Coculture of Human Dendritic and T Cells for the Study of Specific T Cell-Mediated Responses Against Food Allergens

Abstract

Dendritic cells (DCs) connect innate and adaptive immunity by sampling, capturing, processing, and presenting the allergen to distinct subsets of CD4⁺ T cells. In food allergy, this process leads to the generation of allergen-specific Th2 responses and the production of type 2 cytokines that ultimately induce the synthesis of IgE by allergen-specific B cells. In this chapter, we have described different protocols for the isolation of circulating DCs as well as the generation of DC-like cells derived from autologous peripheral monocytes and the human monocytic THP-1 cell line. Coculture of isolated/generated DCs with CD4⁺ T cells obtained from PBMCs of allergic subjects allows the study of antigen-specific T cell immune responses against food allergens. Early responses upon allergen recognition can be determined by the upregulation of activation markers such as CD154 (CD40 ligand) and the detection of type 2 cytokines (IL-4, IL-5, IL-9, and IL-13). Delayed allergen-specific CD4⁺ T cell responses induce the proliferation of these cells and the accumulation of type 2 cytokines in coculture supernatants that can be quantified by different approaches (ELISA, Ella^TM, and multiplex assays). Together, the protocols described in this chapter can be used to investigate the features of food proteins to induce food allergy, the influence of environmental factors to generate Th2-polarization, the function of DCs to generate differential immune responses in allergic versus tolerant individuals, and to assess the immunomodulating properties of potential therapeutic substances.

Keywords: Allergen specific; Cytokine; Dendritic cells; Food allergy; In vitro; T cells.