Nature Methods 3, 629 - 636 (2006)
Published online: 21 July 2006; | doi:10.1038/nmeth903
Enrichment of regulatory CD4+CD25+ T cells by inhibition of phospholipase D signalingNagendra Singh1, Yoichi Seki1, Mariko Takami1, Babak Baban1, Phil R Chandler1, Davood Khosravi1, Xiangjian Zheng2, Mayuko Takezaki1, Jeffrey R Lee1, 3, 4, Andrew L Mellor1, Wendy B Bollag2 & Makio Iwashima11
Immunotherapy Center, Medical College of Georgia, 1120 15th Street, Augusta, Georgia 30912, USA. 2
Program in Regenerative Medicine, Institute of Molecular Medicine and Genetics, Department of Medicine, Medical College of Georgia, 1120 15th Street, Augusta, Georgia 30912, USA. 3
Department of Pathology, Medical College of Georgia, 1120 15th Street, Augusta, Georgia 30912, USA. 4
Department of Pathology, Veterans Affairs Medical Center, 1 Freedom Way, Augusta, Georgia, 30904, USA.
Correspondence should be addressed to Makio Iwashima miwashima@mcg.edu 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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1, 2 and 3 (clone 1D11 (R&D Systems)
