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Supplemental Digital Content is available in the text., Background. Metastatic carcinoma of a renal allograft is a rare but life threatening event with a difficult clinical management. Recent reports suggested a potential role of BK polyomavirus (BKPyV) in the development of urologic tract malignancies in kidney transplant recipients. Methods. We investigated a kidney-pancreas female recipient with an history of BKPyV nephritis who developed a rapidly progressive and widely metastatic donor-derived renal carcinoma 9 years after transplantation. Results. Histology and fluorescence in situ hybridization analysis revealed a donor-derived (XY tumor cells) collecting (Bellini) duct carcinoma. The presence of BKPyV oncogenic large tumor antigen was identified in large amount within the kidney tumor and the bowel metastases. Whole genome sequencing of the tumor confirmed multiple genome BKPyV integrations. The transplanted kidney was removed, immunosuppression was withdrawn, and recombinant interleukin-2 (IL-2) was administered for 3 months, inducing a complete tumor clearance, with no evidence of disease at 6-year follow-up. The immunological profiling during IL-2 therapy revealed the presence of donor-specific T cells and expanded cytokine-producing bright natural killer cells but no donor-specific antibodies. Finally, we found persistently elevated anti-BK virus IgG titers and a specific anti-BKPyV T cell response. Conclusions. This investigation showed evidence for the potential oncogenic role of BKPyV in collecting duct carcinoma in renal allografts and demonstrated that immunosuppression withdrawal and IL-2 therapy can lead to an efficient antitumor cellular mediated rejection possibly via 3 distinct mechanisms including (1) host-versus-graft, (2) host-versus-tumor, and (3) anti-BKPyV responses.

The success of pancreas islet isolation largely depends on donor characteristics, including extracellular matrix composition of which collagen is the main element. We hypothesized that isolation yields are proportional to collagen digestion percentage, and aimed to determine a threshold that predicts isolation success. The amount of pancreas collagen (I-V) was determined using colorimetry prior to and after the digestion process in 52 human islet isolations. Collagen I-V and VI were also assessed histologically. We identified a collagen digestion threshold of ≥ 60% as an independent factor beyond which an islet preparation has a ninefold increased odds of yielding ≥ 250 000 islet equivalents (IEQ) (P = 0.009) and a sixfold increased odds of being transplanted (P = 0.015). Preparations with ≥ 60% collagen digestion (n = 35) yielded 283 017 ± 164 214 IEQ versus 180 142 ± 85 397 in the < 60% collagen digestion group (n = 17) (P = 0.016); respectively 62.9% versus 29.4% of those were transplanted (P = 0.024). Common donor characteristics, initial collagen content, enzyme blend, and digestion times were not associated with collagen digestion percentage variations. Donor age positively correlated with the amount of collagen VI (P = 0.013). There was no difference in islet graft survival between high and low digestion groups. We determined that a 60% pancreas collagen digestion is the threshold beyond which an islet isolation is likely to be successful and transplanted.

Treg therapy is being tested in clinical trials in transplantation and autoimmune diseases, however, the impact of inflammation on Tregs is unclear. In this study, we challenged human Tregs ex-vivo with pro-inflammatory cytokines, TNFα and IL-6. These cytokines enhanced Treg proliferation induced by anti-CD3 and anti-CD28 or CD28 superagonist (CD28SA) while maintaining high expression of FOXP3 and HELIOS, demethylated FOXP3 enhancer, and low expression of cytokines IFNg, IL-4 and IL-17. Blocking TNF receptor signaling using etanercept or deletion of TNF receptor 2 using CRISPR/Cas9 blunted Treg proliferation and attenuated FOXP3 and HELIOS expression, revealing the importance of TNFR2 signaling in Treg proliferation and lineage stability. The robust proliferation induced by CD28SA with IL-6 and TNFα may be adopted for the expansion of therapeutic Tregs. Metabolomics analysis showed that Tregs expanded with CD28SA plus cytokines had more active glycolysis and oxidative phosphorylation, increased energy production, and higher antioxidant potential. Finally, CD28SA plus cytokine-expanded Tregs had comparable suppressive activity in vitro and in vivo in a humanized mouse model of graft-versus-host-disease when compared to Tregs expanded using the conventional protocol. These results demonstrate that human Tregs positively respond to proinflammatory cytokines with enhanced proliferation without compromising their lineage identity or function.

Anti-CD19 chimeric antigen receptor (CD19-CAR)-engineered T cells are approved therapeutics for malignancies. The impact of the hinge domain (HD) and the transmembrane domain (TMD) between the extracellular antigen-targeting CARs and the intracellular signaling modalities of CARs has not been systemically studied. In this study, a series of 19-CARs differing only by their HD (CD8, CD28, or IgG4) and TMD (CD8 or CD28) was generated. CARs containing a CD28-TMD, but not a CD8-TMD, formed heterodimers with the endogenous CD28 in human T cells, as shown by co-immunoprecipitation and CAR-dependent proliferation of anti-CD28 stimulation. This dimerization was dependent on polar amino acids in the CD28-TMD and was more efficient with CARs containing CD28 or CD8 HD than IgG4-HD. The CD28-CAR heterodimers did not respond to CD80 and CD86 stimulation but had a significantly reduced CD28 cell-surface expression. These data unveiled a fundamental difference between CD28-TMD and CD8-TMD and indicated that CD28-TMD can modulate CAR T-cell activities by engaging endogenous partners.

