On the impact of hepatitis C virus and heterologous immunity on alloimmune responses following liver transplantation.

Virus-induced heterologous immunity is considered a barrier to transplantation tolerance. Yet, hepatitis C (HCV)-infected liver transplant (LT) patients occasionally achieve operational tolerance. We investigated the mechanisms through which HCV infection modulates donor-specific T cell responses following LT and the influence of HCV eradication. We generated T cell lines from HCV-infected LT and non-LT patients before and after HCV eradication and quantified alloreactive responses using cell lines expressing single-HLA class-I antigens in the presence/absence of PD-1/CTLA-4 blockade. HCV-specific CD8+ T cells cross-reacted with allogeneic class-I HLA molecules. HCV-positive LT recipients exhibited a higher proportion of CD8+ T cells coexpressing inhibitory receptors (PD-1/CTLA4) than HCV-negative LT, and their expression correlated with CXCL10 plasma levels. This resulted in decreased antidonor and third-party proliferative responses, which were significantly reversed by HCV eradication. PD-1/CTLA-4 blockade increased the proportion of HCV-specific CD8+ T cells reacting against donor only before viral clearance. In conclusion, HCV infection results in the generation of HCV-specific CD8+ T cells capable of reacting against allogeneic HLA molecules. Following LT, this results in a PD-1/CTLA4-dependent decrease in alloimmune responses. Our findings challenge the notion that heterologous immunity is necessarily detrimental in LT and provide an explanation for the association between HCV eradication and immune-mediated allograft damage.

Applicability, safety, and biological activity of regulatory T cell therapy in liver transplantation.

Regulatory T cells (Tregs) are a lymphocyte subset with intrinsic immunosuppressive properties that can be expanded in large numbers ex vivo and have been shown to prevent allograft rejection and promote tolerance in animal models. To investigate the safety, applicability, and biological activity of autologous Treg adoptive transfer in humans, we conducted an open-label, dose-escalation, Phase I clinical trial in liver transplantation. Patients were enrolled while awaiting liver transplantation or 6-12 months posttransplant. Circulating Tregs were isolated from blood or leukapheresis, expanded under good manufacturing practices (GMP) conditions, and administered intravenously at either 0.5-1 million Tregs/kg or 3-4.5 million Tregs/kg. The primary endpoint was the rate of dose- limiting toxicities occurring within 4 weeks of infusion. The applicability of the clinical protocol was poor unless patient recruitment was deferred until 6-12 months posttransplant. Thus, only 3 of the 17 patients who consented while awaiting liver transplantation were dosed. In contrast, all six patients who consented 6-12 months posttransplant received the cell infusion. Treg transfer was safe, transiently increased the pool of circulating Tregs and reduced anti-donor T cell responses. Our study opens the door to employing Treg immunotherapy to facilitate the reduction or complete discontinuation of immunosuppression following liver transplantation.

IL-2 therapy restores regulatory T-cell dysfunction induced by calcineurin inhibitors.

CD4+CD25+FOXP3+ Tregs constitute a heterogeneous lymphocyte subpopulation essential for curtailing effector T cells and establishing peripheral tolerance. Calcineurin inhibitors (CNIs) are among the most effective agents in controlling effector T-cell responses in humans. However, CNIs also reduce the size of the Treg pool. The functional consequences of this negative effect and the mechanisms responsible remain to be elucidated. We report here that CNIs compromise the overall Treg immunoregulatory capacity to a greater extent than would be predicted by the reduction in the size of the Treg compartment, given that they selectively promote the apoptosis of the resting and activated Treg subsets that are known to display the most powerful suppressive function. These effects are caused by reduced access to IL-2, because Tregs remain capable of translocating NFAT even in the presence of high CNI levels. Exogenous IL-2 restores the phenotypic changes and overall gene-expression effects exerted by CNIs and can even promote Treg expansion by enhancing antiapoptotic Bcl-2 expression. In a skin transplant model, the addition of IL-2 synergizes with CNIs treatment, promoting intragraft accumulation of Tregs and prolonged allograft survival. Hence, the combination of IL-2 and CNIs constitutes an optimal immunomodulatory regimen that enhances the pool of suppressive Treg subsets while effectively controlling cytopathic T cells.

