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FOXP3+ regulatory T cells (Tregs) are central to maintaining peripheral tolerance and immune homeostasis. They have the potential to be developed as a cellular therapy to treat various clinical ailments such as autoimmune disorders, inflammatory diseases and to improve transplantation outcomes. However, a major question remains whether Tregs can persist and exert their function effectively in a disease state, where a broad spectrum of inflammatory mediators could inactivate Tregs. In this study, we investigated the potential of mesenchymal stem cell (MSC)-derived exosomes to promote and sustain Tregs function. MSC-conditioned media (MSC-CM) cultured Tregs were more suppressive in both polyclonal and allogeneic responses and were resistant to inflammatory stimulation in vitro compared with the controls. A similar enhancement of Treg function was also observed by culturing Tregs with MSC-derived exosomes alone. The enhanced suppressive activity and stability of Treg cultured in MSC-CM was reduced when exosomes were depleted from MSC-CM. We identified that MSC-derived exosomes could upregulate the expression of LC3(II/I), phosphorylate Jak3 and Stat5 to promote Treg survival, and regulate FOXP3 expression in Tregs. Overall, our study demonstrates that MSC-derived exosomes are capable of enhancing Hucb-Tregs function and stability by activating autophagy and Stat5 signalling pathways. Our findings provide a strong rationale for utilizing MSC-derived exosomes as an effective strategy to enhance Treg function, and improve the overall Tregs-based cell therapy landscape. Hucb-Tregs treated with MSC-CM or MSC-derived exosomes were more suppressive and were resistant to inflammatory stimulation. MSC-derived exosomes could activate autophagy and JAK3-Stat5 signalling pathway to promote Treg survival, and regulate FOXP3 expression. MSC-derived exosomes could be an effective strategy to improve the Tregs-based cell therapy landscape. [ABSTRACT FROM AUTHOR]

We have analysed insulin antibodies in 149 adults with type 1 diabetes and 2859 people without diabetes. We have determined that insulin antibody levels are higher in adults with, versus without, diabetes and that the levels are falling, and more patients are becoming antibody‐negative post islet cell transplantation. [ABSTRACT FROM AUTHOR]

The role of Regulatory T cells (Tregs) in tolerance induction post-transplantation is well-established, but Tregs adoptive transfer alone without combined immunosuppressants have failed so far in achieving clinical outcomes. Here we applied a set of well-designed criteria to test the influence of commonly used immunosuppressants (belatacept, tacrolimus, and mycophenolate) on cord blood-derived Tregs (CB-Tregs). Our study shows that while none of these immunosuppressants modulated the stability and expression of homing molecules by CB-Tregs, belatacept met all other selective criteria, shown by its ability to enhance CB-Tregs-mediated in vitro suppression of the allogeneic response without affecting their viability, proliferation, mitochondrial metabolism and expression of functional markers. In contrast, treatment with tacrolimus or mycophenolate led to reduced expression of functional molecule GITR in CB-Tregs, impaired their viability, proliferation and mitochondrial metabolism. These findings indicate that belatacept could be considered as a candidate in Tregs-based clinical immunomodulation regimens to induce transplant tolerance. [ABSTRACT FROM AUTHOR]

Islet transplantation is an emerging therapy for type 1 diabetes and hypoglycemic unawareness. However, a key challenge for islet transplantation is cellular rejection and the requirement for long-term immunosuppression. In this study, we established a diabetic humanized NOD-scidIL2Rγnull (NSG) mouse model of T-cell-mediated human islet allograft rejection and developed a therapeutic regimen of low-dose recombinant human interleukin-2 (IL-2) combined with low-dose rapamycin to prolong graft survival. NSG mice that had received renal subcapsular human islet allografts and were transfused with 1 × 107 of human spleen mononuclear cells reconstituted human CD45+ cells that were predominantly CD3+ T cells and rejected their grafts with a median survival time of 27 days. IL-2 alone (0.3 × 106 IU/m2 or 1 × 106 IU/m2) or rapamycin alone (0.5-1 mg/kg) for 3 weeks did not prolong survival. However, the combination of rapamycin with IL-2 for 3 weeks significantly prolonged human islet allograft survival. Graft survival was associated with expansion of CD4+CD25+FOXP3+ regulatory T cells (Tregs) and enhanced transforming growth factor-β production by CD4+ T cells. CD8+ T cells showed reduced interferon-γ production and reduced expression of perforin-1. The combination of IL-2 and rapamycin has the potential to inhibit human islet allograft rejection by expanding CD4+FOXP3+ Tregs in vivo and suppressing effector cell function and could be the basis of effective tolerance-based regimens. [ABSTRACT FROM AUTHOR]