BACKGROUND Xenotransplantation has made tremendous progress over the last decade. METHODS We discuss kidney and heart xenotransplantation, which are nearing initial clinical trials. RESULTS Life sustaining genetically modified kidney xenografts can now last for approximately 500 days and orthotopic heart xenografts for 200 days in non-human primates. Anti-swine specific antibody screening, preemptive desensitization protocols, complement inhibition and targeted immunosuppression are currently being adapted to xenotransplantation with the hope to achieve better control of antibody-mediated rejection (AMR) and improve xenograft longevity. These newest advances could probably facilitate future clinical trials, a significant step for the medical community, given that dialysis remains difficult for many patients and can have prohibitive costs. Performing a successful pig-to-human clinical kidney xenograft, that could last for more than a year after transplant, seems feasible but it still has significant potential hurdles to overcome. The risk/benefit balance is progressively reaching an acceptable equilibrium for future human recipients, e.g. those with a life expectancy inferior to two years. The ultimate question at this stage would be to determine if a "proof of concept" in humans is desirable, or whether further experimental/pre-clinical advances are still needed to demonstrate longer xenograft survival in non-human primates. CONCLUSION In this review, we discuss the most recent advances in kidney and heart xenotransplantation, with a focus on the prevention and treatment of AMR and on the recipient's selection, two aspects that will likely be the major points of discussion in the first pig organ xenotransplantation clinical trials.

PURPOSE OF REVIEW Cell-based immunotherapies have made enormous progress over the last decade with the approval of several anti-CD19-chimeric antigen receptor (CAR)-T cell therapies for haemato-oncological diseases. CARs are synthetic receptors comprising an antigen-specific extracellular domain fused to a hinge, transmembrane and intracellular signalling domains. The success obtained with CD19 CAR-T cells rekindled interest in using CAR-T cells to treat HIV seropositive patients. The purpose of this review is to discuss historical and recent developments of anti-HIV CARs. RECENT FINDINGS Since the first description of CD4+-based CARs in the early 90s, new generations of anti-HIV CARs were developed. They target the hetero-trimeric glycoprotein gp120/gp41 and consist of either a CD4+ extracellular domain or a VH/VL segment derived from broadly neutralizing antibodies. Recent efforts were employed in multiplexing CAR specificities, intracellular signalling domains and T cells resistance to HIV. SUMMARY Several new-anti HIV CAR-T cells were successfully tested in preclinical mice models and are now waiting to be evaluated in clinical trials. One of the key parameters to successfully using CAR-T cells in HIV treatment will depend on their capacity to control the HIV reservoir without causing off-targeting activities.

Forkhead box P3-expressing regulatory T (Treg) cells are essential for self-tolerance, with an emerging role in tissue repair and regeneration. Their ability to traffic to tissue and perform complex therapeutic tasks in response to the tissue microenvironment make them an attractive candidate for drug development. Early experiences of Treg cell therapy in patients with graft-versus-host disease, type 1 diabetes, and organ transplantation have shown that it is feasible, safe, and potentially efficacious in some settings. Many ongoing trials in patients with a wide variety of diseases will further enhance our knowledge about the optimal approaches for Treg cell manufacturing and dosing. We review the current preclinical rationale supporting Treg cell therapy in a variety of disease settings ranging from tissue transplantation, autoimmune diseases, and non-immune-mediated inflammatory settings. We point out challenges in development of Treg cell therapy and speculate how synthetic biology can be used to enhance the feasibility and efficacy of Treg cell therapy for autoimmune and autoinflammatory diseases.

Background Tolerogenic dendritic cells (DCs) represent a promising approach to promote transplantation tolerance. In this study, the potential of autologous bone marrow (BM)-derived murine DC to protect rat-to-mouse islets xenografts was analyzed. Methods Tolerogenic DCs were generated by differentiating BM cells in the presence of granulocyte-macrophage colony-stimulating factor and interleukin 10 (IL-10, IL-10 DC). The phenotype of IL-10 DC was characterized in vitro by expression of costimulatory/inhibitory molecules (flow cytometry) and cytokines (Luminex and ELISA), their function by phagocytosis and T-cell stimulation assays. To study transplant tolerance in vivo, rat islets were transplanted alone or in combination with autologous murine IL-10 DC under the kidney capsule of streptozotocin-induced diabetic C57BL/6 mice. Xenograft survival was evaluated by monitoring glycemia, cellular infiltration of xenografts by microscopy and flow cytometry 10 days post-transplantation. Results Compared with control DC, IL-10 DC exhibited lower levels of major histocompatibility complex class II, costimulatory molecules (CD40, CD86, CD205), lower production of pro-inflammatory cytokines (IL-12p70, TNF, IL-6), and higher production of IL-10. Phagocytosis of xenogeneic rat splenocytes was not impaired in IL-10 DC, whereas stimulation of T-cell proliferation was reduced in the presence of IL-10 DC. Xenograft survival of rat islets in diabetic mice co-transplanted with autologous murine IL-10 DC was significantly prolonged from 12 to 21 days, without additional immunosuppressive treatment. Overall, infiltration of xenografts by T cells and myeloid cells was not different in IL-10 DC recipient mice, but enriched for CD8+ T cells and myeloid cells with suppressor-associated phenotype. Conclusions Autologous IL-10-differentiated DC with tolerogenic properties prolong rat-to-mouse islets xenograft survival, potentially by locally inducing immune regulatory cells, indicating their potential for regulatory immune cell therapy in xenotransplantation.

Regulatory T cells (Treg cells) are a small subset of immune cells that are dedicated to curbing excessive immune activation and maintaining immune homeostasis. Accordingly, deficiencies in Treg cell development or function result in uncontrolled immune responses and tissue destruction and can lead to inflammatory disorders such as graft-versus-host disease, transplant rejection and autoimmune diseases. As Treg cells deploy more than a dozen molecular mechanisms to suppress immune responses, they have potential as multifaceted adaptable smart therapeutics for treating inflammatory disorders. Indeed, early-phase clinical trials of Treg cell therapy have shown feasibility, tolerability and potential efficacy in these disease settings. In the meantime, progress in the development of chimeric antigen receptors and in genome editing (including the application of CRISPR–Cas9) over the past two decades has facilitated the genetic optimization of primary T cell therapy for cancer. These technologies are now being used to enhance the specificity and functionality of Treg cells. In this Review, we describe the key advances and prospects in designing and implementing Treg cell-based therapy in autoimmunity and transplantation. Our increased understanding of how regulatory T cells suppress immune responses has led to their use in early-phase clinical trials for inflammatory disorders, with promising results. This Review describes the key advances and prospects in designing and implementing regulatory T cells as multifaceted, adaptable smart therapeutics in autoimmunity and transplantation.

