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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.

Extracellular vesicles (EVs) are a heterogenous group of nanosized, membrane-bound particles which are released by most cell types. They are known to play an essential role in cellular communication by way of their varied cargo which includes selectively enriched proteins, lipids, and nucleic acids. In the last two decades, wide-ranging evidence has established the involvement of EVs in the regulation of immunity, with EVs released by immune and non-immune cells shown to be capable of mediating immune stimulation or suppression and to drive inflammatory, autoimmune, and infectious disease pathology. More recently, studies have demonstrated the involvement of allograft-derived EVs in alloimmune responses following transplantation, with EVs shown to be capable of eliciting allograft rejection as well as promoting tolerance. These insights are necessitating the reassessment of standard paradigms of T cell alloimmunity. In this article, we explore the latest understanding of the impact of EVs on alloresponses following transplantation and we highlight the recent technological advances which have enabled the study of EVs in clinical transplantation. Furthermore, we discuss the rapid progress afoot in the development of EVs as novel therapeutic vehicles in clinical transplantation with particular focus on liver transplantation.

Idiopathic aplastic anemia (AA) has 2 key characteristics: an autoimmune response against hematopoietic stem/progenitor cells and regulatory T-cells (Tregs) deficiency. We have previously demonstrated reduction in a specific subpopulation of Treg in AA, which predicts response to immunosuppression. The aims of the present study were to define mechanisms of Treg subpopulation imbalance and identify potential for therapeutic intervention. We have identified 2 mechanisms that lead to skewed Treg composition in AA: first, FasL-mediated apoptosis on ligand interaction; and, second, relative interleukin-2 (IL-2) deprivation. We have shown that IL-2 augmentation can overcome these mechanisms. Interestingly, when high concentrations of IL-2 were used for in vitro Treg expansion cultures, AA Tregs were able to expand. The expanded populations expressed a high level of p-BCL-2, which makes them resistant to apoptosis. Using a xenograft mouse model, the function and stability of expanded AA Tregs were tested. We have shown that these Tregs were able to suppress the macroscopic clinical features and tissue manifestations of T-cell–mediated graft-versus-host disease. These Tregs maintained their suppressive properties as well as their phenotype in a highly inflammatory environment. Our findings provide an insight into the mechanisms of Treg reduction in AA. We have identified novel targets with potential for therapeutic interventions. Supplementation of ex vivo expansion cultures of Tregs with high concentrations of IL-2 or delivery of IL-2 directly to patients could improve clinical outcomes in addition to standard immunosuppressive therapy.

Cirrhosis-associated immune dysfunction (CAID) contributes to disease progression and organ failure development. We interrogated immune system function in nonseptic compensated and decompensated cirrhotic patients using the TruCulture whole blood stimulation system, a novel technique that allows a more accurate representation than traditional methods, such as peripheral blood mononuclear cell culture, of the immune response in vivo. Thirty cirrhotics (21 decompensated and 9 compensated) and seven healthy controls (HCs) were recruited. Whole blood was drawn directly into three TruCulture tubes [unstimulated to preloaded with heat-killed Escherichia coli 0111:B4 (HKEB) or lipopolysaccharide (LPS)] and incubated in dry heat blocks at 37°C for 24 h. Cytokine analysis of the supernatant was performed by multiplex assay. Cirrhotic patients exhibited a robust proinflammatory response to HKEB compared with HCs, with increased production of interferon-γ-induced protein 10 (IP-10) and IFN-λ1, and to LPS, with increased production of IFN-λ1. Decompensated patients demonstrated an augmented immune response compared with compensated patients, orchestrated by an increase in type I, II, and III interferons, and higher levels of IL-1β, IL-6, and IL-8 post-LPS stimulation. IL-1β, TNF-α, and IP-10 post-HKEB stimulation and IP-10 post-LPS stimulation negatively correlated with biochemical markers of liver disease severity and liver disease severity scores. Cirrhotic patients exposed to bacterial products exhibit an exaggerated inflammatory response orchestrated by IFNs, IL-6, and IL-8. Poststimulation levels of a number of proinflammatory cytokines negatively correlate with markers of liver disease severity raising the possibility that the switch to an immunodeficient phenotype in CAID may commence earlier in the course of advanced liver disease. NEW & NOTEWORTHY Decompensated cirrhotic patients, compared with compensated patients, exhibit a greater exaggerated inflammatory response to bacterial products orchestrated by interferons, IL-6, and IL-8. Postbacterial product stimulation levels of a number of pro-inflammatory cytokines negatively correlate with liver disease severity biomarkers and liver disease severity scores raising the possibility that the switch to an immunodeficient phenotype in cirrhosis-associated immune dysfunction may commence earlier in the course of advanced liver disease.

