Cyclophosphamide inhibits the generation and function of CD8+ regulatory T cells

CD8+ regulatory T cells (Treg) and CD4+CD25+ Treg infiltrate human cancers, thus favoring tumor immune escape. Therefore, in the setting of antitumor therapeutic protocols, it is important to associate antitumor treatment with agents that are able to inhibit Treg function. Cyclophosphamide (CY) has been demonstrated to be effective in counteracting CD4+CD25+ Treg activity. Hence, we tested its inhibitory efficacy on human CD8+ Treg. Because CY is a prodrug, 4-hydroperoxycyclophosphamide (4-HC), a derivative of CY that in aqueous solution is converted to 4-hydroxycyclophosphamide, an active metabolite of CY, was used. 4-HC significantly inhibited CD8+ Treg generation and function but only at the higher tested concentration (0.5 μg/mL), that is, in the therapeutic range of the drug. The lower 4-HC concentration tested (0.1 μg/mL) was almost ineffective. 4-HC inhibitory effects were related to apoptosis/necrosis induction. When CD8+CD28+ non-Treg were analyzed for comparative purposes, significantly lower cytotoxic rates among these cells were observed than among CD8+ Treg, which were differentiated because they did not express the CD28 molecule. These data demonstrate that CD8+ Treg are inhibited through cytotoxic phenomena by CY, thus supporting the use of this drug at adequate concentrations and schedules of administration as a Treg inhibitor in combinatorial chemo- or immunotherapeutic anticancer protocols.