This study investigates the activation of T cells through antibodies targeting the Influenza virus hemagglutinin (HA). The methodology involves measuring luciferase expression as a readout for T cell activation in response to varying concentrations of specific antibodies.
Take serial dilutions of an antibody targeting the Influenza virus hemagglutinin, or HA, a glycoprotein consisting of a globular head and a stalk.
Add the dilutions onto transfected mammalian cells expressing surface-bound HA. The antibodies bind to the HA stalk regions, forming immune complexes.
Introduce engineered T cells expressing Fc receptors and encoding a luciferase reporter under the control of the transcription factor NFAT.
During incubation, the HA-bound antibody binds to the Fc receptor, while the HA head binds to sialic acid, triggering T cell activation and increasing cytosolic calcium concentration.
This activation initiates a signaling cascade that results in the dephosphorylation of NFAT, triggering its nuclear translocation.
NFAT binds to its response elements in the genome, driving luciferase expression.
Add a cell-permeable bioluminescent substrate. Luciferase oxidizes the substrate, producing a signal.
Decreased luminescence with decreasing antibody concentration confirms HA stalk-specific antibody-mediated T cell activation.
To assess the ability of specific antibodies of interest to target the hemagglutinin-expressing cells, replace the supernatant of the virally infected cell cultures with 25 microliters of antibody-dependent cell-mediated cytotoxicity, or ADCC, assay medium.
Next, in a separate 96-well plate, perform 4-fold dilutions of the antibodies of interest in triplicate and fresh ADCC medium starting at 10 micrograms per milliliter according to the schematic, and taking care to include the background and no antibody controls.
When all of the antibody has been diluted, transfer 25 microliters of the diluted antibody to the appropriate corresponding wells in the experimental cell plate and place the plate in the cell culture incubator. After 15 minutes, add 7.5 times 10 to the 4th modified effector Jurkat cells, expressing the appropriate Fc receptor per well in 25 microliters of ADCC medium for a final volume of 75 microliters of ADCC medium per well.
Return the plate to the cell culture incubator for 6 hours, thawing luciferase substrate buffer in a 37-degree Celsius water bath during the last hour of the incubation. When the buffer is ready, add 75 microliters of luciferase substrate buffer to all of the wells except the background control well, and read the plate on a luminometer. The fold induction of antibody-dependent cell-mediated cytotoxicity in the transfected or infected cell cultures can then be calculated.
繁體中文