Background & Aims Hereditary haemorrhagic telangiectasia is characterized by arterio-venous malformations (AVM). It frequently involves the liver without clinical symptoms, but may lead to biliary ischaemia, portal hypertension, or fatal high-output heart failure. The indication of liver transplantation is controversial. Methods Herein, we report the case of a 65-year-old female patient with a ‘double Osler syndrome’ consisting of hereditary haemorrhagic telangiectasia (HHT) and type I hereditary angioedema diagnosed at the age of 25 and 22 years respectively. Results Hereditary angioedema was treated with danazol for several decades until multiple hypoechogenic liver masses were detected. Albeit danazol treatment was replaced by C1 esterase inhibitor infusions, hepatocellular carcinoma was diagnosed at the age of 64 and the patient was listed for liver transplantation. HHT was marked by recurrent epistaxis until the age of 63 when severe intestinal bleeding occurred. At the age of 65, severe dyspnoea (NYHA class IV) developed and rapidly progressive high-output cardiac failure was diagnosed. Despite argon plasma coagulation to control bleeding from intestinal angiodysplasia, and treatment with bevacizumab to inhibit angiogenesis, the patient died from severe gastrointestinal bleeding associated with cardiogenic shock at the age of 66 before being transplanted. Conclusion The indication to list this patient for liver transplantation was debated several times before the diagnosis of hepatocellular carcinoma because of good general condition and low MELD score. Precise guidelines for screening and management of patients with hepatic HHT need to be better defined.

This manuscript presents quantitative findings on the actual effectiveness of terminal complement component 5 (C5) inhibitors and complement component 1 (C1) esterase inhibitors through their formal and common &ldquo, off-label&rdquo, (compassionate) indications. The results emanated from pairwise and network meta-analyses to present evidence until September 2019. Clinical trials (CT) and real-life non-randomized studies of the effects of interventions (NRSI) are consistent on the benefits of C5 inhibitors and of the absence of effects of C1 esterase inhibitors (n = 7484): Mathematically, eculizumab (surface under the cumulative ranking area (SUCRA) &gt, 0.6) and ravulizumab (SUCRA &ge, 0.7) were similar in terms of their protective effect on hemolysis in paroxysmal nocturnal hemoglobinuria (PNH), thrombotic microangiopathy (TMA) in atypical hemolytic uremic syndrome (aHUS), and acute kidney injury (AKI) in aHUS, in comparison to pre-/off-treatment state and/or placebo (SUCRA &lt, 0.01), and eculizumab was efficacious on thrombotic events in PNH (odds ratio (OR)/95% confidence interval (95% CI) in CT and real-life NRSI, 0.07/0.03 to 0.19, 0.24/0.17 to 0.33) and chronic kidney disease (CKD) occurrence/progression in PNH (0.31/0.10 to 0.97, 0.66/0.44 to 0.98). In addition, meta-analysis on clinical trials shows that eculizumab mitigates a refractory generalized myasthenia gravis (rgMG) crisis (0.29/0.13 to 0.61) and prevents new acute antibody-mediated rejection (AMR) episodes in kidney transplant recipients (0.25/0.13 to 0.49). The update of findings from this meta-analysis will be useful to promote a better use of complement inhibitors, and to achieve personalization of treatments with this class of drugs.

Background: Interleukin-1 (IL-1)&beta, and IL-1 receptor antagonist (IL-1Ra) have been proposed as important mediators during chronic liver diseases. We aimed to determine whether the modulation of IL-1&beta, signaling with IL-1Ra impacts on liver fibrosis. Methods: We assessed the effects of IL-1&beta, on human hepatic stellate cells (HSC) and in mouse models of liver fibrosis induced by bile duct ligation (BDL) or carbon tetrachloride treatment (CCl-4). Results: Human HSCs treated with IL-1&beta, had increased IL-1&beta, IL-1Ra, and MMP-9 expressions in vitro. HSCs treated with IL-1&beta, had reduced &alpha, smooth muscle actin expression. These effects were all prevented by IL-1Ra treatment. In the BDL model, liver fibrosis and Kuppfer cell numbers were increased in IL-1Ra KO mice compared to wild type mice and wild type mice treated with IL-1Ra. In contrast, after CCl-4 treatment, fibrosis, HSC and Kupffer cell numbers were decreased in IL-1Ra KO mice compared to the other groups. IL-1Ra treatment provided a modest protective effect in the BDL model and was pro-fibrotic in the CCl-4 model. Conclusions: We demonstrated bivalent effects of IL-1Ra during liver fibrosis in mice. IL-1Ra was detrimental in the CCl-4 model, whereas it was protective in the BDL model. Altogether these data suggest that blocking IL-1-mediated inflammation may be beneficial only in selective liver fibrotic disease.

The microbiological safety of islet preparations is paramount. Preservation medium contamination is frequent, and its impact on islet yield and function remains unclear. Microbiological samples collected during islet isolations from 2006 to 2016 were analyzed and correlated to isolation and allo- and autotransplantation outcomes. Microbial contamination of preservation medium was found in 64.4% of processed donor pancreases (291/452). We identified 464 microorganisms including Staphylococcus (253/464, 54.5%), Streptococcus (31/464, 6.7%), and Candida species (25/464, 5.4%). Microbial contamination was associated with longer warm and cold ischemia times and lower numbers of postpurification islet equivalents, purity, transplant rate, and stimulation index (all P < 0.05). Six percent of the preparations accepted for transplantation showed microbial contamination after isolation (12/200); 9 of 12 were Candida species. Six patients were transplanted with a sample with late microbial growth discovered after the infusion. Insulin independence rate was not affected. This risk of transplanting a contaminated islets preparation was reduced by half following the implementation of an additional sampling after 24 h of islet culture. Pancreas preservation fluid microbial contamination is associated with lower transplant rate and poorer in vitro function, but not with changes in graft survival. Culture medium testing 1 day after isolation reduces the risk of incidental transplantation with contaminated islets.