Regulatory T-cell therapy in liver transplantation.

Modern immunosuppression drug regimens have produced excellent short-term survival after liver transplantation but it is generally accepted that the side effects of these medications remain a significant contributing factor for less satisfactory long term outcomes. The liver has unique tolerogenic properties as evidenced by the higher rates of operational tolerance seen in liver transplant recipients compared to other solid organ transplants, and therefore, liver transplantation offers an attractive setting in which to study tolerizing therapies. CD4+ CD25+ FOXP3+ regulatory T cells (Tregs) are crucial for maintenance of self-tolerance and prevention of autoimmune disease and are therefore an appealing potential candidate for use as a tolerizing cell therapy. In this review, we summarize the evidence from drug withdrawal trials of spontaneous operational tolerance in liver transplantation, the unique immunology of the hepatic microenvironment, the evidence for the use of CD4+ CD25+ FOXP3+ regulatory T cells as a tolerance inducing therapy in liver transplantation and the challenges in producing clinical grade Treg cell products.

Prospective multicenter clinical trial of immunosuppressive drug withdrawal in stable adult liver transplant recipients.

UNLABELLED: Lifelong immunosuppression increases morbidity and mortality in liver transplantation. Discontinuation of immunosuppressive drugs could lessen this burden, but the safety, applicability, and clinical outcomes of this strategy need to be carefully defined. We enrolled 102 stable liver recipients at least 3 years after transplantation in a single-arm multicenter immunosuppression withdrawal trial. Drugs were gradually discontinued over a 6 to 9-month period. The primary endpoint was the development of operational tolerance, defined as successful immunosuppressive drug cessation maintained for at least 12 months with stable graft function and no histopathologic evidence of rejection. Out of the 98 recipients evaluated, 57 rejected and 41 successfully discontinued all immunosuppressive drugs. In nontolerant recipients rejection episodes were mild and resolved over 5.6 months (two nontolerant patients still exhibited mild gradually improving cholestasis at the end of follow-up). In tolerant recipients no progressive clinically significant histological damage was apparent in follow-up protocol biopsies performed up to 3 years following drug withdrawal. Tolerance was independently associated with time since transplantation (odds ratio [OR] 1.353; P = 0.0001), recipient age (OR 1.073; P = 0.009), and male gender (OR 4.657; P = 0.016). A predictive model incorporating the first two clinical variables identified subgroups of recipients with very high (79%), intermediate (30%-38%), and very low (0%) likelihood of successful withdrawal. CONCLUSION: When conducted at late timepoints after transplantation, immunosuppression withdrawal is successful in a high proportion of carefully selected liver recipients. A combination of clinical parameters could be useful to predict the success of this strategy. Additional prospective studies are now needed to confirm these results and to validate clinically applicable diagnostic biomarkers.

A Very Short Course of HBIg+NA Followed by Entecavir or Tenofovir Monotherapy Prevents HBV Recurrence in Low-Risk Liver Transplant Recipients.

