Friday October 11, 2019 from 16:45 to 17:45
Impact of Immune Modulatory Drugs on Xenoantigen-stimulated Regulatory T Cell
Xiaoqian Ma1,2, Xin He1, Sang Li2, Jia Wang2, Cejun Yang1,2, Juan Zhang1,2, Pengfei Rong1,2, Wei Wang1,2.
1The Institute for Cell Transplantation and Gene Therapy, The Third Hospital of Central South University, Changsha, People's Republic of China; 2Engineering and Technology Research Center for Xenotransplantation of Human Province, Changsha, People's Republic of China
Introduction: Immunosuppression strategies that selectively inhibit effector T cells while preserving and even enhancing regulatory T cells (Treg) may prevent rejection while minimizing toxicity. This study aims to give experimental evidence regarding the influence of current using immunosuppressive drugs on xenoantigen-stimulated Treg viability, mitochondrial function, proliferation and function and as a suggestion for the selection of Treg-friendly and xenotransplantation 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 for 2 weeks. 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 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. Mitochondrial function was evaluated by seahorse assay.
Results: 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 affected chemokine receptors. From the CFSE assay, a significant reduction of cell proliferation was observed when CD25- responder cell stimulated with irradiated pig PBMC were treated with ISDs alone or the mixture of ISDs in comparison to the control group. However only MPA significantly inhibited the proliferation of Treg 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. 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. From the OCR assay, TAC and BEL significantly downregulated the maximal respiratory capacity of xenospecific Treg compared to MPA and ISDs mix.
Conclusion: Our results suggested different immune modulatory drugs have different influence on Treg. A combination of multiple immunosuppressants at low-dose would be better able to support Treg while adequately preventing rejection while minimizing toxicity.
NSFC81201171, Hunan Provincial Natural Science Foundation of China 2017JJ3423.