Chronic active antibody-mediated rejection (caAMR) is a major cause of allograft loss after kidney transplantation (1). The BANFF 2013 classification redefined caAMR by the presence of donor-specific anti-HLA antibodies (DSA) together with immuno-histopathological evidence for active vascular lesions of the endothelium (C4d deposits, glomerulitis, peritubular capillaritis) as well as evidence of chronic tissue injury (transplant glomerulopathy, peritubular capillary basement membrane multilayering or arterial intimal fibrosis) (2,3). Humoral immunity, detected by the presence of DSA, and B cells are considered pivotal in the development of caAMR. Gosset et al. showed that circulating DSA are responsible for accelerated allograft fibrosis independently of acute AMR (1). This article is protected by copyright. All rights reserved.

The field of xenotransplantation has fluctuated between great optimism and doubts over the last 50 years. The initial clinical attempts were extremely ambitious but faced technical and ethical issues that prompted the research community to go back to preclinical studies. Important players left the field due to perceived xenozoonotic risks and the lack of progress in pig-to-non-human-primate transplant models. Initial apparently unsurmountable issues appear now to be possible to overcome due to progress of genetic engineering, allowing the generation of multiple-xenoantigen knockout pigs that express human transgenes and the genome-wide inactivation of porcine endogenous retroviruses. These important steps forward were made possible by new genome editing technologies, such as CRISPR/Cas9, allowing researchers to precisely remove or insert genes anywhere in the genome. An additional emerging perspective is the possibility of growing humanized organs in pigs using blastocyst complementation. This article summarizes the current advances in xenotransplantation research in non-human primates and it describes the newly developed genome editing technology tools and interspecific organ generation. This article is protected by copyright. All rights reserved.

Background TAFRO syndrome has been reported in Japan among human herpesvirus 8 (HHV-8)-negative/idiopathic multicentric Castleman’s disease (iMCD) patients. To date, the majority of iMCD patients with TAFRO syndrome originate from Japan. Case presentation Herein, we report a 67-year-old HIV/HHV-8-negative Caucasian iMCD patient diagnosed with TAFRO. He presented with marked systemic inflammation, bicytopenia, terminal renal insufficiency, diffuse lymphadenopathies, and anasarca. Lymph node and bone marrow biopsies revealed atrophic germinal centers variably hyalinized and megakaryocytic hyperplasia with mild myelofibrosis. Several other biopsies performed in kidneys, liver, gastrointestinal tract, prostate, and lungs revealed unspecific chronic inflammation. The patient had a complete response to corticosteroids, tocilizumab, and rituximab. He relapsed twice following discontinuation of rituximab. When reviewing the literature, we found seven other Caucasian cases with TAFRO syndrome. There were no significant differences with those described by the Japanese cohort except for the higher frequency of kidney failure and auto-antibodies in Western patients. Conclusion This case illustrates that patients with TAFRO syndrome can develop non-specific inflammation in several tissue sites. Furthermore, this case and our review of the literature demonstrate that TAFRO syndrome can affect Caucasian and Japanese patients highlighting the importance of evaluating for this syndrome independently of ethnic background.

Replacement of pancreatic β-cells through deceased donor islet transplantation is a proven therapy for preventing recurrent life-threatening hypoglycemia in type 1 diabetes. Although near-normal glucose levels and insulin independence can be maintained for many years following successful islet transplantation, restoration of normal functional β-cell mass has remained elusive. It has recently been proposed that dedifferentiation / plasticity towards other endocrine phenotypes may play an important role in stress-induced β-cell dysfunction in type 2 diabetes. Here we report loss of end-differentiated β-cell phenotype in two intraportal islet allotransplant recipients. Despite excellent graft function and sustained insulin independence, all examined insulin-positive cells had lost expression of the end-differentiation marker, urocortin-3, or appeared to co-express the α-cell marker, glucagon. In contrast, no insulin(+) / urocortin-3(-) cells were seen in non-diabetic deceased donor control pancreatic islets. Loss of end-differentiated phenotype may facilitate β-cell survival during the stresses associated with islet isolation and culture, in addition to sustained hypoxia following engraftment. As further refinements in islet isolation and culture are made in parallel with exploration of alternative β-cell sources, graft sites and ultimately fully-vascularised bioengineered insulin-secreting microtissues, differentiation status immunostaining provides a novel tool to assess whether fully mature β-cell phenotype has been maintained. This article is protected by copyright. All rights reserved.

PURPOSE OF REVIEW Immunological barriers still preclude clinical xenotransplantation. The protective role of CD4(+)CD25(+)Foxp3(+) T regulatory cells (Treg) in allotransplantation is well described and therefore could represent a promising therapeutical tool for xenotransplantation. This review addresses the latest findings on Treg in xenotransplantation research. RECENT FINDINGS In vivo costimulation blockade based strategies including anti CD154 monoclonal antibodies (mAbs) in combination with rapamycin or anti LFA 1 mAb prolonged both concordant and discordant islets xenografts survival in a Treg dependent manner. In vitro IL 10 secretion was shown to be critical for the suppression of xenogeneic responses mediated by Treg. Moreover transgenic expression of inducible costimulator immunoglobulin or PD L1 on porcine endothelial cells inhibited human T cell proliferation in vitro and was associated with the induction of Treg and IL 10 secretion. CXCR3 mediated the recruitment of Treg to pig endothelium. Finally the recruitment of human Treg was enhanced by the immobilization of human CCL17 on pig endothelium. SUMMARY There is increasing evidence for the potential of CD4(+)CD25(+)Foxp3(+) Treg to protect xenografts. Induction of Treg in recipients and/or recruitment of human Treg to pig endothelium may represent novel strategies to prevent cell mediated rejection in pig to human xenotransplantation.

