Centre for Transplant and Renal Research
Westmead Millennium Insititute
Foxp3+ regulatory T cells are essential for the induction and maintenance of tolerance to neonatal porcine islet cell cluster xenografts induced by co-stimulatory blockade
Yi Wen Qian1, Wayne Hawthorne1, Jingjing Wu1, David Liuwantara1, Heather Burns1, Yi Vee Chew1, Stephen Alexander2, Philip O'Connell1, Min Hu1.
1Centre for Kidney Research, Children's Hospital at Westmead, Sydney, Australia; 2Centre for Transplantation and Renal Research, Westmead Millennium Institute, Sydney, Australia
Introduction: Co-stimulatory blockade with CTLA-4 Fc and MR-1 (anti-CD154) antibodies inhibit B7-CD28 pathway and CD40-CD154 pathway respectively, and induce tolerance towards porcine islet cell cluster (NICC) xenografts in mice. However, the role of Foxp3+ regulatory T cells (Tregs) in the induction and maintenance of NICC xenografts tolerance is unclear.
Aim: 1. To investigate the role of Tregs in a NICC xenotransplantation mouse model. 2. To investigate whether Foxp3+ Treg are essential for the induction and maintenance of tolerance to NICC xenografts.
Methods: C57BL/6-DEREG mice were transplanted with 2000 to 4000 NICC under the renal capsule. DEREG mice have diphtheria toxin receptor-eGFP attached to their Foxp3 gene. Foxp3+ Tregs can be selectively depleted with diphtheria toxin (DT), and the eGFP allows for the tracking of Foxp3+ Tregs through the lymphoid and graft tissues. Recipient mice were treated IP with CTLA-4 Fc (500µg) at day 0, and MR-1 (500µg) at day 0, 2, 4, and 6. Tregs were depleted by DT (12ng/gram/mouse) during the induction phase (at 3 days before transplantation), or maintenance phase (at 80 days post transplantation) for 21 days. Rejection was determined by histology and insulin/glucagon staining, and function was determined by porcine c-peptide concentration.
Results: Without DT, tolerance to NICC xenografts was achieved in DEREG mice with histology at 100 days, showing xenografts with intact islets and positive insulin staining and elevated porcine C-peptide (mean=480 pmol/L, n=6). Large numbers of Foxp3+ Treg (GFP+) were observed in NICC xenografts in tolerant mice on day 8, fewer on day 20, then at a greater number on day 100. DT depletion during the induction phase reduced GFP+ Treg from 6.72% ± 0.96% of CD4+ cells in blood lymphocytes to 0.1% ± 0.05% on day 0, and maintained under 2% for the duration of the depletion (day -3 to 18). DT treated mice rejected their NICC graft with a massive cell infiltration, and no insulin or glucagon staining.The proportion of B220+CD25+ cells in double negative T cells increased to 6.0% by day 80 in draining lymph nodes of the tolerant group, compared to 2.2% in Treg depleted group. DT depletion during the maintenance stage maintained GFP+ Treg at less than 2.5% of CD4+ T cell for the duration of the depletion (day 80 to 100). DT treat mice in maintenance phase had massive cell infiltration and poor islet morphology as compared to the tolerant mice. No insulin or glucagon staining were present in their grafts and no porcine C-peptide in their serum.
Conclusion: Foxp3+ Tregs are essential for both the induction and maintenance of tolerance to islet xenografts after co-stimulation blockade by CTLA-4 Fc and anti-CD154 antibodies.
07:00 - 08:00
|Cell & Tissue Xenotransplantation: Tolerance||Foxp3+ regulatory T cells are essential for the induction and maintenance of tolerance to neonatal porcine islet cell cluster xenografts induced by co-stimulatory blockade||Room 109|