In several murine models of transplantation, the "cross-dressing" of recipient antigen presenting cells (APCs) with intact donor major histocompatibility complex (MHC) derived from allograft-released small extracellular vesicles (sEVs) has been recently described as a key mechanism in eliciting and sustaining alloimmune responses. Investigation of these processes in clinical organ transplantation has, however, been hampered by the lack of sensitivity of conventional instruments and assays. We have employed advanced imaging flow cytometry (iFCM) to explore the kinetics of allograft sEV release and the extent to which donor sEVs might induce cross-dressing following liver and kidney transplantation. We report for the first time that recipient APC cross-dressing can be transiently detected in the circulation shortly after liver, but not kidney, transplantation in association with the release of HLA-bearing allograft-derived sEVs. In liver transplant recipients the majority of circulating cells exhibiting donor HLA are indeed cross-dressed cells and not passenger leukocytes. In keeping with experimental animal data, the downstream functional consequences of the transfer of circulating sEVs harvested from human transplant recipients varies depending on the type of transplant and time posttransplant. sEVs released shortly after liver, but not kidney, transplantation exhibit immunoinhibitory effects that could influence liver allograft immunogenicity.

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., CD4+Foxp3+ regulatory T cells isolated from either patients awaiting liver transplantation or recently transplanted recipients can be expanded in large numbers ex vivo and safely infused into recipients on immunosuppression.

Liver transplantation (LT) is a successful treatment for patients with liver failure. However, organ shortage results in over 11% of patients losing their chance of a transplant attributed to liver decompensation (LD) and death. Ischemia/reperfusion injury (IRI) following conventional cold storage (CS) is a major cause of injury leading to graft loss after LT. Normothermic machine perfusion (NMP), a method of organ preservation, provides oxygen and nutrition during preservation and allows aerobic metabolism. NMP has recently been shown to enable improved organ utilization and posttransplant outcomes following a phase I and a phase III randomized trial. The aim of the present study is to assess the impact of NMP on reducing IRI and to define the underlying mechanisms. We transplanted and compared 12 NMP with 27 CS-preserved livers by performing gene microarray, immunoprofiling of hepatic lymphocytes, and immunochemistry staining of liver tissues for assessing necrosis, platelet deposition, and neutrophil infiltration, and the status of steatosis after NMP or CS prereperfusion and postreperfusion. Recipients receiving NMP grafts showed significantly lower peak aspartate aminotransferase (AST) levels than those receiving CS grafts. NMP altered gene-expression profiles of liver tissue from proinflammation to prohealing and regeneration. NMP also reduced the number of interferon gamma (IFN-γ) and interleukin (IL)-17–producing T cells and enlarged the CD4 pos CD25 high CD127 neg FOXP3 pos regulatory T cell (Treg) pool. NMP liver tissues showed less necrosis and apoptosis in the parenchyma and fewer neutrophil infiltration compared to CS liver tissues. Conclusion: Reduced IRI in NMP recipients was the consequence of the combination of inhibiting inflammation and promoting graft regeneration.

Regulatory T cells (Tregs) have unique immunosuppressive properties and are essential to ensure effective immunoregulation. In animal models, Tregs have been shown to prevent autoimmune disorders and establish transplantation tolerance. Therefore, the prospect of harnessing Tregs, either by increasing their frequency or by conferring allospecificity, has prompted a growing interest in the development of immunotherapies. Here, employing a well‐established skin transplant model with a single major histocompatibility complex mismatch, we compared the therapeutic efficacy of adoptively transfer Treg with or without donor specificity and the administration of IL‐2 to promote in vivo expansion of Treg. We showed that IL‐2 treatment preferentially enhances the proliferation of the allospecific Tregs adoptively transferred in an antigen‐dependent manner. In addition, donor‐specific Tregs significantly increased the expression of regulatory‐related marker, such as CTLA4 and inducible costimulator (ICOS), in the skin allograft and draining lymph nodes compared to endogenous and polyclonal transferred Tregs. Importantly, by combining IL‐2 with donor‐specific Tregs, but not with polyclonal Tregs, a synergistic effect in prolonging skin allograft survival was observed. Altogether, our data suggest that this combination therapy could provide the appropriate conditions to enhance the immunoregulation of alloimmune responses in clinical transplantation., Combining adoptive transfer of donor‐specific Tregs with low dose IL‐2 has a synergistic effect in prolonging MHC‐class I–mismatched skin transplant survival due to antigen‐driven Treg expansion.