The major challenge in transplantation medicine remains long-term allograft acceptance, with preserved allograft function under minimal chronic immunosuppression. To safely achieve the goal of sustained donor-specific T and B cell non-responsiveness, research efforts are now focusing on therapies based on cell subsets with regulatory properties. In particular the transfusion of human regulatory T cells (Treg) is currently being evaluated in phase I/II clinical trials for the treatment of graft versus host disease following hematopoietic stem cell transplantation, and is also under consideration for solid organ transplantation. The purpose of this review is to recapitulate current knowledge on naturally occurring as well as induced human Treg, with emphasis on their specific phenotype, suppressive function and how these cells can be manipulated in vitro and/or in vivo for therapeutic purposes in transplantation medicine. We highlight the potential but also possible limitations of Treg-based strategies to promote long-term allograft survival. It is evident that the bench-to-beside translation of these protocols still requires further understanding of Treg biology. Nevertheless, current data already suggest that Treg therapy alone will not be sufficient and needs to be combined with other immunomodulatory approaches in order to induce allograft tolerance.

OBJECTIVE It is generally admitted that the endocrine cell organization in human islets is different from that of rodent islets. However, a clear description of human islet architecture has not yet been reported. The aim of this work was to describe our observations on the arrangement of human islet cells. RESEARCH DESIGN AND METHODS Human pancreas specimens and isolated islets were processed for histology. Sections were analyzed by fluorescence microscopy after immunostaining for islet hormones and endothelial cells. RESULTS In small human islets (40–60 μm in diameter), β-cells had a core position, α-cells had a mantle position, and vessels laid at their periphery. In bigger islets, α-cells had a similar mantle position but were found also along vessels that penetrate and branch inside the islets. As a consequence of this organization, the ratio of β-cells to α-cells was constantly higher in the core than in the mantle part of the islets, and decreased with increasing islet diameter. This core-mantle segregation of islet cells was also observed in type 2 diabetic donors but not in cultured isolated islets. Three-dimensional analysis revealed that islet cells were in fact organized into trilaminar epithelial plates, folded with different degrees of complexity and bordered by vessels on both sides. In epithelial plates, most β-cells were located in a central position but frequently showed cytoplasmic extensions between outlying non–β-cells. CONCLUSIONS Human islets have a unique architecture allowing all endocrine cells to be adjacent to blood vessels and favoring heterologous contacts between β- and α-cells, while permitting homologous contacts between β-cells.

Islet grafts isolated from young donors allow superior functional outcomes but are often associated with poor islet isolation yields. The objective of this study was to comparatively analyze the outcomes of islet isolation between young and older donors. We retrospectively analyzed 564 pancreas isolations performed at our institution. Isolation outcomes were compared between donors aged ≤20 years (n = 42, YD) and >20 years (n = 522, OD). Isolation procedure was identical in both groups. Prepurification percentage of embedded islets was higher in YD (44.3 ± 22.7% vs. 24.9 ± 20.9%, P

BACKGROUND Because metabolic markers are not suitable for early diagnosis of islet graft dysfunction magnetic resonance imaging (MRI) has been used to study islets that were labeled pretransplantation with superparamagnetic iron oxide nanoparticles. However the relation between graft functionality assessed by glycemia and MRI signal remains unclear. METHODS We transplanted hyperglycemic rats intraportally with 2500 ferucarbotran labeled syngeneic (n=10) or allogeneic (n=12) islet equivalents or normoglycemic rats with 5000 xenogeneic human islet equivalents. Images were acquired on a clinical 3 Tesla MRI scanner. RESULTS When rejection occurred on days 4 and 8 in xenogeneic and allogeneic recipients 60 (57 68) and 55 (46 73) of the initial signal remained compared to 93 (71 104) and 82 (59 90) in syngeneic controls (P=0.006 and 0.03). With a cutoff value of 84 on day 4 for the diagnosis of allogeneic rejection sensitivity of 91 and specificity of 70 were obtained. Based on MRI signal on day 4 treatment with antilymphocytic serum from day 4 allowed graft rescue in 75 of recipients. CONCLUSIONS In this model MRI of pretransplantation superparamagnetic iron oxide nanoparticle labeled islet grafts allows timely diagnosis of immune rejection.

Long-term insulin independence after islets of Langerhans transplantation is rarely achieved. The aims of this study were to identify the histological and immunological features of islets transplanted in a type 1 diabetic patient who died of a cerebral hemorrhage after >13 years insulin independence. Islets were pooled from two donors with respectively one and five HLA mismatches. Insulin-positive islets were found throughout the right and left liver, and absent in the pancreas. Two- and three-dimensional analysis showed that islets lost their initial rounded and compact morphology, had a mean diameter of 136 μm and were constituted of an unfolded epithelial band of 39.1 μm. Leukocyte phenotyping showed no evidence of a tolerogenic environment in the islet-containing portal spaces. Finally, HLA typing of microdissected islets showed HLA from the best matched donor in all 23 microdissection samples, compared to 1/23 for the least matched donor. This case report demonstrates that allogeneic islets can survive over 13 years while maintaining insulin independence. Allogeneic islets had unique morphologic features and implanted in the liver regardless of their size. Finally, our results suggest that, in this case, rejection had been prevalent over autoimmunity, although this hypothesis warrants further investigation.

