Contact-Free Co-Culture Model for the Study of Innate Immune Cell Activation During Respiratory Virus Infection

This protocol is designed to investigate crosstalk of epithelial cells and first-line innate immune cells, specifically innate T cells, which is our interest. Primary cells from human sources are used in this technique, allowing individual investigation of physiological crosstalk relevant to what is happening in humans. Innate T cells have been implicated in improving the survival of influenza-infected patients.

Knowing how they get activated could help in developing anti-influenza and antiviral therapy. This method is designed to identify factors that can activate the innate T cells, which may help in developing preventive measures against respiratory virus infection. At day zero, infect human nasal epithelial cells, or HNECs.

First, count cells by adding 150 microliters of 1x trypsin EDTA to the apical chamber of the membrane insert and 350 microliters of 1x trypsin EDTA to the basal chamber of the representative well. Incubate the cells at 37 degrees Celsius for 10 minutes or until the cells detach from the membrane. Flush the cells on the membrane by pipetting up and down and collect the suspension in a 1.5-milliliter centrifuge tube.

Add 200 microliters of DMEM to quench trypsin activity. Then, count cells via trypan blue staining. Calculate the required multiplicity of infection, or MOI, of the virus based on the cell count per well.

Dilute the virus stock accordingly with complete RPMI on ice. For the remaining wells for the infection experiment, add one 1x DPBS into the apical chambers of the membrane inserts. Incubate at 37 degrees Celsius for 10 minutes and remove the 1x DPBS.

Change the basal medium in the membrane inserts to complete RPMI medium by transferring the inserts to a new plate with complete RPMI added to the wells. Add the prepared virus inoculum into the apical chamber of the membrane insert. Incubate at 35 degrees Celsius in a 5%carbon dioxide atmosphere for one hour and remove the viral inoculum from the apical chamber.

Change the basal medium of the membrane insert to fresh RPMI medium and incubate at 35 degrees Celsius in a 5%carbon dioxide atmosphere for 24 to 72 hours. Seed the required number of peripheral blood mononuclear cells, or PBMCs, in complete RPMI medium by directly adding the PBMC suspension into the basal chamber of each well of infected HNECs from day zero. Incubate at 37 degrees Celsius for 24 to 48 hours.

To collect the apical supernatant, add 1x DBPS to each apical chamber and incubate at 37 degrees Celsius for 10 minutes. Then, collect the DPBS in 1.5-milliliter tubes. Aliquot the supernatant for the plaque assay in a new 1.5-milliliter tube and immediately freeze both the stock and the aliquots at minus 80 degrees Celsius.

To collect RNA-form HNECs, transfer the membrane insert to a clean well. Add RNA lysis buffer to the apical chamber and incubate at room temperature for five minutes. Collect the supernatant in a 1.5-milliliter centrifuge tube and store it at minus 80 degrees Celsius until RNA extraction for molecular analysis.

Finally, harvest PBMCs by gently scraping the surface of the well with the broad base of a sterile pipette tip to dislodge the activated PBMCs that may be adherent to the well surface. Collect the basal medium containing PBMCs in a two-milliliter centrifuge tube. Flush the wells twice with 300 microliters of 1x DPBS and collect the wash in the same two-milliliter tube.

Centrifuge the two-milliliter tube and collect the supernatant in a fresh two-milliliter tube without disturbing the cell pellet. Store the aspirated supernatant at minus 80 degrees Celsius for cytokine and chemokine analysis. Resuspend the remaining cell pellet in 200 microliters of 1x DPBS.

This protocol can be used to study innate T cells following influenza infection without the need for epithelial and immune cells from the same donor. A sample of the expected progression of the HNESPCs as they grow on the 3T3 feeder layer is shown here. These were used for differentiation in the air/liquid interface culture to obtain multi-layered HNECs with functional, ciliated, and goblet cells.

Using the HNECs, innate T cell activation can be investigated using flow cytometry. These results show the detection of mucosal-associated invariant T, or MAIT cells, V delta one, gamma delta T cells, and the natural killer T, or NKT cells. These cells were significantly increased in co-culture involving HNECs infected with influenza virus.

This setup can then be applied to other influenza virus strains, or innate immune cell populations to observe their respective activation under co-culture conditions with infected epithelial cells. Accurate calculation of the number of the human nasal epithelial cells to ensure that the right multiplicity of infection is used is important for optimal levels and timing of activation. Crosstalk between epithelial cells and innate immune cells are increasingly gaining attention, and this can be applied further to any other methods involving acute or chronic respiratory immunology.