Inﬂuenza A Virus-Infected Lung Epithelial Cell Co-Culture with Human Peripheral Blood Mononuclear Cells

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 IFN γ ) 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.


Introduction
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]. The objective is to measure and recapitulate the events of early IAV infection in vitro, in a co-culture system with human peripheral blood mononuclear cells (PBMC) and IAV-infected human lung epithelial cells.
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. Personal protective equipment (PPE) should be worn at all times (gloves, lab coat, and eye protection) (see Note 1).

IAV Infection of Human Lung
Epithelial Cell Line, A549 1. 24 h prior to infection, in two T75 flasks, seed 5 Â 10 6 A549 cells in a total volume of 20 mL of media (one flask for IAV infection and the second flask for uninfected control A549s).
2. On the day of infection: leave one flask of A549 cells in the incubator (uninfected control). Wash the other flask with room Typical flow cytometry panel compatible with a four-laser BD LSRII Fortessa flow cytometer, allowing identification of innate and adaptive lymphocyte subsets and assessment of activation measured by intracellular cytokine staining *Batches of SA-conjugated and Tetramerized MR1-5-OP-RU will vary and require titration prior to usage temperature PBS once, cap and gently rotate flask from side to side. Aspirate PBS with glass tissue culture pipette.
3. Thaw virus (PR8) [1] on ice and add 174 μL to 10 mL of room temperature PBS in a 50 mL falcon tube (depending on viral titer of stock) to achieve a multiplicity of infection (MOI) of 10-30 * . Gently pipette this into the T75 containing A549 cells.
5. Remove both T75 flasks from incubator and add 10 mL of cRPMI to the flask containing virus. Cap and gently rotate from side to side. Aspirate media from both flasks.
6. To detach A549 cells, wash flasks once with room temperature PBS, aspirate, and add 2.5 mL of Trypsin versene to each flask. Gently tilt the flask to ensure that the solution coats the entire flask.
8. Add 10 mL of cRPMI to T75 flasks and transfer the contents into two 50 mL falcon tubes. Centrifuge for 5 min at 500 Â g, 25 C. Aspirate supernatant.
9. Resuspend cells in 2 mL of cRPMI and perform cell counts using trypan blue estimation.
10. Adjust the volume of A549 cells so that the final concentration is 2 Â 10 6 cells/mL.

Co-Culture (Start During the 1 h Incubation with Virus)
1. Thaw PBMCs in 37 C water bath and gently pipette dropwise into 9 mL of pre-warmed cRPMI per cryovial and centrifuge at 500 Â g for 5 min (see Note 2).
To check IAV nucleoprotein levels, see Note 3. Add 100 μL of infected and uninfected A549 cells to separate wells in the 96-well plate.

Add 100 μL of uninfected A549s or IAV-infected A549s
(2 Â 10 5 cells) into wells containing PBMC. Leave one well with PBMC only, add 100 uL of cRPMI to this well. Place this plate in the 37 C incubator (5% CO 2 ). 4. After 3-4 h, add brefeldin A (BFA-GOLGI PLUG), 1:2000 to all wells and incubate for a further 6 h in the 37 C incubator (total co-culture 10 h).
5. Remove plate and continue with intracellular cytokine (ICS) staining or place in the 4 C covered in foil to stain the next day.  before any further use. All waste and its container must be disposed as hazardous waste.

Intracellular
2. MAIT cell responses after in vitro influenza co-culture are highly variable between donors. Freshly processed PBMCs may aid in the detection of IFNγ cytokine responses after influenza co-culture.
3. To determine if influenza virus infection of lung epithelial cells is successful after 10 h of culture, intracellular cytokine staining for influenza A virus nucleoprotein is determined by flow cytometry. Follow steps 1-3 and 6-11 of Subheading 3.3 Intracellular cytokine staining.
4. Fixable viability dyes react with exposed amine groups within permeable cells. Therefore, to prevent wasteful reaction with proteins in cytometry buffers, it is recommended to resuspend cells in protein-free media for the viability staining step.