Abstract
Sensing of influenza A virus (IAV) infection by pattern recognition receptors can occur by either direct infection of lung epithelial cells or uptake of virus-infected cells by innate cells such as dendritic cells/monocytes. This triggers a series of downstream events including activation of the inflammasome, the production of cytokines, chemokines, and the upregulation of stress-induced ligands that can lead to the activation of innate cells. These cells include innate lymphocytes such as MAIT, NKT, NK, and γδ T cells. Here we describe a method used to allow activation of human innate lymphocytes in co-culture with an IAV-infected human lung epithelial cell line (A549) to measure ex vivo effector functions (TNF and IFNγ) in a mixed culture environment. We describe (1) infection of the human lung epithelial cell line, (2) co-culture with PBMC, and (3) measurement of activation using intracellular cytokine staining.
Highlights
The innate immune response serves as the first line of defense during viral infections
Sensing of influenza A virus (IAV) infection by pattern recognition receptors (e.g., TLR and RIG-I) can occur by either direct infection of lung epithelial cells or uptake of virusinfected cells by innate cells such as dendritic cells/monocytes. This triggers a series of downstream events including activation of the inflammasome, the production of cytokines, chemokines, and the upregulation of stress-induced ligands that can lead to the activation of innate cells
These cells include innate lymphocytes such as MAIT, NKT, NK, and γδ T cells. These lymphocytes can be activated by non-classical MHC interactions, cytokine-mediated signals or both. This method allows for the activation of human innate lymphocytes in co-culture with IAV-infected human lung epithelial cells (A549) and is used to measure ex vivo effector functions (TNF and IFNγ) in a mixed culture environment [2]
Summary
The innate immune response serves as the first line of defense during viral infections. Sensing of influenza A virus (IAV) infection by pattern recognition receptors (e.g., TLR and RIG-I) can occur by either direct infection of lung epithelial cells or uptake of virusinfected cells by innate cells such as dendritic cells/monocytes This triggers a series of downstream events including activation of the inflammasome, the production of cytokines, chemokines, and the upregulation of stress-induced ligands that can lead to the activation of innate cells. These lymphocytes can be activated by non-classical MHC interactions, cytokine-mediated signals or both This method allows for the activation of human innate lymphocytes in co-culture with IAV-infected human lung epithelial cells (A549) and is used to measure ex vivo effector functions (TNF and IFNγ) in a mixed culture environment [2]. The method described in this chapter comprises three main steps: (1) infection of a human epithelial cell line, (2) co-culture with PBMC to activate the virus responsive cells, and (3) intracellular cytokine staining to measure the extent of functional activation
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