Sep 02, 2021
Influenza A Virus-Infected Lung Epithelial Cell Co-Culture with Human Peripheral Blood Mononuclear Cells
Book Chapter
- Liyen Loh1,2,
- Marios Koutsakos1,
- Katherine Kedzierska1,
- Timothy S. C. Hinks3
- 1Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, The University of Melbourne, Parkville, Australia;
- 2Department of Immunology and Microbiology, University of Colorado Anschutz Medical Campus, Aurora, USA;
- 3Respiratory Medicine Unit, Nuffield Department of Medicine Experimental Medicine, University of Oxford, Oxfordshire, UK
- Springer Nature Books
Protocol Citation: Liyen Loh, Marios Koutsakos, Katherine Kedzierska, Timothy S. C. Hinks 2021. Influenza A Virus-Infected Lung Epithelial Cell Co-Culture with Human Peripheral Blood Mononuclear Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.bmenk3de
Manuscript citation:
Loh L., Koutsakos M., Kedzierska K., Hinks T.S.C. (2020) Influenza A Virus-Infected Lung Epithelial Cell Co-Culture with Human Peripheral Blood Mononuclear Cells. In: Kaipe H., Magalhaes I. (eds) MAIT Cells. Methods in Molecular Biology, vol 2098. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0207-2_9
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 16, 2020
Last Modified: September 02, 2021
Protocol Integer ID: 42158
Keywords: Virus, MAIT cell, Flow cytometry, Tetramer, Infection, Human Epithelial cell,
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.
Attachments
Guidelines
Please read the full protocol before starting any steps.
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 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. 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.
References:
- Koutsakos M, Illing PT, Nguyen THO et al (2019) Human CD8(+) T cell cross-reactivity across influenza A, B and C viruses. Nat Immunol
- Loh L, Wang Z, Sant S et al (2016) Human mucosal-associated invariant T cells contribute to antiviral influenza immunity via IL-18-dependent activation. Proc Natl Acad Sci U S A 113:10,133–10,138
Acknowledgements:
This work was funded by grants to TSCH from the Wellcome Trust (104553/z/14/z, 211050/Z/18/z) and K.K from the National Health and Medical Research Council (NHMRC) Program Grant (1071916). K.K. is an NHMRC Senior Research Fellow (1102792). The content represents only the authors’ views and not those of the European Commission.
Materials
Reagents and Buffers:
1. Complete RPMI (cRPMI): Roswell Park Memorial Media, 10% heat-inactivated fetal calf serum (FCS), 100 U/mL Penicillin, 100 U/mL Streptomycin, and 100 μM MEM Vitamins.
2. Human lung epithelial cell line, A549 (ATCC, VA, USA).
3. PR8 virus (influenza A strain/H1N1/Puerto Rico/1934).
4. Trypsin Versene (In-house preparation).
5. Trypan Blue and Counting Chamber.
6. Brefeldin A—Golgi PLUG (BD, CA, USA).
7. Live/dead Fixable Aqua Dead Cell Stain Kit (ThermoFisher, MA, USA).
8. Cytofix/Cytoperm Fixation/Permeabilization Kit (BD Biosciences, USA).
9. 10% Lysol or 1% Virkon.
10. Fluorescence activated flow cytometry (FACS) buffer: phosphate-buffered saline (PBS), 2 mM EDTA, 0.5% bovine serum albumin (BSA). From a 500 mL bottle of PBS, add 40 mL to a 50 mL falcon containing 2.5 g BSA powder, vortex hard, then filter-sterilize back into PBS bottle using a syringe through a 0.22 um filter.
11. 1% paraformaldehyde (PFA) solution. Dilute 16% paraformaldehyde 1:16 with FACs buffer.
12. Antibodies for flow cytometry (Table 1, shown below).
13. Phosphate-buffered saline (PBS).
Plastic:
1. T75 flasks (Corning, NY, USA).
2. 50 mL Falcon tube (Fischer Scientific, MA, USA).
3. 96-well U-bottom plate polystyrene (Greiner, Germany).