PURPOSE OF REVIEW This article describes biomarkers capable of identifying and predicting operational tolerance in solid organ transplant recipients. We outline the utility of these biomarkers in distinguishing allograft recipients in whom toxic immunosuppressive therapies might safely be minimized or withdrawn, and discuss their value in the appraisal of tolerance induction strategies. Finally, we review the insights derived from biomarker discovery into the cellular mechanisms underlying allograft tolerance. RECENT FINDINGS Important progress has been made in the development of robust signatures of tolerance, in both renal and liver transplant settings. Methodological advances, including high-throughput sequencing and bioinformatic processes, have been brought to bear on biomarker discovery and have heralded improvements in the accuracy with which operational tolerance can be predicted. Although the immunopathological basis for donor-specific tolerance is increasingly recognized to involve a complex interplay between numerous cell types, we review new lines of evidence shedding light on these mechanisms. SUMMARY Significant recent progress in identifying robust tolerance biomarkers has been made. In recognition of the need for rigorous validation of these, the first biomarker-led prospective immunosuppression withdrawal trials are underway. Such projects promise further progress and refinement in tolerance biomarker discovery, and offer hope for the amelioration of the burden associated with immunosuppressive therapies.

// Niloufar Safinia 1,* , Trishan Vaikunthanathan 1,* , Henrieta Fraser 1 , Sarah Thirkell 1 , Katie Lowe 1 , Laura Blackmore 2 , Gavin Whitehouse 2 , Marc Martinez-Llordella 2 , Wayel Jassem 2 , Alberto Sanchez-Fueyo 2 , Robert I. Lechler 1 and Giovanna Lombardi 1 1 MRC Centre for Transplantation, Division of Transplantation Immunology and Mucosal Biology, King’s College London, Guy’s Hospital, London, UK 2 Institute of Liver Studies, King’s College Hospital, London, UK * Co-first author Correspondence to: Niloufar Safinia, email: // Giovanna Lombardi, email: // Keywords : regulatory T cells, tolerance, immunotherapy, immunosuppression, liver transplantation, Immunology and Microbiology Section, Immune response, Immunity Received : July 26, 2015 Accepted : January 01, 2016 Published : January 17, 2016 Abstract 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.

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.

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.

The burden of life-long immunosuppressive medication must be overcome if progress is to be made in long-term outcomes following transplantation. The liver exhibits intrinsic tolerogenic properties that contribute to a unique propensity toward spontaneous acceptance when transplanted. Hence, a proportion of liver transplant recipients develop a state of immunotolerance and display persistently normal allograft function despite the discontinuation of immunosuppression. However, this phenomenon remains elusive for the majority of recipients. Investigations performed in experimental models of spontaneous liver allograft tolerance and in clinical cases of immunosuppression-free liver transplant acceptance have yielded mechanistic insights at the heart of recent strategies toward tolerance prediction and promotion. Results from recent clinical trials signal a shift in how liver allograft tolerance is viewed—not an elusive rarity of academic interest, but a potentially relevant clinical opportunity, which can be safely pursued if appropriately targeted.

Background Sustained virological response rates (SVRs) to directly acting antiviral (DAA) therapy for hepatitis C virus (HCV) are lower in decompensated cirrhosis. Markers of innate immunity predict nonresponse to interferon-based HCV treatment; however, whether they are associated with the response to DAAs in patients with decompensation is not known. Methods Information on demographics, adherence, viral kinetics, and resistance were gathered prospectively from a cohort with decompensated cirrhosis treated with 12 weeks of DAAs. C-X-C motif chemokine-10 (CXCL-10) level and T-cell and natural killer (NK) cell phenotype were analyzed pretreatment and at 4 and 12 weeks of treatment. Results Of 32 patients, 24 of 32 (75%) achieved SVR (responders). Eight of 32 (25%) experienced relapse after the end of treatment (nonresponders). There were no differences in demographics or adherence between groups. Nonresponders had higher CXCL-10; 320 pg/mL (179461) vs 109 pg/mL (88170) in responders (P < .001) and differential CXCL-10 dynamics. Nonresponders had lower NK cell frequency, higher expression of activation receptor NKp30, and lower frequency of the NK subset CD56−CD16+. Conclusions Nonresponders to DAAs displayed a different NK phenotype and CXCL-10 profile to responders. Nonresponders did not have poorer adherence or baseline virological resistance, and this shows that immunological parameters are associated with treatment response to interferon-free treatment for HCV in individuals with decompensated cirrhosis.

