The predominant immune response to allergens in atopic asthma is mediated by Th2 cells resulting from interactions between dendritic cells (DCs) and T cells. In the past few years, we and others have elucidated mechanisms that result in Th2 differentiation. However, the mechanisms that prevent the generation of Th2 responses in the respiratory tract are less well understood. Using a murine model of airway inflammation and two different models of tolerance induced either by administration of high dose of antigen (Ag) or by repeated aerosol exposures to Ag which appear to cause tolerance via different mediators, we are investigating mechanisms that induce or inhibit immune responses in the lungs of mice. Despite advances in our knowledge of T cell differentiation, little is known about how DCs, which are pivotal to lung immune responses, influence the decision between inflammation and tolerance. Our current understanding of T cell and DC biology in the lung lead us to hypothesize that: 1. The induction of inflammation versus tolerance depends on the differential DC phenotype in lung-draining lymph nodes (LNs). 2. Lung LNDCs induce the development of specific T cell subsets that in turn induce either inflammation or tolerance. To address these hypotheses, we will:
Aim I. Characterize DCs in naive, immunized and tolerized mice both phenotypically and functionally. a) DCs and T cells from naive mice or from mice with inflammation or tolerance will be isolated from lung draining LNs and characterized, b) i) DCs from lung-draining LNs of sensitized mice will be transferred to naive wt mice and tested for tolerance induction, c) DCs from LNs of tolerized mice will be transferred to naive mice and tested for inflammation. Bronchoalveolar lavage (BAL) will be performed and lungs will be harvested. CD4+ T cells will be isolated from lung LNs and spleens and restimulated with antigen. End points: lung inflammation, cytokines, serum IgE levels, T cell markers.
Aim II. Determine the ability of differentially matured bone-marrow-derived DCs to break tolerance in the respiratory tract. DCs will be cultured with different maturation stimuli in vitro, pulsed with Ag in vitro and delivered intratracheally (i.t.) into naive or tolerized mice and tested for the ability of the DCs to induce inflammation or break tolerance in mice subjected to either of the two tolerizing conditions. Same end-points will be investigated as in Aim I.
Aim III. Investigate mechanisms of DC-T call interactions in both in vivo and in vitro studies, wt, B7-1-/-, B7-2-/-, CD40-/- mice will be subjected to models of inflammation and tolerance. Lung-draining LNs will be harvested. DCs and CD4+ T cells will be isolated and characterized phenotypically and functionally.
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