Masahiro Ohira

Difference in cytotoxicity against hepatocellular carcinoma between liver and periphery natural killer cells in humans.

In rodents, liver natural killer (NK) cells have been shown to mediate higher cytotoxic activity against tumor cells than do peripheral blood (PB) NK cells. However, such differences between liver and PB NK cells have not been extensively investigated in humans. The phenotypical and functional properties of NK cells extracted from liver perfusates at the time of living donor liver transplantation were investigated. The tumor necrosis factor–related apoptosis‐inducing ligand (TRAIL), a critical molecule for NK cell–mediated anti‐tumor cell killing, was not expressed by freshly isolated PB NK cells or by liver NK cells. Stimulation with interleukin (IL)‐2, significantly up‐regulated the expression of TRAIL on liver NK cells, but this effect was barely observed on PB NK cells. Donor liver NK cells showed the most vigorous cytotoxicity against HepG2, a hepatocellular carcinoma (HCC) cell line, after IL‐2 stimulation (90.5% ± 2.2% at E: T = 10:1), compared with donor and recipient PB NK cells and recipient liver NK cells (64.8% ± 8.2%, 56.1% ± 8.9%, and 34.6% ± 7.5%, respectively). IL‐2 stimulation resulted in an increased expression of killing inhibitory receptors on liver NK cells in parallel with TRAIL expression. Consistently, the cytotoxicities of IL‐2–stimulated donor liver NK cells against self and recipient lymphoblasts were negligible. In conclusion, adoptive transfer of IL‐2–stimulated NK cells extracted from donor liver graft perfusate could mount an anti‐tumor response without causing toxicity against 1‐haplotype identical recipient intact tissues. These findings present a concept to prevent recurrence of HCC after liver transplantation. (HEPATOLOGY 2006;43:362–372.)

Adoptive immunotherapy with liver allograft-derived lymphocytes induces anti-HCV activity after liver transplantation in humans and humanized mice.

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-gamma-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.

Adoptive transfer of TRAIL-expressing natural killer cells prevents recurrence of hepatocellular carcinoma after partial hepatectomy.

Background. Antitumor activity of the liver natural killer (NK) cells reportedly decreases after partial hepatectomy, suggesting that patients with such depressed immune status are susceptible to the recurrence of hepatocellular carcinoma (HCC). We hypothesize that adoptive immunotherapy using activated NK cells can be a novel strategy to improve the depressed immune status in patients with HCC after hepatectomy or partial liver transplantation. In the present study, we have tested this hypothesis by using a mouse model. Methods. Intraportal injection of 1–5×106 Hepa1-6 cells (hepatoma cell line) did not result in liver metastases in untreated B6 mice, but led to the growth of liver metastases after extensive partial hepatectomy. Utilizing this murine HCC metastasis model, we investigated the antitumor activity of both remnant liver and exogenously transferred NK cells. Results. The anti-HCC activity of liver NK cells significantly decreased after partial hepatectomy. The expression of CD69 and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) on liver NK cells was temporarily downregulated. The adoptive transfer of NK cells, including a TRAIL-expressing fraction, extracted from the liver perfusates of poly I:C-stimulated B6 mice inhibited the growth of liver metastasis in B6 or (B6×BALB/c) F1 (B6CF1) mice that underwent hepatectomy and received intraportal Hepa1-6 injection. Conclusions. These findings indicate that adoptive immunotherapy using activated NK cells extracted from normal liver perfusates may be a novel technique for reconstituting the depressed immune status in cases of living donor liver transplantation involving HCC patients, recipients of a partial liver graft.

Clinical-scale isolation of interleukin-2-stimulated liver natural killer cells for treatment of liver transplantation with hepatocellular carcinoma.

