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BACKGROUND AND AIMS: In otherwise near-normal appearing biopsies by routine light microscopy, next-generation pathology (NGP) detected close pairings (immune pairs; iPAIRs) between lymphocytes and antigen-presenting cells (APCs) that predicted immunosuppression weaning failure in pediatric liver transplant (LTx) recipients (Immunosuppression Withdrawal for Stable Pediatric Liver Transplant Recipients [iWITH], NCT01638559). We hypothesized that NGP-detected iPAIRs enrich for true immune synapses, as determined by nuclear shape metrics, intercellular distances, and supramolecular activation complex (SMAC) formation. APPROACH AND RESULTS: Intralobular iPAIRs (CD45 high lymphocyte-major histocompatibility complex II + APC pairs; n = 1167, training set) were identified at low resolution from multiplex immunohistochemistry-stained liver biopsy slides from several multicenter LTx immunosuppression titration clinical trials (iWITH; NCT02474199 (Donor Alloantigen Reactive Tregs (darTregs) for Calcineurin Inhibitor (CNI) Reduction (ARTEMIS); Prospective Longitudinal Study of iWITH Screen Failures Secondary to Histopathology). After excluding complex multicellular aggregates, high-resolution imaging was used to examine immune synapse formation ( n = 998). By enriching for close intranuclear lymphocyte-APC distance (mean: 0.713 μm) and lymphocyte nuclear flattening (mean ferret diameter: 2.1), SMAC formation was detected in 29% of iPAIR-engaged versus 9.5% of unpaired lymphocytes. Integration of these morphometrics enhanced NGP detection of immune synapses (ai-iSYN). Using iWITH preweaning biopsies from eligible patients ( n = 53; 18 tolerant, 35 nontolerant; testing set), ai-iSYN accurately predicted (87.3% accuracy vs. 81.4% for iPAIRs; 100% sensitivity, 75% specificity) immunosuppression weaning failure. This confirmed the presence and importance of intralobular immune synapse formation in liver allografts. Stratification of biopsy mRNA expression data by immune synapse quantity yielded the top 20 genes involved in T cell activation and immune synapse formation and stability. CONCLUSIONS: NGP-detected immune synapses (subpathological rejection) in LTx patients prior to immunosuppression reduction suggests that NGP-detected (allo)immune activity usefulness for titration of immunosuppressive therapy in various settings.

Following transplantation, human CD4+T cells can respond to alloantigen using three distinct pathways. Direct and semi-direct responses are considered potent, but brief, so contribute mostly to acute rejection. Indirect responses are persistent and prolonged, involve B cells as critical antigen presenting cells, and are an absolute requirement for development of donor specific antibody, so more often mediate chronic rejection. Novel in vitro techniques have furthered our understanding by mimicking in vivo germinal centre processes, including B cell antigen presentation to CD4+ T cells and effector cytokine responses following challenge with donor specific peptides. In this review we outline recent data detailing the contribution of CD4+ T follicular helper cells and antigen presenting B cells to donor specific antibody formation and antibody mediated rejection. Furthermore, multi-parametric flow cytometry analyses have revealed specific endogenous regulatory T and B subsets each capable of suppressing distinct aspects of the indirect response, including CD4+ T cell cytokine production, B cell maturation into plasmablasts and antibody production, and germinal centre maturation. These data underpin novel opportunities to control these aberrant processes either by targeting molecules critical to indirect alloresponses or potentiating suppression via exogenous regulatory cell therapy.

Promoting immune tolerance to transplanted organs can minimize the amount of immunosuppressive drugs that patients need to take, reducing lifetime risks of mortality and morbidity. Regulatory T cells (Tregs) are essential for immune tolerance, and preclinical studies have shown their therapeutic efficacy in inducing transplantation tolerance. Here, we report the results of a phase 1/2 trial (ARTEMIS, NCT02474199) of autologous donor alloantigen-reactive Treg (darTreg) therapy in individuals 2 to 6 years after receiving a living donor liver transplant. The primary efficacy endpoint was calcineurin inhibitor dose reduction by 75% with stable liver function tests for at least 12 weeks. Among 10 individuals who initiated immunosuppression withdrawal, 1 experienced rejection before planned darTreg infusion, 5 received darTregs, and 4 were not infused because of failure to manufacture the minimal infusible dose of 100 × 106 cells. darTreg infusion was not associated with adverse events. Two darTreg-infused participants reached the primary endpoint, but an insufficient number of recipients were treated for assessing the efficacy of darTregs. Mechanistic studies revealed generalized Treg activation, senescence, and selective reduction of donor reactivity after liver transplantation. Overall, the ARTEMIS trial features a design concept for evaluating the efficacy of Treg therapy in transplantation. The mechanistic insight gained from the study may help guide the design of future trials.

