Regulatory T-cells within bone marrow-derived stem cells actively confer immunomodulatory and neuroprotective effects against stroke

Abstract

Regulatory T-cells (T_regs) may exert a neuroprotective effect on ischemic stroke by inhibiting both inflammation and effector T-cell activation. Transplantation of human bone marrow-derived stem cells (BMSCs) in ischemic stroke affords neuroprotection that results in part from the cells' anti-inflammatory property. However, the relationship between T_regs and BMSCs in treatment of ischemic stroke has not been fully elucidated. Here, we tested the hypothesis that T_regs within the BMSCs represent active mediators of immunomodulation and neuroprotection in experimental stroke. Primary rat neuronal cells were subjected to an oxygen-glucose deprivation and reperfusion (OGD/R) condition. The cells were re-perfused and co-cultured with T_regs and/or BMSCs. We detected a minority population of T_regs within BMSCs with both immunocytochemistry (ICC) and flow cytometry identifying cells expressing phenotypic markers of CD4, CD25, and FoxP3 protein. BMSCs with the native population of T_regs conferred maximal neuroprotection compared to the treatment conditions containing 0%, 10%, and 100% relative ratio T_regs. Increasing the T_reg population resulted in increased IL6 secretion and decreased FGF-β secretion by BMSCs. This study shows that a minority population of T_regs exists within the therapeutic BMSC population, which serves as robust mediators of the immunomodulatory and neuroprotective effect provided by BMSC transplantation.

Keywords: Adaptive immunity; foxp3; immunotherapy; ischemia.

Figure 1.

T_reg magnetic cell isolation procedure. Whole spleens were isolated from mice and manually dissociated into a single-cell solution to remove extracellular matrix (ECM). The solution was passed through a strainer and then incubated with magnetic bead conjugated antibodies. Labeled cells were then passed through a magnetic field with metal beads. Non-CD4⁺ cells were depleted from the solution and CD4⁺/CD25⁺ cells were sorted from the remaining solution to yield CD4⁺/CD25⁺ T_regs. ECM: extracellular matrix.

Figure 2.

A subpopulation of cells expressing protein markers characteristic of T_regs is identified in BMSCs. (a) Fluorescent antibody labeling with CD4 (red), CD25 (magenta), and FoxP3 (green) shows specificity in a subpopulation of cells, demonstrating the presence of T_regs as a distinct subpopulation in BMSCs. (b) CD4 and CD25 were expressed in a small subpopulation of BMSCs (1.46% and 3.39%, respectively). (c) FoxP3 fluorescence in the stained sample (green) shows bimodal distribution and a relatively large delta compared to the unstained control (black). Bimodal distribution suggests that there is a FoxP3⁺ and a FoxP3-subpopulation in BMSCs. The bimodal FoxP3 histogram was analyzed to quantify the possible FoxP3⁺ subpopulation of BMSCs. The bimodal histogram was separated into a right-shifted FoxP3⁺ population (63.2%) and left-shifted FoxP3-population (36.8%).

Figure 3.

The native population of T_regs in BMSCs relatively increases neuroprotection and reduces IL-6 production. (a) The vertical axis displays the viable neuron (MAP2⁺) cell count normalized to the control group (black) as a percentage. OGD/R condition results in significant decrease in cell viability (black vs. white bar). BMSCs depleted of T_regs confer neuroprotection after OGD/R and BMSC treatment with native population of T_regs increases cell viability. (b) IL-6 secretion, a pro-inflammatory cytokine, by BMSCs was measured via ELISA. IL-6 secretion was significantly increased by depleting BMSC cell transplant of T_regs. OGD: oxygen-glucose deprivation; IL-6: interleukin-6.

Figure 4.

Increasing the ratios of T_regs in BMSC treatment after OGD/R decreases neuroprotection capacity of T_regs. (a) Treatment with BMSCs and the native ratio of T_regs confers significant neuroprotection after OGD/R. Supplemental T_regs in ratios of 1:10 and 1:1 T_regs: BMSCs show significant reduction in neuroprotection relative the native 1:100 ratio. (b) FGF-β, a cytokine secreted by BMSCs promoting proliferation and differentiation, was measured by ELISA. Increasing the ratio of T_regs correlates to a dose-dependent decrease in FGF-β secretion. (c) BMSC IL-6 secretion was measured by ELISA. IL-6 production was significantly increased in the 1:10 T_reg ratio treatment group, while it was significantly decreased in the 1:1 ratio treatment group. OGD: oxygen-glucose deprivation; IL-6: interleukin-6: FGF-β: fibroblast growth factor-beta.

Figure 5.

A graphical depiction of cell culture is depicted showing the interaction between T_regs, BMSCs, and neural cells (astrocytes and neurons). BMSCs are shown to be neuroprotective by promoting neuron survival (green arrows, +) and attenuating astrocyte activation (red arrows, −). T_regs are shown to potentially have a dualistic, concentration-dependent effect on BMSCs. At the native concentration, T_regs relatively decrease BMSC IL-6 production, a potentially deleterious pro-inflammatory cytokine. BMSC FGF-β production, a cytokine related to BMSC survival, proliferation, and differentiation, is reduced in a concentration-dependent manner with T_reg co-culture. BMSC: bone marrow-derived stem cell.