Frank Lehner

A New Population of Myeloid-Derived Suppressor Cells in Hepatocellular Carcinoma Patients Induces CD4+CD25+Foxp3+ T Cells

BACKGROUND & AIMS Several studies have shown that development of hepatocellular carcinoma (HCC) generates a number of immune suppressive mechanisms in these patients. Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of cells that have been shown to inhibit T-cell responses in tumor-bearing mice, but little is known about these cells in humans owing to a lack of specific markers. In this study, we have investigated the frequency and function of a new population of MDSC denoted here as CD14(+)HLA-DR(-/low) in HCC patients. We have also identified a novel, MDSC-mediated immune regulatory pathway in these patients. METHODS We have directly isolated and characterized MDSCs for phenotype and function from peripheral blood (n = 111) and tumor (n = 12) of patients with HCC. RESULTS The frequency of CD14(+)HLA-DR(-/low) cells in peripheral blood mononuclear cells (PBMC) from HCC patients was significantly increased in comparison with healthy controls. CD14(+) HLA-DR(-/low) cells were unable to stimulate an allogeneic T-cell response, suppressed autologous T-cell proliferation, and had high arginase activity, a hallmark characteristic of MDSC. Most important, CD14(+)HLA-DR(-/low) cells from HCC patients induced a CD4(+)CD25(+)Foxp3(+) regulatory T-cell population when cocultured with autologous T cells. CONCLUSION CD14(+)HLA-DR(-/low) cells are a new population of MDSC increased in blood and tumor of HCC patients. We propose a new mechanism by which MDSC exert their immunosuppressive function, through the induction of CD4(+)CD25(+)Foxp3(+) regulatory T cells in cocultured CD4(+) T cells. Understanding the mechanism of action of MDSC in HCC patients is important in the design of effective immunotherapeutic protocols.

Myeloid Derived Suppressor Cells Inhibit Natural Killer Cells in Patients with Hepatocellular Carcinoma via the NKp30 Receptor

Several immune suppressive mechanisms that evade the host immune response have been described in patients with hepatocellular carcinoma (HCC); one of these mechanisms is expansion of myeloid‐derived suppressor cells (MDSCs). MDSCs have been shown to inhibit T cell responses in tumor‐bearing mice, but little is known about these cells in humans. Here, we have analyzed and characterized the effect of MDSCs on the innate immune system, in particular, their interaction with natural killer (NK) cells in patients with HCC. MDSCs from patients with HCC inhibited autologous NK cell cytotoxicity and cytokine secretion when cultured together in vitro. This suppression was dependent on cell contact, but did not rely on the arginase activity of MDSCs, which is a hallmark function of these cells. However, MDSC‐mediated inhibition of NK cell function was dependent mainly on the NKp30 on NK cells. Conclusion: Our study suggests a new role for MDSCs in patients with HCC in disarming the innate immune system and further contributing to the immune suppressor network in these patients. These findings have important implications when designing immunotherapy protocols. (HEPATOLOGY 2009.)

Hepatitis E virus infection as a cause of graft hepatitis in liver transplant recipients

Hepatitis E virus (HEV) infection induces self‐limiting liver disease in immunocompetent individuals. Cases of chronic hepatitis E have recently been identified in organ transplant recipients. We questioned if chronic hepatitis E plays a role in graft hepatitis after liver transplantation in a low endemic area. Two hundred twenty‐six liver transplant recipients, 129 nontransplanted patients with chronic liver disease, and 108 healthy controls were tested for HEV antibodies. HEV RNA was investigated in all sera from transplanted patients. HEV antibodies were detected in 1 healthy control (1%), 4 patients with chronic liver disease (3%), and 10 liver transplant recipients (4%). Three liver transplant patients also tested positive for HEV RNA. Two of them developed persistent viremia with HEV genotype 3. The patients were anti‐HEV immunoglobulin G–negative and HEV RNA–negative before transplantation and had an episode of acute hepatitis 5 or 7 months after transplantation, which led to advanced liver fibrosis after 22 months in 1 patient. Seroconversion to anti‐HEV occurred not before 4 months after the first detection of HEV RNA. The possibility of reverse zoonotic transmission was experimentally confirmed by the infection of 5 pigs with a patients serum. The pigs showed histological inflammation in the liver, and HEV RNA was detectable in different organs, including muscle. In conclusion, the prevalence of HEV infection in Central European liver transplant recipients is low; however, chronic hepatitis E may occur and needs to be considered in the differential diagnosis of graft hepatitis. The diagnosis of HEV infection should be based on HEV RNA determination in immunosuppressed patients. We suggest that immunocompromised individuals should avoid eating uncooked meat and contact with possibly HEV‐infected animals. Liver Transpl 16:74–82, 2010.

