NHMRC Early Career Fellow and Senior Lecturer
Westmead Millennium Institute, University of Sydney
Co-stimulation Blockade Inducing Tolerance to Porcine Islet Xenografts Produced Foxp3+ Regulatory T cells with High IL-10 Production and Expressing MHC class II that Were Capable of Dominant Tolerance
Min Hu1, Jingjing Wu1, Wayne Hawthorne1, Heather Burns1, Peta Phillips1, David Liuwantara1, Stephen Alexander2, Shounan Yi1, Philip O’Connell1.
1Centre for Transplant and Renal Research, Westmead Millennium Institute for Medical Research, University of Sydney, Westmead, Australia; 2Centre for Kidney Research, the Children’s Hospital at Westmead, University of Sydney,, Westmead, Australia
Introduction: Neonatal porcine islet cell clusters (NICC) xenotransplantation is potential therapy for treatment of type 1 diabetes. However cellular rejection and high level of immunosuppression remain major barriers for clinical application. We have previously demonstrated using CTLA-4Fc and anti-CD154 mAb for blocking B7-CD28 and CD40-CD40L(CD154) costimulatory pathways induced islet xenograft tolerance in CBA mouse recipients that was associated with increased CD4+ T cell apoptosis. However, this is not clear whether Foxp3+ regulatory T cells (Tregs) play a role, and whether CTLA-4Fc treatment inhibits Foxp3+ Treg function in xenotransplantation.
Aim: 1) To investigate the role of Foxp3+ Tregs in a mouse model of NICC xenografts tolerance induced by co-stimulation blockade through B7-CD28 and CD40-CD40L co-stimulatory pathway using CTLA-4Fc and anti-CD154 mAb. 2) To evaluate the function of Foxp3+ Tregs from these tolerant recipient mice by assessment of their ability to transfer dominant tolerance to NICC xenografts. 3) To characterize Foxp3+ Tregs from tolerant recipient mice.
Results: NICC xenografts at day > 100 from recipient BALB/c mice which were treated by CTLA4-Fc and anti-CD154 Ab showed intact islets and strong positive insulin staining. In contrast, BALB/c recipients without treatment rejected their NICC xenografts within 32 days of transplantation which were confirmed by histology showing no intact islets. CD4+Foxp3+ double positive cells were observed in the tolerant NICC xenografts by fluorescence staining. In contract, rejecting NICC xenografts from control group had heavily infiltrated by CD4+ cells with no Foxp3+ cells at the time rejection. Flow cytometric analysis demonstrated Foxp3+ Tregs expanded in local tolerant NICC xenografts and draining lymph node, as well as systemically. Tolerant NICC xenograft expressed elevated levels of TGF-β, IFN-γ and IL-10 by real time PCR. Isolated Foxp3+ Tregs from spleens of tolerant recipient mice (tolerant-Tregs) produced high levels of IL-10, expressed MHC class II, and had diverse T cell receptor Vβ (TRBV) repertoires with a clonal expansion in CDR3 of TRBV14. Assessment of Tregs function in vivo, an adoptive transfer NOD-SCID mouse model showed these tolerant-Tregs had the capacity to transfer dominant tolerance and exhibited more potent regulatory function to NICC xenografts than naïve-Tregs. Further the potent regulatory function of tolerant-Tregs than naïve-Tregs are specific to NICC xenografts, but not to C57BL/6 islet allografts.
Conclusions: Foxp3+ Tregs play important role in NICC xenografts tolerance induced by treatment of CTLA-4Fc and anti-CD154 mAb. Foxp3+ Tregs were activated and had more potent regulatory function in vivo in xenorecognition despite of B7-CD28 and CD40-CD40L costimulatory blockade by CTLA-4Fc and anti-CD154 mAb. These tolerant-Tregs had the capacity to transfer dominant tolerance and specifically exhibited more potent regulatory function to NICC xenografts.
*M.H. and J.W. contributed equally to this work.
07:00 - 08:00
|Orals: Tolerance, Zoonosis||Co-stimulation Blockade Inducing Tolerance to Porcine Islet Xenografts Produced Foxp3+ Regulatory T cells with High IL-10 Production and Expressing MHC class II that Were Capable of Dominant Tolerance||Room 109|
15:30 - 17:00
|Immunology||Modification of the “One Study” Panels for Whole Blood Immunophenotypic Monitoring for Transplant Clinical Trials and Islet Transplant patients at Westmead||Room 111-112|