(2017), IXA 2017- Abstracts of the 14th Congress of the International Xenotransplantation Association, Baltimore, USA. Xenotransplantation, 24: n/a, e12328. doi:10.1111/xen.12328
Treatment of Xenoangene-Expanded Cord Blood-Derived Regulatory T Cells with Beletacept Enhances their Suppressive Capacity in the Xenogeneic Response
Xiaoqian Ma1, Sang Li1, Jia Wang1, Pengfei Rong1, Cejun Yang1, Juan Zhang1, Wei Wang1.
1Institute for Cell Transplantation and Gene Therapy, the 3rd Xiangya Hospital of Central South University, Changsha, People's Republic of China
Introduction: Xenoantigen-stimulated Treg have shown promising capacity in xenotransplantation. But establishing graft tolerance is difficult to obtain with a single infusion of Treg. This study aims to give experimental evidence regarding the influence of beletacept and other current using immunosuppressive drugs on xenospecific Treg viablity, proliferation and function as a suggestion for the selection of Treg-friendly and xenotransplantaion suitable immunosuppressive regimens.
Methods: Human cord blood derived CD4+CD25+CD127low Treg were cultured with IL-2, rapamycin and irradiated porcine PBMC as xenoantigen stimulation. Treg phenotype and suppressive capacity were assessed after xenoantigen stimulation. The effects of major ISDs as tacrolimus (TAC), mycophenolate (MPA) and new ISD beletacept (BEL) and the mixture of three ISDs (ISDs mix) on xenospecific Treg were test. Cell viability was measured by MTS assay. Proliferation was evaluated by CFSE proliferation kit. MLR was used to assess the impact of ISDs to the function of xenospecific Treg and seven days later the supernatant was collected to perform CBA assay.
Results: Xeno MLR assay and phenotype analysis showed we have induced xenospecific Treg successfully. Then the doses leading to 50% of Treg viability (LD50) (0.3ug/ml for MPA) from MTS assays was selected for the following assay. Since BEL and TAC did not affect the viability of xenospecific Treg severely and the suggested maximum therapeutic concentration of TAC and BEL were selected and used for the in vitro analysis. No matter Treg were treated with ISD alone or the mixture of ISDs, the immunosuppressive treatments have no effect on the expression of Treg functional molecules but affect chemokine receptors. From the CFSE assay, a significant reduction of cell proliferation was observed when CD25- responder cell were cultured with irradiated pig PBMC and ISDs alone or the mixture of the three drugs in comparison to the control group. But when Treg cultured with irradiated pig PBMC and ISD alone or ISDs mix, only MPA and the ISDs mixture affected the xenospecific Treg proliferation but not BEL or TAC. For xeno-MLR assays, TAC and MPA did not have any negative effect on the suppressive capacity of xenospecific Treg but rather BEL strengthened it at the ratio of responder :Treg 64:1.The results of CBA assay showed IL6, TNF-α, IL17A and IFN-γ was significantly reduced when activated CD25-responder cell stimulated by the irradiated pig PBMC were treated by ISDs or Treg respectively compared to the non-treated control group. Further when activated CD25- responder cells co-cultured with Treg and ISDs, BEL and Treg have synergetic effect to reduction of IL6, TNF-α, IL17A and IFN-γ which explained why BEL could strengthen the suppressive ability of Treg.
Conclusion: Our results suggested BEL combined with xenospecific Treg may be the best choice for xenotransplantation clinical use
The study was supported by NSFC 81201171and 81471715..