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