Equipment:
1. Flow Cytometer, BD LSR FORTESSA, or equivalent.
2. Water Bath.
Table 1 (Antibodies):
Example flow cytometric activation panel and IAV nucleoprotein expression
| Marker | Fluorophore | Laser | Clone | Dilution | |
| Surface stain activation | |||||
| Live Dead | Aqua | Violet | 1/800 (stain in PBS prior to surface stain) | ||
| MRI-5-OP-RU Tet | SA-BV421 | Violet | *Titrate 1/200-1/400 | ||
| CD19 | APC-H7 | Red | H1B19 | 1/100 | |
| CD14 | APC-H7 | Red | MΦP9 | 1/100 | |
| CD8α | PerCP-Cy5.5 | Blue | SK1 | 1/50 | |
| TCRγδ | FITC | Blue | 2F11 | 1/50 | |
| CD4 | BV650 | Violet | OKT4 | 1/200 | |
| CD161 | BV605 | Violet | HP-3G10 | 1/50 | |
| CD3 | PE-CF594 | Yellow/Green | UCHT1 | 1/200 | |
| TCR Vα7.2 | PE | Yellow/Green | 3C10 | 1/200 | |
| CD56 | PE-Cy7 | Yellow/Green | NCAM16.2 | 1/100 | |
| Intracellular stain activation | |||||
| TNF | APC | Red | MAb11 | 1/50 | |
| IFNγ | AF700 | Red | B27 | 1/100 | |
| Intracellular stain IAV-NP infection | |||||
| IAV nucleoprotein | FITC | Blue | 1331 | 1/100 |
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
Safety warnings
Personal protective equipment (PPE) should be worn at all times (gloves, lab coat, and eye protection).
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet).
Before start
Biological Hazards—Human PBMC samples are classified as non-infectious. Influenza A virus—PR8-strain (H1N1) is a lab-adapted strain of IAV virus. Work should be risk assessed, and we recommend controls which include but are not restricted to the following: Lab coat, safety glasses, and gloves should be worn when performing this protocol. Work with human PBMCs and virus in a Class II biohazard cabinet. Use filter tips when working with virus. Decontaminate all pipette tips that have been used for human and virus work in 10% lysol or 1% Virkon when working in the biohazard cabinet. After use, the biohazard hood should be decontaminated by wiping down with 70% ethanol and by UV sterilization for 15 min before any further use. All waste and its container must be disposed as hazardous waste.
IAV Infection of Human Lung Epithelial Cell Line, A549
IAV Infection of Human Lung Epithelial Cell Line, A549
24:00:00 prior to infection, in two T75 flasks, seed 5 x 106 A549 cells in a total volume of 20 mL media .
Note
One flask for IAV infection and the second flask for uninfected control A549s.
On the day of infection:
Leave one flask of A549 cells in the incubator (uninfected control).
Wash the other flask with Room temperature PBS once, cap and gently rotate flask from side to side. Aspirate PBS with glass tissue culture pipette.
Thaw virus (PR8) On ice and add 174 µL virus to 10 mL 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.
Note
Example calculation of MOI 10:
An MOI of 10 using 1x106 PBMC per well requires 1x107 virus particles/well.
The volume required/well of a 1x109 plaque forming units (pfu)/mL virus titer is:
1x107 pfu/1x109 pfu/mL = 0.01 mL/well or 10 μL/well.
Incubate flask horizontally for 01:00:00 in the 37 °C incubator (5% CO2).
Remove both T75 flasks from incubator and add 10 mL cRPMI to the flask containing virus. Cap and gently rotate from side to side. Aspirate media from both flasks.
To detach A549 cells, wash flasks once with Room temperature PBS , aspirate, and add 2.5 mL Trypsin versene to each flask. Gently tilt the flask to ensure that the solution coats the entire flask.
Incubate for 00:05:00 in the 37 °C incubator (5% CO2).
Add 10 mL cRPMI to T75 flasks and transfer the contents into two 50 mL falcon tubes.
Centrifuge 500 x g, 25°C, 00:05:00 .
Aspirate supernatant.
Resuspend cells in 2 mL cRPMI and perform cell counts using trypan blue estimation.
Adjust the volume of A549 cells so that the final concentration is 2x106 cells/mL.
Co-Culture (Start During the 1 h Incubation with Virus)
Co-Culture (Start During the 1 h Incubation with Virus)
Thaw PBMCs in 37 °C water bath .
Gently pipette dropwise into 9 mL pre-warmed cRPMI per cryovial.
Centrifuge 500 x g, 00:05:00 .
Note
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.
Aspirate media and count cells.
Resuspend PBMCs at 10x106 cells/mL in cRPMI.
