*Purpose: Lifelong treatment with immunosuppressive drugs (IS) to prevent graft rejection in transplant recipients is accompanied by side effects. Some recipients are considered to be immunologically tolerant towards their graft, as they have been safely withdrawn from IS late after liver transplantation (LTx). If tolerant (TOL) LTx recipients can be recognized and withdrawn earlier after LTx, related side effects may be reversed or avoided. Several studies have examined peripheral blood markers to identify TOL LTx recipients, but an accurate tolerance profile has not been determined yet. In this study, we evaluated CD137-expressing donor-specific T-cells upon a short-term stimulation as a potential identifier of tolerance.

*Methods: TOL LTx recipients (n=10) withdrawn from IS for on average 4.2 years, and a control group on regular IS regimen (CTRL; n=12) matched for time after LTx (on average 15.2 years), primary disease, age, gender and CMV serostatus were included. Recipient peripheral blood mononuclear cells (PBMCs) were stimulated overnight in vitro with donor(-like) or third party splenocytes with the same number but different HLA mismatches as with donor. With flow cytometry, responses towards splenocytes were analyzed by quantifying expression of CD137, an activation-induced marker for antigen-specific T-cells. Naïve and memory T-cells were characterized by CCR7 and CD45RA and regulatory T-cells (Tregs) were characterized by FoxP3 and CD45RA expression.

*Results: Phenotypically, total CD4 and CD8 T-cells, resting Tregs and activated Tregs did not differ between TOL and CTRL. Donor-specific CD4 and CD8 T-cell responses (ratio CD137+ T-cells: donor/unstimulated) were significantly lower in TOL compared to CTRL (mean CD4 TOL 1.4 vs CTRL 3.1 p=0.036; mean CD8 TOL 1.1 vs CTRL 2.4 p=0.049), whereas this was not observed for third party stimulation. In addition, less donor-reactive effector memory (EM) cells were observed within CD4 and CD8 T-cells in TOL compared to CTRL.

*Conclusions: In TOL LTx recipients circulating donor-reactive CD4 and CD8 T-cells, in particular the EM T-cell compartment that are capable of migrating to the graft, are less frequent. This selective loss of EM T-cells may underlie hypo responsiveness to the graft in TOL. Overall, these results indicate that quantification of donor-specific T-cell reactivity by measuring activation induced CD137 hypo responsiveness may enable identification of TOL LTx recipients.

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