In contrast to other solid organ transplantations, liver grafts have tolerogenic properties. Animal models indicate that donor leukocytes transferred into the recipient after liver transplantation (LTX) play a relevant role in this tolerogenic phenomenon. However, the specific donor cell types involved in modulation of the recipient alloresponse are not yet defined. We hypothesized that this unique property of liver grafts may be related to their high content of organ-specific natural killer (NK) and CD56 þ T cells. Here, we show that a high proportion of hepatic NK cells that detach from human liver grafts during pretransplant perfusion belong to the CD56bright subset, and are in an activated state (CD69 þ ). Liver NK cells contained perforin and granzymes, exerted stronger cytotoxicity against K562 target cells when compared with blood NK cells, and secreted interferon-c, but no interleukin-10 or T helper 2 cytokines, upon stimulation with monokines. Interestingly, whereas the CD56bright subset is classically considered as noncytolytic, liver CD56bright NK cells showed a high content of cytolytic molecules and degranulated in response to K562 cells. After LTX, but not after renal transplantation, significant numbers of donor CD56dim NK and CD56 þ T cells were detected in the recipient circulation for approximately 2 weeks. In conclusion, during clinical LTX, activated and highly cytotoxic NK cells of donor origin are transferred into the recipient, and a subset of them mixes with the recirculating recipient NK cell pool. The unique properties of the transferred hepatic NK cells may enable them to play a role in regulating the immunological response of the recipient against the graft and therefore contribute to liver tolerogenicity. Liver Transpl 16:895-908, 2010. V C 2010 AASLD.Received December 23, 2009; accepted March 28, 2010. It is generally recognized that after clinical liver transplantation (LTX), the incidence of chronic rejection is lower than after transplantation of other organ grafts. Furthermore, in about 20% of LTX recipients, immunosuppressive therapy can be withdrawn without occurrence of graft rejection.1 Various animal models are spontaneously tolerant to LTX, even though they reject other organs.2,3 Furthermore, cotransplantation of a liver allograft can prevent rejection of other organ grafts from the same donor. 4,5 The mechanisms responsible for this relative tolerogenicity of the liver have only been partially elucidated. A number of observations in animal models indicate that the immune cells present in the liver graft may play a relevant role in the induction of tolerance. With LTX, so-called passenger leukocytes from the donor are transferred into the recipient and can establish a condition of chimerism of variable proportions and duration.6,7 Independent studies from different groups have shown that in rat models,

Hepatic NK cells constitute $40% of hepatic lymphocytes and are phenotypically and functionally distinct from blood NK cells. Whether hepatic NK cells derive from precursors in the BM or develop locally from hepatic progenitors is still unknown. Here, we identify all five known sequential stages of NK-cell development in the adult human liver and demonstrate that CD34 1 hepatic progenitors can generate functional NK cells. While early NK-cell precursors (NKPs) were similar in liver and blood, hepatic stage 3 NKPs displayed immunophenotypical differences, suggesting the onset of a liver-specific NK-cell development. Hepatic stage 3 NKPs were RORC neg and did not produce IL-17 or IL-22, excluding them from the lymphoid tissue-inducer (LTi) subset. In vitro culture of hepatic NKPs gave rise to functional NK cells exhibiting strong cytotoxicity against K562 targets. To determine whether hepatic NKPs are stably residing in the liver, we analyzed donor and recipient-derived cells in transplanted livers. Shortly after liver transplantation all donor NKPs in liver grafts were replaced by recipient-derived ones, indicating that hepatic NKPs are recruited from the bloodstream. Together, our results show that NKPs are continuously recruited from peripheral blood into the liver and can potentially differentiate into liverspecific NK cells.

Summary Whether or not Natural Killer (NK) cells affect the immune response to solid organ allografts is still controversial. Main determinants of NK‐cell activation are specific HLA/killer‐cell immunoglobulin‐like receptors (KIR) interactions that, in transplantation, may induce NK‐cell alloreactivity. So far, in liver transplantation (LTX) donor‐versus‐recipient alloreactivity has not been investigated; in addition, studies of predicted recipient‐versus‐donor NK‐cell alloreactivity have led to contradicting results. We typed a cohort of LTX donors and recipients for HLA‐C/Bw4 and KIRs. We estimated the effect of NK‐cell alloreactivity, as predicted by classically used models, in the donor‐versus‐recipient direction. The results indicate that HLA/KIR mismatches in the donor‐versus‐recipient direction do not predict graft rejection nor graft or patient survival, suggesting that donor‐derived NK cells do not play a major role in LTX outcome. In addition, when considering predicted NK‐cell alloreactivity in the reverse direction (recipient‐versus‐donor), we first confirmed that donor HLA‐C genotype was not associated with acute rejection, graft or patient survival and secondly we found that none of the models describing NK‐cell alloreactivity could predict LTX outcome. Overall our observations suggest that, in contrast to what is shown in haematopoietic stem cell transplantation, donor‐derived NK cells may not contribute in preventing liver graft rejection, and that recipient‐versus‐donor NK‐cell alloreactivity does not predict LTX outcome.

Our data do not support a graft-protective role for HLA-G after LTX, but show that end-stage liver diseases are associated with HLA-G expression on hepatocytes, which may determine a negative feedback to protect the liver against immunological damage.