**5.3 The impact of regulatory T cells on the operational tolerance**

The establishment of immune tolerance after solid organ transplantation (SOT) is the key therapeutic challenge in the post-operative period. In most cases, it is induced by the continuous application of immunosuppressive therapy. However, in some patients, a discontinuation of the regimen arrives, due to infections, cancer etc. Surprisingly, in some cases changes in immunological parameters, indicating the development of a state of immune tolerance were found despite the lack of immune suppression. This particular situation is defined as operational tolerance and is characterized by the absence of any clinical and histological signs of rejection in therapy free patients [114]. The operational tolerance (OT) is reported after transplantation of different solid organs, but is frequent in LT [115, 116]. It is supposed that tolerogenic properties of the liver and residential lymphocytes play a key role in this process [117]. The exact mechanisms are not fully clarified, but the intensive research highlights that it depends on multiple factors. Among them are regulatory T cells, particular gene expression profile and serum levels of HLA-G.

Regulatory T cells are the first parameter associated with OT. In patients with spontaneous tolerance they are increased independently of the age of recipient [118]. According to the study of Koshiba et al., not only the proportion CD4 + CD25high + T cells was increased in the tolerant patients' peripheral lymphocytes and suppressed MLR specifically to the donor antigen, but also FOXP3 expressing cells were present within the tolerant liver [119]. Pons and colleagues describe sustained increase in CD4 + CD25+ and CD4 + CD25hi cells in patients with operational tolerance in comparison with non-OT patients in a bimonthly evaluation intervals until M16 [1].

Interestingly, in LT Tregs show a particular dynamic. In one of the earliest studies, pre-transplantation levels of Tregs were higher in patients than in controls. Lowest levels were observed at month 3 after Tx, followed by a relative increase at 12 months and at later time points [120].

The main question is why these cells are elevated in recipients? In fact, up to now reports discussing this topic in the literature are insufficient. The detailed study of Demirkiran and colleagues examined the presence and allosuppressive activity of CD4 + CD25 + Foxp3+ Tregs in perfusates of human liver grafts and monitored the cells presence in the circulation of recipients after liver Tx. The authors show an increased proportion of CD4 + CD25 + CTLA4+ T cells compared with healthy control blood. The increased percentages of Foxp3+ cells, which were negative for CD127, confirmed the enrichment of Tregs in perfusates. They suppressed proliferation and IFN-γ production of donor and recipient T cells. In vivo within the first weeks after Tx, up to 5% of CD4 + CD25 + CTLA4+ T cells in recipient blood were derived from the donor liver, indicating that a substantial number of donor Tregs detach from the liver graft during perfusion and continue to migrate into the recipient after Tx. These donor Tregs suppress the direct pathway alloresponses and may in vivo contribute to chimerism-associated tolerance early after liver Tx [121]. In a small number of patients, we found a pick in the percentage of Tregs at day 7 (D7) followed by a decrease until D30 being always around and above healthy controls values independently of the diagnosis or age. The simultaneous routine measurement of liver function laboratory parameters revealed that the increase in Tregs precedes albumin synthesis restoration [122]. In another study Baumann et al. evidence that the benign clinical course of subclinical rejection (SCR) compared to acute clinical rejection (ACR) is associated with intrahepatic T cell infiltration patterns showing less cytotoxic T cells and more CD4 + FOXP3+ Tregs. They demonstrate that in patients with SCR the pattern of infiltrating T cells is characterized by a stronger accumulation of CD4+ cells, an increasing CD4+/CD8+ ratio, and an increasing CD4+ forkhead box P3 (FOXP3) + regulatory T cell (Treg)/CD8+ ratio, which was not seen in acute clinical rejection. These intrahepatic T cell patterns were not reflected in the peripheral blood [123]. Cumulatively, these data suggest the presence of particular sort of cellular chimerism, associated with liver transplantation.

The chimerism is a specific phenomenon characterized by the presence of cells from one individual in another. Microchimerism is reported in hematopoietic stem cells transplantation as a result of migration of passenger lymphocytes from the graft into recipient and in pregnancy (foetus). In SOT, microchimerism is described in mice first [124]. Among multiple reports showing than donor mononuclear cells migrate from the graft in the recipient, Jonsson et al. find that the peak levels of chimerism are within the first 48 hours after transplantation and the range reaches 20% of total peripheral blood mononuclear cells [125, 126]. In a concise and very interesting review, Abrol and colleagues assume that increased expression of chemokines in the liver attracts alloreactive T cells that are subsequently destroyed by coming in contact with various liver cells inherently programmed towards tolerance induction [127]. Donor specific hypo-responsiveness, down regulation of T helper type I cytokine (IFN-γ) and no change in T helper type 2 cytokine (IL10) in the *in vitro* mixed lymphocyte reaction in recipients who achieved operational tolerance were also reported [128]. Indeed, this topic needs more studies directed to the detailed evaluation of different cellular subsets. Probably, Tregs might be part of passenger leucocytes or they might be secondary induced by the modified hepatic environment.
