Imaging antigen-induced PI3K activation in T cells

Harriague J and Bismuth G
Source: Nat Immunol
Publication Date: (2002)
Issue: 3(11): 1090-1096
Research Area:
Immunotherapy / Hematology
Cells used in publication:
T cell, human peripheral blood unstim.
Species: human
Tissue Origin: blood
Nucleofector® I/II/2b

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. The authors have engineered an eGFP protein fused to the PH-domain of Akt (GFP-Akt-PH) and nucleofected 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


Imaging antigen-induced PI3K activation in T cells. Harriague J, Bismuth G. Departement de Biologie Cellulaire, Institut Cochin, INSERM U567, CNRS UMR 7627, Universite Rene Descartes, 22 rue Mechain, 75014 Paris, France. Activation of phosphoinositide 3-kinase (PI3K) at the immunological synapse between a T cell and an antigen-presenting cell (APC) has not been demonstrated. Using fluorescent-specific probes, we show here that the formation of an immunological synapse led to sustained production of 3'-phosphoinositides in the T cell, whereby phosphatidylinositol-3,4,5-trisphosphate (PIP3) but not phosphatidylinositol-3,4-bisphosphate was localized to the cell membrane. The accumulation of PIP3 after T cell activation preceded the increase in intracellular calcium. Neither the formation of conjugates between T cells and APCs nor signaling events such as phosphotyrosine accumulation and calcium increase changed substantially when PI3K was inhibited, and only a limited reduction in synthesis of interleukin 2 occurred. In T cell-APC conjugates, PIP3 accumulated at the T cell-APC synapse as well as in the rest of the T cell plasma membrane, which indicated unusual regulation of PI3K activity during antigen presentation.