The effects of 5-azacytidine on the function and number of regulatory T cells and T-effectors in myelodysplastic syndrome.

Expansion of regulatory T cells occurs in high-risk myelodysplastic syndrome and correlates with a poor prognosis. DNA methyltransferase inhibitors, particularly 5-azacytidine, have been shown to increase the survival of patients with high-risk myelodysplastic syndrome. It is not entirely clear whether this improvement in patients' survival is related to the effects of DNA methyltransferase inhibitors on the immune system and/or the direct effect of these drugs on the dysplastic clone. In this study we investigated the effect of 5-azacytidine on the function and proliferation capability of regulatory T cells and T-helper cells. The number and function of CD4(+) T-cell subsets in 68 patients with intermediate-2/high-risk myelodysplastic syndrome were serially assessed at diagnosis and following treatment. The in-vitro effects of 5-azacytidine on CD4(+) T-cell subsets isolated from both healthy donors and patients with myelodysplastic syndrome were also investigated. The number of peripheral blood regulatory T cells was significantly higher in myelodysplastic syndrome patients than in healthy donors and responders to treatment (P=0.01). The absolute numbers of T-helper 1 and T-helper 2, but not T-helper 17, cells were significantly reduced following 12 months of treatment (P=0.03, P=0.03). The in vitro addition of 5-azacytidine to CD4(+) T cells reduced the proliferative capacity of regulatory T cells (P=0.03). In addition, the 5-azacytidine-treated regulatory T cells had reduced suppressive function and produced larger amounts of interleukin-17. The FOXP3 expression in 5-azacyti-dine-treated T-effectors was also increased. Interestingly, these FOXP3(+)/interleukin-17(+) cells originated mainly from effector T cells rather than regulatory T cells. Our data suggest that 5-azacytidine has profound effects on CD4(+) T cells, which correlate with disease status after treatment. Furthermore, despite the demethylation of the FOXP3 promoter and increased FOXP3 expression following 5-azacytidine treatment, these phenotypic regulatory T cell-like cells lack the regulatory function and cytokine profile of regulatory T cells. These findings are important in correlating the clinically relevant immunomodulatory effects of 5-azacytidine.

Functional characterization of CD4+ T cells in aplastic anemia.

The role of CD4(+) T cells in the pathogenesis of aplastic anemia (AA) is not well characterized. We investigate CD4(+) T-cell subsets in AA. Sixty-three patients with acquired AA were studied. Th1 and Th2 cells were significantly higher in AA patients than in healthy donors (HDs; P = .03 and P = .006). Tregs were significantly lower in patients with severe AA than in HDs (P < .001) and patients with non-severe AA (P = .01). Th17 cells were increased in severe AA (P = .02) but normal in non-severe AA. Activated and resting Tregs were reduced in AA (P = .004; P = .01), whereas cytokine-secreting non-Tregs were increased (P = .003). Tregs from AA patients were unable to suppress normal effector T cells. In contrast, AA effector T cells were suppressible by Tregs from HDs. Th1 clonality in AA, investigated by high-throughput sequencing, was greater than in HDs (P = .03). Our results confirm that Th1 and Th2 cells are expanded and Tregs are functionally abnormal in AA. The clonally restricted expansion of Th1 cells is most likely to be antigen-driven, and induces an inflammatory environment, that exacerbate the functional impairment of Tregs, which are reduced in number.