Idiopathic aplastic anemia (AA) is an immune-mediated and serious form of bone marrow failure. Akin to other autoimmune diseases, we have previously shown that in AA regulatory T-cells (Tregs) are reduced in number and function. The aim of this study was to further characterize Treg subpopulations in AA and investigate the potential correlation between specific Treg subsets and response to immunosuppressive therapy (IST) as well as their in-vitro expandability for potential clinical use. Using mass cytometry (CyTOF) and an unbiased multidimensional analytical approach, we identified two specific human Treg subpopulations (Treg A and Treg B) with distinct phenotypes, gene-expression, expandability and function. Treg subpopulation B, predominates in IST responder patients, has a memory/activated phenotype (with higher expression of CD95, CCR4 and CD45RO within FOXP3hi, CD127lo Tregs), expresses the IL- 2/STAT5 pathway and cell-cycle commitment genes. Furthermore, in-vitro expanded Tregs become functional and with the characteristics of Treg subpopulation B. Collectively, this study identifies human Treg subpopulations that can be used as predictive biomarkers for response to IST in AA and potentially other autoimmune diseases. We also show that Tregs from AA patients are IL-2 sensitive and expandable in-vitro, suggesting novel therapeutic approaches such as low dose IL-2 therapy and/or expanded autologous Tregs and meriting further exploration.

International audience; BACKGROUND AND AIMS: The beneficial effect of one graft on another has been reported in combined transplantation but the associated mechanisms and biological influence of each graft have not yet been established. METHODS: In multiple analyses, we explored the PBMC phenotype and signature of 45 immune-related messenger RNAs and 754 microRNAs from a total of 235 patients, including combined liver-kidney transplant recipients (CLK), patients with a liver (L-STA) or kidney (K-STA) graft only under classical immunosuppression and patients with tolerated liver (L-TOL) or kidney grafts (K-TOL). RESULTS: CLK show an intermediary phenotype with a higher percentage of peripheral CD19(+) CD24(+) CD38(Low) memory B cells and Helios(+) Treg cells, two features associated with tolerance profiles, compared to L-STA and K-STA (P \textless 0.05, P \textless 0.01). Very few miRNA were significantly differentially expressed in CLK vs. K-STA and even fewer when compared to L-STA (35 and 8, P \textless 0.05). Finally, CLK are predicted to share common miRNA targets with K-TOL and even more with L-TOL (344 and 411, P = 0.005). Altogether CLK display an intermediary phenotype and gene profile, which is closer to that of liver transplant patients, with possible similarities with the profiles of tolerant patients. CONCLUSION: These data suggest that CLK patients show the immunological influence of both allografts with liver having a greater influence

Achieving drug-free tolerance or successfully using only small doses of immunosuppression is a major goal in organ transplantation. To investigate the potential mechanisms by which some kidney transplant recipients can achieve operational tolerance, we compared the expression profiles of microRNA in peripheral blood mononuclear cells of operationally tolerant patients with those of stable patients treated with conventional immunosuppression. B cells from operationally tolerant patients overexpressed miR-142-3p. The expression of miR-142-3p was stable over time and was not modulated by immunosuppression. In Raji B cells, overexpression of miR-142-3p modulated nearly 1000 genes related to the immune response of B cells, including potential miR-142-3p targets and molecules previously identified in the blood of operationally tolerant patients. Furthermore, our results suggested that a negative feedback loop involving TGF-β signaling and miR-142-3p expression in B cells may contribute to the maintenance of tolerance. In summary, miR-142-3p expression in peripheral blood mononuclear cells correlates with operational tolerance. Whether upregulation of miR-142-3p modulates inflammatory responses to promote tolerance or is a result of this tolerance state requires further study.

Positive and negative costimulation by members of the CD28 family is critical for the development of productive immune responses against foreign pathogens and their proper termination to prevent inflammation-induced tissue damage. In addition, costimulatory signals are critical for the establishment and maintenance of peripheral tolerance. This paradigm has been established in many animal models and has led to the development of immunotherapies targeting costimulation pathways for the treatment of cancer, autoimmune disease, and allograft rejection. During the last decade, the complexity of the biology of costimulatory pathways has greatly increased due to the realization that costimulation does not affect only effector T cells but also influences regulatory T cells and antigen-presenting cells. Thus, costimulation controls T-cell tolerance through both intrinsic and extrinsic pathways. In this review, we discuss the influence of costimulation on intrinsic and extrinsic pathways of peripheral tolerance, with emphasis on members of the CD28 family, CD28, cytotoxic T-lymphocyte antigen-4 (CTLA-4), and programmed death-1 (PD-1), as well as the downstream cytokine interleukin-1 (IL-2).

