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