Increased hepatotoxicity of tumor necrosis factor-related apoptosis-inducing ligand in diseased human liver

Tumor necrosis factor–related apoptosis‐inducing ligand (TRAIL) induces apoptosis in tumor cells but not in most normal cells and has therefore been proposed as a promising antitumor agent. Recent experiments suggested that isolated primary human hepatocytes but not monkey liver cells are susceptible to certain TRAIL agonists, raising concerns about the use of TRAIL in cancer treatment. Whether TRAIL indeed exerts hepatotoxicity in vivo and how this is influenced by chemotherapeutic drugs or liver disease are completely unknown. Employing different forms of recombinant TRAIL, we found that the cytokine can induce proapoptotic caspase activity in isolated human hepatocytes. However in marked contrast, these different TRAIL preparations induced little or no cytotoxicity when incubated with tissue explants of fresh healthy liver, an experimental model that may more faithfully mimic the in vivo situation. In healthy liver, TRAIL induced apoptosis only when combined with histone deacetylase inhibitors. Strikingly, however, TRAIL alone triggered massive apoptosis accompanied by caspase activation in tissue explants from patients with liver steatosis or hepatitis C viral infection. This enhanced sensitivity of diseased liver was associated with an increased expression of TRAIL receptors and up‐regulation of proapoptotic Bcl‐2 proteins. Conclusion: These results suggest that clinical trials should be performed with great caution when TRAIL is combined with chemotherapy or administered to patients with inflammatory liver diseases. (HEPATOLOGY 2007.)

A Randomized, Controlled Study to Assess the Conversion From Calcineurin-Inhibitors to Everolimus After Liver Transplantation—PROTECT

Posttransplant immunosuppression with calcineurin inhibitors (CNIs) is associated with impaired renal function, while mTor inhibitors such as everolimus may provide a renal‐sparing alternative. In this randomized 1‐year study in patients with liver transplantation (LTx), we sought to assess the effects of everolimus on glomerular filtration rate (GFR) after conversion from CNIs compared to continued CNI treatment. Eligible study patients received basiliximab induction, CNI with/without corticosteroids for 4 weeks post‐LTx, and were then randomized (if GFR > 50 mL/min) to continued CNIs (N = 102) or subsequent conversion to EVR (N = 101). Mean calculated GFR 11 months postrandomization (ITT population) revealed no significant difference between treatments using the Cockcroft‐Gault formula (−2.9 mL/min in favor of EVR, 95%‐CI: [−10.659; 4.814], p = 0.46), whereas use of the MDRD formula showed superiority for EVR (−7.8 mL/min, 95%‐CI: [−14.366; −1.191], p = 0.021). Rates of mortality (EVR: 4.2% vs. CNI: 4.1%), biopsy‐proven acute rejection (17.7% vs. 15.3%), and efficacy failure (20.8% vs. 20.4%) were similar. Infections, leukocytopenia, hyperlipidemia and treatment discontinuations occurred more frequently in the EVR group. No hepatic artery thrombosis and no excess of wound healing impairment were noted. Conversion from CNI‐based to EVR‐based immunosuppression proved to be a safe alternative post‐LTx that deserves further investigation in terms of nephroprotection.

Caspase activation is associated with spontaneous recovery from acute liver failure.