We report the results of a prospective randomized controlled trial in liver transplantation assessing the efficacy and safety of antithymocyte globulin (ATG-Fresenius) plus tacrolimus monotherapy at gradually decreasing doses. Patients were randomized to either: (a) standard-dose tacrolimus plus steroids;or (b) peritransplant ATG-Fresenius plus reduced-dose tacrolimus monotherapy followed by weaning of tacrolimus starting 3 months after transplantation. The primary end-point was the achievement of very low-dose tacrolimus (every-other-day or once daily dose with

γδ T cells are innate-type lymphocytes that preferentially act as regulators of local effector immune responses. Recent reports found an altered distribution of the two main subpopulations of blood γδ T cells (Vδ1 and Vδ2) in operationally tolerant liver transplant recipients. Based on this, γδ T cells subset quantification was proposed as a biomarker of immunologic risk in liver transplantation. The specific characteristics of γδ T cell subsets in transplantation remain however unknown. We have investigated here the phenotype, repertoire and functional properties of γδ T cell subsets in a large population of allograft recipients. Our results indicate that alterations in the γδ T cell compartment are not restricted to tolerant liver recipients. In fact, most immunosuppressed liver and kidney recipients also display an enlarged peripheral blood γδ T cell pool mainly resulting from an expansion of Vδ1 T cells exhibiting an oligoclonal repertoire and different phenotypic and cytokine production traits than Vδ2 T cells. We propose that persistent viral infections are likely to contribute to these alterations. Our data provide novel insight in the biology of γδ T cells and a rationale for exploring these lymphocytes in more depth into the pathogenesis of viral infections in transplantation.

Regulatory T cells (T(reg) cells) are central to the maintenance of immune homeostasis. However, little is known about the stability of T(reg) cells in vivo. In this study, we demonstrate that a substantial percentage of cells had transient or unstable expression of the transcription factor Foxp3. These 'exFoxp3' T cells had an activated-memory T cell phenotype and produced inflammatory cytokines. Moreover, exFoxp3 cell numbers were higher in inflamed tissues in autoimmune conditions. Adoptive transfer of autoreactive exFoxp3 cells led to the rapid onset of diabetes. Finally, analysis of the T cell receptor repertoire suggested that exFoxp3 cells developed from both natural and adaptive T(reg) cells. Thus, the generation of potentially autoreactive effector T cells as a consequence of Foxp3 instability has important implications for understanding autoimmune disease pathogenesis.

Acute cellular rejection occurs frequently during the first few weeks following liver transplantation. During this period, its molecular phenotype is confounded by peri- and postoperative proinflammatory events. To unambiguously define the molecular profile associated with rejection, we collected sequential biological specimens from 55 patients at least 3 years after liver transplantation who developed rejection during trials of intentional immunosuppression withdrawal. We analyzed liver tissue and blood samples obtained before initiation of drug withdrawal and at rejection, alongside blood samples collected during the weaning process. Gene expression profiling was conducted using whole-genome microarrays and real-time polymerase chain reaction. Rejection resulted in distinct blood and liver tissue transcriptional changes in patients who were either positive or negative for hepatitis C virus (HCV). Gene expression changes were mostly independent from pharmacological immunosuppression, and their magnitude correlated with severity of histological damage. Differential expression of a subset of genes overlapped across all conditions. These were used to define a blood predictive model that accurately identified rejection in HCV-negative, but not HCV-positive, patients. Changes were detectable 1-2 mo before rejection was diagnosed. Our results provide insight into the molecular processes underlying acute cellular rejection in liver transplantation and help clarify the potential utility and limitations of transcriptional biomarkers in this setting.

Defective immune regulation plays a permissive role enabling effector cells to initiate and perpetuate tissue damage, eventually resulting in autoimmune disease. Numerical and functional regulatory T-cell (Treg) impairment has been previously reported in autoimmune liver disease (AILD; including autoimmune hepatitis and autoimmune sclerosing cholangitis ASC). However, in these early reports, Tregs were phenotypically defined as CD4+CD25+ or CD4+CD25high cells. In the current study, we reexamined phenotypic and functional properties of Tregs by adopting a more refined definition of these cells that also includes negativity or low level of expression of CD127. We studied 43 AILD patients and 22 healthy subjects (HSs) and found that CD4+CD25+CD127− Tregs were decreased in the former. This decrease was more marked in patients with active disease than in those in remission. In AILD, Treg frequencies correlated inversely with parameters of disease activity and were not affected by immunosuppressive treatment. We also document, for the first time, that, in AILD, bona-fide Tregs produce less interleukin (IL)−10 and are impaired in their ability to suppress CD4+CD25− target cell proliferation, a feature that in HSs, but not in AILDs, is dependent, at least in part, on IL-10 secretion. Decreased IL-10 production by Tregs in AILD is linked to poor responsiveness to IL-2 and phospho signal transducer and activator of transcription 5 up-regulation. Conclusion: Tregs are numerically impaired in AILD, this impairment being more prominent during active disease. Notably, defective IL-10 production, resulting from low Treg responsiveness to IL-2, contributes to Treg functional impairment. (Hepatology 2015;62:863–875)

