Cell contact, prostaglandin E(2) and transforming growth factor beta 1 play non-redundant roles in human mesenchymal stem cell induction of CD4+CD25(High) forkhead box P3+ regulatory T cells

Abstract

Adult human mesenchymal stromal or stem cells (MSC) can differentiate into a variety of cell types and are candidate cellular therapeutics in regenerative medicine. Surprisingly, these cells also display multiple potent immunomodulatory capabilities, including allosuppression, making allogeneic cell therapy a possibility. The exact mechanisms involved in regulatory T cell induction by allogeneic human MSC was examined, using purified CD4+ populations and well-characterized bone marrow-derived adult human MSC. Allogeneic MSC were shown to induce forkhead box P3 (FoxP3)+ and CD25+ mRNA and protein expression in CD4+ T cells. This phenomenon required direct contact between MSC and purified T cells, although cell contact was not required for MSC induction of FoxP3 expression in an unseparated mononuclear cell population. In addition, through use of antagonists and neutralizing antibodies, MSC-derived prostaglandins and transforming growth factor (TGF)-beta1 were shown to have a non-redundant role in the induction of CD4+CD25+FoxP3+ T cells. Purified CD4+CD25+ T cells induced by MSC co-culture expressed TGF-beta1 and were able to suppress alloantigen-driven proliferative responses in mixed lymphocyte reaction. These data clarify the mechanisms of human MSC-mediated allosuppression, supporting a sequential process of regulatory T cell induction involving direct MSC contact with CD4+ cells followed by both prostaglandin E(2) and TGF-beta1 expression. Overall, this study provides a rational basis for ongoing clinical studies involving allogeneic MSC.

Fig 1

Characterization and differentiation potential of human mesenchymal stromal cells (MSC). (a) Cell surface markers expressed by human MSC were determined by flow cytometry. Isotype controls are represented by open histograms, specific cell surface markers by closed histograms. The capacity of MSC to differentiate along mesenchymal lineages was also determined by: (b) phase-contrast microscopy (magnification × 200) of (i) control, undifferentiated MSC; (ii) adipogenic differentiated MSC, determined by oil red O staining; (iii) osteogenic differentiated MSC determined by alizarin red S staining; or (c) glycosaminoglycan content, an indicator of chondrogenic differentiation, determined using a 1,9-dimethylmethylene blue assay and a picogreen DNA assay for undifferentiated and differentiated MSC (n = 2). Differentiation conditions are described in Methods. Data are represented as the mean ± standard error ratio of GAG/DNA (µg/µg). *P < 0·05 compared with undifferentiated MSC.

Fig 2

Exposure to mesenchymal stromal cells (MSC) increases CD4⁺ T cell expression of CD25 and forkhead box P3 (FoxP3). CD4⁺ T cells were cultured in the absence (CD4) or presence of MSC (CD4+MSC), purified and examined by either (a) semi-quantitative reverse transcription– polymerase chain reaction (RT–PCR) for the expression of glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) or FoxP3 mRNA after 24 h co-culture; or (b) quantitative real-time PCR for FoxP3 mRNA. **P < 0·01 compared with CD4⁺ T cells alone. Data representative of three independent determinations. Intracellular expression of FoxP3 or surface CD25 from parallel experiments were determined by flow cytometry from purified CD4⁺ cell populations (c). CD4⁺FoxP3⁺ or CD4⁺CD25^High cells were determined from purified CD4⁺ T cells following 72 h culture in the absence (top panel) or presence of MSC (lower panel). Dot plots are representative of three independent experiments. The numbers in the upper right quadrants indicate the percentage of double-positive cells (large font) with mean fluorescent intensity (MFI) above (small font).

Fig 3

Prevention of T cell–mesenchymal stromal cells (MSC) contact reduced forkhead box P3 (FoxP3) and CD25^High induction in CD4⁺ T cells. CD4⁺ T cells or unseparated peripheral blood mononuclear cells (PBMC) were cultured with MSC for 72 h or separated in co-culture using a transwell system with MSC in the upper chamber and CD4⁺ T cells or unseparated PBMC in the lower. FoxP3 mRNA concentration in CD4⁺ cells was determined by quantitive reverse transcription–polymerase chain reaction (qRT–PCR) (a). Intracellular FoxP3 or surface CD25 protein expression by CD4⁺ T cells (b) or unseparated PBMC (c) was determined by flow cytometry. CD4⁺ T cells (CD4) and unseparated PBMC cultured alone are shown for comparison. Data are representative of three experiments. The numbers in the upper right quadrants indicate the percentage of double-positive cells (large font) with MFI above (small font).

Fig 4

Transforming growth factor (TGF)-β1 and prostaglandin E₂ (PGE₂) play a non-redundant role in mesenchymal stromal cells (MSC) induction of forkhead box P3 (FoxP3) and CD25^High expression by CD4⁺ T cells. (a) FoxP3 mRNA expression determined by real-time polymerase chain reaction (PCR) of CD4⁺ T cells purified from culture alone (CD4) or in the presence of MSC and/or the cyclooxygenase-2 antagonist (Indo) (40 µM) and/or neutralizing specific antibody against TGF-β1 (αTGF-β1) (4 µg/ml). MSC induced significant FoxP3 expression in CD4⁺ T cells. Addition of anti-TGF-β1 or indomethacin (40 µM) to CD4⁺ T cells co-cultured with MSC significantly reduced FoxP3 mRNA expression). Statistical analysis was performed by one-way anova with Tukey's multiple comparisons; results are expressed as mean concentration ± standard error. *P < 0·05; **P < 0·01). Analysis of intracellular FoxP3 (b) and CD25^High surface expression (c), by purified CD4⁺ T cell from parallel co-cultures. Data are representative of three experiments. The numbers in the upper right quadrants indicate the percentage of double-positive cells (large font) with MFI given above (small font).

Fig 5

Purified CD4⁺ CD25⁺ T cells induced by mesenchymal stromal cells (MSC), expressed transforming growth factor (TGF)-β1 and suppressed alloresponses. TGF-β1 mRNA was increased in purified CD4⁺ T cells cultured previously in the presence of MSC determined by reverse transcription–polymerase chain reaction (RT–PCR) (a) and quantified by real-time PCR for TGF-β1 mRNA (b). Real-time PCR showed a significant (***P < 0·001) increase in TGF-β1 expression by CD4⁺ T cells co-cultured previously with MSC for 24 h compared with CD4⁺ T cells alone. Results are representative of three independent experiments each performed in duplicate, expressed as mean concentration ± standard error (s.e.). Purified CD4⁺CD25⁺ T cells induced by MSC co-culture suppressed alloantigen-driven proliferation in mixed lymphocyte reaction (MLR) (c). Microbead purified CD4⁺CD25⁺ T cells induced from co-cultured with MSC were then cultured with or without MHC mismatched donor cells (P1 or P2) in two-way MLR at a 1:1 ratio for 96 h. Allodriven proliferation was reduced (*P = 0·034) significantly in the presence of CD4⁺CD25⁺ T cells measured by [³H]-thymidine incorporation and expressed as mean cpm ± s.e. Results are representative of three independent experiments each performed in triplicate.