Human mesenchymal stromal cells enhance the immunomodulatory function of CD8(+)CD28(-) regulatory T cells

Abstract

One important aspect of mesenchymal stromal cells (MSCs)-mediated immunomodulation is the recruitment and induction of regulatory T (Treg) cells. However, we do not yet know whether MSCs have similar effects on the other subsets of Treg cells. Herein, we studied the effects of MSCs on CD8(+)CD28(-) Treg cells and found that the MSCs could not only increase the proportion of CD8(+)CD28(-) T cells, but also enhance CD8(+)CD28(-)T cells' ability of hampering naive CD4(+) T-cell proliferation and activation, decreasing the production of IFN-γ by activated CD4(+) T cells and inducing the apoptosis of activated CD4(+) T cells. Mechanistically, the MSCs affected the functions of the CD8(+)CD28(-) T cells partially through moderate upregulating the expression of IL-10 and FasL. The MSCs had no distinct effect on the shift from CD8(+)CD28(+) T cells to CD8(+)CD28(-) T cells, but did increase the proportion of CD8(+)CD28(-) T cells by reducing their rate of apoptosis. In summary, this study shows that MSCs can enhance the regulatory function of CD8(+)CD28(-) Treg cells, shedding new light on MSCs-mediated immune regulation.

Figure 1

MSCs increase the percentage of CD8⁺CD28⁻ T cells. Representative flow cytometry dot plots of the percentage of CD28⁻ T cells by gating on CD8⁺ T cells among the purified CD3⁺ T cells (a) or among the purified CD3⁺CD8⁺ T cells (c) in the absence or presence of MSCs 3 or 6 days at the ratio of 5∶1, and higher percentages of CD28⁻ T cells were observed when cocultured with MSCs (b, d). The results are representative of four independent experiments. The bar graphs indicate the means±s.d., statistically significant differences are indicated as follows: *P<0.05 and **P<0.01. MSC, mesenchymal stem cell.

Figure 2

The proliferation and activation of naive CD4⁺ T cells is markedly inhibited by the CD8⁺CD28⁻ T cells educated by MSCs. The T-cell proliferative response was evaluated by the CFSE dilution of the CFSE-labeled CD4⁺ T cells in the presence of anti-CD3 (200 ng/ml) and anti-CD28 (1 μg/ml). The CD8⁺CD28⁻ T cells and MSCs-pre-treated CD8⁺CD28⁻ T cells were added respectively at the ratio of 1∶1, and their inhibitory effects on T-cell proliferation were analyzed (a, b). The CD8⁺CD28⁻ T cells and MSCs-pre-treated CD8⁺CD28⁻ T cells were cocultured with naive CD4⁺ T cells at a ratio of 1∶1 in the presence of a stimulus for 24 h, and their effects on T-cell activation were determined by the expression of CD69 (c, d) and CD25 (e, f) on CD4⁺ T cells. The gray histograms correspond to the isotype controls. The bar graphs indicate the means±s.d. Statistically significant differences are indicated as follows: *P<0.05 and **P<0.01, n=3. CFSE, 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester; MSC, mesenchymal stem cell.

Figure 3

MSCs enhance the ability of CD8⁺CD28⁻ T cells to inhibit IFN-γ production by responder CD4⁺ T cells and induce the apoptosis of activated CD4⁺ T cells. Flow cytometry plots represent the levels of IFN-γ secreted by the CD4⁺ T cells. The CD8⁺CD28⁻ T cells and MSCs-pre-treated CD8⁺CD28⁻ T cells were added respectively at a ratio of 1∶1, and IFN-γ production was significantly downregulated by MSCs-pre-treated CD8⁺CD28⁻ T cells (a, b). The purified activated T cells (CD4⁺CD25⁺ T cells) were cocultured with CD8⁺CD28⁻ T cells and MSCs-pre-treated CD8⁺CD28⁻ T cells at a ratio of 1∶1, and the cytotoxity of CD8⁺CD28⁻ T cells was evaluated by Annexin V-positive cells within the total CD4⁺ T-cell population (c, d) The bar graphs indicate the means±s.d.; statistically significant differences are indicated as follows: *P<0.05 and **P<0.01, n=3. MSC, mesenchymal stem cell.

Figure 4

MSCs affect the functions of the CD8⁺CD28⁻ T cells partially by upregulating the expression of IL-10 and FasL. An analysis of the effector molecules that expressed by the CD8⁺CD28⁻ T cells in the presence or absence of MSCs, including TGF-β (a), IL-10 (b), FasL (c), PD-L1 (d), TRAIL (e) and CTL-A4 (f). The frequency and mean fluorescence intensity (MFI) of IL-10 and FasL were moderately increased by MSCs (b, c). The results are representative of three independent experiments. The bar graphs indicate the means±s.d. Statistically significant differences are indicated as follows: *P<0.05 and **P<0.01. CTLA-4, cytotoxic T-lymphocyte antigen 4; MSC, mesenchymal stem cell; TRAIL, tumor necrosis factor-related apoptosis inducing ligand.

Figure 5

MSCs increase the percentage of CD8⁺CD28⁻ T cells by decreasing the apoptosis. The kinetics of the relative percentages of CD8⁺CD28⁻ T cells were detected in purified CD8⁺ T cells cocultured with or without MSCs (a). MSCs increased the percentages of CD8⁺CD28⁻ T cells neither shift CD8⁺CD28⁺ T cells to CD8⁺CD28⁻ T cells (b), nor prior to promote the proliferation of CD8⁺CD28⁻ T cells (c), but decreased the apoptosis of CD8⁺CD28⁻ T cells (d). The bar graphs indicate the means±s.d.; statistically significant differences are indicated as follows: *P<0.05 and **P<0.01, n=3.

Figure 6

MSCs-mediated increase in the frequency of CD8⁺CD28⁻ T cells and the decrease of CD8⁺CD28⁻ T cells apoptosis were partially via IL-6. The frequency of viable CD8⁺CD28⁻ T cells was increased after cocultured with MSCs, and anti-IL-6 mAb partially reversed the MSCs-mediated effects on CD8⁺CD28⁻ T cell survival (a, b). Similarly, the frequency of CD8⁺CD28⁻ T cells among purified CD8⁺ T cells were increased in the presence of MSCs, and that increase was partially blocked by anti-IL-6 mAb (c). The bar graphs indicate the means±s.d.; statistically significant differences are indicated as follows: *P<0.05 and **P<0.01, n=3. mAb, monoclonal antibody; MSC, mesenchymal stem cell.