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Lck Mediates Signal Transmission from CD59 to the TCR/CD3 Pathway in Jurkat T Cells

2014; Public Library of Science; Volume: 9; Issue: 1 Linguagem: Inglês

10.1371/journal.pone.0085934

1932-6203

Anna M. Lipp, Kata Juhász, Christian Paar, Christoph Ogris, Paul Eckerstorfer, Roland Thuenauer, Jürgen Hesse, Benedikt Nimmervoll, Hannes Stockinger, Gerhard J. Schütz, Ulrich Bodenhofer, Zsolt Balogi, Alois Sonnleitner,

Monoclonal and Polyclonal Antibodies Research

The glycosylphosphatidylinositol (GPI)-anchored molecule CD59 has been implicated in the modulation of T cell responses, but the underlying molecular mechanism of CD59 influencing T cell signaling remained unclear. Here we analyzed Jurkat T cells stimulated via anti-CD3ε- or anti-CD59-coated surfaces, using time-resolved single-cell Ca(2+) imaging as a read-out for stimulation. This analysis revealed a heterogeneous Ca(2+) response of the cell population in a stimulus-dependent manner. Further analysis of T cell receptor (TCR)/CD3 deficient or overexpressing cells showed that CD59-mediated signaling is strongly dependent on TCR/CD3 surface expression. In protein co-patterning and fluorescence recovery after photobleaching experiments no direct physical interaction was observed between CD59 and CD3 at the plasma membrane upon anti-CD59 stimulation. However, siRNA-mediated protein knock-downs of downstream signaling molecules revealed that the Src family kinase Lck and the adaptor molecule linker of activated T cells (LAT) are essential for both signaling pathways. Furthermore, flow cytometry measurements showed that knock-down of Lck accelerates CD3 re-expression at the cell surface after anti-CD59 stimulation similar to what has been observed upon direct TCR/CD3 stimulation. Finally, physically linking Lck to CD3ζ completely abolished CD59-triggered Ca(2+) signaling, while signaling was still functional upon direct TCR/CD3 stimulation. Altogether, we demonstrate that Lck mediates signal transmission from CD59 to the TCR/CD3 pathway in Jurkat T cells, and propose that CD59 may act via Lck to modulate T cell responses.