Myeloid Derived Suppressor Cell Expansion Correlates With Increased regulatory T Cells In Myelodysplastic Syndrome
Authors: Kittang Astrid Olsnes , Kordasti Shahram , Sand Kristoffer Evebø , Perezabellan Pilar , Seidl Thomas , Rye Kristin Paulsen , Hagen Karen Marie , Hallan Geir , Kulasekararaj Austin G , Bruserud Øystein , Mufti Ghulam J. 
Published: October 2019

Abstract

Background
T cell mediated immune dysregulation is an established feature of Myelodysplastic syndrome (MDS). We have shown previously that the number of T regulatory cells (Tregs) are increased in high risk MDS and associate with disease progression. In low risk disease however, T helper-17 cells (Th17) are increased which correlates with augmented bone marrow apoptosis. Nevertheless, the role of other components of immune system in MDS pathogenesis is still emerging. Myeloid derived suppressor cells (MDSCs) are a group of immature myeloid cells with a potent immunosuppressive effect which are expanded in an inflammatory environment. MDSCs suppress T cells by depriving them of arginine and cysteine, as well as the secretion of Interleukin (IL)-10, Transforming Growth Factor (TGF)-β and induction of Tregs. While, the role of MDSCs in negatively regulating the anti-tumour immune responses and in determining the T cell polarity have been demonstrated in solid tumours, this information is lacking in MDS. The aim of this study was to investigate the number and function of MDSCs in MDS and potential contribution to immune dysregulation in MDS.

Material and Methods
MDSCs and Treg numbers were assessed in peripheral blood (PB) and bone marrow (BM). In total we have analysed PB from 24 MDS patients of whom 12 patients had concurrent BM samples. PB samples from 11 age-matched healthy donors (HD) were also included. The WHO subtypes were: RC, RARS or RCMD (n=16, 67%) and 8 patients (33%) had RAEB. Cell surface staining and flow-cytometry was performed following red cell-lysis using the following markers: Live/dead dye, (eBioscience), Lineage markers (CD3, CD16, CD19, CD20, CD56,), CD33, CD34, HLA-DR, CD11b, CD15, CD66b, CD14, CX3CR1, CXCR3 and CXCR4. MDSCs were stained for intracellular TGF-β and IL-10 following fixation and permeabilisation, and they also stained positive for Arginase-1. To evaluate MDSCs function, CFSE stained CD3+CD4+CD25- (T-effectors) from MDS patients were stimulated by anti-CD3/CD28 antibodies. Cells were cultured with: T-effectors alone, T-effectors + CD3+/CD4+/CD25high (Tregs), (ratio 2:1); T-effectors + Tregs + HLA-DR-/CD14+ (MDSC) (ratio 2:1:1).

Results
MDSCs in the PB were higher in patients with RAEB compared to both healthy donors (HD) and non-RAEB MDS (2.6% v 1.03% p=0.0004) & (2.6% v 2% p=0.02). The absolute numbers of MDSCs were also higher in RAEB compared to non-RAEB subtypes (0.17 x 109/L v 0.08 x 109/L, p=0.02) and in Intermediate (INTR)/High (HR)/Very high (VHR) risk groups in comparison with Very low (VLR)/ Low (LR) risk group (0.31 x 109/L v 0.08 x 109/L, p=0.01). MDSCs frequency in MDS patients was higher in BM than in PB (45% v 2.6%, p=0.002). The PB MDSCs were mainly Monocytic (M)-MDSCs (0.6 % in PB v 0.3% in BM, p=0.02). M-MDSCs were also expressing higher CX3CR1, (MFI 350 v 8849, p=0.008) and higher CXCR4 (MFI 1846 v 2498, p=0.0156) compared to G-MDSCs.
Granulocytic (G)-MDSC percentages were increased in INT/HR/VHR compared to VLR/LR (0.7% v 0.2%, p=0.0089) and HD (0.2% v 0.7%, p=0.0385). G-MDSCs were also more frequent in the peripheral blood of RAEB compared to non-RAEB subtypes (0.5% v 0.1% p=0.0299). G-MDSCs from MDS patients produce higher amount of IL-10/TGF-β compared to Monocytic (M)- MDSCs, evaluated by intracellular staining (IL-10 R-MFI, 7.9 v 3.8, p=0.003 and TGF-β R-MFI 15 v 4.8, p=0.024).
To evaluate the suppressive effect of MDSCs, autologous Tregs and T-effectors from MDS patients (one INTR and one LR) were co-cultured in the presence and absence of MDSCs. Presence of MDSCs, not only increased the T-effector suppression (figure 1) but also induced Tregs proliferation. In patients with RAEB we also found a positive correlation between absolute counts of MDSC and CD27+FOXP3+CD4+Tregs (Spearman R=0.9, p=0.0374).

Discussion
Our data show that MDSCs are increased in MDS patients, particularly in high-risk disease, compared to healthy donors. The expanded MDSCs express chemokine receptors and secrete IL-10 and TGF-β, and potentiate the suppressive effect of Tregs. In RAEB patients, numbers of MDSCs are positively correlated with Treg numbers. Data suggests that increased MDSCs play an important role in immunopathogenesis of MDS by suppressing the immune-surveillance against the dysplastic clone. Inhibition of MDSCs (i.e. by arginase inhibitor) could reverse the immunosuppressive environment and re-establish immune-surveillance in MDS.

Disclosures:
No relevant conflicts of interest to declare.