Ohira M, Ishiyama K, Tanaka Y, Doskali M, Igarashi Y, Tashiro H, et al. Adoptive immunotherapy with liver allograft-derived lymphocytes induces anti-HCV activity after liver transplantation in humans and humanized mice. J Clin Invest 2009;119:3226-3235. (Reprinted with permission of the American Society for Clinical Investigation; permission conveyed through Copyright Clearance Center, Inc.)
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After liver transplantation in HCV-infected patients, the virus load inevitably exceeds pretransplantation 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. © 2010 American Society for Clinical Investigation.
End-stage liver disease and hepatocellular carcinoma (HCC) related to chronic hepatitis C virus (HCV) infection have become the principal indication for liver transplantation.1 Unfortunately, HCV infection universally recurs after transplantation, due to reinfection of the liver graft by circulating virions within a few days after transplant.2 Posttransplant HCV recurrence is associated with accelerated progression to cirrhosis, HCC, graft loss, retransplantation, and death. The best way to prevent posttransplant HCV recurrence is to eradicate the virus prior to transplantation. However, the combination of pegylated interferon alpha (IFN-α) and ribavirin is difficult to use in this context, because most patients have end-stage liver disease and do not tolerate this therapy well. Pegylated IFN-α and ribavirin can also be used after transplantation. Nevertheless, the viral eradication rates are only of the order of 25%-45%, due to multiple factors including high viral levels, immune suppression, poor tolerance, and the risk of graft rejection which leads to dose reductions or early treatment interruptions.1
In the November 11 issue of The Journal of Clinical Investigation, Ohira et al. report an interesting immunotherapeutic approach for preventing posttransplant HCV recurrence, based on adoptive transfer of interleukin 2 (IL-2)/anti-CD3 monoclonal antibody (OKT3)-treated liver allograft–derived lymphocyte pools enriched in natural killer (NK) and natural killer T (NKT) cells.3 The study was a phase 1 clinical trial in which 14 patients with cirrhosis who have HCC, including seven patients with chronic HCV genotype 1 infection, six patients with chronic hepatitis B virus (HBV) infection, and one uninfected patient were treated with liver allograft-derived lymphocytes. Five patients with cirrhosis who had chronic HCV infection served as controls. The primary endpoint of the study was prevention of HCC recurrence. Liver-resident lymphocytes were extracted from the donors' liver graft perfusates. These lymphocytes harbored substantially more NK and NKT cells than peripheral lymphocytes from the same donors. Liver-resident lymphocytes were cultured 3 days with IL-2, and OKT3 was added to the culture medium 1 day before administration in order to prevent graft-versus-host disease by inactivating CD3+ alloreactive T cells. The activated liver NK cell–enriched lymphocytes were then administered to the liver transplant recipients in a single intravenous injection. The administration of IL-2/OKT3-treated liver lymphocytes was well tolerated. After a mean follow-up of 23.4 months (range, 10.7-32.9 months), the 14 liver transplant recipients were alive without recurrence of HCC, whereas their circulating lymphocytes showed significantly enhanced cytotoxicity against HepG2 cells, an HCC cell line, as compared to the untreated controls.
Because the study was not firmly conclusive regarding prevention of HCC, the authors decided to focus on the seven HCV-infected patients who received liver allograft–derived lymphocytes and the five HCV-infected controls. During the first months after liver transplantation, HCV RNA levels were significantly lower on average in the treated recipients than in the controls. HCV RNA became undetectable (<50 international units [IU]/mL) after immunotherapy in two treated patients 4 weeks after liver transplantation, but in none of the controls. In one of the treated patients, HCV RNA was still undetectable 20 months after liver transplantation, whereas HCV infection recurred 2 months after transplantation in the second one.
In order to assess the anti-HCV activity of IL-2/OKT3-treated liver lymphocytes in vitro, the same cells were cocultured with hepatic cells containing HCV replicons. Treatment of liver lymphocytes with IL-2 and OKT3 strongly promoted their anti-HCV activity, which appeared to be at least in part dependent on the ability of both NK and NKT cells to secrete IFN-γ. This finding was in keeping with the observation that, 2 weeks after liver transplantation, the number of IFN-γ–secreting cells in peripheral blood was significantly higher in the lymphocyte-treated than in the control liver transplant recipients.
In order to confirm that IL-2/OKT3-treated liver lymphocytes have anti-HCV activity in vivo, additional experiments were performed in the chimeric urokinase-type plasminogen activator–severe combined immunodeficient (uPA-SCID) mouse model with engrafted human hepatocytes (“humanized” mouse) infected with HCV. In this model, intraperitoneal injection of IL-2/OKT3-treated liver lymphocytes 2 weeks after HCV inoculation prevented the development of HCV infection. This effect was partially countered by anti-IFN-γ–neutralizing antibodies, a finding that confirmed the important role played by this cytokine in the observed anti-HCV activity. No toxicity on human hepatocytes was observed in the model.
