Tolerance

Saturday October 12, 2019 from 08:00 to 08:30

Room: Room A.017

301.2 Human CD27+HLADR+ Treg show xeno-antigen specific protection of porcine neonatal islet xenograft in humanized mice model

Award Winner

Xiaoqian Ma, People's Republic of China has been granted the TTS-IXA Congress Scientific Award

Xiaoqian Ma, People's Republic of China

The Institute for Cell Transplantation and Gene Therapy, The Third Hospital of Central South University

Abstract

Human CD27+HLADR+ Treg show xeno-antigen specific protection of porcine neonatal islet xenograft in humanized mice model

Xiaoqian Ma1,2, Lu Cao1, Wayne Hawthorne2, Shounan Yi2, Philip O'Connell2.

1The Institute for Cell Transplantation and Gene Therapy, The Third Hospital of Central South University, Changsha, People's Republic of China; 2Centre for transplant and Renal Research,, Westmead Institute for Medical Research, University of Sydney, Sydney, Australia

Introduction: Polyclonal regulatory T cells (Treg) have been shown suppressive ability for xenoimmune responses. However, to achieve effective suppression and avoid opportunistic infection and malignancy for clinical xenotransplantation, xenoantigen-specific Treg are required to be developed. Our previous study has shown that CD27+HLA-DR+ Treg subset separated from xenoantigen stimulated human Treg (XnTreg) were potent and xenoantigen-specific in suppression of the xenogeneic response in vitro. In this study we further investigated their xenoantigen specificity in protection against neonatal porcine islet cell clusters (NICC) xenograft rejection in vivo.
Methods: Human CD4+CD25+CD127- Treg isolated from healthy donor peripheral blood mononuclear cells (PBMC) were expanded for 3 or 4 cycles with anti-CD3/CD28 beads and irradiated porcine PBMC as xenoantigen stimulated Treg (XnTreg). Human XnTreg were separated by cell sorting with Treg cell surface markers CD27 and HLADR into non-selected, CD27+HLADR+ and non-CD27+HLADR+ Treg subsets prior to cotransfer of NICC recipient NOD-SCID IL2rg-/- mice with autologous PBMC at a 1:25 ratio of Treg (4x10^5): PBMC (1x10^7). Serum, spleen and NICC xenografts were harvested from recipient mice at day 60 after human cell cotransfer for analysis of xenograft survival and Treg in vivo function.
Results: Human PBMC engraftment was confirmed by flow cytometry, with 35.2-52.4% of human CD45+ cells in the spleen in all groups, by day 60 after adoptive transfer. The majority (> 92%) of these cells were CD3+ cells. Mice reconstituted with human PBMC without Treg rejected their xenografts completely within 35 days. However adoptive transfer with the CD27+HLADR+ Treg can prevent islet xenograft rejection. In human PBMC and CD27+HLADR+ Treg double transfer group, immunohistochemical analysis revealed more intact and insulin positive-staining NICC grafts compared with non-selected Xn Treg group at day 60. Only very few insulin positive-staining cells whereas a large number of human CD3 and CD8 infiltrated the islet graft in non-CD27+HLADR+ Treg group. However in CD27+HLADR+ Treg group there were few CD3 and CD8 human leukocytes surrounded but not infiltrated the islet graft. The porcine C-peptide results also confirmed the finding from immunohistochemistry. After glucose stimulation, the porcine C-peptide was 551.8 ± 140.2 pmol/L in CD27+HLADR+ Treg group, with only 232.5 ± 60.4 pmol/L and 130.9 ± 40.7 pmol/L in Xn Treg group and non-CD27+HLADR+ Treg group.  
Conclusion: Compared with the previously reported Treg number (2x 10^6), only 1/5 number of the CD27+HLADR+ Treg can prevent the islet xenograft rejection which suggested this memory like Treg may be more specific to the xenograft and have potential as an effective cell therapy in immunomodulation in xenotransplantation.

NSFC81201171, Hunan Provincial Natural Science Foundation of China 2017JJ3423.



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