Effect of Treatment with Azacitidine on CD4+CD25+Foxp3+ Regulatory T Cells in Myelodysplastic Syndrome Survivors
Authors: Tajima Fumihito , Adachi Koji , Kawatani Toshio 
Published: October 2019

Abstract
Background: Regulatory T cells (Treg) play an important role in immune surveillance of malignancies. Treg expansion occurs frequently in high-risk myelodysplastic syndrome (MDS) and in progression of this disease. Azacitidine (AZA) is increasingly used for treatment of MDS. In this study, we examined changes in the number of circulating Treg in peripheral blood of patients with MDS treated by AZA and we evaluated the clinical significance of these changes.
Patients and Methods: Patients with MDS and acute myeloid leukemia with myelodysplasia-related changes (AML-MRC) according to the WHO 2008 classification who were initially treated between January 2015 and December 2015 and followed up for more than six months were enrolled in the study. AZA was given intravenously at a dose of 75 mg/m2 daily for 5 or 7 consecutive days every 4 weeks. The response was assessed using the IWG response criteria for MDS. A total of 29 patients with AML-MRC (n=5), refractory anemia with excess blasts (RAEB) (n=7), refractory cytopenia with multilineage dysplasia (RCMD) (n=16), and myelodysplastic/myeloproliferative neoplasms (MDS/MPN) (n=1) were included in the study. IPSS was intermediate-1 (n=15), intermediate-2 (n=6), and high (n=8). Eighteen patients did not need treatment, including transfusion (untreated group). Eleven patients were treated with AZA (AZA group). After obtaining informed consent, blood was drawn from all patients at initial diagnosis. In the 11 patients treated with AZA, blood was also drawn after 6 completed cycles. CD4+CD25+Foxp3+ Treg cells, CD3+CD8+ T cells, and CD3-CD56+ NK cells were analyzed using flow cytometry and the absolute numbers of these cells were calculated.
Results: The median age was 74 years (range: 44-88) and 22 of the patients were male. The median period of observation was 283 days. In the AZA group, 3 patients died and one had recurrence (AZA-ineffective group), and 7 reached CR or PR (AZA-effective group). The absolute number of Treg was higher in AML/MRC + MDS/MPN than in RCMD (32.5 ± 19.6 vs. 16.4 ± 11.9 /μL, p=0.0234). Treg% values in peripheral blood before treatment in the AZA (1.17±0.80%), AZA-effective (1.15±0.54%), and AZA-ineffective (1.21±1.24%) groups were all significantly higher than that in the untreated group (0.49±0.26%, p=0.0022, p=0.0004, p=0.0241, respectively). Similarly, the absolute numbers of Treg were significantly higher in the AZA (31.9±17.0 /μL), AZA-effective (24.3±11.5 /μL), and AZA-ineffective (45.1±18.2 /μL) groups than in the untreated group (14.9±8.1 /μL, p=0.0011, p=0.0296, p=0.00003, respectively), and significantly higher in the AZA-ineffective group than in the AZA-effective group (p=0.0434). After 6 months of treatment in the AZA group, there were significant decreases in %Treg (1.17±0.80% to 0.65±0.48%, p=0.0373) and absolute number of Treg (31.9 ± 17.0 to 19.7 ± 13.6 /μL, p=0.0258). These changes were not significant in the AZA-effective group, but the absolute number of Treg significantly decreased in the AZA-ineffective group (45.1 ± 18.2 to 16.23 ± 9.54 /μL, p=0.0194). No Treg values after treatment differed significantly from those in the untreated group. The count of CD8+ T cells was unchanged by AZA treatment, but was moderately higher in the AZA group compared to the untreated group (378.6 ± 504.6 vs. 205.4 ± 140.2 /μL, p=0.0455). The counts of CD3-CD56+ NK cells did not differ significantly between the untreated group and the AZA group before treatment. However, after AZA treatment, this count became significantly higher than that in the untreated group (311.0 ± 263.5 vs. 159.2 ± 105.5 /μL, p = 0.0371). The count of NK cells did not reach a high level in the AZA-ineffective group, but was significantly higher (300.9 ± 183.5 /μL, p=0.0030) in the AZA-effective group.
Conclusion: The number of Treg in MDS cases requiring treatment was higher than that in MDS cases for which treatment was unnecessary. In particular, Treg was high in MDS with a poor prognosis. Treg was decreased by treatment with AZA and became equivalent to that in the untreated group. In contrast, there was increase in the number of NK cells after AZA treatment, which was found to be particularly high in cases with a good prognosis. These results suggest that an immunological mechanism associated with the prognosis of MDS may be improved by treatment with AZA. Further examination of this mechanism and the influence of AZA on the immunological effect is required.

Disclosures
No relevant conflicts of interest to declare.