Acute liver failure (ALF) has various causes and is characterized by rapid hepatocyte dysfunction with development of encephalopathy in the absence of preexisting liver disease. Whereas most patients require liver transplantation to prevent the high mortality, some patients recover spontaneously and show complete liver regeneration. Because of the low incidence of ALF, however, the molecular mechanisms of liver dysfunction and regeneration are largely unknown. In this study, we investigated the role of apoptosis and caspases in 70 ALF patients using novel biomarkers that allow the detection of caspase activation in serum samples. Compared with healthy individuals, activation of caspases was strongly enhanced in ALF patients. Interestingly, patients with spontaneous recovery from ALF revealed a significantly higher activation of caspases than patients that required transplantation or died, although in the latter patients extensive DNA fragmentation and signs of nonapoptotic cell death were observed. In the spontaneous survivors, increased caspase activation was accompanied by elevated levels of tumor necrosis factor alpha (TNF‐α) and interleukin‐6 (IL‐6), important cytokines involved in liver regeneration. Conclusion: Our data suggest that caspase activation and apoptosis are involved in ALF of patients with spontaneous recovery, whereas caspase‐independent cell death might be more relevant in irreversible forms of liver failure. These findings might be important for therapeutic options of ALF but also suggest that measurement of caspase activation might be of prognostic value to predict the outcome of acute liver failure. (HEPATOLOGY 2008.)

Increase of infectious complications in ABO-incompatible kidney transplant recipients—a single centre experience

BACKGROUND Due to the shortage of deceased donors ABO-incompatible (ABOi) living kidney transplantation has become a popular alternative to deceased kidney transplantation. In recent years, recipient desensitization with a combination of anti-CD20 treatment (rituximab), antigen-specific immunoadsorptions (IA) and intravenous immunoglobulin (IVIG), led to promising short-term and intermediate-term results. However, little is known about the impact of this intensified desensitization protocol on the risk of surgical and infectious complications. METHODS We retrospectively analysed 21 consecutive recipients who underwent ABOi renal transplantation. Pre-transplant desensitization included administration of rituximab (375 mg/m(2)), mycophenolate mofetil (MMF), tacrolimus and prednisolone 4 weeks prior of scheduled transplantation as well as IA and IVIG. Forty-seven patients who underwent ABO-compatible (ABOc) renal transplantation served as the control group. Medical records and electronic databases were reviewed for patient and graft survival, renal function, rate of rejections, viral and bacterial infections as well as for surgical complications (SCs) post-transplantation. RESULTS All patients showed an immediate graft function. During a mean follow-up of 15.7 ± 8.3 months (interquartile range 11.9) patient survival was 95 and 98% in the ABOi and ABOc group, respectively. Allograft survival and function, as assessed by serum creatinine levels and calculated glomerular filtration rate at 1 year, did not differ between ABOi and ABOc recipients. Furthermore, the rate of biopsy-proven acute rejections was comparable between the two groups. However, there was a trend towards more SCs within the ABOi group (29 versus 11%, non-significant). In addition, the rate of viral infections including cytomegalovirus, Herpes simplex virus, Varicella zoster virus and polyoma virus was significantly increased among the ABOi recipients (50 versus 21%; P = 0.038) despite comparable tacrolimus trough levels and MMF and steroid doses. CONCLUSIONS Our results, in line with the extended experience of other groups, demonstrate favourable short-term allograft survival and function after ABOi renal transplantation after desensitization with antigen-specific IA, IVIG and rituximab. However, the intensified desensitization was associated with an increased risk of infectious complications. This observation prompted us to briefly escalate the desensitization protocol in ABOi kidney recipients in our centre.

Five-year outcomes in kidney transplant patients converted from cyclosporine to everolimus: the randomized ZEUS study.

