Selective depletion of CD4+CD25+Foxp3+ regulatory T cells by low-dose cyclophosphamide is explained by reduced intracellular ATP levels.

CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells have been shown to play important roles in mediating cancer development. Although cyclophosphamide (CY) has shown promise as a drug to selectively target Treg cells with low-dose in vivo, the underlying molecular mechanism remains unclear. In this report, we provide evidence that ATP, the energy molecule and signal element, accounts for the selective depletion of Treg cells by low-dose CY. Relative to conventional T cells or other cell types, ATP levels were much lower in Treg cells. This was due to Treg cells that downregulate one microRNA, miR-142-3p, and upregulate ecto-nucleoside triphosphate diphosphohydrolase CD39. The transfection of miR-142-3p or the blockade of CD39 could increase intracellular ATP levels of Treg cells, consequently decreasing the sensitivity of Treg cells to low-dose CY. On the other hand, the transfection of miR-142-3p inhibitor or the addition of soluble CD39 to the cultured CD4(+)CD25(-) T cells resulted in the decrease of intracellular ATP levels and increase of sensitivity of conventional T cells to low-dose CY. Furthermore, we found that the low levels of ATP attenuated the synthesis of glutathione, leading to the decrease of CY detoxification, thus increasing the sensitivity of Treg cells to low-dose CY. Therefore, we here identify a molecular pathway through which low-dose CY selectively ablates Treg cells. Our findings also imply that low levels of ATP are probably related to Treg cell function.