Understanding the immunological phenotype of transplant recipients is important to improve outcomes and develop new therapies. Immunophenotyping of whole peripheral blood (WPB) by flow cytometry is a rapid method to obtain large amounts of data relating to the outcomes of different transplant treatments with limited patient impact. Healthy individuals and patients with type 1 diabetes (T1D) enrolled in islet transplantation were recruited and WPB was collected. 46 fluorochrome-conjugated mouse-anti-human antibodies were used (43 of 46 antibodies were titrated). BD cytometer setup and tracking beads were used to characterize and adjust for cytometer performance. Antibody cocktails were pre-mixed <60 minutes before staining. Multicolour panels were designed based on fluorochrome brightness, antigen density, co-expression, and fluorochrome spillover into non-primary detectors in each panel on a 5 laser flow cytometer. WPB sample staining used 50–300 μl WPB for each panel and was performed within 2 hours of blood sample collection. Samples were acquired on a BD-LSRFortessa. The operating procedures, including specimen collection, antibody cocktails, staining protocol, flow-cytometer setup and data analysis, were standardized. The staining index of 43 antibodies and the spillover spreading matrix for each panel was calculated. The final concentrations for the 46 antibodies used was determined for staining of WPB samples. Absolute cell-count and 7 leukocyte profiling panels consisting of subsets and/or status of granulocytes, monocytes, dendritic, B, NK, and T cells including regulatory T cells (Tregs) and NKT were designed and established on a 5 laser BD-LSR Fortessa. 13 T1D patients, including 4 islet transplant recipients and 8 healthy controls, were evaluated. The ability to reproducibly measure immune subsets and immune-profiles of islet transplant patients up to 18 months post transplantation has been established as a tool to measure immune cell reconstitution after transplantation. [ABSTRACT FROM AUTHOR]

Abstract: Background: Hypoxia‐induced damage is one of the key factors associated with islet graft dysfunction. Mesenchymal stem cells (MSCs) could be used to enhance the therapeutic effect of islet transplantation due to their paracrine potential such as exosomes. In this study, we investigated whether exosomes from human umbilical cord‐derived MSC‐conditioned medium (hu‐MSC‐CM) could increase the survival and function of neonatal porcine islet cell clusters (NICCs) exposed to hypoxia. Methods: Neonatal porcine islet cell clusters were cultured with hu‐MSC‐CM, with or without exosomes, and native medium RPMI‐1640 (Control) under hypoxic conditions (1% O2). The effects of exosomes on NICCs viability and function in vitro were examined by FACS, the Loops system, and the Extracellular Flux assay, respectively. Results: Compared with NICCs cultured in RPMI‐1640 medium and hu‐MSC‐CM without exosomes, the survival ratio, viability, and function increased in NICCs cultured in hu‐MSC‐CM with exosomes. Conclusions: This study found that hu‐MSC‐CM could protect NICCs from hypoxia‐induced dysfunction, and exosomes played an important role in hypoxic resistance, suggesting a potential strategy to improve islet transplantation outcomes. [ABSTRACT FROM AUTHOR]