ZEUS study was an open‐label, 12‐month, multicenter study in which 300 de novo kidney transplant recipients were randomized to continue receiving cyclosporine (CsA) or convert to everolimus at 4.5 months posttransplant. Five‐year follow‐up data were available for 245/269 patients (91.1%) who completed the core 12‐month study (123 everolimus, 109 CsA). At 5 years, adjusted estimated GFR was 66.2 mL/min/1.73 m2 with everolimus versus 60.9 mL/min/1.73 m2 with CsA; the mean difference was 5.3 mL/min/1.73 m2 in favor of everolimus (95% CI 2.4, 8.3; p < 0.001 [intent‐to‐treat population]). In a post hoc analysis of patients remaining on study drug at 5 years (everolimus 77, CsA 86), mean difference was 8.2 mL/min/1.73 m2 (95% CI 4.3, 12.1; p < 0.001) in favor of everolimus. The cumulative incidence of biopsy‐proven acute rejection postrandomization was 13.6% with everolimus versus 7.5% with CsA (p = 0.095), largely accounted for by grade I rejection (16/21 patients and 7/11 patients, respectively). Postrandomization, graft loss, mortality, serious adverse events and neoplasms were similar in both arms. In conclusion, conversion of kidney transplant patients to everolimus at 4.5 months posttransplant is associated with a significant improvement in renal function that is maintained to at least 5 years. The increase in early mild acute rejection did not affect long‐term graft function.

Conversion from cyclosporine to everolimus at 4.5 months posttransplant: 3-year results from the randomized ZEUS study.

The long‐term effect of conversion from calcineurin inhibitor (CNI) therapy to an mTOR inhibitor requires clarification. Following completion of the 12‐month, open‐label, multicenter ZEUS study, in which 300 kidney transplant recipients were randomized to continue cyclosporine (CsA) or convert to everolimus at 4.5 months posttransplant, outcomes were assessed at month 36 (n = 284; 94.7%). CNI therapy was reintroduced in 28.4% of everolimus patients by month 36. The primary efficacy endpoint, estimated glomerular filtration rate (Nankivell, ANCOVA) was significantly higher with everolimus versus the CsA group at month 24 (7.6 mL/min/1.73 m2, 95%CI 4.3, 11.0 mL/min/1.73 m2; p < 0.001) and month 36 (7.5 mL/min/1.73 m2, 95%CI 3.6, 11.4 mL/min/1.73 m2; p < 0.001). The incidence of biopsy‐proven acute rejection from randomization to month 36 was 13.0% in the everolimus arm and 4.8% in the CsA arm (p = 0.015). Patient and graft survival, as well as incidences of malignancy, severe infections and hospitalization, were similar between groups. Kidney transplant patients who are converted from CsA to everolimus at month 4.5 and who remain on everolimus thereafter may achieve a significant improvement in renal function that is maintained to 3 years. There was a significantly higher rate of rejection in the everolimus arm but this did not exert a deleterious effect by 3 years posttransplant.

Successful Outcome Of Acute Graft-versus-host Disease In A Liver Allograft Recipient By Withdrawal Of Immunosuppression

BACKGROUND Graft-versus-host disease (GVHD) after liver transplantation is uncommon, and the outcome is almost always fatal. Since 1987, about 30 cases have been described, and patient survival is mostly exceptional. METHODS A 29-year-old man underwent retransplantation due to chronic cholestatic syndrome, 5 years after his first liver transplantation. Indication for the first liver transplantation was acute liver failure caused by exsiccosis. After the second transplantation, the patient had an initially uneventful course, developing thrombocytopenia at day 21 followed by skin rash and septic complications. Diagnosis of acute GVHD was made by using serological techniques for HLA-A and HLA-DRB and subsequently by fluorogenic sequence-specific primed polymerase chain reaction. In addition, donor lymphocytes were marked by immunohistochemical methods via biopsies of the skin. Immunosuppressive therapy was withdrawn to allow the patients own immune system to eliminate donor cells. RESULTS By withdrawing the immunosuppressive therapy, clinical and morphological signs of GVHD vanished. The patient is doing well without recurrence 13 months after transplantation. CONCLUSION Withdrawal of immunosuppressive therapy is a promising approach in the treatment of acute GVHD to allow the patients immune system to reconstitute itself, reject offending lymphocytes, and avoid lethal septic complications.