Abstract Background Kidney transplantation is the gold-standard treatment for patients with kidney failure. However, one-third of patients awaiting a kidney transplant are highly sensitized to human leukocyte antigens (HLA), resulting in an increased waiting time for a suitable kidney, more acute and chronic rejection, and a shorter graft survival compared to non-highly sensitised patients. Current standard immunosuppression protocols do not adequately suppress memory responses, and so alternative strategies are needed. Autologous polyclonally expanded regulatory T cells (Tregs) have been demonstrated to be safe in transplant settings and could be a potential alternative to modulate memory immune alloresponses. Methods The aim of this trial is to determine whether adoptive transfer of autologous Tregs into HLA sensitised patients can suppress memory T and B cell responses against specific HLA antigens. This is a two-part, multi-centre, prospective clinical trial, comprising an observational phase (Part 1) aiming to identify patients with unregulated cellular memory responses to HLA (Pure HLA Proteins) followed by an interventional phase (Part 2). The first 9 patients identified as being eligible in Part 1 will undergo baseline immune monitoring for 2 months to inform statistical analysis of the primary endpoint. Part 2 is an adaptive, open labelled trial based on Simon’s two-stage design, with 21 patients receiving Good Manufacturing Practice (GMP)-grade polyclonally expanded Tregs to a dose of 5–10 × 106 cells/kg body weight. The primary EP is suppression of in vitro memory responses for 2 months post-infusion. 12 patients will receive treatment in stage 1 of Part 2, and 9 patients will receive treatment in stage 2 of Part 2 if ≥ 50% patients pass the primary EP in stage 1. Discussion This is a prospective study aiming to identify patients with unregulated cellular memory responses to Pure HLA Proteins and determine baseline variation in these patterns of response. Part 2 will be an adaptive phase IIa clinical trial with 21 patients receiving a single infusion of GMP-grade polyclonally expanded Tregs in two stages. It remains to be demonstrated that modulating memory alloresponses clinically using Treg therapy is achievable. Trial registration EudraCT Number: 2021–001,664-23. REC Number: 21/SC/0253. Trial registration number ISRCTN14582152.

Regulatory T cells are fundamental for the induction and maintenance of immune homeostasis, with their dysfunction resulting in uncontrolled immune responses and tissue destruction predisposing to autoimmunity, transplant rejection and several inflammatory and metabolic disorders. Recent discoveries have demonstrated that metabolic processes and mitochondrial function are critical for the appropriate functioning of these cells in health, with their metabolic adaptation, influenced by microenvironmental factors, seen in several pathological processes. Upon activation regulatory T cells rearrange their oxidation-reduction (redox) system, which in turn supports their metabolic reprogramming, adding a layer of complexity to our understanding of cellular metabolism. Here we review the literature surrounding redox homeostasis and metabolism of regulatory T cells to highlight new mechanistic insights of these interlinked pathways in immune regulation.