Pathogen-induced immune responses prevent the establishment of transplantation tolerance in experimental animal models. Whether this occurs in humans as well remains unclear. The development of operational tolerance in liver transplant recipients with chronic hepatitis C virus (HCV) infection allows us to address this question. We conducted a clinical trial of immunosuppression withdrawal in HCV-infected adult liver recipients to elucidate (i) the mechanisms through which allograft tolerance can be established in the presence of an ongoing inflammatory response and (ii) whether anti-HCV heterologous immune responses influence this phenomenon. Of 34 enrolled liver recipients, drug withdrawal was successful in 17 patients (50%). Tolerance was associated with intrahepatic overexpression of type I interferon and immunoregulatory genes and with an expansion of exhausted PD1/CTLA4/2B4-positive HCV-specific circulating CD8(+) T cells. These findings were already present before immunosuppression was discontinued and were specific for HCV infection. In contrast, the magnitude of HCV-induced proinflammatory gene expression and the breadth of anti-HCV effector T cell responses did not influence drug withdrawal outcome. Our data suggest that in humans, persistent viral infections exert immunoregulatory effects that could contribute to the restraining of alloimmune responses, and do not necessarily preclude the development of allograft tolerance.

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. (Hepatology 2013;58:1824-1835).

Stable Foxp3 expression is crucial for regulatory T (Treg) cell function. We observed that antigen-driven activation and inflammation in the central nervous system (CNS) promoted Foxp3 instability selectively in the autoreactive Treg cells that expressed high Foxp3 levels before experimental autoimmune encephalitis induction. Treg cells with a demethylated Treg cell-specific demethylated region in the Foxp3 locus down-regulated Foxp3 transcription in the inflamed CNS during the induction phase of the response. Stable Foxp3 expression returned at the population level with the resolution of inflammation or was rescued by IL-2:anti-IL-2 complex treatment during the antigen priming phase. Thus, a subset of fully committed self-antigen-specific Treg cells lost Foxp3 expression during an inflammatory autoimmune response and may be involved in inadequate control of autoimmunity. These results have important implications for Treg cell therapies, and give insights into the dynamics of the Treg cell network during auto-reactive CD4+ T cell effector responses in vivo.

The maintenance of immune tolerance requires the deletion of self-reactive T cells in the thymus. The expression of tissue-specific antigen genes (TSAs) by thymic epithelial cells is critical for this process and depends on the activity of the Autoimmune Regulator (Aire) protein, however, the molecular mechanism(s) Aire uses to target TSA gene loci are unknown. Here we identified two Aire-interacting proteins – activating transcription factor 7 interacting protein (ATF7ip) and methyl CpG binding protein 1 (MBD1) –that are required for Aire’s targeting of TSA geneloci. Moreover, Mbd1−/− mice developed pathological autoimmunity and had a defect in Aire-dependent thymic TSA gene expression underscoring the critical importance of Aire’s interaction with the ATF7ip-MBD1 protein complex in maintaining central tolerance.

The transcription factor DEC1 is induced by CD28 ligation and is required for optimal CD4+ T cell responses and the development of EAE., During the initial hours after activation, CD4+ T cells experience profound changes in gene expression. Co-stimulation via the CD28 receptor is required for efficient activation of naive T cells. However, the transcriptional consequences of CD28 co-stimulation are not completely understood. We performed expression microarray analysis to elucidate the effects of CD28 signals on the transcriptome of activated T cells. We show that the transcription factor DEC1 is highly induced in a CD28-dependent manner upon T cell activation, is involved in essential CD4+ effector T cell functions, and participates in the transcriptional regulation of several T cell activation pathways, including a large group of CD28-regulated genes. Antigen-specific, DEC1-deficient CD4+ T cells have cell-intrinsic defects in survival and proliferation. Furthermore, we found that DEC1 is required for the development of experimental autoimmune encephalomyelitis because of its critical role in the production of the proinflammatory cytokines GM-CSF, IFN-γ, and IL-2. Thus, we identify DEC1 as a critical transcriptional mediator in the activation of naive CD4+ T cells that is required for the development of a T cell–mediated autoimmune disease.