Abstract: Background: Neonatal pig islet‐like cell clusters (NICC) are an attractive source of insulin‐producing tissue for potential transplantation treatment of type 1 diabetic patients. However, a considerable loss of NICC after their transplantation due to apoptosis resulted from islet isolation and instant blood‐mediated inflammatory reaction remains to be overcome. Methods: EGM2 medium depleted with hydrocortisone and supplemented with 50 mmol/L isobutylmethylxanthine, 10 mmol/L nicotinamide, and 10 mmol/L glucose was used to culture NICC at day 1, the day after isolation and changed every other day. NICC cultured with EGM2 or control Ham's F‐10 medium were collected at day 7 of culture for the following assays. The viability of NICC was evaluated by AO/EB staining and FACS. Static assay and oxygen consumption rate analysis were performed to assess the function of NICC. Insulin and glucagon gene expression were measured by real‐time PCR. Tubing loops model and TUNEL assay were performed to confirm the apoptosis‐resistant ability of NICC cultured with modified EGM2 medium. Serum starvation and hypoxia treatment were used to test the tolerant capability of NICC in the microenvironment of hypoxia/nutrient deficiency in vitro. The molecules involved in apoptosis pathways in NICC were analyzed by Western blotting. Results: Compared with Ham's F‐10 medium, culturing NICC with EGM2 medium led to increased number and viability of NICC with higher stimulation index, upregulated gene expression of both insulin and glucagon, and enhanced mitochondria function. Furthermore, fewer modified EGM2 medium cultured NICC were found under apoptosis when evaluated in an in vitro tubing loop model of IBMIR. Moreover, EGM2 medium cultured NICC demonstrated much less apoptotic cells under either serum starvation or hypoxia condition than their Ham's F‐10 medium cultured counterparts. The enhanced capability of EGM2 cultured NICC to resist apoptosis was associated with their elevated protein levels of anti‐apoptotic Bcl‐2 family member Mcl‐1. Conclusion: Culturing NICC with EGM2 provides a simple and effective approach not only to increase NICC yield, viability, and maturation but also to enhance their resistance to apoptosis to preserve the initial graft mass for successful islet xenotransplantation. [ABSTRACT FROM AUTHOR]

Background A high immunosuppressive burden is required for long-term islet xenograft survival in non-human primates even using genetically modified donor pigs. Aims We aimed to investigate the capacity of baboon regulatory T cells (Treg) to suppress islet xenograft rejection, thereby developing a potential immunoregulatory or tolerance therapy that could be evaluated in NHP models of xenotransplantation. Materials & Methods Baboon Treg expanded with stimulation by porcine peripheral blood mononuclear cells ( PBMC) were characterized by cell phenotyping and suppressive activity assays in vitro. Their function in vivo was evaluated in neonatal porcine islet cell clusters ( NICC) transplanted NOD- SCID IL-2rγ−/− ( NSG) mice receiving baboon PBMC alone or with expanded autologous Treg. Results The majority of expanded Treg coexpressed Foxp3 and CD39 and were highly suppressive of the baboon anti-pig xenogeneic T cell response in vitro. Reconstitution of mice with baboon PBMC alone resulted in NICC xenograft rejection within 35 days. Cotransfer with baboon PBMC and Treg prolonged islet xenograft survival beyond 100 days, correlating with Treg engraftment, intragraft CD39 and Foxp3 gene expression, and reduced graft infiltrating effector T cells and reduced interferon-γ production. Discussion & Conclusion Our data supports the capacity of ex vivo expanded CD39+ baboon Treg to suppress islet xenograft rejection in primatized mice, suggesting it has potential as an adjunctive immunotherapy in preclinical NHP models of xenotransplantation. [ABSTRACT FROM AUTHOR]