Tumor recurrence is the main limitation of liver transplantation (LT) in patients with hepatocellular carcinoma (HCC) and can be promoted by immunosuppressants. However, there is no prevention or treatment for HCC recurrence after LT. Here we describe a clinical-scale method for an adoptive immunotherapy approach that uses natural killer (NK) cells derived from deceased donor liver graft perfusate to prevent tumor recurrence after LT. Liver mononuclear cells (LMNCs) that were extracted from deceased donor liver graft perfusate contained a high percentage of NK cells (45.0 ± 4.0%) compared with peripheral blood mononuclear cells (PBMCs) (21.8 ± 5.2%) from the same donor. The CD69 activation marker and the natural cytotoxicity receptors, NKp44 and NKp46, were expressed at high levels in freshly isolated liver NK cells. Furthermore, interleukin-2 (IL-2)-stimulated NK cells showed greater upregulation of activation markers and the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which is critical for NK cell-mediated antitumor cell death and increased production of interferon. Moreover, IL-2 stimulation induced LMNCs to exhibit a strong cytotoxicity against NK-susceptible K562 target cells compared with PBMCs (p < 0.01). Finally, we also showed that the final product contained a very low T-cell contamination (0.02 ± 106 cells/kg−1), which reduces the risk of graft-versus-host disease (GVHD). Collectively, our results suggest that the adoptive transfer of IL-2-stimulated NK cells from deceased donor liver graft perfusate could be a promising treatment for LT patients with HCC.

Successful hepatitis B vaccination in liver transplant recipients with donor-specific hyporesponsiveness.

Currently, patients are prescribed lifelong treatment with hepatitis B immunoglobulin (HBIg) after liver transplantation (LT) for hepatitis B virus (HBV)‐related diseases in order to prevent reinfection with HBV. Active immunization with an HBV vaccine would be a preferable alternative; however, the immunosuppressive environment in LT recipients is believed to elicit a poor response to vaccination. Minimizing the exposure of the HBV‐infected LT recipients to immunosuppressants would be beneficial in inducing adaptive immunity against HBV by vaccination. In this study, in addition to efforts to minimize immunosuppression, prophylaxis with HBV vaccination combined with continuous HBIg administration was performed in 17 LT recipients who had undergone transplantation attributable to HBV‐related diseases. During the observation period, the overall response rate to HBV vaccination was 64.7%. The immune status of the recipients was evaluated by a mixed lymphocyte reaction assay in response to allostimulation. Patients showing a donor‐specific hyporesponse with a well‐maintained response to the third‐party stimulus always achieved a sustained immune response to the vaccine, whereas patients showing a hyporesponse to both the donor and the third‐party stimulus were unable to do so. Thus, inducing an anti‐donor‐specific immunosuppressive status by minimizing immunosuppression should enable post‐transplant HBV vaccination to be a promising prophylactic strategy.

Possibility of Adoptive Immunotherapy With Peripheral Blood-derived CD3―CD56+ and CD3+CD56+ Cells for Inducing Antihepatocellular Carcinoma and Antihepatitis C Virus Activity

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-&ggr;. 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.

Significant correlation between spleen volume and thrombocytopenia in liver transplant patients: A concept for predicting persistent thrombocytopenia

Interferon (IFN) therapy with or without ribavirin treatment is well established as a standard antiviral treatment for hepatitis C virus (HCV)–infected patients. However, susceptibility to thrombocytopenia is a major obstacle for initiating or continuing this therapy, particularly in liver transplant (LTx) recipients with HCV. Studies have reported that splenectomy performed concurrently with LTx is a feasible strategy for conditioning patients for anti‐HCV IFN therapy. However, the relationship between the severity of splenomegaly and alterations in the blood cytopenia in LTx recipients remains to be clarified. Here, we analyzed the relationship between spleen volume (SV) and thrombocytopenia in 45 patients who underwent LTx at Hiroshima University Hospital. The extent of pre‐LTx splenomegaly [the SV to body surface area (BSA) ratio in an individual] was inversely correlated with both the post‐LTx white blood cell count and platelet (PLT) count (P < 0.001). Furthermore, the PLT count of patients with thrombocytopenia (PLT count ≤ 5 × 104/mm3) increased significantly in the group without splenomegaly (SV/BSA value < 400) versus that in the group with splenomegaly (P = 0.005). Thus, if both splenomegaly and thrombocytopenia coexist (PLT count ≤ 5 × 104/mm3 and SV/BSA value ≥ 400), persistent thrombocytopenia is predictable after LTx. Liver Transpl 15:208–215, 2009.

