**7. Clinical relevance of regulatory T cells for liver transplantation**

Slowly, but without doubt, regulatory T cells are getting involved in the diagnostic process of many pathological conditions, expressed by deviations in immune tolerance – autoimmune [142, 143], tumors [144, 145], recurrent pregnancy loss [146, 147], primary immune deficiencies [110, 148] etc.

Current data indicate that Tregs have the potential to be a potential biomarker for the monitoring of the posttransplantation period [2]. Specifically, in liver transplanted patients Tregs are object of intensive research mostly because they are inherently involved in the operational tolerance and are part of natural liver toleragenic mechanisms. Despite the abundant data showing the benefit of Tregs determination during posttransplantation period, there are still many unresolved questions. Some of them are related to the definition of Tregs phenotype that should be used. Although identified as FoxP3 + CD4+ T cells, the expression of FoxP3 was demonstrated less informative than the promotore demethylation because it distinguishes true Tregs from transiently FOXP3+ activated T cells [149]. For diagnostic purposes, Tregs are often defined as CD127-CD25 + CD4+, but recent advances in the field showed a population CD25- [150], which is not fully characterized regarding CD127. Another direction that needs to be elucidated are Tregs in biopsies and peripheral blood. In Barcelona consensus (2016) several studies are shown with ambiguous results [151], that does not provide clear evidences for the relevance of Tregs measurement in posttransplantation period. The third direction is the significantly decreased expression of CD25 in relation to the immunosuppressive therapy and the consecutive inability to find out CD25hiTreg cells in the periphery. Although Tregs are highly informative regarding the operational tolerance [152], the question is still unresolved and more studies are required to determine the value of Tregs in the monitoring of post-transplantation period.

It seems, that Tregs are more promising as a therapeutic approach for the control of immune activation and development of a state of immune tolerance. Early experimental studies demonstrated association between them and the delay in islet allograft rejection and long term survival [153, 154]. During last five years, together with other approaches [155], Tregs attract the medical interest in the field of GVHD and solid organ transplantation [156, 157]. Todo et al. in 2016 report phase I results clinical trial with ex vivo expanded recipient polyclonal Tregs in living kidney transplants. Despite variability in recipient's renal disease, the expansion protocol produced Tregs which met all release criteria, expressing >98% CD4 + CD25+ with <1% CD8+ and CD19+ contamination and > 80% FOXP3 expression with stable demethylation in the FOXP3 promoter. Within recipients, expanded Tregs amplified circulating Treg levels in a sustained manner. Clinically, all doses of Treg therapy tested were safe with no adverse infusion related side effects, infections or rejection events up to two years post-transplant [158]. Another study undertook a direct comparison of the *in vitro* and *in vivo* functional activities of the different memory and naïve Treg subpopulations showing that the naive Treg is the Treg population that exhibits the ideal biological features of a Treg therapeutic while the highly suppressive memory Tregs should be purposefully excluded from a Treg therapeutic due to their low lineage delity, low proliferative capacity, and greater pro-inflammatory potential [159]. Recently published results from The ONE study demonstrates that regulatory cell therapy is achievable and safe in living-donor kidney transplant recipients, and is associated with fewer infectious complications, but similar rejection rates in the first year. Therefore, immune cell therapy is a potentially useful therapeutic approach in recipients of kidney transplant to minimize the burden of general immunosuppression [160].

Finally, the immune capacity of the liver depends on the local hepatic and immune cells that transitory populate it. The current research provides abundant data about the intercellular tolerogenic mechanisms. However, some points need to be better clarified from the scientific and medical point of view. Among them are the fine-tuning of common immunosuppressive therapeutics regarding regulatory T cells, biochemical mechanisms of interactions between hepatocytes and immune cells, whether immune parameters of activation/suppression might provide information in advance about the liver function, the impact of individual immunogenetic variations on the recovery and operational tolerance etc. We think that the immune system should be considered as important player in the liver tolerance network and involved in the post-transplantation period monitoring.