Background For xenotransplantation to truly succeed, we must develop immunomodulatory strategies to suppress the xenoimmune response but by minimizing immunosuppression over the long term. Regulatory macrophages (Mreg) have been shown to suppress polyclonal T-cell proliferation in vitro and prolong allograft survival in vivo. However, the question of whether they are capable of suppressing xenoimmune responses remains unknown. This study assessed the potential of human Mreg to be used as an effective immunomodulatory method in xenotransplantation. Methods CD14+ monocytes selected from human peripheral blood mononuclear cells ( PBMC) were cultured with macrophage colony-stimulating factor (M- CSF) for 7 days with IFN-γ added at day 6 for Mreg induction. Mreg phenotyping was performed by flow cytometric analysis, and the in vitro suppressive function was assessed by mixed lymphocyte reaction ( MLR) using irradiated pig PBMC as the xenogeneic stimulator cells, human PBMC as responder cells, and autologous Mreg as suppressor cells. To assess mRNA expression of Mreg functional molecules indoleamine-2,3-dioxygenase ( IDO), IL-10, inducible nitric oxide synthase ( iNOS) and TGF-β were measured by real-time PCR. Supernatants were collected from the MLR cultures for IDO activity assay by high-performance liquid chromatography ( HPLC). The effects of the IDO inhibitor 1-D/L-methyl-tryptophan (1- MT), iNOS inhibitor NG-monomethyl-l-arginine (L- NMMA), and anti- IFN-γ or anti- TGF-β monoclonal antibody ( mAb) treatment on Mreg suppressive capacity were tested from the supernatants of the MLR assays. Results We demonstrated that induced Mreg with a phenotype of CD14low CD16−/low CD80low CD83−/low CD86+/hi HLA- DR+/hi were capable of suppressing proliferating human PBMC, CD4+, and CD8+ T cells, even at a higher responder:Mreg ratio of 32:1 in a pig-human xenogeneic MLR. The strong suppressive potency of Mreg was further correlated with their upregulated IDO expression and activity. The IDO upregulation of Mreg was associated with an increased production of IFN-γ, an IDO stimulator, by xenoreactive responder cells in the xenogeneic MLR. While no effect on Mreg suppressive potency was detected by addition of the iNOS inhibitor L- NMMA or anti- TGF-β mAb into the MLR assays, inhibition of IDO activity by neutralizing IFN-γ or by IDO inhibitor 1- MT substantially impaired the capacity of Mreg to suppress the xenogeneic response, indicating the importance of upregulated IDO activity in Mreg-mediated suppression of the xenogeneic response in vitro. Conclusion This study demonstrates that human Mreg are capable of suppressing the xenoimmune response in vitro via IDO-involved mechanism(s), suggesting their potential role as an effective immunomodulatory tool in xenotransplantation. [ABSTRACT FROM AUTHOR]

The bone marrow cavity (BMC) has recently been identified as an alternative site to the liver for islet transplantation. This study aimed to compare the BMC with the liver as an islet allotransplantation site in diabetic monkeys. Diabetes was induced in Rhesus monkeys using streptozocin, and the monkeys were then divided into the following three groups: Group1 (islets transplanted in the liver with immunosuppressant), Group 2 (islets transplanted in the tibial BMC), and Group 3 (islets transplanted in the tibial BMC with immunosuppressant). The C-peptide and blood glucose levels were preoperatively measured. An intravenous glucose tolerance test (IVGTT) was conducted to assess graft function, and complete blood cell counts were performed to assess cell population changes. Cytokine expression was measured using an enzyme-linked immune sorbent assay (ELISA) and MILLIPLEX. Five monkeys in Group 3 exhibited a significantly increased insulin-independent time compared with the other groups (Group 1: 78.2 ± 19.0 days; Group 2: 58.8 ± 17.0 days; Group 3: 189.6 ± 26.2 days) and demonstrated increases in plasma C-peptide 4 months after transplantation. The infusion procedure was not associated with adverse effects. Functional islets in the BMC were observed 225 days after transplantation using the dithizone (DTZ) and insulin/glucagon stains. Our results showed that allogeneic islets transplanted in the BMC of diabetic Rhesus monkeys remained alive and functional for a longer time than those transplanted in the liver. This study was the first successful demonstration of allogeneic islet engraftment in the BMC of non-human primates (NHPs). [ABSTRACT FROM AUTHOR]

