Increased circulating of myeloid-derived suppressor cells in myelodysplastic syndrome

Myelodysplastic syndrome (MDS) is a group of clonal hematopoietic stem cell disorders, characterized by varying degrees of peripheral cytopenia caused by ineffective dysplasia of the myeloid lineages. MDS also has a high risk of progression to acute myeloid leukemia. But the role of immune abnormalities in the pathogenesis of MDS is still not clear. Myeloid-derived suppressor cells (MDSCs) constitute a heterogeneous population of immature cells derived from the bone marrow. In the recent years, MDSCs were reported to play an important role in suppressing lymphocytes in tumor-bearing animal models and cancer patients. This could be one of the mechanisms on tumor immune evasion, which aggravates the development and growth of tumors. In a mice model, MDSCs are characterized by coexpression of GR1 and CD11b. In humans, the phenotype of MDSCs was accepted as Lin- HLA-DR- CD33+.1 In the present study, we investigated the level of circulating MDSCs (Lin- HLA-DR- CD33+) in patients with MDS and evaluated the association between MDSCs and malignancy in MDS. METHODS Patients Thirty-five patients with MDS (24 men and 11 women, median age 59 years (age range 21-83), including refractory anemia (n=3); refractory anemia with ringed sideroblasts (n=3); refractory cytopenia with multilineage dysplasia (n=9); refractory anemia with excess blast-I (n=4); refractory anemia with excess blast-II (n=16)), and without other systemic diseases, were enrolled in the present study. All the patients underwent diagnosis in the Department of Hematology, General Hospital of Tianjin Medical University from March 2011 to April 2012, according to the diagnostic criteria of MDS proposed between 2007 and 2008. After treatment for three months, 14 MDS patients were investigated again for their MDSCs changes. Twenty normal healthy individuals were selected as controls (9 men and 11 women, median age 34 years (age range 26-82)). The study was approved by the Ethics Committee of Tianjin Medical University. Informed written consent was obtained from all the patients and normal individuals in accordance with the Declaration of Helsinki. Flow cytometric analysis Peripheral blood samples were collected in EDTA-anticoagulant tubes from the patients and normal individuals. The number of circulating MDSCs was measured by using flow cytometry (FCM) assay. The markers used in the assay were anti-CD33-APC, anti-LINFITC (CD3, CD14, CD16, CD19, CD20, CD56) and anti-HLA-DR-PE antibodies (BD Biosciences, USA). The number of stem cells from the bone marrow, which was collected in heparin-anticoagulant tubes, was measured by FCM using anti-CD34-PerCP antibodies (BD Biosciences). Data acquisition and analysis were carried out by using FACS-Calibur flow cytometer (BD Biosciences), with the Cell Quest software (Becton Dickinson, version 3.1). Statistical analysis Non-parametric statistical analyses were used in analyzing the data. Statistical analysis was performed by using the Mann-Whitney U test or Wilcoxon signed-rank test for the unpaired data between the two groups. P <0.05 was considered as statistically significant. Statistical analysis was performed using SPSS11.5 (SPSS Inc, Chicago, IL, USA). RESULTS Circulating MDSCs increases in MDS and correlates with high risk according to IPSS/WPSS score MDS patients' median MDSCs were 1.61% which was higher than that of the controls (0.83%, P=0.033). The median MDSCs of high-risk MDS patients (including intermediate-2-risk and high-risk group) based on the IPSS score was 4.45%, which was significantly higher than that of the low-risk MDS patients (including low-risk and intermediate-1-risk group; 0.63%, P=0.003). Furthermore, the median MDSCs of high-risk MDS patients (including high-risk and very high-risk group) based on the WPSS score was 4.39%, which was higher than that of low-risk MDS patients (including very low-risk, low-risk and intermediate-risk group, 0.47%, P=0.002, Figure 1A-1C).Figure 1.: The median MDSCs of high-risk MDS patients. A: The percentage of MDSCs was compared between patients with MDS and healthy controls (P=0.033). B: The percentage of MDSCs was compared between high-risk and low-risk MDS patients based on the IPSS score (P=0.003). C: The percentage of MDSCs was compared between high-risk and low-risk MDS patients based on the WPSS score (P=0.002). D: After therapy, the ratio of MDSCs decreased in the remission group, and increased in PD. Meanwhile, the ratio of MDSCs in SD was similar as in pre-therapy. E: