Simple Assays for Measuring Innate Interactions with Fungi
In recent decades, there has been a steady rise in immunocompromised populations and consequently a dramatic increase in the clinical relevance of normally non-pathogenic and commensal fungi such as Aspergillus fumigatus and Candida albicans. Understandin
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1. Introduction The study of the interactions between the innate and adaptive arms of the immune system can provide significant insights into the mechanisms for the generation of an appropriate and protective immune response to pathogenic challenge. In accordance with this, complete immunity to the majority of pathogenic organisms, including Candida albicans, often requires the activation of antigen-presenting cells and T cells of the innate and adaptive arms of the immune system, respectively (1). Dendritic cells (DCs) are the most effective of the antigen-presenting cells and hence serve as a critical link to T-cell activation. The ability to generate relatively large numbers of DCs from progenitor cells in the bone marrow and to maintain these cells in vitro has been critically important for defining roles of DCs in health and disease. Herein, we describe
Alexandra C. Brand and Donna M. MacCallum (eds.), Host-Fungus Interactions: Methods and Protocols, Methods in Molecular Biology, vol. 845, DOI 10.1007/978-1-61779-539-8_17, © Springer Science+Business Media, LLC 2012
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the culture of bone marrow-derived DCs or BMDCs (2, 3), and the isolation of splenic T cells. We then study the proliferation of and cytokine production by splenic T cells in response to co-culture with BMDCs primed with C. albicans. This co-culture condition is referred to as a mixed leukocyte reaction. Rapid expansion of pathogen-specific T cells is often triggered by pathogen-primed DCs. While the quantification of T-cell proliferation generally requires the pulsing of the T cells with [3H] thymidine (4, 5), we employ an alternative method which instead utilises carboxyfluorescein diacetate succinimidyl ester (CFDA-SE). This labelling method is safer because it eliminates the use of a radioactive substance. An additional advantage is that in conjunction with flow cytometry, CFDA-SE labelling facilitates the simultaneous quantification of T-cell proliferation and viability. In principle, CFDA-SE passively diffuses through cell membranes. It is retained in the cytoplasm of the labelled cells following the removal of acetates by intracellular esterases and covalent coupling of the succinimidyl group to intracellular molecules. The subsequent equal division of the highly fluorescent CFSE content between mother and daughter cells permits the tracking of up to seven cell divisions (6). Flow cytometry also provides additional insights into the proliferating T cells through the use of phenotyping antibodies such as anti-CD4 and anti-CD8 antibodies (7, 8). For example, CD8+ T cells have the ability to kill tumour cells and cells infected with intracellular pathogens. In contrast, most CD4+ T cells do not have cytolytic ability and their key role is to help to activate other immune cells. CD4+ T cells are divided into at least three helper subsets (Th1, Th2, and Th17) that secrete signature cytokines (gamma interferon, interleukin 4, and interleukin 17, respectively) to promote immune responses tailored made for the pathogen
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