For each sample, aliquot 100 µL cells (1x10^6 PBMC) into three wells of a 96-well U-bottom plate.
Note
These wells will correspond to Media Control, uninfected A549 + PBMC, and IAV-infected A549 + PBMC, respectively.
Check IAV nucleoprotein levels:
Note
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. (Steps 25-27 followed by steps 31-38, shown here in subsequent substeps for convenience)
Spin down plate by centrifuging at 400 x g, 4°C, 00:05:00 . Discard supernatant in waste container containing 10% Lysol or 1% Virkon in class II biosafety cabinet.
Stain cells with live/dead discrimination marker Aqua (1:800) final volume of 50 μL/well. Use PBS as a diluent. Incubate at Room temperature in the dark for 00:15:00 .
Note
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.
15m
Centrifuge plate at 400 x g, 4°C, 00:05:00 . Discard supernatant.
Resuspend the cells in 100 µL cold cytofix/perm solution and incubate On ice in the dark for 00:20:00 .
20m
Wash cells with 100 µL diluted (1:10 in dH2O) perm/wash buffer . Centrifuge for 450 x g, 4°C, 00:05:00 .
Resuspend cells in 50 µL intracellular cytokine stain , see (Table 1) below.
Table 1 (Antibodies):
Example flow cytometric activation panel and IAV nucleoprotein expression
| Marker | Fluorophore | Laser | Clone | Dilution | |
| Surface stain activation | |||||
| Live Dead | Aqua | Violet | 1/800 (stain in PBS prior to surface stain) | ||
| MRI-5-OP-RU Tet | SA-BV421 | Violet | *Titrate 1/200-1/400 | ||
| CD19 | APC-H7 | Red | H1B19 | 1/100 | |
| CD14 | APC-H7 | Red | MΦP9 | 1/100 | |
| CD8α | PerCP-Cy5.5 | Blue | SK1 | 1/50 | |
| TCRγδ | FITC | Blue | 2F11 | 1/50 | |
| CD4 | BV650 | Violet | OKT4 | 1/200 | |
| CD161 | BV605 | Violet | HP-3G10 | 1/50 | |
| CD3 | PE-CF594 | Yellow/Green | UCHT1 | 1/200 | |
| TCR Vα7.2 | PE | Yellow/Green | 3C10 | 1/200 | |
| CD56 | PE-Cy7 | Yellow/Green | NCAM16.2 | 1/100 | |
| Intracellular stain activation | |||||
| TNF | APC | Red | MAb11 | 1/50 | |
| IFNγ | AF700 | Red | B27 | 1/100 | |
| Intracellular stain IAV-NP infection | |||||
| IAV nucleoprotein | FITC | Blue | 1331 | 1/100 |
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
Incubate On ice in the dark for 00:30:00 .
30m
Wash cells with 150 µL perm/wash buffer . Centrifuge for 450 x g, 4°C, 00:05:00 .
Repeat with a second wash with 200 µL FACs buffer . Centrifuge for 450 x g, 4°C, 00:05:00 .
Resuspend cells in 100 µL 1% PFA and transfer to bullet tubes.
Note
Keep samples in the dark and at cold until acquisition on the flow cytometer.
For suggested flow cytometric gating strategy see Fig. 1. below:
Fig. 1
Flow cytometry gating strategy for MAIT cells and other lymphocyte subsets
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 (2x105 cells) into wells containing PBMC. Leave one well with PBMC only, add 100 µL cRPMI to this well. Place plate in the 37 °C incubator (5% CO2).
After 03:00:00 –04:00:00 , add brefeldin A (BFA-GOLGI PLUG), 1:2000 to all wells.
Incubate for a further 06:00:00 in the 37 °C incubator (10:00:00 total co-culture ).
Remove plate and continue with intracellular cytokine (ICS) staining or place in the 4 °C covered in foil to stain the next day.
Intracellular Cytokine Staining
Intracellular Cytokine Staining
1h 35m
1h 35m
Spin down plate by centrifuging at 400 x g, 4°C, 00:05:00 . Discard supernatant in waste container containing 10% Lysol or 1% Virkon in class II biosafety cabinet.
Stain cells with live/dead discrimination marker Aqua (1:800) final volume of 50 μL/well. Use PBS as a diluent. Incubate at Room temperature in the dark for 00:15:00 .
Note
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.
