‐OVA IgG immune complexes resulted in enhanced airway inflammation, eosinophilia and Th2 cytokine release, mediated through enhanced antigen‐specific T‐cell proliferation in vivo. Finally, antigen‐specific IgG in the serum of sensitized mice led to a significant increase of antigen‐specific CD4⁺ T‐cell proliferation induced by WT, but not FcγR‐deficient, lung DC. We conclude that FcγR‐mediated enhanced antigen presentation and T‐cell stimulation by lung DC has a significant impact on inflammatory responses following allergen challenge in asthma.     

Both B‐1a and B‐1b cells exposed to Mycobacterium tuberculosis lipids differentiate into IgM antibody‐secreting cells

Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis. The cellular immune response to mycobacteria has been characterized extensively, but the antibody response remains underexplored. The present study aimed to examine whether host or bacterial phospholipids induce secretion of IgM, and specifically anti‐phospholipid IgM, antibodies by B cells and to identify the responsible B‐cell subset. Here we show that peritoneal B cells responded to lipid antigens by secreting IgM antibodies. Specifically, stimulation with M. tuberculosis H37Rv total lipids resulted in significant induction of total and anti‐phosphatidylcholine IgM. Similarly, IgM antibody production increased significantly with stimulation by whole Mycobacterium bovis bacillus Calmette–Guérin. The B‐1 subset was the dominant source of IgM antibodies after exposure to cardiolipin. Both CD5⁺ B‐1a and CD5^? B‐1b cell subsets secreted total IgM antibodies after exposure to M. tuberculosis H37Rv total lipids in vitro. Overall, our results suggest that the poly‐reactive B‐1 cell repertoire contributes to non‐specific anti‐phospholipid IgM antibody secretion in response to M. tuberculosis lipids.     

Human α1‐antitrypsin modifies B‐lymphocyte responses during allograft transplantation

B‐lymphocyte activities are associated with allograft rejection. Interleukin‐10 (IL‐10) ‐expressing B cells, however, exhibit regulatory attributes. Human α1‐antitrypsin (hAAT), a clinically available anti‐inflammatory circulating glycoprotein that rises during acute‐phase responses, promotes semi‐mature dendritic cells and regulatory T (Treg) cells during alloimmune responses. Whether B lymphocytes are also targets of hAAT activity has yet to be determined. Here, we examine whether hAAT modulates B‐cell responses. In culture, hAAT reduced the lipopolysaccharide‐stimulated Ki‐67⁺ B‐cell population, IgM release and surface CD40 levels, but elevated IL‐10‐producing cells 1.5‐fold. In CD40 ligand‐stimulated cultures, hAAT promoted a similar trend; reduction in the Ki‐67⁺ B‐cell population and in surface expression of CD86, CD80 and MHCII. hAAT increased interferon‐γ‐stimulated macrophage B‐cell activating factor (BAFF) secretion, and reduced BAFF‐receptor levels. Draining lymph nodes of transgenic mice that express circulating hAAT (C57BL/6 background) and that received skin allografts exhibited reduced B‐lymphocyte activation compared with wild‐type recipients. BSA‐vaccinated hAAT transgenic mice exhibited 2.9‐fold lower BSA‐specific IgG levels, but 2.3‐fold greater IgM levels, compared with wild‐type mice. Circulating Treg cells were 1.3‐fold greater in transgenic hAAT mice, but lower in B‐cell knockout (BKO) and chimeric hAAT–BKO mice, compared with wild‐type mice. In conclusion, B cells are cellular targets of hAAT. hAAT‐induced Treg cell expansion appears to be B‐cell‐dependent. These changes support the tolerogenic properties of hAAT during immune responses, and suggest that hAAT may be beneficial in pathologies that involve excessive B‐cell responses.