Foxp3-modified bone marrow mesenchymal stem cells promotes liver allograft tolerance through the generation of regulatory T cells in rats

Abstract

Background: The transcription factor forkhead box P3 (Foxp3) is a master regulatory gene necessary for the development and function of CD4(+)CD25(+) regulatory T cells (Tregs). Mesenchymal stem cells (MSC) have recently emerged as promising candidates for cell-based immunosuppression/tolerance induction protocols. Thus, we hypothesized that MSC-based Foxp3 gene therapy would improve immunosuppressive capacity of MSC and induce donor-specific allograft tolerance in rat's liver allograft model.

Methods: The present study utilized a lentivirus vector to overexpress the therapeutic gene Foxp3 on MSC. In vivo, Injections of 2 × 10(6) MSC, FUGW-MSC or Foxp3-MSC into the portal vein were carried out immediately after liver transplantation.

Results: Successful gene transfer of Foxp3 in MSC was achieved by lentivirus carrying Foxp3 and Foxp3-MSC engraftment in liver allograft was confirmed by fluorescence microscopy. Foxp3-MSC treatment significantly inhibited the proliferation of allogeneic ACI CD4(+) T cells to splenocytes (SC) from the same donor strain or third-party BN rat compared with MSC. Foxp3-MSC suppressive effect on the proliferation of CD4(+) T cells is contact dependent and associated with Programmed death ligand 1(PD-L1) upregulation in MSC. Co-culture of CD4(+) T cells with Foxp3-MSC results in a shift towards a Tregs phenotype. More importantly, Foxp3-MSC monotherapy achieved donor-specific liver allograft tolerance and generated a state of CD4(+)CD25(+)Foxp3(+) Tregs-dependent tolerance.

Conclusion: Foxp3-engineered MSC therapy seems to be a promising and attractive cell therapy approach for inducing immunosuppression or transplant tolerance.

Fig. 1

Successful gene transfer of Foxp3 in MSC and Foxp3-MSC engraftment in liver allograft. a MSC were infected with control lentivirus (FUGW, Green) and GFP-Foxp3 lentivirus (Foxp3, Green), Nucleus were stained by DAPI (Blue). MSC were efficiently transduced with >95 % GFP-positive cells. b The expression of Foxp3 was detected by western blot in Foxp3-MSC and FUGW-MSC lysis at 96 h after infection. The production of Foxp3-GFP fusion protein was confirmed in Foxp3-MSC lysis compared with FUGW-MSC lysis. c Liver allograft sections were analyzed by fluorescence microscopy for the presence of GFP-Foxp3-MSC, substantial numbers of GFP-Foxp3-MSC liver graft were observed in liver allograft (upper panel, original magnification ×400; lower panel, original magnification ×100)

Fig. 2

Foxp3 transduction improved the immunosuppressive activity of MSC and induced the expansion of Foxp3⁺ Tregs. a The immunogenicity of Foxp3-MSC and effect of Foxp3-MSC on T cell proliferative response in vitro. Results are mean ± SE of three independent experiments. *p < 0.05 vs PBS; ^# p < 0.05 vs MSC; ^& p < 0.05 vs Foxp3-MSC. b Foxp3 transduction significantly improved the immunosuppressive activity of MSC. Results are mean ± SE of three independent experiments. *p < 0.05 vs MSC. c The expression of cell surface molecule in MSCs altered by Foxp3 transduction. Isotype FUGW-MSC are represented by open histograms and Foxp3-MSC by closed histograms. d, e Blocking experiments with neutralizing mAb of Nrp-1 and PD-L1 on CD4⁺ T cell proliferative response. Results are representative of three independent experiments. *p < 0.05 vs PBS; ^# p < 0.05 vs Foxp3-MSC + IgG. f Frequency of CD4⁺CD25⁺Foxp3⁺ T cells. Results are representative of five independent experiments. *p < 0.05 vs PBS; ^# p < 0.05 vs MSC. g Representative dot plots of CD25 and Foxp3 staining of CD4⁺-gated cells. Percentages of CD4⁺CD25⁺Foxp3⁺ T cells are indicated in the top right corners of each dot plot

Fig. 3

Foxp3-MSC monotherapy achieves donor-specific liver allograft tolerance. a Kaplan–Meier survival curves for untreated, donor-derived MSC, FUGW-MSC and Foxp3-MSC, recipient-derived Foxp3-MSC and third party Foxp3-MSC groups. b Kaplan–Meier survival curves for the Foxp3-MSC, Foxp3-MSC+IgG and Foxp3-MSC+α-CD25 groups. c Histology of liver allografts in ACI recipients on POD 7 (H&E, original magnification ×100). d RAI scores according to the Banff scheme. Results are mean ± SE of five independent experiments. *p < 0.05 vs untreated; ^# p < 0.05 vs MSC. e, f Changes in the serum ALT, AST and TBIL concentrations in all 4 groups 7 days after LT. Results are mean ± SE of three independent experiments. *p < 0.05 vs untreated; ^# p < 0.05 vs MSC. g Representative skin biopsy samples were obtained on day 7 and 100 after skin transplantation (H&E, original magnification ×100)

Fig. 4

Frequency of Tregs generated in ACI recipients. a Representative dot plots of splenic Tregs. Percentages of CD4⁺CD25⁺Foxp3⁺ T cells are indicated in the top right corners of each dot plot. b Graphic results of frequency of splenic CD4⁺CD25⁺Foxp3⁺ T cells. Results are representative of five independent experiments. *p < 0.05 vs untreated; ^# p < 0.05 vs MSC. c Graphical representation of numbers of Foxp3⁺ T cells (cells/mm²) within the liver allografts. The results are representative of six separate experiments. *p < 0.05 vs untreated; ^# p < 0.05 vs MSC. d Representative immunohistochemistry staining of intragraft Foxp3⁺ T cells. (IHC, original magnification ×100)