Microsurgical hepatic artery reconstruction during living‐donor liver transplantation by using head‐mounted surgical binocular system

We have described our experience with arterial reconstruction during living‐donor liver transplantation by using Varioscope® AF3 – a head‐mounted surgical binocular system with automatic focusing and continuous zoom magnification from 3.6× to 7.2×. From July 1996 to December 2006, 91 grafts were implanted in 89 living‐donor liver transplantation recipients, including two that required retransplantation. For microsurgical reconstruction of the graft hepatic artery, a conventional operating microscope was used in the first 10 transplants and Varioscope, in the subsequent 81. The time required to complete arterial reconstruction while using a conventional operating microscope and Varioscope was 78.6 ± 44.6 min and 35.5 ± 15.5 min, respectively. No arterial complications, including hepatic artery thrombosis, occurred in any of the 89 patients during the observation period. In living‐donor liver transplantation, successful hepatic artery reconstruction can be safely carried out using Varioscope.

Using recipient's middle hepatic vein for drainage of the right paramedian sector in right liver graft.

Background. Congestion in the right paramedian sector of a right liver graft without a middle hepatic vein (MHV) may lead to graft dysfunction. To solve this problem, we have developed a technique for reconstructing the MHV tributaries of the right liver grafts by using the preserved recipient’s native MHV trunk. Methods. Between 2005 and 2007, among 34 right liver graft liver transplant patients with significant MHV tributaries (>5 mm in diameter), 21 patients underwent right liver graft living-donor liver transplantation: draining MHV tributaries with recipient’s native MHV trunk. We evaluated the patency of the reconstructed MHV tributaries, graft regeneration, and graft survival. Results. The 3-month patency rates of the reconstructed V8 and V5 were 92% and 76%, respectively. The 1-year survival rate and the regeneration index of the right paramedian sector 6 months after transplantation were higher in patients with reconstructed MHV tributaries than that for patients without reconstructed MHV tributaries. Conclusion. The use of the recipient’s MHV trunk for the reconstruction of the MHV tributaries of the right liver grafts is considered to be a valuable and a feasible strategy in right liver graft living-donor liver transplantation.

Fc-Gamma Receptor Polymorphisms Predispose Patients to Infectious Complications After Liver Transplantation

We investigated the impact of polymorphisms in host innate immunoregulatory genes on the development of infectious complications after liver transplantation (LT). The single‐nucleotide polymorphisms (SNPs) of C1QA [276A/G], FCGR2A [131H/R], and FCGR3A [158F/V], genes encoding the Fc gamma receptor (FcγR), were analyzed in 89 living donor LT recipients in relation to the occurrences of postoperative infectious complications within 30 days after LT. Consistent with a lower affinity of the isoform encoded by FCGR3A [158F] to both IgG1 and IgG3, a significantly higher incidence of bloodstream infections (BSI) was observed in the FCGR3A [158F/V or F/F] than in the FCGR3A [158V/V] individuals. The combination of FCGR2A and FCGR3A SNPs further stratified the incidence of BSI, regardless of C1QA SNP. The predominant causative pathogen of BSI in the FCGR3A [158F/F or F/V] patients was gram‐positive cocci (73.3%), of which one third was methicillin‐resistant Staphylococcus aureus. No differences were observed in the incidence of fungal infections or in cytomegalovirus infections with respect to the three gene polymorphisms. Our findings indicate that FcγR SNPs are predisposing factors for BSI and can predict mortality after LT. This study provides a foundation for further prospective studies on a larger scale.