Pediatric Mesenchymal Stem Cells Exhibit Immunomodulatory Properties Toward Allogeneic T and B Cells Under Inflammatory Conditions

Abstract

Mesenchymal stem cells from pediatric patients (pMSCs) are an attractive cell source in regenerative medicine, due to their higher proliferation rates and better differentiation abilities compared to adult MSCs (aMSCs). We have previously characterized the immunomodulatory abilities of pMSCs on T cells under co-culture. It has also been reported that aMSCs can inhibit B cell proliferation and maturation under inflammatory conditions. In this study, we therefore aimed to clarify the immunomodulatory effect of pMSCs toward T and B cells in an inflammatory microenvironment. Bone marrow derived pMSCs were primed to simulate inflammatory conditions by exposure with 50 ng/mL of IFN-γ for 3 days. To analyze the interaction between pMSCs and T cells, CD3/CD28 stimulated peripheral blood mononuclear cells (PBMCs) were co-cultured with primed or unprimed pMSCs. To investigate B cell responses, quiescent B cells obtained from spleens by CD43 negative selection were stimulated with anti-IgM, anti-CD40, IL-2, and co-cultured with either IFN-γ primed or unprimed pMSC. pMSC phenotype, B and T cell proliferation, and B cell functionality were analyzed. Gene expression of indoleamine 2,3-dioxygenease (IDO), as well as the expression of HLA-ABC, HLA-DR and the co-stimulatory molecules CD80 and CD86 was upregulated on pMSCs upon IFN-γ priming. IFN-γ did not alter the immunomodulatory abilities of pMSCs upon CD4⁺ nor CD8⁺ stimulated T cells compared to unprimed pMSCs. IFN-γ primed pMSCs but not unprimed pMSCs strongly inhibited naïve (CD19⁺CD27^-), memory (CD19⁺CD27⁺), and total B cell proliferation. Antibody-producing plasmablast (CD19⁺CD27^highCD38^high) formation and IgG production were also significantly inhibited by IFN-γ primed pMSCs compared to unprimed pMSCs. Collectively, these results show that pMSCs have immunomodulatory effects upon the adaptive immune response which can be potentiated by inflammatory stimuli. This knowledge is useful in regenerative medicine and allogeneic transplantation applications toward tailoring pMSCs function to best modulate the immune response for a successful implant engraftment and avoidance of a strong immune reaction.

Keywords: B cell; T cell; allogeneic; immunomodulation; inflammatory microenvironment; mesenchymal stem cell.

Figure 1

Effect of IFN-γ on the expression of immune regulatory and co-stimulatory markers on pMSCs upon IFN-γ stimulation. pMSCs treated with 50 ng/mL IFN-γ for 3 days or unprimed (-IFN-γ) pMSCs were analyzed for IDO gene expression by qRT-PCR, and stained for HLA-ABC, HLA-DR, CD80, and CD86 expression and analyzed by FACS. (A) Expression of gene encoding for IDO relative to GAPDH. N = 3 pMSCs donors, single replicates. (B) Representative flow cytometry histograms of HLA-DR, HLA-ABC, CD80, and CD86 expression (blue line) on pMSCs based on the unstained control (dot line) with and without the addition of IFN-γ. (C) Percentage of positive cells and Mean Fluorescence Intensity (MFI) for HLA-ABC, HLA-DR, CD80, and CD86 markers. N = 3 different pMSC donors in triplicates. Results are shown as means ± SD. *p < 0.05.

Figure 2

pMSCs are immunomodulatory toward T cells in a dose-dependent manner. CD3/CD28 stimulated PBMCs were co-cultured with IFN-γ primed (+IFN-γ) or unprimed (-IFN-γ) pMSCs at 1:2.5, 1:5, 1:10, and 1:20 pMSCs:PBMCs ratios. Flow cytometric analysis was performed after 5 days of co-culture, and CD4⁺ and CD8⁺ proliferating T cells were detected by CFSE. (A) Representative FACS plots and histograms showing the gating strategy for stimulated CD4⁺ and CD8⁺ T cells alone or in co-culture with unprimed or IFN-γ primed pMSCs. (B) CD4⁺ and (C) CD8⁺ T cell proliferation in co-culture with unprimed or IFN-γ primed pMSCs. Results are expressed as the relative proliferation measured as the 1/Mean Fluorescence Intensity (MFI) of CFSE normalized to the (+CD3/CD28) stimulated control. N = 3 different pMSC donors with N = 3 different PBMC donors in triplicates. Results are represented as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

IFN-γ primed pMSCs significantly reduce the proliferation rates on naïve, memory and total numbers of B cells compared to unprimed cells. Stimulated (+anti-CD40, anti-IgM, and IL-2) B cells were co-cultured with either IFN-γ primed or unprimed pMSCs. B cells were retrieved and analyzed by flow cytometry after 7 days. (A) FACS gating strategy of viable CD19⁺ B cells that were classified according to the expression of CD27 into memory (CD27⁺), naïve (CD27⁻) B cells, or plasmablasts (CD27^high CD38^high). (B) Relative proliferation of B cells when co-cultured with IFN-γ primed or unprimed pMSCs. Results are shown as the 1/MFI (CFSE) of CD19⁺ viable cells (total B cells), CD27⁺ cells (memory), and CD27⁻ cells (naïve) relative to the B cell stimulated condition. N = 3 B cell donors co-cultured with 1 pMSC donor in duplicates or triplicates. Figures show means ± SD. ***p < 0.001.

Figure 4

IFN-γ primed pMSCs but not unprimed significantly decrease plasmablast differentiation and IgG production. (A) Plasmablast frequencies were measured upon 7 days of co-culture of stimulated B cells with IFN-γ primed or unprimed pMSCs. Results are expressed as the % of viable CD19⁺ CD27^high CD38^high cells. (B) IgG concentration (ng/mL) was detected in the supernatants of stimulated B cells and IFN-γ primed or unprimed pMSCs by ELISA. N = 3 different B cell donors co-cultured with 1 pMSC donor Results are shown as means ± SD. *p < 0.05; **p < 0.01.