After liver transplantation in HCV-infected patients, the virus load inevitably exceeds pre-transplantation levels. This phenomenon reflects suppression of the host-effector immune responses that control HCV replication by the immunosuppressive drugs used to prevent rejection of the transplanted liver. Here, we describe an adoptive immunotherapy approach, using lymphocytes extracted from liver allograft perfusate (termed herein liver allograft-derived lymphocytes), which includes an abundance of NK/NKT cells that mounted an anti-HCV response in HCV-infected liver transplantation recipients, despite the immunosuppressive environment. This therapy involved intravenously injecting patients 3 days after liver transplantation with liver allograft-derived lymphocytes treated with IL-2 and the CD3-specific mAb OKT3. During the first month after liver transplantation, the HCV RNA titers in the sera of recipients who received immunotherapy were markedly lower than those in the sera of recipients who did not receive immunotherapy. We further explored these observations in human hepatocyte-chimeric mice, in which mouse hepatocytes were replaced by human hepatocytes. These mice unfailingly developed HCV infections after inoculation with HCV-infected human serum. However, injection of human liver-derived lymphocytes treated with IL-2/OKT3 completely prevented HCV infection. Furthermore, an in vitro study using genomic HCV replicon-containing hepatic cells revealed that IFN-γ-secreting cells played a pivotal role in such anti-HCV responses. Thus, our study presents what we believe to be a novel paradigm for the inhibition of HCV replication in HCV-infected liver transplantation recipients. IntroductionLiver failure and hepatocellular carcinoma (HCC) due to chronic hepatitis C infection are the most common indications for liver transplantation (LT), and the incidences of both have been projected to increase further in the future. Recurrent HCV infection of the allograft is universal, occurs immediately after LT, and is associated with accelerated progression to cirrhosis, graft loss, and death (1, 2). This reflects the suppression of those host-effector immune responses that usually control HCV replication, suggesting that the immunosuppressive environment may play a major role in the rapid progression of recurrent HCV infection after LT (3, 4). Further, the immunosuppressive condition described above is considered to increase the incidence of cancer recurrence after LT in HCC patients. We recently proposed the novel strategy of adjuvant immunotherapy for preventing the recurrence of HCC after LT; this immunotherapy involves intravenously injecting LT recipients with activated liver allograft-derived NK cells (5, 6). Since the immunosuppressive regimen currently used after LT reduces the adaptive immune components but effectively maintains the innate components of cellular immunity (7-9), the augmenta-

We recently showed that interleukin (IL)-2-stimulated CD56+ cells derived from the liver exert vigorous cytotoxicity against hepatocellular carcinoma (HCC) by their binding to the tumor necrosis factor-related apoptosis-inducing ligand expressed on natural killer cells and the corresponding death receptors, and exhibit inhibitory effects on hepatitis C virus (HCV) replication by production of a high level of interferon-γ. These findings prompted us to develop a technique to increase the number of such innate components of cellular immunity from peripheral blood mononuclear cells (PBMCs) so that, they can be easily applied for immunotherapy clinically. We expanded CD3⁻CD56+ and CD3+CD56+ cells ex vivo from PBMCs of human volunteers by using media containing IL-2 and anti-CD3 monoclonal antibody. Among the various culture media used, autoserum supplemented X-VIVO 15 most efficiently supported PBMCs expansion and maintained the viability of the expanded cells (approximately 60-fold expansion after 28-d culture). Cultivation of PBMCs in this medium resulted in the highest proportion of CD3⁻CD56 cells among the propagated lymphocytes (approximately 40% after 28-d culture). An experiment using genomic HCV replicon-containing hepatic cells showed that the CD3⁻CD56+ cell-enriched expansion strongly inhibited HCV replication when compared with freshly isolated PBMCs. The additional anti-CD3 monoclonal antibody pulse stimulation induced anti-HCV activity even in the CD3+CD56+ cells among the propagated PBMCs. Further, cytotoxic assay showed that the expansion of CD3+CD56+ and CD3⁻CD56+ cells resulted in vigorous cytotoxicity against HCC. In conclusion, CD56+ cells obtained from the PBMCs show anti-HCV activity in addition to anti-HCC activity.

Patients requiring liver transplantation (LT) frequently experience renal insufficiency (RI), which affects their survival. Although calcineurin inhibitor-sparing immunosuppressive regimens (CSRs) are well known to prevent RI, the immune state in recipients receiving CSR remains to be intensively investigated. Among 60 cases of living-donor LT at our institute, 68% of the patients had none to mild RI (non-RI group) and 32% of the patients had moderate to severe RI (RI group). The RI group received a CSR comprising reduced dose of tacrolimus, methylprednisolone, and mycophenolate mofetil, while the non-RI group received a regimen comprising conventional dose of tacrolimus and methylprednisolone. One year after LT, the mean estimated glomerular filtration rate (eGFR) in the RI group had significantly improved, although it was still lower than that of the non-RI group. Serial mixed lymphocyte reaction assays revealed that antidonor T-cell responses were adequately suppressed in both groups. Thus, we provide evidence that CSR leads to improvement of eGFR after LT in patients with RI, while maintaining an appropriate immunosuppressive state.

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AimThe anti‐tumor effects of natural killer (NK) cells vary among individuals. Tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) expressed on liver NK cells is a marker of anti‐tumor cytotoxicity against hepatocellular carcinoma (HCC) in immune cell therapy. This study aimed to develop a liver immune status index (LISI) that predicts low TRAIL expression and validates its ability to predict recurrence after initial hepatectomy for primary HCC.MethodsA functional analysis of liver NK cells co‐cultured with interleukin‐2 for 3 days was performed of 40 liver transplant donors. The LISI, which predicted low TRAIL expression (25% quartile: <33%) in liver NK cells, was calculated using multiple logistic regression analysis. Next, 586 initial hepatectomy cases were analyzed based on the LISI.ResultsOur model was based on the Fibrosis‐4 index+0.1 (odds ratio [OR], 1.33), body mass index (OR, 0.61), and albumin levels+0.1 (OR, 0.54). The area under the receiver operating characteristic curve (AUC) of the LISI for low TRAIL expression was 0.89. Stratification of the recurrence rates (RR) revealed that LISI was an independent predictive factor of RR (moderate risk: hazard ratio, 1.44; high risk: hazard ratio, 3.02). The AUC was similar for the LISI, albumin–indocyanine green evaluation grade, albumin–bilirubin score, and geriatric nutritional risk index for predicting RR. Among the vascular invasion cases, the LISI was more useful than the other indexes.ConclusionOur model facilitates the prediction of RR in high‐risk patients by providing LISI to predict the anti‐tumor effects of NK cells.