Presented at:
World Transplant Congress 2006
Boston, MA, TX, July 22-27, 2004
Advances in immune monitoring in renal transplantation
Sagoo P, Hernandez-Fuentes MP, Flavia R, Jimenez E, Perucha E, Stevenson S, Milton R, Warrens A, Lechler RI
King's College London, United Kingdom; Imperial College London, United Kingdom.
Given the morbidity and mortality associated with long-term immunosuppression, an assay indicating the status of a recipient’s immune response to donor would prove an invaluable resource, allowing the level of immunosuppression administered to be based on the patients
immune reactivity. We are currently conducting parallel studies supported by the ITN and European Union to develop several routine laboratory techniques to asses donor-specific responses generated via direct and indirect pathways of allo-antigen presentation in renal transplant recipients.
Patient groups used to define expected responses are as follow: Stable renal function on (1) CNI or (2) non-CNI based immunosuppression, (3) Patients undergoing chronic rejection (CR) and (4) Patients with stable function off immunosuppression - tolerant recipients. Quantitative assessments of donor-specific responses are made by IFNγ ELISpot, where responder frequencies generated by the indirect pathway are made by stimulating recipient PBMCs with a donor-antigen preparation. We have been comparing the suitability of different preparations for this assay; purified peptides of HLA molecule variable regions, membrane proteins from donor-cell lysates and soluble HLA-monomers. Direct pathway donor-reactive CD8 and CD4 T cell frequencies are studied by stimulation with donor-derived APCs using ELISpot and CFSE analysis. Depletion of CD4+CD25+ cells from directly and indirectly primed responder populations in these assays is used to detect Treg-mediated regulation of anti-donor responses.
Indirect alloantigen presentation occurs most efficiently using soluble purified HLA-peptides. Given the limitation of this strategy to comprehensively monitor anti-donor responses, we have optimized the membrane protein preparation to obtain similar responses. Interim data analysis showed donor-specific hypo-responsiveness in the direct pathway was detected in 33% of stable group 1 patients, in which group donor-specific regulation in the indirect pathway was detected in 21% of patients. We expect that with more patients analyzed by the study end-point, a clinically important answer should be obtained.
Donor-specific assays developed for this study should significantly further our understanding of immune-reactivity and regulatory mechanisms during CR and tolerance and may provide a basis for identifying tolerance in renal transplant recipients.