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INTRA-GRAFT MOLECULAR PATHWAYS PREDICT OPERATIONAL TOLERANCE IN LIVER TRANSPLANTATION

Felix Bohne¹, Marc Martínez-Llordella¹, Juan-José Lozano², Rosa Miquel¹, Carlos Benítez¹, María-Carlota Londoño¹, Tommaso M. Manzia³, Roberta Angelico³, Dorine W. Swinkels⁴, Harold Tjalsma⁴, Marta López¹, Juan Gonzales-Abraldes¹, Eliano Bonaccorsi-Riani¹, Elmar Jaeckel⁵, Richard Taubert⁵, Jacques Pirenne⁶, Antoni Rimola¹, Giuseppe Tisone³, Alberto Sánchez-Fueyo¹

¹Liver Unit, Hospital Clinic Barcelona, 0IBAPS, CIBEREHD, University of Barcelona, Barcelona, Spain; ²Bioinformatics Platform, CIBEREHD, Barcelona, Spain; ³Liver Transplant Unit, Surgical Clinic, University of Rome “Tor Vergata”, Rome, ITALY.; ⁴Department of Laboratory Medicine, Laboratory of Genetic , Endocrine & Metabolic diseases (830 LGEM), Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; ⁵Department of Gastroenterology, Hepatology & Endocrinology, Medical School of Hannover, Hannover, Germany; ⁶University Hospitals Leuven, Leuven, Belgium

Body: Complete cessation of immunosuppressive therapy (IS) is feasible in selected liver transplant recipients who are considered operationally tolerant (TOL) and exhibit specific blood cell gene expression patterns. The intragraft  molecular pathways associated with liver operational tolerance however have not been thoroughly analysed before.
Study Design: 75 stable liver recipients were enrolled in a prospective European multi-center IS withdrawal clinical trial. IS was gradually discontinued over a 6-9 month period and recipients were then followed-up for 12 months and considered TOL if no rejection was detected over the entire duration of the study. Liver biopsies were obtained at baseline and at the end of the study in TOL recipients, and at baseline and at the time of rejection in non-TOL recipients. For the current study we employed liver tissue samples collected from 33 TOL and 42 non-TOL recipients at baseline, 18 non-TOL recipients at the time of rejection, and 7 TOL at the end of the study.
Methods: We conducted transcriptional profiling on all liver samples employing Illumina and Affymetrix microarrays and real-time PCR. Additionally, serum samples were used to assess iron metabolism parameters.   
Results:  At baseline, TOL and non-TOL grafts differed in 168 genes (FDR<25%). The most significant among them were HAMP and TFRC (FDR=0%), both of them critical for the regulation of iron metabolism. These results were replicated using real time qPCR. The lower HAMP expression levels noted in Non-TOL grafts correlated with lower serum hepcidin and ferritin levels and reduced intra-graft iron stores. These findings were stable over time and could be employed to accurately predict the outcome of the weaning process.
Conclusions: These results point for the first time to a critical role of iron homeostasis in the regulation of intra-graft alloimmune responses in humans and provide a set of novel biomarkers to conduct drug-weaning trials in liver transplantation.