Abstract
Antigen stimulation of lymphocytes induces upregulation of phospholipase D (PLD) activity, but the biological significance of PLD-mediated signaling in T cells has not been well established. Here we demonstrate that PLD signaling is essential for proliferation of mouse CD8+ T cells and CD4+CD25− T cells, but is not required for proliferation of CD4+CD25+ regulatory T cells. We exploited this observation to develop an efficient method to enrich for regulatory T cells starting from preparations of total CD4+ T lymphocytes. Inhibition of PLD signaling blocked effector T-cell proliferation after T cell–antigen receptor (TCR) engagement, but had no significant effect on the proliferation of CD4+CD25+ T cells with regulatory functions. Consequently, cells expanded in vitro for one week by antigen receptor stimulation with PLD signal inhibition were markedly enriched for regulatory T cells.
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Acknowledgements
We thank A. Weiss and D.H. Munn for critical reading of the manuscript, T. Tada for encouragement, M. Frohman and Y. Kanaho for reagents, MCG flow Cytometry Core and Histology core for support, and A. Sridhar, M. Takezaki and D. McCool for assistance. This work is supported by US National Institutes of Health grants AI055022 and AI049398 (to M.I), and AR45212 and HL70046 (to W.B.B.).
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N.S. and M.I. are authors of a pending patent (application number US 60-625403), which covers the work described in this article.
Supplementary information
Supplementary Fig. 1
Original pictures of western blots and agarose gels used in this study. (PDF 656 kb)
Supplementary Fig. 2
Transfection efficiency of siRNA construct against PLD and its effect. (PDF 341 kb)
Supplementary Fig. 3
Effect of 1-but on T-cell division and death. (PDF 658 kb)
Supplementary Fig. 4
A schematic presentation of the procedure utilized to enrich T cells in the presence of 1-alcohol. (PDF 504 kb)
Supplementary Fig. 5
Role of cytokines in CD41-but mediated T cell suppression. (PDF 242 kb)
Supplementary Fig. 6
Tissue architectures of recipient mice of scurfy and BM3 T cells. (JPG 297 kb)
Supplementary Fig. 7
Effect of 1-but treatment on CD69 expression and cytokine production by sorted CD4 T cells. (PDF 911 kb)
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Singh, N., Seki, Y., Takami, M. et al. Enrichment of regulatory CD4+CD25+ T cells by inhibition of phospholipase D signaling. Nat Methods 3, 629–636 (2006). https://doi.org/10.1038/nmeth903
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DOI: https://doi.org/10.1038/nmeth903
