Yuka Igarashi

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.

Blockade of invariant TCR-CD1d interaction specifically inhibits antibody production against blood group A carbohydrates

Previously, we detected B cells expressing receptors for blood group A carbohydrates in the CD11b(+)CD5(+) B-1a subpopulation in mice, similar to that in blood group O or B in humans. In the present study, we demonstrate that CD1d-restricted natural killer T (NKT) cells are required to produce anti-A antibodies (Abs), probably through collaboration with B-1a cells. After immunization of wild-type (WT) mice with human blood group A red blood cells (A-RBCs), interleukin (IL)-5 exclusively and transiently increased and the anti-A Abs were elevated in sera. However, these reactions were not observed in CD1d(-/-) mice, which lack NKT cells. Administration of anti-mouse CD1d blocking monoclonal Abs (mAb) prior to immunization abolished IL-5 production by NKT cells and anti-A Ab production in WT mice. Administration of anti-IL-5 neutralizing mAb also diminished anti-A Ab production in WT mice, suggesting that IL-5 secreted from NKT cells critically regulates anti-A Ab production by B-1a cells. In nonobese diabetic/severe combined immunodeficient (NOD/SCID/γc(null)) mice, into which peripheral blood mononuclear cells from type O human volunteers were engrafted, administration of anti-human CD1d mAb prior to A-RBC immunization completely inhibited anti-A Ab production. Thus, anti-CD1d treatment might constitute a novel approach that could help in evading Ab-mediated rejection in ABO-incompatible transplant recipients.

Engraftment of human hepatocytes in the livers of rats bearing bone marrow reconstructed with immunodeficient mouse bone marrow cells.

Abstract:  Background:  Previously, we created, a chimeric mouse (humanized mouse), a severe combined immunodeficiency (SCID) mouse whose liver was >90% repopulated with human (h)‐hepatocytes, which are useful for the testing of drug metabolism and toxicity, as well as a hepatitis B virus and hepatitis C virus‐susceptible animal model. However, their small body size and small total blood volume limited the utilization for analytical purposes, which led us to develop a method to create a chimeric rat bearing h‐hepatocyte‐repopulated liver.

Rho inhibitor prevents ischemia–reperfusion injury in rat steatotic liver

BACKGROUND & AIMS Hepatic stellate cells are thought to play a role in modulating intrahepatic vascular resistance based on their capacity to contract via Rho signaling. We investigated the effect of a Rho-kinase inhibitor on ischemia-reperfusion injury in the steatotic liver. METHODS Steatotic livers, induced by a choline-deficient diet in rats, were subjected to ischemia-reperfusion injury. Hepatic stellate cells isolated from steatotic livers were analyzed for contractility and Rho signaling activity. The portal pressure of the perfused rat liver and the survival rate after ischemia-reperfusion were also investigated. RESULTS Hepatic stellate cells from steatotic livers showed increased contractility and upregulation of Rho-kinase 2 compared with those from normal livers. Furthermore, endothelin-1 significantly enhanced the contractility and phosphorylation level of myosin light chain and cofilin in hepatic stellate cells isolated from steatotic livers. A specific Rho-kinase inhibitor, fasudil, significantly suppressed the contractility and decreased the phosphorylation levels of myosin light chain and cofilin. Serum levels of endothelin-1 were markedly increased after IR in rats with steatotic livers, whereas fasudil significantly decreased endothelin-1 serum levels. Rats with steatotic livers showed a significant increase in portal perfusion pressure after ischemia-reperfusion and a significant decrease in survival rate; fasudil treatment significantly reduced these effects. CONCLUSIONS Activation of Rho/Rho-kinase signaling in hepatic stellate cells isolated from steatotic livers is associated with an increased susceptibility to ischemia-reperfusion injury. A Rho-kinase inhibitor attenuated the activation of hepatic stellate cells isolated from steatotic livers and improved ischemia-reperfusion injury in steatotic rats.

Adoptive transfer of allogeneic liver sinusoidal endothelial cells specifically inhibits T-cell responses to cognate stimuli.

Although it is well known that liver allografts are often accepted by recipients, leading to donor-specific tolerance of further organ transplants, the underlying mechanisms remain unclear. We had previously used an in vitro model and showed that mouse liver sinusoidal endothelial cells (LSECs) selectively suppress allospecific T-cells across major histocompatibility complex (MHC) barriers. In the present study, we established an in vivo model for evaluating the immunomodulatory effects of allogeneic LSECs on corresponding T-cells. Allogeneic BALB/cA LSECs were injected intraportally into recombination activating gene 2 γ-chain double-knockout (RAG2/gc-KO, H-2b) mice lacking T, B, and natural killer (NK) cells. In order to facilitate LSEC engraftment, the RAG2/gc-KO mice were injected intraperitoneally with monocrotaline 2 days before the adoptive transfer of LSECs; this impaired the host LSECs, conferring a proliferative advantage to the transplanted LSECs. After orthotopic allogeneic LSEC engraftment, the RAG2/gc-KO mice were immune reconstituted intravenously with C57BL/6 splenocytes. After immune reconstitution, mixed lymphocyte reaction (MLR) assay using splenocytes from the recipients revealed that specific inhibition of host CD4+ and CD8+ T-cell proliferation was greater in response to allostimulation with irradiated BALB/cA splenocytes rather than to stimulation with irradiated third party SJL/jorllco splenocytes. This inhibitory effect was attenuated by administering anti-programmed death ligand 1 (PD-L1) monoclonal antibody during immune reconstitution in the above-mentioned mice, but not in RAG2/gc-KO mice engrafted with Fas ligand (FasL)-deficient BALB/cA LSECs. Furthermore, engraftment of allogeneic BALB/cA LSECs significantly prolonged the survival of subsequently grafted cognate allogeneic BALB/cA hearts in RAG2/gc-KO mice immune reconstituted with bone marrow transplantation from C57BL/6 mice. In conclusion, murine LSECs have been proven capable of suppressing T-cells with cognate specificity for LSECs in an in vivo model. The programmed death 1/PD-L1 pathway is likely involved in these suppressive effects.

