TGF-β Enhances the Anti-inflammatory Effect of Tumor- Infiltrating CD33+11b+HLA-DR Myeloid-Derived Suppressor Cells in Gastric Cancer: A Possible Relation to MicroRNA-494

Abstract

Background: Accumulation of myeloid-derived suppressor cells (MDSCs) constitutes a key mechanism of tumor immune evasion in gastric cancer (GC). Therefore, searching for more accurate prognostic factors affecting their immunosuppressive role has become a growing interest in cancer immunotherapy research. Increased expression of microRNA-494 was noticed in MDSCs from tumor-bearing mice, suggesting another new therapeutic objective for cancer treatment. It was also discovered that tumor-derived transforming growth factor beta (TGF-β) is responsible for the up-regulation of microRNA-494 in MDSCs. The purpose of this study was to address the effect of recombinant (rTGF-β) on the anti-inflammatory activity of MDSCs in GC and its possible association with micro-RNA-494 expression in tumor tissue.

Methods: Freshly obtained GC tumor tissue samples and peripheral blood were used for isolation of CD33+11b+HLADR- MDSCs cells from 40 GC patients and 31 corresponding controls using flow cytometry. MDSCs were co-cultured with isolated autologous T cells to assess proliferation and cytokine production in the presence and absence of rTGF-β. Real-time PCR and Enzyme linked immunosorbent assay were used to evaluate tumor expression of miRNA-494 and TGF-β respectively.

Results: Results showed that rTGF-β markedly increased the suppressive ability of tumor MDSCs on proliferation of autologous T cells and interferon gamma production. However, no inhibitory effect was observed for MDSCs from circulation. In addition, infiltration of MDSCs in tumors is associated with the prognosis of GC. MiRNA-494 was also extensively expressed in tumor samples with a significant correlation to MDSCs.

Conclusion: These results indicate that tumor-derived MDSCs but not circulatory MDSCs have an immunosuppressive effect on T cells, potentially involving TGF-β mediated stimulation. Results also suggest a role for miRNA-494 in GC progression. Therefore, control of TGF-β and miRNA-494 may be used as a treatment strategy to downregulate the immunosuppressive effect of MDSCs.<br />.

Keywords: Gastric cancer; T cell suppression; microRNA-494; myeloid-derived suppressor cells; transforming growth factor beta.

Figure 1

Comparison between GC Patients and Control Group in CD11b+CD33+HLA- MDSCs (%): Percentages of CD33+11b+HLA-DR- MDSCs cells from GC patients and controls: a: Isolated cells from PBMCs from a GC patient were stained with anti-HLA-DR, anti-CD33, and anti-CD11b. b: MDSCs cells in healthy control subject. C: Data represented as means ± SDs of percentages of CD11b+CD33+HLA- cells using flow cytometry, where isolated cells from patients (left) and controls (right) were stained with anti-HLA-DR, followed by anti-CD33 and anti-CD11b. Data were summarized as dot plot. Each dot represents the expression percentage of MDSCs for one individual. The level of significance was set at *p ≤0.05

Figure 2

Relation of Circulating CD11b+CD33+HLA- MDSCs to GC Characteristics. a: Comparison between (stage I + II) and (stage III + IV) in percentages of circulating CD11b+CD33+HLA- cells: Data represented as means ± SDs and were summarized as dot plot. Each dot represents the percentage of MDSCs for one individual. The level of significance was set at *p ≤0.05. b: Comparison between Grade I, II, and grade III in percentages of circulating CD11b+CD33+HLA- cells. c: Comparison between (patients with no metastasis) and (patients with metastatic disease) in percentages of circulating MDSCs

Figure 3

a: Comparison between CD11b+CD33+HLA- cells in different types of samples. b: Comparison between TGF-β levels (pg/ml). c: Correlation analysis between CD11b+CD33+HLA- cells in tumor tissues and TGF-β level in both circulation and tumor tissues. Correlation was conducted by Spearman coefficient. d: Comparison between (stage I + II) and (stage III + IV) in percentages of tumor derived CD11b+CD33+HLA- cells: Data represented as means ± SDs and were summarized as dot plot. Each dot represents the percentage of MDSCs for one individual. The level of significance was set at *p ≤0.05

Figure 4

a: Comparison between GC patients and control group in TGF-β levels in circulation (pg/ml). b: Comparison between (stage I + II) and (stage III + IV) in TGF-β levels in circulation (pg/ml). Data represented as means ± SDs and were summarized as dot plot. Each dot represents the level for one individual. The level of significance was set at *p ≤0.05

Figure 5

. a: Comparison between GC patients and control group in microRNA-494 expression (folds). b: microRNA-494 expression (folds) in tumor tissues and their surrounding tissue (n=40). c: Comparison between (stage I + II) and (stage III + IV) in microRNA-494 expression (folds). Data represented as means ± SDs and were summarized as dot plot. Each dot represents the expression for one individual. d: Correlation analysis between CD11b+CD33+HLA- cells in tumor tissues and circulation vs microRNA-494 expression (folds). e: microRNA-494 expression (folds) vs TGF-β level. Correlation was conducted by spearman coefficient. The level of significance was set at *p ≤0.05

Figure 6

a: CD11b+CD33+HLA- cells were mixed with CFSE-labeled T cells (responder T cells), and the proliferation of responder T cells was assessed by the percentage of CFSE diluting responder T cells. n = 40. b: rTGF-β effect on the suppressive activity of tumor derived CD11b+CD33+HLA- cells: c and d: T cell proliferation was assessed by MTT. Data represented as means ± SDs and were summarized as dot plot. Each dot represents the level for one individual. The level of significance was set at *p ≤0.05

Figure 7

rTGF--β effect on the suppressive activity of MDSCs on IFN-γ release from responder T cells. Data represented as means ± SDs and were summarized as dot plot. Each dot represents the serum level of IFN-γ for one individual. Left dot plot shows unstimulated IFN-γ (pg/ml). Right dot plot shows cytokine level after addition of rTGF-β. The level of significance was set at *p ≤0.05