Summary: Background: Dysregulated inflammatory responses and oxidative stress are key pathogenic drivers of chronic inflammatory diseases such as liver cirrhosis (LC). Regulatory T cells (Tregs) are essential to prevent excessive immune activation and maintain tissue homeostasis. While inflammatory cues are well known to modulate the function and stability of Tregs, the extent to which Tregs are influenced by oxidative stress has not been fully explored. Methods: The phenotypic and functional properties of CD4+CD25+CD127lo/- Tregs isolated from patients with LC were compared to healthy controls (HC). Treg redox state was investigated by characterizing intracellular reactive oxygen species (ROS), NADPH oxidase-2 (Nox2) activity, mitochondrial function, morphology, and nuclear factor-erythroid 2-related factor (Nrf2) antioxidant signalling. The relevance of Nrf2 and its downstream target, Heme-oxygenase-1 (HO-1), in Treg function, stability, and survival, was further assessed using mouse models and CRISPR/Cas9-mediated HO-1 knock-out. Findings: Circulating Tregs from LC patients displayed a reduced suppressive function, correlating with liver disease severity, associated with phenotypic abnormalities and increased apoptosis. Mechanistically, this was linked to a dysregulated Nrf2 signalling with resultant lower levels of HO-1, enhanced Nox2 activation, and impaired mitochondrial respiration and integrity. The functional deficit in LC Tregs could be partially recapitulated by culturing control Tregs in patient sera. Interpretation: Our findings reveal that Tregs rely on functional redox homeostasis for their function, stability, and survival. Targeting Treg specific anti-oxidant pathways may have therapeutic potential to reverse the Treg impairment in conditions of oxidative damage such as advanced liver disease. Funding: This study was funded by the Wellcome Trust (211113/A/18/Z).

Background & aimsA substantial proportion of pediatric liver transplant recipients develop subclinical chronic allograft injury. We studied whether there are distinct patterns of injury based on histopathologic features and identified associated immunologic profiles.MethodsWe conducted a cross-sectional study of 157 stable, long-term pediatric recipients of transplanted livers (70 boys; > 6 years old at time of transplantation; mean, 8.9 ± 3.46 years after liver transplantation) who underwent liver biopsy analysis from August 13, 2012, through May 1, 2014. Participants had received livers from a living or deceased donor and had consistently normal results from liver tests. Liver biopsy specimens were scored by a central pathologist; an unsupervised hierarchical cluster analysis of histologic features was used to sort biopsy samples into 3 clusters. We conducted transcriptional and cytometric analyses of liver tissue samples and performed a systems biology analysis that incorporated clinical, serologic, histologic, and transcriptional data.ResultsThe mean level of alanine aminotransferase in participants was 27.6 ± 14.57 U/L, and the mean level of γ-glutamyl transferase was 17.4 ± 7.93 U/L. Cluster 1 was characterized by interface activity (n = 34), cluster 2 was characterized by periportal or perivenular fibrosis without interface activity (n = 45), and cluster 3 had neither feature (n = 78). We identified a module of genes whose expression correlated with levels of alanine aminotransferase, class II donor-specific antibody, portal inflammation, interface activity, perivenular inflammation, portal and perivenular fibrosis, and cluster assignment. The module was enriched in genes that regulate T-cell-mediated rejection (TCMR) of liver and other transplanted organs. Functional pathway analysis showed overrepresentation of TCMR gene sets for cluster 1 but not clusters 2 or 3.ConclusionIn an analysis of biopsies from an apparently homogeneous group of stable, long-term pediatric liver transplant recipients with consistently normal liver test results, we found evidence of chronic graft injury (inflammation and/or fibrosis). Biopsy samples with interface activity had a gene expression pattern associated with TCMR.

CD4+CD25+Foxp3+ Tregs are known to acquire tissue-specific features and exert cytoprotective and regenerative functions. The extent to which this applies to liver-resident Tregs is unknown. In this study, we aimed to explore the phenotypic and functional characteristics of adult murine liver resident Tregs during homeostasis. Additionally, we investigated their role in ameliorating liver inflammation and tissue damage. Quantification of Foxp3+CD4+CD25+ cells comparing different tissues showed that the liver contained significantly fewer resident Tregs. A combination of flow cytometry phenotyping and microarray analysis of intra-hepatic and splenic Tregs under homeostatic conditions revealed that, although intra-hepatic Tregs exhibited the core transcriptional Treg signature, they expressed a distinct transcriptional profile. This was characterized by reduced CD25 expression and increased levels of pro-inflammatory Th1 transcripts Il1b and Ifng. In vivo ablation of Tregs in the Foxp3-DTR mouse model showed that Tregs had a role in reducing the magnitude of systemic and intra-hepatic inflammatory responses following acute carbon tetrachloride (CCl₄) injury, but their absence did not impact the development of hepatocyte necrosis. Conversely, the specific expansion of Tregs by administration of IL-2 complexes increased the number of intra-hepatic Tregs and significantly ameliorated tissue damage following CCl₄ administration in C57BL/6 mice. The cytoprotective effect observed in response to IL-2c was associated with the increased expression of markers known to regulate Treg suppressive function. Our results offer insight into the transcriptome and complex immune network of intra-hepatic Tregs and suggest that strategies capable of selectively increasing the pool of intra-hepatic Tregs could constitute effective therapies in inflammatory liver diseases.

