Enrichment of regulatory CD4⁺CD25⁺ T cells by inhibition of phospholipase D signaling

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.