A novel and effective method to generate human porcine-specific regulatory T cells with high expression of IL-10, TGF-\u03b21 and IL-35

Mingqian Li,Judith Eckl,Christiane Geiger,Dolores J Schendel,Heike Pohla

doi:10.1038/s41598-017-04322-3

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+CD127low/− 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.

Highlights

The shortage of human organs and cells remains a major obstacle for human organ transplantation
We present a new method for generation of highly suppressive xeno-reactive Treg that secrete high amounts of IL-10, IL-35 and TGF-β1 through cocultivation of naïve CD4+ T cells with porcine-antigen-loaded Tolerogenic Dendritic cells (DC) (tolDC)
DC (C5-DC)[30], fresh CD14+ monocytes were isolated from healthy donor peripheral blood mononuclear cells (PBMC) (Supporting Information Fig. S1a) and anti-inflammatory or inflammatory cytokines were added to induce tolerogenic or immunogenic phenotypes, respectively

Summary

Introduction

The shortage of human organs and cells remains a major obstacle for human organ transplantation. Ex vivo expansion of natural Treg (nTreg) is expensive and laborious[16], while, in contrast, antigen-specific induced Treg exhibit highly potent suppression at low cell numbers[17]. IL-35 was found to be constitutively expressed at high levels in mouse Foxp3+ Treg[21], and several groups showed that IL-35 promotes regulatory B cell and Treg proliferation and mediates suppressive functions[21,22,23]. IL-27 is an immune regulatory cytokine that induces Th17 cells to produce IL-1025, 26, but it exerts anti-tumor effects by enhancing survival of tumor-antigen-specific CD8+ T cells[27] and suppressing Treg by blocking the expression of Foxp[3] via STAT1 and STAT3 activation[28, 29]

Methods

Results

Conclusion