Following transplantation, human CD4+T cells can respond to alloantigen using three distinct pathways. Direct and semi-direct responses are considered potent, but brief, so contribute mostly to acute rejection. Indirect responses are persistent and prolonged, involve B cells as critical antigen presenting cells, and are an absolute requirement for development of donor specific antibody, so more often mediate chronic rejection. Novel in vitro techniques have furthered our understanding by mimicking in vivo germinal centre processes, including B cell antigen presentation to CD4+ T cells and effector cytokine responses following challenge with donor specific peptides. In this review we outline recent data detailing the contribution of CD4+ T follicular helper cells and antigen presenting B cells to donor specific antibody formation and antibody mediated rejection. Furthermore, multi-parametric flow cytometry analyses have revealed specific endogenous regulatory T and B subsets each capable of suppressing distinct aspects of the indirect response, including CD4+ T cell cytokine production, B cell maturation into plasmablasts and antibody production, and germinal centre maturation. These data underpin novel opportunities to control these aberrant processes either by targeting molecules critical to indirect alloresponses or potentiating suppression via exogenous regulatory cell therapy. [ABSTRACT FROM AUTHOR]

Background and aimsThe 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.MethodsIn 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).ResultsCLK 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 < 0.05, P < 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 < 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.ConclusionThese data suggest that CLK patients show the immunological influence of both allografts with liver having a greater influence.

The use of calcineurin inhibitors (CNI) after liver transplantation is associated with post-transplant nephrotoxicity. Conversion to myc-ophenolate mofetil (MMF) monotherapy improves renal function, but is related to graft rejection in some recipients. Our aim was to identify variables associated with rejection after conversion to MMF monotherapy. Conversion was attempted in 40 liver transplant recipients. Clinical variables were determined and peripheral mononuclear blood cells were immunophenotyped during a 12-month follow-up. Conversion was classified as successful (SC) if rejection did not occur during the follow-up. MMF conversion was successful with 28 patients (70%) and was associated with higher glomerular filtration rates at the end of study. It also correlated with increased time elapsed since transplantation, low baseline CNI levels (Tacrolimus ≤ 6.5 ng/mL or Cyclosporine ≤ 635 ng/mL) and lower frequency of tacrolimus use. The only clinical variable independently related to SC in multivariate analysis was low baseline CNI levels (p = 0.02, OR: 6.93, 95%, CI: 1.3-29.7). Mean baseline fluorescent intensity of FOXP3+ T cells was significantly higher among recipients with SC. In conclusion, this study suggests that baseline CNI levels can be used to identify recipients with higher probability of SC to MMF monotherapy. Clinicaltrials.gov identification: NCT01321112.

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.

Hepatic expression of iron homeostasis genes and serum iron parameters predict the success of immunosuppression withdrawal following clinical liver transplantation, a phenomenon known as spontaneous operational tolerance. In experimental animal models, spontaneous liver allograft tolerance is established through a process that requires intra-hepatic lymphocyte activation and deletion. Our aim was to determine if changes in systemic iron status regulate intra-hepatic lymphocyte responses. We used a murine model of lymphocyte-mediated acute liver inflammation induced by Concanavalin A (ConA) injection employing mice fed with an iron-deficient (IrDef) or an iron-balanced diet (IrRepl). While the mild iron deficiency induced by the IrDef diet did not significantly modify the steady state immune cell repertoire and systemic cytokine levels, it significantly dampened inflammatory liver damage after ConA challenge. These findings were associated with a marked decrease in T cell and NKT cell activation following ConA injection in IrDef mice. The decreased liver injury observed in IrDef mice was independent from changes in the gut microflora, and was replicated employing an iron specific chelator that did not modify intra-hepatic hepcidin secretion. Furthermore, low-dose iron chelation markedly impaired the activation of isolated T cells in vitro. All together, these results suggest that small changes in iron homeostasis can have a major effect in the regulation of intra-hepatic lymphocyte mediated responses.