Introduction Severe aplastic anemia (SAA) is an immune mediated bone marrow failure syndrome characterized, among other features, by reduced numbers and dysfunctional regulatory T cells, as shown by our group. Using mass cytometry, we identified an immune signature that predicts clinical response toimmunosuppression (IST)consisting of two Treg subsets (A and B), differentially expressing CD45RA, CD95, CD7, CD28, and CCR4(Kordasti Blood 2016). Treg B are more suppressive and enriched in cell cycle related proteins, suggesting their proliferative potential. The aim of this study was to investigate the in vitro expandability of Treg subpopulations as well as their function, stability, plasticity, clonality and gene signature following expansion with the aim of using autologous in vitro expanded Tregs as a therapeutic option for AA patient. Methods and results Treg expansion, phenotype, and function Total Tregs from AA and HDs are sensitive to low dose IL-2 as shown by STAT5 phosphorylation (p < 0.001, n = 6 AA and 2 HDs). Moreover Tregs from AA and HDs can be expanded at a comparable rate (fold increase 33x vs 21x, p = n.s. n = 6 AA and 8 HDs) in a Treg promoting culture (stimulation with anti CD3/CD28 beads and IL-2 1,000 IU/ml for 4 weeks with all-trans retinoic acid and rapamycin). The expanded total Tregs have the phenotype of Treg B. To investigate the potential differences between Treg A and B in terms of function and expandability, Treg A (CD4+CD25hiCD127loCD45RAhiCD95-CCR4lo) and B (CD4+CD25hiCD127loCD45RAloCD95+CCR4hi) were sorted from 6 HDs and expanded for 4 weeks (as above). On average Treg A and B expanded equally (Treg A 45x, and B 33x fold increase, p = n.s.). After expansion Treg A up-regulate CD95 expression (p = 0.005) similar to Treg B phenotype. Expanded Treg A and B both suppress proliferation of autologous Tconv (n = 3, p < 0.001) as well as the IFN-g and TNF-a secretion. Only Treg B suppressed IL-17A secretion (n = 3, p < 0.05) (figure 1). Treg-specific demethylated region (TSDR) Treg stability was assessed by DNA methylation analysis by deep ampliconbisulphitesequencing of the TSDR. TSDR CpG sites in expanded HDs and AA Tregs are > 98% unmethylated, confirming their stability. Interestingly, TSDR sites were less methylated in Treg B compared to Treg A (5% v 35%), but it decreases to a similar level as Treg B after expansion. Treg plasticity after expansion To investigate the plasticity, total Tregs, Treg A, and Treg B were cultured for 5 days in a Th17 polarizing culture (containing IL-1b,IL-6, IL-2). After 5 days of culture, only total Tregs were secreting IL-17A (p = 0.013), but neither Treg A, nor Treg B. Despite IL-17A secretion, Tregs were still able to suppress autologous Tconv proliferation (n = 3, p= 0.001). T cell receptor diversity To track the clonal origin of Treg A and B, we performed amplification and sequencing of CDR3 using immunoSEQ Platform, as already described. Our results show that both AA and HDs expanded Tregs show a comparable level of TCR VbCDR3 diversity (average clonality score 0.12); moreover Tregs, Treg A, and Treg B are polyclonal, pre and post expansion (clonality score 0.01 for all the subsets). Finally, Treg A and B do not share any TCR sequence and do not have any overlap in their TCR repertoire, suggesting they originate from different clones. Gene expression Expanded Tregs (total, A and B) were compared to established human Treg gene signature (Ferraro PNAS 2014). Treg B aremore enriched in Treg genes compared Treg A (Tregs B vs A false discovery rate (FDR) < 0.0001). IL-7 and CD80 are among the top 5 expressed genes in all 3 subpopulations, which are important for Tregs' motility and homeostasis. Conclusions In summary, AA Tregs can be expanded in vitro and expanded Tregs are more similar to Treg B, which are lacking in SAA as shown before. Although we were not able to sort Treg subpopulations from AA patients due to very low numbers, the expanded Treg A and B from HDs demonstrate a functional Treg profile with minimal plasticity. Interestingly the main difference between Treg A and B is the ability of Treg B to suppress IL-17A secretion. This is important, as IL-17 plays a crucial role in AA pathophysiology (Peffault de Latour Blood 2010). Overall, the current study suggests that in vitro expansion of AA Tregs is feasible. The expanded Tregs show the phenotype of the most functional Treg subset and likely to control the inflammatory response inAA, hence pave the way toward a novel cellular therapy for SAA. Disclosures Marsh: Alexion pharmaceuticals: Honoraria.