15m
Centrifuge plate at 400 x g, 4°C, 00:05:00 . Discard supernatant.
Add 50 µL surface phenotype stain (Table 1 below) to each well.
Table 1 (Antibodies):
Example flow cytometric activation panel and IAV nucleoprotein expression
| Marker | Fluorophore | Laser | Clone | Dilution | |
| Surface stain activation | |||||
| Live Dead | Aqua | Violet | 1/800 (stain in PBS prior to surface stain) | ||
| MRI-5-OP-RU Tet | SA-BV421 | Violet | *Titrate 1/200-1/400 | ||
| CD19 | APC-H7 | Red | H1B19 | 1/100 | |
| CD14 | APC-H7 | Red | MΦP9 | 1/100 | |
| CD8α | PerCP-Cy5.5 | Blue | SK1 | 1/50 | |
| TCRγδ | FITC | Blue | 2F11 | 1/50 | |
| CD4 | BV650 | Violet | OKT4 | 1/200 | |
| CD161 | BV605 | Violet | HP-3G10 | 1/50 | |
| CD3 | PE-CF594 | Yellow/Green | UCHT1 | 1/200 | |
| TCR Vα7.2 | PE | Yellow/Green | 3C10 | 1/200 | |
| CD56 | PE-Cy7 | Yellow/Green | NCAM16.2 | 1/100 | |
| Intracellular stain activation | |||||
| TNF | APC | Red | MAb11 | 1/50 | |
| IFNγ | AF700 | Red | B27 | 1/150 | |
| Intracellular stain IAV-NP infection | |||||
| IAV nucleoprotein | FITC | Blue | 1331 | 1/100 |
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
Incubate for 00:30:00 On ice , in the dark.
30m
Wash cells once with 150 µL FACs buffer . Centrifuge for 1500 rpm, 4°C, 00:05:00 . Flick off supernatant in discard container in biohazard cabinet.
Resuspend the cells in 100 µL cold cytofix/perm solution and incubate On ice in the dark for 00:20:00 .
20m
Wash cells with 100 µL diluted (1:10 in dH2O) perm/wash buffer . Centrifuge for 450 x g, 4°C, 00:05:00 .
Resuspend cells in 50 µL intracellular cytokine stain , see (Table 1) below.
Table 1 (Antibodies):
Example flow cytometric activation panel and IAV nucleoprotein expression
| Marker | Fluorophore | Laser | Clone | Dilution | |
| Surface stain activation | |||||
| Live Dead | Aqua | Violet | 1/800 (stain in PBS prior to surface stain) | ||
| MRI-5-OP-RU Tet | SA-BV421 | Violet | *Titrate 1/200-1/400 | ||
| CD19 | APC-H7 | Red | H1B19 | 1/100 | |
| CD14 | APC-H7 | Red | MΦP9 | 1/100 | |
| CD8α | PerCP-Cy5.5 | Blue | SK1 | 1/50 | |
| TCRγδ | FITC | Blue | 2F11 | 1/50 | |
| CD4 | BV650 | Violet | OKT4 | 1/200 | |
| CD161 | BV605 | Violet | HP-3G10 | 1/50 | |
| CD3 | PE-CF594 | Yellow/Green | UCHT1 | 1/200 | |
| TCR Vα7.2 | PE | Yellow/Green | 3C10 | 1/200 | |
| CD56 | PE-Cy7 | Yellow/Green | NCAM16.2 | 1/100 | |
| Intracellular stain activation | |||||
| TNF | APC | Red | MAb11 | 1/50 | |
| IFNγ | AF700 | Red | B27 | 1/150 | |
| Intracellular stain IAV-NP infection | |||||
| IAV nucleoprotein | FITC | Blue | 1331 | 1/100 |
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
Incubate On ice in the dark for 00:30:00 .
30m
Wash cells with 150 µL perm/wash buffer . Centrifuge for 450 x g, 4°C, 00:05:00..
Repeat with a second wash with 200 µL FACs buffer . Centrifuge for 450 x g, 4°C, 00:05:00..
Resuspend cells in 100 µL 1% PFA and transfer to bullet tubes.
Note
Keep samples in the dark and at cold until acquisition on the flow cytometer.
For suggested flow cytometric gating strategy see Fig. 1. below:
Fig. 1
Flow cytometry gating strategy for MAIT cells and other lymphocyte subsets