International audience; BACKGROUND.: Insulin independence after islet transplantation is generally achieved after multiple infusions. However, single infusion would increase the number of recipients. Our aim was to evaluate the results of islet-after-kidney transplantation according to the number of infusions. METHODS.: Islets were isolated at the Geneva University, shipped, and transplanted into French patients from the Swiss-French GRAGIL network, on the "Edmonton" immunosuppression protocol between 2004 and 2010. RESULTS.: Nineteen patients were transplanted with 33 preparations. Fifteen patients reached 24 months follow-up; eight subjects were single-graft recipients and seven were double-graft recipients. Finally, single-graft recipients received a median of 5312 islet equivalents/kg (5186-6388) vs. 10,564 (10,054-11,375) for double-graft recipients (P=0.0003) with similar islet mass at first infusion. Insulin independence was achieved in five of eight single-graft subjects (62.5%) versus five of seven in double-graft subjects (71.4%), not significant. Median insulin independence duration was 4.7 (3.1-15.2) months after one infusion vs. 19 (9.6-20.8) months after two infusions (not significant). At 24 months posttransplant, comparing single- with double-graft patients, insulin doses were 0.23 (0.11-0.34) U/kg vs. 0.02 (0.0-0.23) U/kg, P=0.11; HbA1c was 6.5% (5.9%-6.8%) vs. 6.2% (5.9%-6.3%), P=0.16; and basal C-peptide was 302 (143-480) pmol/L vs. 599 (393-806) pmol/L, P=0.05. Only 37.5% of single-graft patients had a β-score ≥4 compared with 100% of double-graft patients (P=0.03). Two recipients experienced postinfusion bleeding, and two patients (13%) showed renal dysfunction in the absence of biopsy-proven rejection. CONCLUSIONS.: One infusion achieves good glycemic control and sometimes insulin independence. However, double-graft patients remain insulin-free longer, tend to have lower HbA1c, and show better graft function 24 months after transplant.

Exercise, obesity and type 2 diabetes are associated with elevated plasma concentrations of interleukin-6 (IL-6). Glucagon-like peptide-1 (GLP-1) is a hormone that induces insulin secretion. Here we show that administration of IL-6 or elevated IL-6 concentrations in response to exercise stimulate GLP-1 secretion from intestinal L cells and pancreatic alpha cells, improving insulin secretion and glycemia. IL-6 increased GLP-1 production from alpha cells through increased proglucagon (which is encoded by GCG) and prohormone convertase 1/3 expression. In models of type 2 diabetes, the beneficial effects of IL-6 were maintained, and IL-6 neutralization resulted in further elevation of glycemia and reduced pancreatic GLP-1. Hence, IL-6 mediates crosstalk between insulin-sensitive tissues, intestinal L cells and pancreatic islets to adapt to changes in insulin demand. This previously unidentified endocrine loop implicates IL-6 in the regulation of insulin secretion and suggests that drugs modulating this loop may be useful in type 2 diabetes.

Extended pancreatectomy is associated with the risk of surgical diabetes. Islet autotransplantation is successful in the prevention of diabetes after pancreas resection for chronic pancreatitis (CP), with insulin independence rates of 50% at 1 year. The aim of the present study is to demonstrate the safety and efficiency of islet autotransplantation after extended left pancreatectomy for benign disease.Between 1992 and 2009, 25 patients underwent extended pancreatectomy and islet autotransplantation for benign disease. Of these, 15 patients were operated for focal lesions located at the neck of the pancreas (14 benign tumors and 1 traumatic pancreatic section), the remainder being CP cases. After unequivocal diagnosis of benignity, the rest of the pancreas was processed and infused into the portal vein. Metabolic results were analyzed and isolation results were compared with those obtained from patients with CP or donors with brain death (DBD).There was no mortality and a low morbidity (Streptococcus mitis bacteremia in 1 patient), no portal thrombosis or pancreatic fistula occurred. Median follow-up was 90 months. Actuarial patient survival was 100% at 10 years. Actuarial insulin independence was 94% at 10 years. All patients had positive basal and stimulated C-peptide levels and normal HbA1c. Mean islet yields were 5455 IEQ/gram vs. 1457 in CP (P=0.001) and 3738 in DBD (P=0.003).Islet autotransplantation after extensive pancreatic resection for benign disease is a safe and successful procedure. Islet yields after isolation, which are equivalent to the live donor situation, are significantly better than those from DBD donors.

International audience; BACKGROUND: It has been suggested that the age of human organ donors might influence islet isolation and transplantation outcome in a negative way due to a decrease of in vivo function in islets isolated from older donors. METHODS: We retrospectively analyzed 332 islet isolations according to donor age. We determined isolation outcome by islet yields, transplantation rates, and [beta]-cell function in vitro. Transplanted patients were divided into two groups depending on donor age (n=25 and n=31 patients for 45-year-old donors, respectively). We assessed islet graft function by C-peptide/glucose ratio, [beta] score, secretory units of islets in transplantation index, and insulin independence rate at 1, 6, and 12 months after transplantation. RESULTS: There was no difference in islet yields between the two groups (251,900+/-14,100 and 244,600+/-8400 islet equivalent for 45-year-old donors, respectively). Transplantation rates and stimulation indices were similar in both groups as well. All islet graft function parameters were significantly higher at 1-month follow-up in patients who had received islets from younger donors. At 6-month follow-up after second or third injection and at 12-month follow-up, secretory units of islets in transplantation indices and C-peptide/glucose ratios were significantly higher in patients with donors aged 45 years or younger. CONCLUSIONS: These data suggest that, despite similar outcomes of the isolation procedure, islet graft function is significantly influenced by donor age. These results may have important consequences in the definition of pancreas allocation criteria.

OBJECTIVE Streptozotocin (STZ) is the most widely used diabetogenic agent in animal models of islet transplantation. However, the immunomodifying effects of STZ and the ensuing hyperglycemia on lymphocyte subsets, particularly on T regulatory cells (Tregs), remain poorly understood. RESEARCH DESIGN AND METHODS This study evaluated how STZ-induced diabetes affects adaptive immunity and the consequences thereof on allograft rejection in murine models of islet and skin transplantation. The respective toxicity of STZ and hyperglycemia on lymphocyte subsets was tested in vitro. The effect of hyperglycemia was assessed independently of STZ in vivo by the removal of transplanted syngeneic islets, using an insulin pump, and with rat insulin promoter diphtheria toxin receptor transgenic mice. RESULTS Early lymphopenia in both blood and spleen was demonstrated after STZ administration. Direct toxicity of STZ on lymphocytes, particularly on CD8+ cells and B cells, was shown in vitro. Hyperglycemia also correlated with blood and spleen lymphopenia in vivo but was not lymphotoxic in vitro. Independently of hyperglycemia, STZ led to a relative increase of Tregs in vivo, with the latter retaining their suppressive capacity in vitro. The higher frequency of Tregs was associated with Treg proliferation in the blood, but not in the spleen, and higher blood levels of transforming growth factor-β. Finally, STZ administration delayed islet and skin allograft rejection compared with naive mice. CONCLUSIONS These data highlight the direct and indirect immunosuppressive effects of STZ and acute hyperglycemia, respectively. Thus, these results have important implications for the future development of tolerance-based protocols and their translation from the laboratory to the clinic.

