Distinct Profile and Epigenetic Modulation Of STAT Signaling In FOXP3+ T Regulatory Cells Among The Various MDS Subtypes

T regulatory cells (Tregs) safeguard against autoimmunity, but may compromise antitumor immunity. Aberrant immune signaling via signal transducer and activator of transcription (STAT) proteins is involved in disease pathobiology in numerous malignancies. Azacytidine treatment affects Treg levels, particularly in responders, but its effect on STAT signaling is unknown. We explored the alterations of STAT signaling profile (SP) of Tregs during azacytidine treatment and their association with clinical and biological parameters.

Peripheral blood mononuclear cells of 19 late-stage MDS patients were obtained before and 15 days after azacytidine initiation. According to WHO, one had RAEB-I (5%), 9(47%) patients had RAEB-II, 7(37%) CMML-II and 2(10%) AML/MDS. Based on the IWG criteria patients were divided into responders (CR, PR and hematologic improvement, n=8) and non-responders (stable disease and failure, n=11). We applied a modified protocol of phospho-specific flow cytometry to measure either basal (untreated) or potentiated (after stimulation with IL-6 and IL-2 for 15’) phospho-STAT1, STAT3 and STAT5 levels with simultaneous staining for FOXP3. The following potentiated, i.e. target/stimuli, nodes were studied: pSTAT1/IL-6, pSTAT3/IL-6 and pSTAT5/IL-2. Comparisons were performed by paired, unpaired t-test or one-way ANOVA as appropriate. Clustering of SPs was performed with hierarchical cluster analysis and was correlated with response, disease subtype, and cytogenetics by using ÷² or Fisher Exact tests.

The pretreatment levels of Tregs were significantly higher in non-responders compared to responders (8.4%±0.42% vs 5.5%±0.46%, p=0.004). By contrast, azacytidine treatment significantly upregulated Tregs at d15 only in responders (7.1±0.47%, p=0.03), whereas non-responders exhibited a mild, but non-significant increase (9.7%±1.22%, p=0.18). Unsupervised clustering of the Treg SPs revealed 4 clusters. No differences were noticed in the SP of Tregs in relation to treatment response, cytogenetics, IPSS, WPSS and IPSS-R. By contrast, the disease subtypes were nonrandomly distributed among the clusters. Most CMML-2 patients segregated in cluster 4, which displayed high basal levels of phospho-STAT1 and 5, but weak potentiated responses, whereas the cluster 2 was enriched in RAEB-II patients and was characterized by very high basal levels of pSTAT5 and relatively intense response of pSTAT3 in IL-6 (Fig 1). Intriguingly, the pretreatment levels of Helios+ Tregs were significantly higher in patients of cluster 2 (37.9%±7.7% of total Tregs) compared to patients in clusters 1, 3 and 4 (23.1±2.3%, 22.8%±2.4% and 22.2%±2.1%, respectively, p=0.035) indicating a higher level of Treg activity and, potentially, immune suppression in these patients. In addition, the clustering of the ratios of Treg SPs after/before azacytidine treatment revealed a distinct epigenetic modulation of Treg signaling via STATs in CMML-2 compared to all other patients (p=0.036), characterized by downregulation of pSTAT1 and pSTAT5 basal levels and upregulation of potentiated responses at day 15 after first azacytidine administration (Fig 2).

Figure 1

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Pretreatment SPs of Tregs in 19 patients.

Figure 1

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Pretreatment SPs of Tregs in 19 patients.

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Figure 2

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Ratio of the SPs of Tregs 15 days after/before azacytidine initiation in 19 patients.

Figure 2

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Ratio of the SPs of Tregs 15 days after/before azacytidine initiation in 19 patients.

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Collectively, consistent with recent data (Constantini B et al, 2012), we observed both arithmetical and functional alterations of Tregs in MDS patients after azacytidine treatment. Moreover, both the pretreatment SP and the differential modulation of STAT signaling in the Tregs of CMML-2 and RAEB-II patients, argue for a potentially diverse epigenetic regulation of tumor immunity among the various MDS subtypes.