Measuring the Activation of Fc-Mediated Effector Functions by HA Antibodies

Overview

This video demonstrates an assay measuring the antibody-mediated activation of T cell effector functions. Adding an influenza virus hemagglutinin (HA)-specific monoclonal antibody to transfected mammalian cells expressing HA results in the formation of immune complexes with HA. Upon introducing engineered T cells expressing Fc receptors and a luciferase reporter, the T cells are activated by the antibody-HA complexes, and this activation is detected by adding a luciferase substrate and measuring the produced bioluminescence.

Protocol

1. Expression of Influenza Virus Hemagglutinin via Transfection

1. Plate Human Embryonic Kidney (HEK 293T) cells at a density of 2 x 10⁴ cells/well in a white tissue culture treated 96-well plate and let it sit for 4 h in a 37 °C incubator (with 5% CO₂).
2. Transfect cells with 100 ng of DNA coding for viral hemagglutinin and 0.2 μL of transfection reagent per well in a total volume of 50 μL.
   NOTE: Both plasmid and transfectant reagent are diluted in 1x Opti-Minimum Essential Media (MEM) Reduced Serum Medium.
3. After incubating plates in a 37 °C incubator with 5% CO₂ for 18 h, remove transfection media.

2. Assessing the FcRg/NFAT-mediated Activation of Luciferase Activity by Monoclonal Antibodies or Sera

1. First, replace the growth media of transfected cells with 25 µL of assay media (1x RPMI 1640 supplemented with 4% (vol/vol) low IgG fetal bovine sera).
2. In a separate 96-well plate, perform four-fold dilutions of the antibodies of interest starting at 30 μg/mL using the assay media. Perform dilutions in triplicates, and an example plate layout is illustrated in Figure 1A. Ensure that a control that excludes antibodies (no antibody control) and a background control (assay buffer alone) is included.
   NOTE: For both the ‘no antibody' and background controls, add 25 µL of assay buffer instead of the antibody.
3. Transfer 25 μL of the serially diluted antibodies from Step 2.2 to corresponding wells on the plate of transfected cells.
4. Incubate plate in a 37 °C incubator (with 5% CO₂) for 15 min.
5. Add 25 μL of the appropriate modified effector Jurkat cell expressing murine FcgRIV or human FcgRIIIa (75,000 cells/well). The total volume for each well should be 75 µL, and the starting final concentration of the antibody is at 10 µg/mL (Figure 1B).
   NOTE: For the background control, add 25 µL of assay buffer instead of effector cells. To examine antibody-dependent cell-mediated phagocytosis, a modified Jurkat cell expressing the human FcgRIIa can be used.
6. Incubate the plate in a 37 °C incubator with 5% CO₂ for 6 h.
7. Thaw and warm luciferase substrate buffer in 37 °C water for about 1 h before use.
   NOTE: To make the luciferase substrate buffer, reconstitute the lyophilized luciferase assay substrate in the provided assay buffer. The reconstituted substrate buffer can be stored at -30 °C to -10 °C for up to six weeks.
8. Add 75 μL/well of luciferase substrate buffer to all wells except for the background control.
9. Read on a luminometer.
10. Calculate fold induction with the following equation: Fold induction = (Induced value-Background value) / (No mAb value-Background value).

Representative Results

Figure 1: A schematic of a sample plate layout and composition of the experimental groups. (A) The starting concentration for each antibody sample is at 10 µg/mL and serially diluted across the plate (column 1 to column 12) 4-fold. Each sample is done in triplicates. Row G is reserved for the no mAb control, and Row H is the background. (B) The final volume for each experimental group before the addition of luciferase substrate buffer is 75 µL. Of note, the sample and ‘no mAb groups’ have 25 µL of effector cells added, while the background control groups have 75 µL of assay media.

Materials

Name	Company	Catalog Number	Comments
HEK 293T cells	ATCC	CRL-3216	Human embryonic kidney cells
Lipofectamine 2000	Life Technologies	11668019	Transfection reagent
1X Opti-MEM Reduced Serum Medium	ThermoFisher Scientific	51985-034
White tissue culture treated 96-well plate	Corning, Inc.	3917	Assay plates
10X MEM	Gibco	11430-030
Jurkat cell expressing murine FcgRIV	Promega	M1201	Kit provides includes cells, Bio-Glo Luciferase assay system, 1X RPMI and low IgG serum
Jurkat cell expressing human FcgRIIIa	Promega	G7010	Kit provides includes cells, Bio-Glo Luciferase assay system, 1X RPMI and low IgG serum
Jurkat cell expressing human FcgRIIa	Promega	G9901	Kit provides includes cells, Bio-Glo Luciferase assay system, 1X RPMI and low IgG serum
Luminometer	BioTek	Synergy H1 Multi-Mode reader	Luminescence plate reader
Bio-Glo Luciferase Assay System	Promega	G7940	Contains luciferase assay buffer and luciferase assay substrate