BACKGROUND: Anti-CD154 (MR1) monoclonal antibody (mAb) and rapamycin (RAPA) treatment both improve survival of rat-to-mouse islet xenograft. The present study investigated the effect of combined RAPA/MR1 treatment on rat-to-mouse islet xenograft survival and analyzed the role of CD4(+)CD25(+)Foxp3(+) T regulatory cells (Treg) in the induction and maintenance of the ensuing tolerance. METHODOLOGY/PRINCIPAL FINDINGS: C57BL/6 mice were treated with MR1/RAPA and received additional monoclonal anti-IL2 mAb or anti CD25 mAb either early (0-28 d) or late (100-128 d) post-transplantation. Treg were characterised in the blood, spleen, draining lymph nodes and within the graft of tolerant and rejecting mice by flow cytometry and immunohistochemistry. Fourteen days of RAPA/MR1 combination therapy allowed indefinite islet graft survival in >80% of the mice. Additional administration of anti-IL-2 mAb or depleting anti-CD25 mAb at the time of transplantation resulted in rejection (100% and 89% respectively), whereas administration at 100 days post transplantation lead to lower rejection rates (25% and 40% respectively). Tolerant mice showed an increase of Treg within the graft and in draining lymph nodes early post transplantation, whereas 100 days post transplantation no significant increase of Treg was observed. Rejecting mice showed a transient increase of Treg in the xenograft and secondary lymphoid organs, which disappeared within 7 days after rejection. CONCLUSIONS/SIGNIFICANCES: These results suggest a critical role for Treg in the induction phase of tolerance early after islet xenotransplantation. These encouraging data support the need of developing further Treg therapy for overcoming the species barrier in xenotransplantation.

The role of T regulatory cells (Treg) in the induction and maintenance of allograft tolerance is being studied to a great extent. In contrast, little is known on their potential to prevent graft rejection in the field of xenotransplantation, where acute vascular rejection mediated by cellular and humoral mechanisms and thrombotic microangiopathy still prevents long-term graft survival. In this regard, the induction of donor-specific tolerance through isolation and expansion of xenoantigen-specific recipient Treg is currently becoming a focus of interest. This review will summarize the present knowledge concerning Treg and their potential use in xenotransplantation describing in particular CD4(+)CD25(+)Foxp3(+) T cells, CD8(+)CD28(-) Treg, double negative CD4(-)CD8(-) T cells, and natural killer Treg. Although only studied in vitro so far, human CD4(+)CD25(+)Foxp3(+) Treg is currently the best characterized subpopulation of regulatory cells in xenotransplantation. CD8(+)CD28(-) Treg and double negative CD4(-)CD8(-) Treg also seem to be implicated in tolerance maintenance of xenografts. Finally, one study revealing a role for natural killer CD4(+)Valpha14(+) Treg in the prolongation of xenograft survival needs further confirmation. To our opinion, CD4(+)CD25(+)Foxp3(+) Treg are a promising candidate to protect xenografts. In contrast to cadaveric allotransplantation, the donor is known prior to xenotransplantation. This advantage allows the expansion of recipient Treg in a xenoantigen specific manner before transplantation.

BACKGROUND Toll like receptors are key players in sterile inflammation phenomena and can link the innate and adaptive immune systems by enhancing graft immunogenicity. They are also considered mediators of types 1 and 2 diabetes development. The aim of the present study was to assess the role of Toll like receptor 4 (TLR4) in mediating the inflammatory and immune responses to pancreatic islets thereby promoting inflammatory destruction and immune rejection of islet grafts. METHODS Experiments were conducted in murine and human in vitro systems and in vivo murine islet transplant models using species specific anti TLR4 monoclonal antibodies. In vitro mixed lymphocyte islet reaction experiments were performed to assess T cell activation and proliferation. In vivo both a syngeneic (B6 to B6) marginal mass islet transplant model to assess the impact of TLR4 blockade on islet engraftment and an allogeneic (DBA1 to B6) model were used. RESULTS In vitro TLR4 blockade decreased lipopolysaccharide mediated ß cell apoptosis and T cell activation and proliferation against allogeneic islets. In vivo TLR4 blockade resulted in significantly better syngeneic marginal mass islet engraftment and in indefinite allogeneic islet graft survival. Tolerance was not observed because donor specific skin graft rechallenge in nonrejecting animals resulted in rejection of both skin and islets but without accelerated rejection as compared to naive animals. CONCLUSION Taken together our data indicate that TLR4 blockade leads to a significant improvement of syngeneic islet engraftment and of allogeneic islet graft survival. A mechanism of graft accommodation with concurrent inhibition of donor specific immune memory is likely to be involved.

In the field of regenerative medicine, one of the ultimate goals is to generate functioning organs from pluripotent cells, such as ES cells or induced pluripotent stem cells (PSCs). We have recently generated functional pancreas and kidney from PSCs in pancreatogenesis- or nephrogenesis-disabled mice, providing proof of principle for organogenesis from PSCs in an embryo unable to form a specific organ. Key when applying the principles of in vivo generation to human organs is compensation for an empty developmental niche in large nonrodent mammals. Here, we show that the blastocyst complementation system can be applied in the pig using somatic cell cloning technology. Transgenic approaches permitted generation of porcine somatic cell cloned embryos with an apancreatic phenotype. Complementation of these embryos with allogenic blastomeres then created functioning pancreata in the vacant niches. These results clearly indicate that a missing organ can be generated from exogenous cells when functionally normal pluripotent cells chimerize a cloned dysorganogenetic embryo. The feasibility of blastocyst complementation using cloned porcine embryos allows experimentation toward the in vivo generation of functional organs from xenogenic PSCs in large animals.

Mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs) have been extensively investigated in small animal models to treat both acute and chronic liver injuries. Mechanisms of action are not clearly elucidated but may include their ability to differentiate into hepatocyte-like cells, to reduce inflammation, and to enhance tissue repair at the site of injury. This approach is controversial and evidence in large animals is missing. Side effects of MSC infusion such as the contribution to a fibrotic process have been reported in experimental settings. Nevertheless, MSCs moved quickly from bench to bedside and over 280 clinical trials are registered, of which 28 focus on the treatment of liver diseases. If no severe side-effects were observed so far, long-term benefits remain uncertain. More preclinical data regarding mechanisms of action, long term safety and efficacy are warranted before initiating large scale clinical application. The proposal of this review is to visit the current state of knowledge regarding mechanisms behind the therapeutic effects of MSCs in the treatment of experimental liver diseases, to address questions about efficacy and risk, and to discuss recent clinical advances involving MSC-based therapies.

Background & Aims: Mesenchymal stem cell (MSC) transplantation was shown to be effective for the treatment of liver fibrosis, but the mechanisms of action are not yet fully understood. We transplanted encapsulated human MSCs in two mouse models of liver fibrosis to determine the mechanisms behind the protective effect. Methods: Human bone marrow-derived MSCs were microencapsulated in novel alginate-polyethylene glycol microspheres. In vitro, we analyzed the effect of MSC-conditioned medium on the activation of hepatic stellate cells and the viability, proliferation, cytokine secretion, and differentiation capacity of encapsulated MSCs. The level of fibrosis induced by bile duct ligation (BDL) or carbon tetrachloride (CCl4) was assessed after intraperitoneal transplantation of encapsulated MSCs, encapsulated human fibroblasts, and empty microspheres. Results: MSC-conditioned medium inhibited hepatic stellate cell activation and release of MSC secreted anti-apoptotic (IL-6, IGFBP-2) and anti-inflammatory (IL-1Ra) cytokines. Viability, proliferation, and cytokine secretion of microencapsulated MSCs were similar to those of non-encapsulated MSCs. Within the microspheres, MSCs maintained their capacity to differentiate into adipocytes, chondrocytes, and osteocytes. 23% (5/22) of the MSC clones were able to produce anti-inflammatory IL-1Ra in vitro. Microencapsulated MSCs significantly delayed the development of BDL-and CCl4-induced liver fibrosis. Fibroblasts had an intermediate effect against CCl4-induced fibrosis. Mice transplanted with encapsulated MSCs showed lower mRNA levels of collagen type I, whereas levels of matrix metalloproteinase 9 were significantly higher. Human IL-1Ra was detected in the serum of 36% (4/11) of the mice transplanted with microencapsulated MSCs. Conclusions: MSC-derived soluble molecules are responsible for an anti-fibrotic effect in experimental liver fibrosis. (C) 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

The aim of this study was to assess the expression of different types of cadherins in human islets and their role in human β-cell apoptosis. Expression of E-, N-, and P-cadherins was studied by immunofluorescence on pancreas sections and islet cells, and by Western blotting on protein extracts of isolated islets and islet cells. The effects of specific cadherins on cell adhesion and apoptosis were studied using chimeric proteins containing functional E-, N-, or P-cadherin ectodomains fused to Fc fragment of Ig (E-cad/Fc, N-cad/Fc, and P-cad/Fc) and immobilized on glass substrate. β-Cells were identified by immunofluorescence for insulin and apoptotic cells by terminal deoxynucleotide transferase-mediated 2′-deoxyuridine, 5′-triphosphate nick-end labeling. By immunofluorescence, we showed that E- and N-, and not P-, cadherins were expressed at the surface of islet cells. By triple staining, we showed that E-cadherin was expressed at similar extent in β- and α-cells, whereas N-cadherin was preferentially expressed in β-cells. These results were confirmed by Western blot analysis using protein extracts from fluorescence-activated cell sorting-sorted β- and non-β-cells. Adhesion tests showed that the affinity of islet cells for E-cad/Fc and N-cad/Fc and not for P-cad/Fc was increased compared with control. By terminal deoxynucleotide transferase-mediated 2′-deoxyuridine, 5′-triphosphate nick-end labeling, we showed that the percentage of apoptotic cells was lower in aggregated β-cells compared with single β-cells and that attachment to E-cad/Fc and N-cad/Fc and not to P-cad/Fc decreased apoptosis of single β-cells compared with control. Our results show that at least E- and N-cadherins are expressed at the surface of human β-cells and that these adhesion molecules are involved in the maintenance of β-cell viability.

Bone marrow was recently proposed as an alternative and potentially immune-privileged site for pancreatic islet transplantation. The aim of the present study was to assess the survival and rejection mechanisms of free and encapsulated xenogeneic islets transplanted into the medullary cavity of the femur, or under the kidney capsule of streptozotocin-induced diabetic C57BL/6 mice. The median survival of free rat islets transplanted into the bone marrow or under the kidney capsule was 9 and 14 days, respectively, whereas that of free human islets was shorter, 7 days (bone marrow) and 10 days (kidney capsule). Infiltrating CD8(+) T cells and redistributed CD4(+) T cells, and macrophages were detected around the transplanted islets in bone sections. Recipient mouse splenocytes proliferated in response to donor rat stimulator cells. One month after transplantation under both kidney capsule or into bone marrow, encapsulated rat islets had induced a similar degree of fibrotic reaction and still contained insulin positive cells. In conclusion, we successfully established a small animal model for xenogeneic islet transplantation into the bone marrow. The rejection of xenogeneic islets was associated with local and systemic T cell responses and macrophage recruitment. Although there was no evidence for immune-privilege, the bone marrow may represent a feasible site for encapsulated xenogeneic islet transplantation.