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Common variable immunodeficiency (CVID) is characterized by hypogammaglobulinemia and clinical manifestations such as infections, autoimmunity, and malignancy. We sought to determine if responsiveness to interleukin-21 (IL-21), a key cytokine for B cell differentiation, correlates with distinct clinical phenotypes in CVID. CVID subjects were recruited through the Canadian Primary Immunodeficiency Evaluative Survey registry. Peripheral blood mononuclear cells were cultured with anti-CD40 ± interferon-gamma, interleukin-4 (IL-4), IL-21, and/or IL-4+IL-21. B cell subpopulations and IgG production were determined at baseline and day 7 by flow cytometry and ELISA. Clinical complications were compared using contingency tables. CVID subjects exhibited decreased CD27+ B cells and IgG production after 7 days of stimulation with anti-CD40+IL-21 (p 2% class-switched memory B cells at baseline. The IL-4 and IL-21 in vitro assays distinguish two groups of CVID subjects and can be used with baseline B cell subpopulation phenotyping to better identify patients experiencing more vs. fewer clinical non-infectious complications and potentially to modulate therapy.

IVIg is widely used as an immunomodulatory therapy. We have recently demonstrated that IVIg protects against airway hyperresponsiveness (AHR) and inflammation in mouse models of allergic airways disease (AAD), associated with induction of Foxp3+ regulatory T cells (Treg). Using mice carrying a DTR/EGFP transgene under the control of the Foxp3 promoter (DEREG mice), we demonstrate in this study that IVIg generates a de novo population of peripheral Treg (pTreg) in the absence of endogenous Treg. IVIg-generated pTreg were sufficient for inhibition of OVA-induced AHR in an Ag-driven murine model of AAD. In the absence of endogenous Treg, IVIg failed to confer protection against AHR and airway inflammation. Adoptive transfer of purified IVIg-generated pTreg prior to Ag challenge effectively prevented airway inflammation and AHR in an Ag-specific manner. Microarray gene expression profiling of IVIg-generated pTreg revealed upregulation of genes associated with cell cycle, chromatin, cytoskeleton/motility, immunity, and apoptosis. These data demonstrate the importance of Treg in regulating AAD and show that IVIg-generated pTreg are necessary and sufficient for inhibition of allergen-induced AAD. The ability of IVIg to generate pure populations of highly Ag-specific pTreg represents a new avenue to study pTreg, the cross-talk between humoral and cellular immunity, and regulation of the inflammatory response to Ags.

The regulatory properties of B cells have been studied in autoimmune diseases; however, their role in allergic diseases is poorly understood. We demonstrate that Semaphorin 4C (Sema4C), an axonal guidance molecule, plays a crucial role in B cell regulatory function. Mice deficient in Sema4C exhibited increased airway inflammation after allergen exposure, with massive eosinophilic lung infiltrates and increased Th2 cytokines. This phenotype was reproduced by mixed bone marrow chimeric mice with Sema4C deficient only in B cells, indicating that B lymphocytes were the key cells affected by the absence of Sema4C expression in allergic inflammation. We determined that Sema4C-deficient CD19+CD138+ cells exhibited decreased IL-10 and increased IL-4 expression in vivo and in vitro. Adoptive transfer of Sema4c−/− CD19+CD138+ cells induced marked pulmonary inflammation, eosinophilia, and increased bronchoalveolar lavage fluid IL-4 and IL-5, whereas adoptive transfer of wild-type CD19+CD138+IL-10+ cells dramatically decreased allergic airway inflammation in wild-type and Sema4c−/− mice. This study identifies a novel pathway by which Th2-mediated immune responses are regulated. It highlights the importance of plasma cells as regulatory cells in allergic inflammation and suggests that CD138+ B cells contribute to cytokine balance and are important for maintenance of immune homeostasis in allergic airways disease. Furthermore, we demonstrate that Sema4C is critical for optimal regulatory cytokine production in CD138+ B cells.

