Modulation of the IL-6/STAT3 Signaling Axis in CD4+ T Cells As a Potential Immune Mechanism of Action of Azacytidine in High-Risk Myelodysplastic Syndromes

• Published in: Blood 2019-11, Vol.134 (Supplement_1), p.2988-2988
• Main Authors: Lamprianidou, Eleftheria, Kordella, Chrysoula, Kazachenka, Anastasiya, Zoulia, Emmanouela, Bernard, Elsa, Filia, Anastasia, Laidou, Stamatia, Vassilakopoulos, Theodoros P., Papageorgiou, Sotirios, Pappa, Vassiliki, Galanopoulos, Athanasios, Viniou, Nora-Athina, Nakou, Evangelia, Hadzidimitriou, Anastasia, Kassiotis, George, Papaemmanuil, Elli, Mitroulis, Ioannis, Kotsianidis, Ioannis
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2019-126504

Azacytidine (AZA), the mainstay of therapy in high risk Myelodysplastic syndromes (HR-MDS), affects CD4+ T-cell polarization and function, but the effect of these changes on tumor immunity is unclear. Signal transducer and activator of transcription (STAT) proteins are key regulators of differentiation and polarization of CD4+ T-cells in both health and cancer, but the STAT signaling architecture of CD4+ T-cell subsets in HR-MDS and its modulation by AZA are currently unknown. We applied single-cell phosphospecific flow cytometry in peripheral blood mononuclear cells from 67 HR-MDS patients at various time-points during AZA therapy. Unsupervised clustering of pretreatment STAT signaling profiles (SPs) of CD4+ T-cells revealed three signaling clusters (SCs), mainly differing in the potentiated responses of STAT3 to IL-6 stimulation (IL-6/STAT3 node). Compared to SC#1 and SC#3, patients in SC#2 displayed higher IL-6/STAT3 levels, higher levels of naïve (TN, p=0.05) and lower levels of PD1+ (p=0.04) and central memory ( TCM, p=0.04) CD4+ T-cells, and longer median overall survival (mOS, p=0.028, fig 1A). Moreover, comparisons of single signaling nodes revealed that the IL-6/STAT3 node correlated inversely with PD1+ (p=0.02) and IL-4+ (p=0.04) and positively with naïve CD4+ (p=0.04) and IFNγ+CD8+ T-cells (p=0.01). No other differences in clinicobiologic parameters, CD4+ and CD8+ T-cell subpopulations (FOXP3, IFNγ, IL-4, IL-17, Perforin and Helios) were noted among the 3 SCs and all other single nodes. To assess the effect of AZA on STAT signaling, we clustered the fold fold-change of pre- versus 6-month post-AZA SPs in CD4+ T-cells. Again the IL6/STAT3 node was the only differentiator among the clusters, and, by single node analysis, downregulation of IL6/STAT3 at 6th cycle (n=26) was associated with better response to AZA (p=0.02) and longer mOS (p=0.03), compared to upregulation of the same node (n=22); the latter also accompanied by an increase of IFNγ+CD8+ cells after AZA, (p=0.02, fig 1B). Further supporting a direct and beneficial modulation of the IL-6/STAT3 axis in CD4+ T-cells by AZA, the kinetics of IL-6/STAT3 during AZA therapy revealed a marked downregulation of the former node both at day15 (p=0.04) and cycle 6 after AZA (p=0.04) in responders (n=5), while no changes were observed in non-responders (n=7). We further compared the transcriptional profiles of isolated bone marrow CD4+ T-cells between responders (n=4) and non-responders to AZA (n=4) b