A novel and effective method to generate human porcine-specific regulatory T cells with high expression of IL-10, TGF-β1 and IL-35

Abstract

Organ transplantation remains the most effective treatment for patients with late stage organ failure. Transgenic pigs provide an alternative organ donor source to the limited availability of human organs. However, cellular rejection still remains to be the obstacle for xenotransplantation. Superior to other methods, antigen-specific regulatory T cells (Treg) alleviate cellular rejection with fewer side effects. Here we demonstrate the use of a fast method to provide tolerogenic dendritic cells (tolDC) that can be used to generate effective porcine-specific Treg cells (PSTreg). TolDC were produced within three days from human monocytes in medium supplemented with anti-inflammatory cytokines. Treg were generated from naïve CD4⁺ T cells and induced to become PSTreg by cocultivation with porcine-antigen-loaded tolDC. Results showed that PSTreg exhibited the expected phenotype, CD4⁺CD25⁺CD127^low/- Foxp3⁺, and a more activated phenotype. The specificity of PSTreg was demonstrated by suppression of effector T cell (Teff) activation markers of different stages and inhibition of Teff cell proliferation. TolDC and PSTreg exhibited high expression of IL-10 and TGF-β1 at both protein and RNA levels, and PSTreg also highly expressed IL-35 at RNA levels. Upon restimulation, PSTreg retained the activated phenotype and specificity. Taken together, the newly developed procedure allows efficient generation of highly suppressive PSTreg.

Figure 1

DC generation from monocytes with anti-inflammatory cytokines exhibited tolerogenic phenotype and produced anti-inflammatory cytokines. TolDC and C5-DC were harvested after maturation and characterized by flow cytometry, anti-CD14, anti-CD83, anti-CD80, anti-CD86 anti-B7-H1 and anti-B7-DC antibodies were used. (a) Histogram, (b) dot chart of MFI ratio tolDC to C5-DC. Data represent 26 independent experiments from 9 donors (CD86: n = 8). Wilcoxon signed-rank test was used to determine p values. (c) IL-10, IL-12p70, TGF-β1 and IFN-γ quantification in DC supernatants using BD CBA Flex Set and ELISA. These data represent 10–19 independent experiments from 9 donors. Culture medium plus maturation cocktail without cells were used as control. Turkey test was used to determine the statistical significance. ***p < 0.001. RNA was isolated from tolDC and C5-DC, and quantified by RT-PCR, (d) Relative RNA level of IL-35 and related genes, IL-10, TGFB1 in tolDC, C5-DC as control. ACTB, G6PDH or CYPB mRNA were determined from the same sample and used as endogenous reference genes. These data represent 7 independent experiments from 6 donors. Wilcoxon signed-rank test was used to determine p values. Error bars: s.e.m.

Figure 2

TolDC and C5-DC successfully express porcine antigen following electroporation of porcine-specific ivtRNA. MHC-I expression was used as surrogate marker for porcine antigen expression. (a) Flow cytometry analysis at different time points after electroporation using a porcine MHC-I-specific monoclonal antibody. Mock-electroporated tolDC and C5-DC were used as controls (grey peaks). (b) in % of cells, (c) as MFI.

Figure 3

High IL-10, TGF-β1 and IL-35 secreting PS Treg generated with porcine antigen loaded DC. CD4⁺ T cells were isolated and cocultured with PS antigen-expressing DC for 10 days in the presence of IL-2 w/o rapamycin. (a,b) Foxp3⁺ cells in PSTreg and NTreg in the alive cell population and in the CD3⁺CD4⁺ cell population after 10 day coculture. For comparison PSTeff and NTeff were analyzed. Data represent 14 independent experiments with cells from 8 donors. (c) Relative quantification of Foxp3, SATB1 and GARP mRNA of PSTreg and NTreg by RT-PCR. Data represent 8 independent experiments from 6 donors. (d) Supernatants of coculture were harvested for analyzing IL-10, TGF-β1, and IFN-γ. Data represent 20 experiments from 7 donors. (e) Relative expression of IL-10- and TGFB1-mRNA expression. Data represent 8 experiments from 6 donors. (f) Relative expression of IL-35-, IL-27-, and IL-12p70 related mRNA. Data represents 8 independent experiments from 6 donors. (c,e,f) represent NTreg and PSTeff as control for PSTreg and NTeff as control for NTreg. ACTB, G6PDH or CYPB mRNA were determined from the same sample and used as endogenous reference genes. ***p < 0.001, **p < 0.01, *p < 0.05. NS, non-significant, p > 0.05. ANOVA with Bonferroni’s Multiple Comparison Test or turkey test was used to determine the statistical significance of Treg percentage (a,b), and cytokine production on protein level (d). Wilcoxon signed-rank test was used to determine statistical significance of mRNA fold change (c,e,f). Error bars: s.e.m.

Figure 4

PSTreg show specific immunosuppressive activity. Three different assays were used to test PSTreg suppressive function towards PSTeff. PS/NTreg and PS/NTeff were seeded in 96 U-well plates with CD3/CD28 beads at different ratios, NTeff and PSTeff were set up as controls. Anti-CD154 and anti-CD25 were used to determine the Teff activation suppression, and CellTrace™ CFSE Cell Proliferation Kit was used to determine the inhibition of Teff proliferation. (a) Suppression of CD154 after 7 h at different ratios. (c) Suppression of CD25 after 96 h. (e) Inhibition of Teff proliferation at different ratios. (n = 3). (b) Suppression of CD154 at ratio 1:1, (d) shows suppression of CD25 at ratio 1:1, Data represents 15 independent experiments from 8 donors and 10 independent experiments from 6 donors, respectively. (f) Inhibition of Teff proliferation at ratio 1:1, (e) Data represents 8 independent experiments from 5 donors. ***p < 0.001, **p < 0.01, *p < 0.05. NS, not significant (p > 0.05). ANOVA with Bonferroni’s Multiple Comparison Test was used to determine the statistical significance. Error bars: s.e.m.

Figure 5

PSTreg express high amounts of IL-10 and TGF-β1. Supernatants from Treg and Teff cocultures were harvested after 7 h and 96 h. IL-10 (a), TGF-β1 (b) and IFN-γ (c) were quantified with BD™ CBA Flex Set analysis and ELISA. Data represents 4-14 independent experiments from 4–8 donors. ***p < 0.001, **p < 0.01, *p < 0.05, NS, non-significant, p > 0.05. ANOVA with Bonferroni’s Multiple Comparison Test was used to determine the statistical significance. The cells of the functional assay were also harvested after coculture for 96 h, and RNA was isolated and the indicated mRNA was quantified by RT-PCR. Relative expression of IL-10, TGFB1 (d) and IL-35 related mRNA (e) in PP to other groups was shown, PN, NP, NN as control. Data represents 7 independent experiments from 6 donors. ACTB, G6PDH or CYPB mRNA were determined from the same sample and used as endogenous reference genes. Wilcoxon signed-rank test was used to determine the p values.

Figure 6

Flowchart of PSTreg generation.