Foxp3+ CD4+ T helper cells called regulatory T (T reg) cells play a key role in controlling reactivity to self-antigens and onset of autoimmunity. T reg cells either arise in thymus and are called natural T reg (nT reg) cells or are generated in the periphery through induction of Foxp3 and are called inducible T reg (iT reg) cells. The relative contributions of iT reg cells and nT reg cells in peripheral tolerance remain unclear as a result of an inability to separate these two subsets of T reg cells. Using a combination of novel TCR transgenic mice with a defined self-antigen specificity and conventional mouse models, we demonstrate that a cell surface molecule, neuropilin-1 (Nrp-1), is expressed at high levels on nT reg cells and can be used to separate nT reg versus iT reg cells in certain physiological settings. In addition, iT reg cells generated through antigen delivery or converted under homeostatic conditions lack Nrp-1 expression. Nrp-1lo iT reg cells show similar suppressive activity to nT reg cells in controlling ongoing autoimmune responses under homeostatic conditions. In contrast, their activity might be compromised in certain lymphopenic settings. Collectively, our data show that Nrp-1 provides an excellent marker to distinguish distinct T reg subsets and will be useful in studying the role of nT reg versus iT reg cells in different disease settings.© 2012 Yadav et al.

Contains fulltext : 108418.pdf (Publisher’s version ) (Open Access) Following organ transplantation, lifelong immunosuppressive therapy is required to prevent the host immune system from destroying the allograft. This can cause severe side effects and increased recipient morbidity and mortality. Complete cessation of immunosuppressive drugs has been successfully accomplished in selected transplant recipients, providing proof of principle that operational allograft tolerance is attainable in clinical transplantation. The intra-graft molecular pathways associated with successful drug withdrawal, however, are not well defined. In this study, we analyzed sequential blood and liver tissue samples collected from liver transplant recipients enrolled in a prospective multicenter immunosuppressive drug withdrawal clinical trial. Before initiation of drug withdrawal, operationally tolerant and non-tolerant recipients differed in the intra-graft expression of genes involved in the regulation of iron homeostasis. Furthermore, as compared with non-tolerant recipients, operationally tolerant patients exhibited higher serum levels of hepcidin and ferritin and increased hepatocyte iron deposition. Finally, liver tissue gene expression measurements accurately predicted the outcome of immunosuppressive withdrawal in an independent set of patients. These results point to a critical role for iron metabolism in the regulation of intra-graft alloimmune responses in humans and provide a set of biomarkers to conduct drug-weaning trials in liver transplantation.

A proportion of transplant recipients can spontaneously accept their grafts in the absence of immunosuppression (operational tolerance). Previous studies identified blood transcriptional and cell-phenotypic markers characteristic of either liver or kidney tolerant recipients. However, the small number of patients analyzed and the use of different transcriptional platforms hampered data interpretation. In this study we directly compared samples from kidney and liver tolerant recipients in order to identify potential similarities in immune-related parameters. Liver and kidney tolerant recipients differed in blood expression and B-cell immunophenotypic patterns and no significant overlaps were detectable between them. Whereas some recipients coincided in specific NK-related transcripts, this observation was not reproducible in all cohorts analyzed. Our results reveal that certain immune features, but not others, are consistently present across all cohorts of operationally tolerant recipients. This provides a set of reproducible biomarkers that should be explored in future large-scale immunomonitoring trials.

Positive and negative costimulation by members of the CD28 family is critical for the development of productive immune responses against foreign pathogens and their proper termination to prevent inflammation-induced tissue damage. In addition, costimulatory signals are critical for the establishment and maintenance of peripheral tolerance. This paradigm has been established in many animal models and has led to the development of immunotherapies targeting costimulation pathways for the treatment of cancer, autoimmune disease, and allograft rejection. During the last decade, the complexity of the biology of costimulatory pathways has greatly increased due to the realization that costimulation does not affect only effector T cells but also influences regulatory T cells and antigen-presenting cells. Thus, costimulation controls T-cell tolerance through both intrinsic and extrinsic pathways. In this review, we discuss the influence of costimulation on intrinsic and extrinsic pathways of peripheral tolerance, with emphasis on members of the CD28 family, CD28, cytotoxic T-lymphocyte antigen-4 (CTLA-4), and programmed death-1 (PD-1), as well as the downstream cytokine interleukin-1 (IL-2).