BACKGROUND: Monoprophylaxis with third-generation nucleos(t)ide analogues (NAs) can be safely adopted in hepatitis B virus (HBV)-positive, liver transplantation (LT) patients after at least 6 months of HBV immunoglobulin (HBIg)+NA. We investigated the efficacy of earlier initiation of post-LT entecavir (ETV) or tenofovir (TDF) monoprophylaxis. METHODS: Between September 2011 and January 2017, all consecutive hepatitis B surface antigen (HBsAg)-positive transplanted patients were scheduled to receive HBIg with ETV or TDF for a period related to the risk for HBV reinfection: 1. low-risk patients (HBeAg-negative and HBV DNA < 12 IU/mL before LT) were due to withdraw from HBIg once HBsAg had become negative after a minimum of 7 days of HBIg+NA; 2. high-risk patients were due to receive HBIg for at least 6 months, after which they continued with third-generation NA monotherapy, only. RESULTS: Twenty patients with a median interquartile range (IQR) follow-up of 46 (64-39) months were enrolled in the study (40% receiving ETV, 60% receiving TDF). Two low-risk patients refused early HBIg withdrawal and were therefore treated and analyzed along with the high-risk group. Eventually, there were 2 groups: group A, which included 12 low-risk patients, and group B, which included 8 patients (six high-risk, 2 low-risk). After transplantation, group A and B patients received HBIg+NA for a median (IQR) time of 7 (9-7) days and 9 (13-5) months, respectively. All 20 recipients demonstrated HBV DNA < 12 IU/mL and stable graft function during follow-up. Two patients (10%), 1 from each group, had HBsAg relapse. Notably, both patients who relapsed had hepatocellular carcinoma (HCC) diagnosed before LT and showed very low levels (< 0.25 IU/mL) of HBsAg after recurrence. CONCLUSION: In low-risk HBsAg-positive recipients, HBIg may be safely discontinued within 2 weeks of LT and replaced by ETV or TDF monotherapy.

Multiparametric Analysis of Circulating Exosomes and Other Small Extracellular Vesicles by Advanced Imaging Flow Cytometry.

Extracellular vesicles (EVs) are responsible for a multitude of physiological functions, including immunomodulation. A heterogenous mixture of small EV (sEV) subsets, including putative exosomes, is derived when commonly used "exosome" isolation techniques are employed. Subset diversity relates in part to their different intracellular origins, and can be associated with distinct functional properties. Recent progress in the EV field has enabled the categorization of such subsets based on their surface composition. For the first time, we combine such emerging subset-specific markers with advanced imaging flow cytometry (iFCM) to perform high-throughput, multiparametric, vesicle-by-vesicle characterization, and functional assessment of specific small EV subsets, and exosomes in particular. The approach allows researchers to address three important applications. First, it is known that different isolation techniques result in the divergent recovery of particular vesicle subsets. Taking three commonly used "exosome" isolation techniques as test cases (ultracentrifugation, size-exclusion chromatography, and polymer-based precipitation), the capacity for convenient and accurate isolate compositional analysis by iFCM is demonstrated. The approach was able to corroborate and to quantify the known skewing of subtype recovery among different isolation approaches. Second, exosomes are a particularly widely studied EV subset. Applying exosome-specific markers to samples collected from an optimal clinical transplantation model, we verify the capacity for iFCM to detect exosomes in circulation, to establish their tissue of origin, and to provide insights as to their functional immunological potential. Finally, we describe a technique for establishing whether the transfer of a molecule of interest to a target cell is exosomally mediated. In so doing, we highlight the approach's utility in assessing the functional impact of circulating exosomes and in identifying their targets. In conclusion, we set out a new methodological approach by which small extracellular vesicle subsets, exosomes in particular, can be conveniently and comprehensively investigated, thereby offering novel phenotypic and functional insights.

Entecavir or tenofovir monotherapy prevents HBV recurrence in liver transplant recipients: A 5-year follow-up study after hepatitis B immunoglobulin withdrawal.

BACKGROUND: Recent data suggest that oral third-generation nucleos(t)ide analogs (NA) monoprophylaxis following hepatitis B immunoglobulin (HBIg) withdrawal may be effective to prevent HBV reinfection after liver transplantation (LT). PATIENTS AND METHODS: Between 01/2010 and 03/2012, all HBV monoinfected and HBV/HDV co-infected LT patients followed in our centre withdrew HBIg ±â€¯NA and were commenced on either ETV or TDF as monotherapy. RESULTS: Seventy-seven patients were included in the study (55% TDF, 45% ETV). Group A comprised 69 HBV monoinfected patients and Group B 8 HBV/HDV co-infected patients. After HBIg withdrawal, Groups A and B patients were followed for 69 (range 13-83) months and 61 (range 31-78) months, respectively. No Group B patients had HBsAg or HBV DNA recurrence, while 6 (9%) Group A patients became HBsAg-positive after a median of 18 (range 1-40) months. The cumulative 5-year incidence of HBsAg recurrence was 9%. All 6 patients demonstrated undetectable HBV-DNA levels and stable graft function during 30 months of additional follow-up. In 3/6 patients, seroconversion was transitory, while the remaining 3 showed HBsAg levels <0.13 IU/mL over the entire period of observation. Pre-LT HCC emerged as the strongest predictor of HBsAg recurrence. CONCLUSION: HBIG can be safely discontinued in HBsAgpositive LT recipients and replaced by ETV or TDF monotherapy.