The change of MDSCs before and after therapy in the remission group was lower than that of the PD group (P=0.024).Circulating MDSCs decreases in remission patients with MDS Low-risk MDS patients had accepted hemopoietic factor and supportive treatments, while the high-risk group was treated by decitabine. After therapy for three months, 14 MDS patients were investigated again for MDSCs changes in the blood. According to the standardized response criteria for MDS in 2000, these patients were divided into three groups: remission group (includes complete remission and partial remission), stable disease group (SD) and disease progression group (PD). We found that the ratio of MDSCs decreased in the remission group and increased in PD. The change of MDSCs before and after therapy in the remission group was lower than that of the PD group (P=0.024). This suggested that the change of circulating MDSCs might correlate to the prognosis of MDS (Figure 1D and 1E). Correlation between MDSCs and clinical signs in MDS patients There was a negative correlation between MDSCs and hemoglobin (HB) in MDS patients (r=-0.214, P=0.022). No significant correlation was observed between MDSCs and neutrophils, reticulocytes or thrombocytes. There was a positive correlation between MDSCs and CD34+ cells (r=0.274, P=0.042) and lactate dehydrogenase in MDS (r=0.406, P=0.001). DISCUSSION In the recent years, it has been generally accepted that MDS occurs as a result of multistep leukemogenesis of malignant clones called leukemic stem cells.2 Leukemic stem cells could be escaping from immune-mediated alterations and leading to a growth advantage, which in-turn mechanisms in MDS to progress as acute myeloid leukemia. Hence, the abnormal immune function plays a key role in MDS pathogenesis. Studies on immune cells in MDS patients have shown that the number and co-stimulatory molecules of dendritic cells (DC) were altered. It suggested that inflammatory injury related DC increased more significantly when compared with antitumor immunity related DC in MDS.3 Some researchers found that the number of Th1 decreased and Th2 increased in MDS patients, which could suppress the immune responses.4 Furthermore, changes of T regulatory cells (T-reg) and Th17 cells were observed in the early or late MDS stages, which could enhance the efficiency of immune tolerance. In addition, it has been shown that these kinds of immune cells were also accumulated at tumor situ in other cancer types, suggesting a similar immune pathogenetic pathway between MDS and solid tumors.5 MDSCs originate from immature myeloid cells in bone marrow. Tumor-derived factors, such as interleukin-6, granulocyte/macrophage colony-stimulating factor, stem cell factor, and vascular endothelial growth factor, could promote the expansion of MDSCs, which in turn increases the level of MDSCs in peripheral blood. Further studies have shown that the population of MDSCs was significantly increased, and often associated with advanced-stage disease in many kinds of cancers, like breast cancer, lung cancer, colon cancer, etc.1 Recently, it has been proved that MDSCs play an important role in tumor immune evasion and progression. MDSCs could be beneficial in setting up tissue microenvironment, which promotes the growth of tumor cells by secreting cytokines (such as vascular endothelial growth factor, tumor necrosis factor-β). Moreover, MDSCs could suppress the functions of immune cells, which lead to immune tolerance. For example, MDSCs could suppress the function of T cells through the ARG1and ROS pathway, suppress the natural killer cells activation by inhibiting perforin, alter the differentiation of DC and expand the T-reg pool, which would suppress immune responses.1 In our study, we investigated the level of circulating MDSCs (Lin- HLA-DR- CD33+) in patients with MDS. Our results showed that the level of circulating MDSCs was increased in MDS. And there was a negative correlation between MDSCs and hemoglobin in MDS. No significant correlation was seen between MDSCs and neutrophils, reticulocytes or thrombocytes, which might be due to the differentiation of myeloid cells in MDS subtypes. MDSCs showed a positive correlation to CD34+ cells and lactate dehydrogenase in MDS, which hint at the tumor load in patients. In conclusion, the level of circulating MDSCs is increased in MDS, and is correlated to the malignancy of this disease. Our data indicate that the increased level of circulating MDSCs might involve in MDS pathogenesis. Therefore, it could be a novel target for treating MDS.