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Novel systemic treatments for hepatocellular carcinoma (HCC) are strongly needed. Immunotherapy is a promising strategy that can induce specific antitumor immune responses. Understanding the mechanisms of immune resistance by HCC is crucial for development of suitable immunotherapeutics. We used immunohistochemistry on tissue-microarrays to examine the co-expression of the immune inhibiting molecules PD-L1, Galectin-9, HVEM and IDO, as well as tumor CD8+ lymphocyte infiltration in HCC, in two independent cohorts of patients. We found that at least some expression in tumor cells was seen in 97% of cases for HVEM, 83% for PD-L1, 79% for Gal-9 and 66% for IDO. In the discovery cohort (n = 94), we found that lack of, or low, tumor expression of PD-L1 (p < 0.001), Galectin-9 (p < 0.001) and HVEM (p < 0.001), and low CD8+TIL count (p = 0.016), were associated with poor HCC-specific survival. PD-L1, Galectin-9 and CD8+TIL count were predictive of HCC-specific survival independent of baseline clinicopathologic characteristics and the combination of these markers was a powerful predictor of HCC-specific survival (HR 0.29; p <0.001). These results were confirmed in the validation cohort (n = 60). We show that low expression levels of PD-L1 and Gal-9 in combination with low CD8+TIL count predict extremely poor HCC-specific survival and it requires a change in two of these parameters to significantly improve prognosis. In conclusion, intra-tumoral expression of these immune inhibiting molecules was observed in the majority of HCC patients. Low expression of PD-L1 and Galectin-9 and low CD8+TIL count are associated with poor HCC-specific survival. Combining immune biomarkers leads to superior predictors of HCC mortality.

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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,

CD4 type 1 T regulatory (Tr1) cells have a crucial role in inducing tolerance. Immune regulation by these cells is mainly mediated through the secretion of high amounts of IL-10. Several studies have suggested that this regulatory population may be involved in tumor-mediated immune-suppression. However, direct evidence of a role for Tr1 cells in human solid tumors is lacking. Using isolated cells from individuals with hepatocellular carcinoma (HCC; = 39) or liver metastases from colorectal cancer (LM-CRC; = 60) we identify a CD4FoxP3IL-13IL-10 T cell population in tumors of individuals with primary or secondary liver cancer that is characterized as Tr1 cells by the expression of CD49b and the lymphocyte activation gene 3 () and strong suppression activity of T cell responses in an IL-10 dependent manner. Importantly, the presence of tumor-infiltrating Tr1 cells is correlated with tumor infiltration of plasmacytoid dendritic cells (pDCs). pDCs exposed to tumor-derived factors enhance IL-10 production by Tr1 cells through up-regulation of the inducible co-stimulatory ligand (ICOS-L). These findings suggest a role for pDCs and ICOS-L in promoting intra-tumoral immunosuppression by Tr1 cells in human liver cancer, which may foster tumor progression and which might interfere with attempts of immunotherapeutic intervention.