Background Semaphorins are important molecules in embryonic development and multiple semaphorins have been identified as having key roles in immune regulation. To date, there is little known about Semaphorin 4C (Sema4C) in immune biology. We report for the first time that Sema4C is inducible in human and murine B-cells and may be important for normal B-cell development. Methods Human tonsillar B-cells were studied following activation via anti-CD40 antibodies in the presence or absence of representative Th1, Th2, and regulatory cytokines. Murine B-cells from WT and Sema4C−/− mice were similarly stimulated. B-cell phenotyping in WT and Sema4C mutant mice was performed by flow cytometry and lymphoid architecture was studied by immunohistochemistry. Sema4C expression and synapse formation were analyzed by confocal microscopy. Results Gene array studies performed on human tonsillar B-cells stimulated to produce IgE revealed that Sema4C was among the top genes expressed at 24 h, and the only semaphorin to be increased under Th2 conditions. Validation studies demonstrated that human and murine B-cells expressed Sema4C under similar conditions. Sema4C−/− mice had impaired maturation of B-cell follicles in spleens and associated decreases in follicular and marginal zone B-cells as well as impaired IgG and IgA production. In keeping with a potential role in maturation of B-cells, Sema4C was expressed predominantly on CD27+ human B-cells. Within 72 h of B-cell activation, Sema4C was localized to one pole in a synapse-like structure, in association with F-actin, B-cell receptor, and Plexin-B2. Cell polarization was impaired in Sema4C−/− mice. Conclusion We have identified a novel immune semaphorin induced in human and murine B-cells under Th2 conditions. Sema4C appears to be a marker for human memory B-cells. It may be important for B-cell polarization and for the formation of normal splenic follicles.

Methods Wild-type and Sema4C mice were sensitized intraperitoneally using 100 μL OVA (0.5 mg/mL ovalbumin and 4 mg/mL aluminum hydroxide in PBS) on days 0 and 14, and were challenged intranasally using 20 μL OVA (10 mg/mL ovalbumin in PBS) from days 28 to 30. Sacrifice and analysis of Airway Hyperresponsiveness via flexiVent was performed on day 31. Serum IgE and IL-10 expression levels were measured by ELISA. B cells were phenotyped by fluorescence-activated cell sorting (FACS). B cell motility was measured by migration assays.

The dendritic cell immunoreceptor (DCIR) is an inhibitory C-type lection receptor possessing immunoregulatory properties. We have shown that DCIR serves as a receptor for IVIg. In this study, we sought to determine its requirement in modulating IVIg mechanism of action by using a murine model of allergic of airways disease. Wild type (WT) and DCIR−/− mice were sensitized and challenged with ovalbumin (OVA), and lung histopathology, bronchoalveolar lavage and phenotyping of inflammatory cells were performed. We further studied the effect of IVIg on allergen uptake and processing of CD11c+ cells in vitro. Briefly, bone marrow-derived dendritic cells from WT, DCIR−/− and Fcr2b−/− mice were treated with IVIg prior to exposure to OVA-ALEXA-647 or DQ-OVA. DCIR−/− mice exhibit exacerbated pulmonary inflammation and eosinophilia compared to WT mice; this phenotype is rescued by IVIg treatment as in WT mice. Furthermore, IVIg significantly impairs the antigen uptake and processing machinery of dendritic cells from WT and DCIR−/− derived BMDCs but not that of Fcr2b−/−-derived BMDCs. In conclusion, DCIR is important in regulating allergic airway inflammation and its presence is not essential for IVIg to function. DCIR appears to be required for normal regulation of pulmonary inflammation.

