Although the liver is less immunogenic than other solid organs, most liver transplant recipients receive lifelong immunosuppression. A subset of liver recipients may be operationally tolerant and not require immunosuppression however we have no biomarkers to identify these patients. We showed that donor-specific tolerance occurs in recipients of orthotopic liver transplants (OLT) after post-transplant total lymphoid irradiation (TLI). The mechanism of this tolerance induction involves apoptosis of intragraft T cells and the subsequent increase of functional T regulatory cells both in the graft and the periphery. Micro RNAs (miRNA) are small noncoding RNA molecules that regulate the posttranscriptional expression of target genes. MiRNAs are important biomarkers of many diseases thus we examined the miRNA profile during the tolerant state. Four groups (n=3) of OLT were studied, three groups analyzed at day seven (d7) post-transplant: syngeneic (syn) recipients (DA⇒DA) with TLI, allogeneic (allo) recipients (DA⇒Lewis) with TLI, untreated allo recipients (DA⇒Lewis), and one group, allo recipients (DA⇒Lewis) treated with TLI analyzed at day >100 (d100) post-transplant. Normal DA rat livers were used as the calibrator and a control group. Untreated allo recipients reject their grafts within 12 days whereas TLI-treated and syn recipients have long-term graft survival.

Unsupervised hierarchical clustering of miRNA expression classified samples into two main groups; allo livers obtained on d7 irrespective of TLI treatment clustered together and syn livers clustered with the allo TLI d100 group. Similarly, principal component analysis demonstrated five clear clusters with the allo untreated (rejecting d7) and allo TLI (tolerant d7) groups in close proximity supporting that miRNA profiling does not distinguish between these groups early post-transplant. In contrast, the allo TLI group at d100 was closely related to the normal and syn liver grafts. Analyses of 60 miRNAs that were selected based on significant (p<0.05) differential expression, between the allo rejecting livers and the allo tolerant livers confirmed that there were no significant differences between the tolerant and syngeneic groups. Thus taken together, our results indicate that the miRNA profile in allografts with established tolerance is remarkably similar to the profile observed in normal and syn grafts. A miRNA profile may hold promise as a biomarker of the tolerant state.

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