The authors concluded that adoptive immunotherapy using IL-2/OKT3-treated liver lymphocytes containing abundant NK and NKT cells could mount strong anti-HCV responses in HCV-infected liver transplant recipients and could be proposed as a useful therapy to prevent posttransplant HCV recurrence.3
NK cells are an important component of the innate immune response due to their ability to lyse virus-infected cells and to activate and recruit cells involved in adaptive immune responses. The ability of IFN-γ produced by NK cells to partially control HCV replication via a noncytolytic mechanism has been demonstrated.4, 5 However, recombinant IFN-γ administered to HCV-infected patients failed to significantly reduce HCV replication. The role of NKT cells in controlling HCV replication remains unclear. In this context, the results presented by Ohira et al., which suggest that IL-2/OKT3-treated liver lymphocytes from donor livers containing abundant NK and NKT cells are able to partially (and inconstantly) control HCV replication after liver transplantation through a noncytolytic mechanism at least partly mediated by IFN-γ, are not fully surprising. Can these properties be used to develop therapies based on adoptive transfer of immunity for the prevention of HCV recurrence after liver transplantation?
Adoptive immunity has been proposed as a therapy for cancers and, more recently, for HBV infection. Cancer development is under close surveillance by NK cells and other components of the immune system, but cancer cells have developed counterattack strategies against immune effectors.6 IL-2 activation of NK cells results in enhanced cytotoxic activity against cancer cells, including cells that were resistant to NK cells. As a result, IL-2 has been used as an immunostimulant in patients with advanced forms of cancer.7 The effect of IL-2 on NK cell activity can be further enhanced by a number of other factors. Adoptive transfer studies have been performed in animal models and in patients with advanced cancers. Although some efficacy has been reported in animals, studies assessing adoptive transfer of autologous and allogeneic NK cells for cancer immunotherapy have been disappointing, showing inconstant and often modest antitumor effects.6
Adoptive transfer of immunity against HBV has also been recently considered as a potential tool to control chronic infection. Animal and human studies have shown that immunity to HBV could be adoptively transferred in the context of immune suppression.8 Several reports suggest that it is possible to achieve sustained control of HBV infection through adoptive transfer of immunity to HBV from anti-hepatitis B surface antigen–positive donors to immunosuppressed HBV-infected recipients.8
HCV recurrence is responsible for high morbidity and mortality after liver transplantation.1 Therefore, prevention of posttransplant HCV-related liver disease is a priority. One approach is to treat HCV recurrence after transplantation and try to control or, better, eradicate infection. The use of pegylated IFN-α and ribavirin has yielded disappointing results, because liver transplant recipients represent a difficult-to-treat population.1 Whether the results of pegylated IFN-α and ribavirin therapy could be improved by combining it with approaches such as that proposed by Ohira et al. remains to be explored. It should be emphasized, however, that in their study, only two patients out of seven were able to control HCV infection, one of them delaying HCV recurrence by a few weeks only. The HCV RNA level reductions were modest in the remaining patients, and transient in most cases.3 In addition, the procedure is cumbersome, because liver lymphocytes have to be recovered from liver perfusates and cultured for 3 days in the presence of IL-2 and OKT3 before being injected into the patients. The ideal number and rhythm of injections also remain to be determined.
A better approach is to eradicate infection before transplantation, as definitive HCV clearance is associated with no likelihood of graft reinfection. Again, pegylated IFN-α and ribavirin therapy has been disappointing in this setting, because most candidates for liver transplantation have advanced liver disease and poor predictors of sustained viral eradication.1 The current development of potent direct-acting antiviral (DAA) drugs should radically change this situation.9–12 These drugs are indeed able to profoundly inhibit HCV replication upon administration, and combining several DAAs that share no cross-resistance has been shown to efficiently prevent virological breakthroughs due to viral resistance.10, 13, 14 It is thus conceivable that, even if the liver still harbors HCV-containing cells, viral production can be shut down to such a low level that no virions are exported in peripheral blood. In that case, removing a still-infected liver and transplanting the new liver into a virus-free environment should considerably reduce the incidence of HCV recurrence after transplantation. If small residual amounts of circulating HCV virions are present and still able to reinfect the liver, adjunctive therapy aimed at preventing hepatocyte reinfection may be necessary. Coverage of the peritransplant and posttransplant period with the same cocktail of DAAs must be recommended but may not be sufficient. Additional inhibition of virion entry into cells by means of polyclonal immunoglobulins, monoclonal antibodies, or small molecule inhibitors may be needed. Approaches based on immune therapy, such as adoptive transfer of immunity, could also be of value in this context. The current and future options to prevent HCV recurrence after liver transplantation are shown in Fig. 1. They are not mutually exclusive.
Studies combining the administration of cocktails of DAAs in the pretransplant and early posttransplant period with posttransplant adoptive immunotherapy should now be conducted in HCV-infected patients awaiting liver transplantation in order to determine the ideal schedule of administration. In this respect, the report by Ohira et al. provides invaluable information on the type of cell preparations that should be used and the expected effect.3 The goal of 0% HCV recurrence after liver transplantation is achievable; the combination of potent direct-acting antiviral therapy and posttransplant adoptive immunotherapy appears as a valuable option.