Protein supplements and adolescent athletes: A pilot study investigating the risk knowledge, motivations and prevalence of use.

AIM: Protein-based sports supplements are among the more common types of nutrition supplements consumed by athletes; however, there is currently limited data investigating the knowledge, motivations and occurrence of use among the adolescent population (13-18 years). This pilot study looks to obtain initial data regarding the use of protein supplements in this population. METHODS: This study investigates the understanding and occurrence of protein supplement use in 87 adolescent athletes based in an Australian capital city who compete in a variety of sports. Sources of information, regularity of use, purchasing habits, associated risk knowledge and supplement beliefs were examined using a self-reported, written questionnaire. RESULTS: A total of 60% (n = 52) of athletes reported using protein supplements, with a positive relationship between age and use (P < 0.05); 48 participants (55%) perceived risks associated with protein supplement consumption, with the most common risk reported as 'I don't know' (22%). Coaches were found to initiate protein supplement use more than other figures in the athlete's life (50%) and were the primary source of information regarding supplements (58%). It was found that 19% of adolescent athletes obtained information about protein supplements from the Internet, and 17% of all consumers purchase their supplements online. CONCLUSIONS: The evident lack of knowledge regarding protein supplements demonstrates a necessity for further education of athletes, coaches and families regarding the responsible purchasing and use of protein supplements in the current landscape of sports nutrition. Future research should further explore the role of the Internet in protein supplement purchase and education.

Successful expansion of functional and stable regulatory T cells for immunotherapy in liver transplantation.

Strategies to prevent organ transplant rejection whilst minimizing long-term immunosuppression are currently under intense investigation with regulatory T cells (Tregs) nearing clinical application. The clinical trial, ThRIL, recently commenced at King's College London, proposes to use Treg cell therapy to induce tolerance in liver transplant recipients, the success of which has the potential to revolutionize the management of these patients and enable a future of drug-free transplants. This is the first report of the manufacture of clinical grade Tregs from prospective liver transplant recipients via a CliniMACS-based GMP isolation technique and expanded using anti-CD3/CD28 beads, IL-2 and rapamycin. We report the enrichment of a pure, stable population of Tregs (>95% CD4(+)CD25(+)FOXP3(+)), reaching adequate numbers for their clinical application. Our protocol proved successful in, influencing the expansion of superior functional Tregs, as compared to freshly isolated cells, whilst also preventing their conversion to Th17 cells under pro-inflammatory conditions. We conclude with the manufacture of the final Treg product in the clinical research facility (CRF), a prerequisite for the clinical application of these cells. The data presented in this manuscript together with the much-anticipated clinical results from ThRIL, will undoubtedly inform the improved management of the liver transplant recipient.

Immunosuppression withdrawal following liver transplantation.

Current immunosuppression regimens in liver transplantation provide excellent short-term survival rates but have many deleterious long-term side effects. They are therefore associated with the higher mortality and morbidity seen in liver transplant recipients compared to the general population and the notion that many liver transplant recipients are over-immunosuppressed is widely accepted. Liver allografts show a greater resistance to alloimmune responses than other solid organ transplants and recent research suggests up to 60% of highly selected recipients could wean off immunosuppression completely. In this review, we look at the evidence from immunosuppression withdrawal trials, the potential benefits of immunosuppression withdrawal and the identification of tolerant transplant recipients.