The increase in airway smooth muscle (ASM) mass is a major structural change in asthma. This increase has been attributed to ASM cell (ASMC) hyperplasia and hypertrophy. The distance between ASMC and the epithelium is reduced, suggesting migration of smooth muscle cells toward the epithelium. Recent studies have suggested a role of chemokines in ASMC migration toward the epithelium; however, chemokines have other biological effects. The objective of the current study is to test the hypothesis that chemokines (eotaxin, RANTES, IL-8, and MIP-1α) can directly influence ASMC mass by increasing the rate of proliferation or enhancing the survival of these cells. Human ASMCs were exposed to different concentrations of eotaxin, RANTES, IL-8, or MIP-1α. To test for proliferation, matched control and stimulated ASMC were pulsed with [3H]thymidine, or ASMCs were stained with BrdU and then analyzed with flow cytometry. Apoptosis was measured using Annexin V staining and flow cytometry. Expression of phosphorylated p42/p44 and MAPKs was assessed by Western blot. In a concentration-dependent manner, chemokines including eotaxin, RANTES, IL-8, and MIP-1α increased ASMC’s [3H]thymidine incorporation and DNA synthesis. IL-8, eotaxin, and MIP-1α decreased the rate of apoptosis of ASMCs compared with the matched controls. A significant increase in phosphorylated p42/p44 MAPKs was seen after treating ASMCs with RANTES and eotaxin. Moreover, inhibition of p42/p44 MAPK phosphorylation reduced the level of chemokine-induced ASM proliferation. We conclude that chemokines might contribute to airway remodeling seen in asthma by enhancing the number and survival of ASMCs.

We have previously found that mice with Semaphorin 4C (Sema4C) deficient B cells exhibited exaggerated airway damage and pulmonary inflammation during allergic airways disease (AAD). We aimed to determine the mechanisms by which Sema4C on B-cells regulates AAD. Following allergen exposure, Sema4C-/- AAD mice had significantly fewer CD138high IL-10+ B cells (CD138high Breg) and significantly increased CD138high IL4+ B cells in spleen. In vitro, Sema4C-/- B cells consistently failed to differentiate into CD138high Breg under Th2 culture conditions. Adoptive transfer of Sema4C-/- plasma cells induced exacerbated Th2 responses, with airway eosinophilia and increased IL-4+ inflammatory cell infiltrates in the lungs. In contrast, transfer of CD138high Breg to WT mice with AAD dramatically alleviated airway inflammation. Co-culturing Sema4C-/- B cells with Sema4C transfected cells (Hek293-Sema4C) corrected impaired CD138high Breg development. Co-culture of B cells with Hek293-Sema4C decreased IgE production for both WT and Sema4C-/- B cells. Sema4C is essential for the development of CD138high Breg and may play a crucial role in the auto-regulation of B cells in allergic inflammation.

We have shown that induced regulatory T cells (iTreg) are necessary and sufficient to rescue antigen-challenged mice in a murine model of allergic airways disease (AAD). Intravenous immunoglobulin (IVIg) treatment of wild-type mice sensitized and challenged to antigen induces Treg in a dendritic-cell (DC) dependent mechanism. In the present study, DEREG mice (which co-express diphtheria toxin (DT) receptor with Foxp3, allowing for selective transient depletion of Treg) were treated with IVIg after depletion of all endogenous Treg with DT. This induces a phenotypically distinct population of functional iTreg. This increase in iTreg is associated with significant augmentation of systemic regulatory B cells (Breg), which express IL-10 and are CD138-high Blimp-low plasmablasts. Under these conditions, all aspects of AAD are ameliorated. However, in animals with an intact endogenous Treg compartment, Breg are not significantly increased by IVIg treatment. We postulate a balance between regulatory cells which favors Treg, and may shift to Breg in the absence of endogenous Treg. Further work will delineate the Breg:Treg balance and how these regulatory subsets contribute to suppression of allergic inflammation.

Background IVIg is a polyclonal IgG preparation with potent immune-modulating properties. We demonstrated that IVIg protects against airway hyperreactivity (AHR) and airway inflammation in mouse models of allergic airway disease, accompanied by peripheral induction of Foxp3 regulatory T-cells (iTreg). The requirement of IVIginduced iTreg and their antigen-specificity in attenuation of AHR and airway inflammation remains unknown.