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Imaging antigen-induced PI3K activation in T cells

Nature Immunology volume 3, pages 1090–1096 (2002)Cite this article

Abstract

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.

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Figure 1: Membrane localization of GFP–Akt-PH in a T cell hybridoma.
Figure 2: Inhibition of Akt membrane relocalization by wortmannin.
Figure 3: Dynamics of Akt and GFP distribution after T cell–APC interaction.
Figure 4: Accumulation of PIP3 after antigen recognition.
Figure 5: PI3K activity and active synapse formation after antigen recognition.
Figure 6: Distribution of Akt in human T cell-DC conjugates.
Figure 7: Dynamics of Akt relocalization during human T cell-DC interaction.

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Acknowledgements

We thank E. Donnadieu, C. Randriamampita and A. Trautmann for their comments on the manuscript; C. Arrieumerlou for discussions; and M.O. Lhuillier for the movies. Supported by the Fondation pour la Recherche Médicale (J.H.) and grants from Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique and the Ligue Nationale contre le Cancer.

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  1. Département de Biologie Cellulaire, Institut Cochin, INSERM U567, CNRS UMR 7627, Université René Descartes, 22 rue Méchain, Paris, 75014, France

    Julie Harriague & Georges Bismuth

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Correspondence to Georges Bismuth.

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Supplementary information

Web Movie 1.

GFP-Akt-PH translocation in T cell-APC conjugates. L625.7 cells were plated on glass coverslips mounted on Petri dishes and incubated overnight at 37 °C with antigen [1 μg/ml tt(830-843) peptide] before the addition of GFP-Akt-PH-expressing T8.1 cells previously loaded with Fura-2/AM. Transmitted light, fluorescence and intracellular calcium were measured every 10 s for 20 min. Transmitted light images are superimposed on fluorescence images showing Akt distribution. The black arrow indicates the conjugate shown in Fig. 1. Insert, left side, calcium response in the same conjugates. (AVI 4605 kb)

Web Movie 2.

Inhibition of Akt membrane relocalization by wortmannin. T8.1 cells were incubated with Fura-2/AM and 100 nM wortmannin at 37 °C for 20 min before being added to antigen-pulsed L625.7 cells. Transmitted light images are superimposed on calcium (left) and fluorescence images (right). Images were acquired every 10 sec for 18 min. (AVI 1683 kb)

Web Movie 3.

Delocalization of Akt in a T cell-APC conjugate after PI3K inhibition. T8.1 cells were incubated with Fura-2/AM and added to L625.7 cells pulsed with antigen. Then, 7 min after the beginning of acquisition, 1 ml of buffer containing 100 nM wortmannin was added (+WTN). Transmitted light images are superimposed on calcium images (left) and fluorescence images (right). Images were acquired every 10 sec for 16 min. (AVI 1547 kb)

Web Movie 4.

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. (AVI 1806 kb)

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Harriague, J., Bismuth, G. Imaging antigen-induced PI3K activation in T cells. Nat Immunol 3, 1090–1096 (2002). https://doi.org/10.1038/ni847

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