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. 2020 Jul;22(2):236-244.
doi: 10.22074/cellj.2020.6313. Epub 2019 Oct 14.

Helios, CD73 and CD39 Induction in Regulatory T Cells Exposed to Adipose Derived Mesenchymal Stem Cells

Affiliations

Helios, CD73 and CD39 Induction in Regulatory T Cells Exposed to Adipose Derived Mesenchymal Stem Cells

Maryam Fakhimi et al. Cell J. 2020 Jul.

Abstract

Objective: Mesenchymal stem cells (MSCs) have prominent immunomodulatory roles in the tumor microenvironment. The current study intended to elucidate Treg subsets and their cytokines after exposing naïve T lymphocytes to adiposederived MSCs (ASCs).

Materials and methods: In this experimental study, to obtain ASCs, breast adipose tissues of a breast cancer patient and a normal individual were used. Magnetic cell sorting (MACS) was employed for purifying naïve CD4+ T cells from peripheral blood of five healthy donors. Naïve CD4+ T cells were then co-cultured with ASCs for five days. The phenotype of regulatory T cells (Tregs) and production of interleukine-10 (IL-10), transforming growth factor beta (TGF-β) and IL-17 were assessed using flow cytometry and ELISPOT assays, respectively.

Result: CD4+CD25-FOXP3+CD45RA+ Tregs were expanded in the presence of cancer ASCs but CD4+CD25+Foxp3+CD45RA+ regulatory T cells were up-regulated in the presence of both cancer- and normal-ASCs. This up-regulation was statistically significant in breast cancer-ASCs compared to the cells cultured without ASCs (P=0.002). CD4+CD25+ FOXP3+Helios+, CD4+CD25- FOXP3+Helios+ and CD25+ FOXP3+CD73+CD39+ Tregs were expanded after co-culturing of T cells with both cancer-ASCs and normal-ASCs, while they were statistically significant only in the presence of cancer-ASCs (P<0.05). Production of IL-10, IL-17 and TGF-β by T cells was increased in the presence of either normal- or cancer-ASCs; however, significant effect was only observed in the IL-10 and TGF-β of cancer-ASCs (P<0.05).

Conclusion: The results further confirm the immunosuppressive impacts of ASCs on T lymphocytes and direct them to specific regulatory phenotypes which may support immune evasion and tumor growth.

Keywords: Adipose-Derived Mesenchymal Stem Cell; Breast Cancer; Immunomodulatory Effects; Regulatory T Cells.

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Conflict of interest statement

There is no conflict of interest in this study.

Figures

Fig 1
Fig 1
Morphological characterization of adipose-derived mesenchymal stem cells (ASCs) as well as flow cytometry analysis. A. ASCs were appeared with spindle shape in culture (scale bar: 100 μm) and B. The flow cytometry analysis of ASCs illustrate that more than 90% of all cancer and normal ASCs were positive for stem cell specific markers including CD44, CD105, CD73 and CD90, but they were negative for the expression of hematopoietic specific markers such as CD14, CD45 and CD34.
Fig 2
Fig 2
Purity of the isolated naïve CD4+ T cells was evaluated using flow cytometry for the expression of CD4 and CD45RA (solid histograms). Results illustrate more than 96% purity for CD4+ and CD45RA+ cells. Solid and lined histograms represent isotype control and unstained cells, respectively.
Fig 3
Fig 3
The percentage of CD45RA+CD4+CD25+FOXP3+ and CD45RA+CD4+CD25- FOXP3+ Tregs population. After five days culturing of naïve CD4+ T cells with adipose-derived mesenchymal stem cells (ASCs), the percentage of A. CD45RA+CD4+CD25+FOXP3+ T cells and B. CD45RA+CD4+CD25-Foxp3+ T cells was evaluated by flow cytometry method. The results illustrate mean ± SEM of cell percentages. **; P<0.01 compared to the control group.
Fig 4
Fig 4
Percentage of the Helios+ Tregs population. After five days culturing of naïve CD4+ T cells with adipose-derived mesenchymal stem cells (ASCs). Percentage of A. The CD4+CD25+FOXP3+Helios+ T cells was evaluated by flow cytometry method and B. Percentage of the CD4+CD25--FOXP3+Helios+ T cells population was also determined. The results illustrate mean ± SEM of cell percentages. **; P<0.01 compared to the control group.
Fig 5
Fig 5
Percentage of the CD39+CD73+ in CD25+FOXP3+ and CD25-FOXP3+ T cells population subsequent to co-culturing with adipose-derived mesenchymal stem cells (ASCs). After five days culturing of naïve CD4+ T cells with ASCs, percentage of CD39+CD73+ T cells in A. CD25+FOXP3+ population and B. CD25-FOXP3+ population was evaluated by flow cytometry method. The results illustrate mean ± SEM. **; P<0.01 compared to the control group.
Fig 6
Fig 6
Producing interleukine-10 (IL-10), transforming growth factor beta (TGF-β) and IL-17 by T cell after co-culturing with adipose-derived mesenchymal stem cells (ASCs). After five days culturing of naïve CD4+ T cells with ASCs, IL-10, TGF-β and IL-17 produced by T cells were evaluated using ELISPOT method. The results illustrate mean ± SEM. *; P<0.05, **; P<0.01 and ***; P<0.001 compared to naïve T cells which were not cocultured with ASCs (control group).

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