Kinetics of Cellular and Humoral Immunity in a Successful Case of Positive Crossmatch Kidney Transplantation: A Case Report

A positive crossmatch remains one of the major barriers to successful kidney transplantation. Highly sensitized patients are at greater risk of hyperacute rejection and subsequent graft loss after transplantation. Although recent advances in desensitization therapy allow kidney transplantation in these patients, the success rate is quite low. Herein, we have reported a successful case of positive crossmatch living donor kidney transplantation using a desensitization protocol with an immune monitoring assay. A 42-year-old woman with end-stage renal disease due to IgA nephropathy had been on hemodialysis for 36 months. She showed positive T-cell and B-cell cytotoxic crossmatches with her husband owing to pretransplantation blood transfusions. We performed a preconditioning regimen comprising a single dose of rituximab (375 mg/m(2)) combined with double-filtration plasmapheresis (DFPP) followed by low doses of intravenous immunoglobulin (DFPP/IVIG treatment). Tacrolimus (target trough level, 5-10 ng/mL) and mycophenolate mofetil (1500 mg/body) were started 2 weeks before the DFPP/IVIG treatment. After 6 DFPP/IVIG sessions, the crossmatch became negative. An induction quadruple immunosuppression protocol included tacrolimus, mycophenolate mofetil, basiliximab, and methylprednisolone. After the transplantation, the patients immune status was evaluated regularly by mixed lymphocyte reactions (MLR) using an intracellular carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeling technique (CFSE-MLR assay) and immunosuppressant therapy was adjusted accordingly. During the observation period, neither antibody-mediated rejection nor acute cellular rejection was encountered in this patient.

Impact of adjuvant immunotherapy using liver allograft-derived lymphocytes on bacteremia in living-donor liver transplantation.

Background. Bacteremia is one of the leading causes of mortality in living-donor liver transplant (LDLT) recipients. Lymphocytes, including natural killer cells, are believed to play a role in the first line of defense against invading infectious microbes. Methods. From January 2004 to December 2009, 114 consecutive LDLT recipients were studied for postoperative bacteremia. The impact of adjuvant immunotherapy using activated liver allograft-derived lymphocytes on bacteremia was retrospectively evaluated by a one-to-one match using propensity score to overcome bias due to the different distribution of covariates for the two groups. Results. After one-to-one matching, 21 patients who did not receive adjuvant immunotherapy and 21 who did not receive adjuvant immunotherapy had the same preoperative and operative characteristics. Six (28.6%) of the 21 patients who did not receive adjuvant immunotherapy had bacteremia, whereas only one (4.8%) of the 21 patients who received adjuvant immunotherapy had bacteremia; thus, the incidence of bacteremia in patients who had received adjuvant immunotherapy was significantly lower than that in patients who had not received adjuvant immunotherapy (P=0.038). Conclusions. Adjuvant immunotherapy using liver allograft-derived lymphocytes may be a promising modality for reducing the postoperative bacteremia after LDLT.

Complex Vascular Reconstruction Using Donor's Vessel Grafts in Orthotopic Liver Transplantation

The vascular abnormalities of recipients are associated with reconstructive difficulties with an increased risk of postoperative complications. We performed an orthotopic liver transplantation that required a complex vascular reconstruction using donor vascular grafts. A patient with hepatitis B virus cirrhosis received a liver from a brain-dead donor. Dynamic computed tomography revealed complete obstruction of the portal vein due to thrombosis as well as narrowing of the hepatic arteries. We employed orthotopic liver transplantation using the piggy-back technique with complex reconstruction of the portal vein and the hepatic arteries. For portal vein reconstruction, we used the donors iliac vein as an interpositional conduit from the recipients gastric coronary vein to graft the portal vein. The hepatic arteries of the graft were reconstructed at the back-table before anastomosis to the side of superior mesenteric artery using an interpositional conduit of the donors external iliac artery. All postoperative studies revealed good graft function with an excellent blood flow through all vascular anastomoses during the first year postoperatively.

Evidence for the Immunosuppressive Potential of Calcineurin Inhibitor-Sparing Regimens in Liver Transplant Recipients with Impaired Renal Function

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.