Although the authors of several studies report elevated numbers of immunosuppressive regulatory T cells (Tregs) in hematologic and solid malignancies, the underlying mechanism is not fully clarified. Cancer is associated with oxidative stress mediated through reactive oxygen species produced by malignant cells, granulocytes, tumor-associated macrophages, and myeloid-derived suppressor The authors of several studies have demonstrated the presence of elevated levels of Tregs in cancer patients, a phenomenon that may promote tumor progression and influence the course of disease. [3][4][5][6] Increasing evidence indicates that tumor cells can induce Tregs directly or indirectly through their microenvironment by, for example, nitric oxide (NO) production, COX-2/PGE 2 expression, or interleukin (IL)-10. 7,8 Malignant cells and, more importantly, cells recruited or induced by tumor cells within the microenvironment, such as tumor-associated macrophages, activated granulocytes, and myeloid-derived suppressor cells, produce significant amounts of reactive oxygen species (ROS). 9,10 The detrimental effect of ROS such as hydrogen peroxide (H 2 O 2 ) and reactive nitrogen species such as NO on natural killer (NK) and T cells is well established and described in malignant and chronic inflammatory diseases. 11-14 Paradoxically, Treg levels can be increased in this hostile (for lymphocytes) milieu.We studied the sensitivity of freshly isolated Tregs from peripheral blood to oxidative stress-induced cell death and resulting functional alterations. Furthermore, we evaluated the antioxidative capacity and components of the antioxidative machinery to gain a better understanding of our observations. We demonstrate for the first time that Tregs show reduced sensitivity to oxidative stress-induced cell death and maintained their suppressive activity even at H 2 O 2 levels that are lethal for half of the conventional effector CD4 ϩ CD25 Ϫ/low T-cell population. Greater expression levels of surface thiols and a stronger intracellular antioxidative capacity observed in Tregs may contribute to their reduced sensitivity to oxidative stress.
Methods
Cell isolationPeripheral blood mononuclear cells from healthy donors were isolated by Ficoll-Paque separation (Amersham Biosciences, Sunnyvale, CA). Tregs and conventional CD4 ϩ T cells were purified with the use of a CD4 ϩ CD25 bright Treg isolation kit (purity Ͼ90%) and frequencies of memory (63.20% Ϯ 13.81%) and naive (36.40% Ϯ 13.42%) Tregs were in the expected range ( Figure S1, available on the Blood website; see the Supplemental Materials link at the top of the online article). CD45RA ϩ and CD45RA Ϫ subpopulations (purity Ͼ 95%) were purified by the use of anti-CD45RA/RO microbeads (Miltenyi Biotec, Auburn, CA). Granulocytes were isolated from the pellet obtained after Ficoll-Paque gradient centrifugation, followed by erythrocyte lysis.This study received institutional review board approval from the Karolinska Institutet, and informed consent was obtained from donors in accordance with t...
