Nucleofection of primary T cells to study the immune synapse

The discovery of the ›immune synapse‹ formed between a T cell and an antigen-presenting cell has allowed a new understanding of the signalling processes involved in T cell activation. Phosphoinositide-3-kinase (PI3K) is one of the central molecules to T cell activation. It phosphorylates the lipid phosphatidylinositol to generate the mono, bis (PIP₂) and triphosphate (PIP₃). The kinase AKT/PKB can bind to PIP₂ and PIP₃ at its pleckstrin homology (PH) domain.

Harriague and Bismuth (2002) have engineered an eGFP protein fused to the PH-domain of Akt (GFP-Akt-PH) and transfected it into T cells. Upon incubation with dendritic cells and antigen, a rapid formation of an immune synapse and a targeting of the fusio-protein to this site of interaction was observed (see movie below).

GFP-Akt-PH translocation in human T cells interacting with DCs.

DCs were plated on polylysine-coated glass coverslips in the presence of 0.1 µg/ml SEE. Human primary T cells, transiently transfected with GFP-Akt-PH, were added. Left, transmitted light images; right, distribution of Akt. Images were acquired every 10 s for 17 min.

(Courtesy of J. Harriague and G. Bismuth, Université René Descartes, Institute Cochin, Départment de Biologie Cellulaire, Paris, France; with kind permission of Nature Immunology)

Reference:

 Harriague J and Bismuth G (2002) Nature Immunol 3(11): 1090-1096