Objective: Chronic low-grade inflammation has long been recognized as the central link between obesity and type 2 diabetes (T2D). The novel subset of T helper (Th) cells, Th22, plays an emerging role in chronic inflammation. We investigated the potential association between Th22 and the pathogenesis of obesity and T2D. Methods: Ninety T2D inpatients (T2D group), 30 healthy participants with BMI ranged from 19 to 23.9 kg/m2 (CTL group) and 30 metabolically healthy obese controls with BMI ≥ 30 kg/m2 (MHO group) were employed in our study. Peripheral frequencies of Th22 and Th1 and Th17 cells were determined by flow cytometry based on their specific cytokine patterns. Cytokine levels in fresh plasma were quantified by ELISA. Results: Compared to that in CTL group (1.18±0.06%, n = 28), peripheral frequency of Th22 cells was significantly increased in MHO group (1.88±0.10%, n = 30) and in T2D group (2.247±0.10%, n = 89). There was a consistent notable increase in plasma interleukin (IL)-22 of T2D patients [47.56 (30.55–76.89) pg/mL] as compared with that of MHO group [36.65 (29.52–55.70) pg/ml; *P<0.0001] and CTLs [36.33 (31.93–40.62) pg/mL; *P<0.0001]. Furthermore, other than Th1/Th17, previously frequently described participants in obesity and T2D, there was a strong correlation between Th22 frequency and the homeostasis model of assessment for insulin resistance index (r = 0.6771, *P<0.0001) and HOMA for β-cell function (r = −0.7264, *P<0.0001). Conclusions: There were increased Th22 frequencies and IL-22 levels in obesity and T2D. Elevated Th22 and IL-22 also aided in the differentiation of MHO from T2D patients. The notable correlation implied that Th22 might play a more determinant role in both insulin resistance and β-cell impairment. [ABSTRACT FROM AUTHOR]

Beta cell replacement therapy has been proposed as a novel therapy for the treatment of type 1 diabetes. The proof of concept has been demonstrated with successful islet allotransplantation. Islet xenotransplantation has been proposed as an alternative, more reliable, and infinite source of beta cells. The advantages of islet xenotransplantation are the ability to transplant a well differentiated cell that is responsive to glucose and the potential for genetic modification which focuses the treatment on the donor rather than the recipient. The major hurdle remains overcoming the severe cellular rejection that affects xenografts. This review will focus on the major advances that have occurred with genetic modification and the successful therapeutic strategies that have been demonstrated in nonhuman primates. Novel approaches to overcome cell-mediated rejection including biological agents that target selectively costimulation molecules, the development of local immunosuppression through genetic manipulation, and encapsulation will be discussed. Overall, there has been considerable progress in all these areas, which eventually should lead to clinical trials. [ABSTRACT FROM AUTHOR]

Hypoxia and islet inflammation are involved in β-cell failure in type 2 diabetes (T2D). Elevated plasma LPS levels have been verified in patients with T2D, and hypoxia occurs in islets of diabetic mice. Activation of inflammasomes in ischemic or hypoxic conditions was identified in various tissues. Here, we investigated whether hypoxia activates the inflammasome in β cells and the possible mechanisms involved. In mouse insulinoma cell line 6 (MIN6), hypoxia (1% O 2 ) primes the NLRP3 inflammasome along with NF-κB signaling activation. Our results demonstrate that hypoxia can activate the NLRP3 inflammasome in LPS-primed MIN6 to result in initiating the β cell inflammatory response and cell death in vitro. Reactive oxygen species (ROS) and the thioredoxin-interacting protein (TXNIP) are up-regulated in response to hypoxia. Finally, the role of the ROS-TXNIP axis in mediating the activation of the NLRP3 inflammasome and cell death was characterized by pretreating with the ROS scavenger N-acetylcysteine (NAC) and performing TXNIP knockdown experiments in MIN6. Our data indicate for the first time that the inflammasome is involved in the inflammatory response and cell death in hypoxia-induced β cells through the ROS-TXNIP-NLRP3 axis in vitro. This provides new insight into the relationship between hypoxia and inflammation in T2D. [ABSTRACT FROM AUTHOR]

Background & Aims Bile acids (BAs) play a crucial role in dietary fat digestion and in the regulation of lipid, glucose, and energy metabolism. Thyroid-stimulating hormone (TSH) is a hormone produced by the anterior pituitary gland that directly regulates several metabolic pathways. However, the impact of TSH on BA homeostasis remains largely unknown. Methods We analyzed serum BA and TSH levels in healthy volunteers under strict control of caloric intake. Thyroidectomized rats were administered thyroxine and injected with different doses of TSH. Tshr −/− mice were supplemented with thyroxine, and C57BL/6 mice were injected with Tshr -siRNA via the tail vein. The serum BA levels, BA pool size, and fecal BA excretion rate were measured. The regulation of SREBP-2, HNF-4α, and CYP7A1 by TSH were analyzed using luciferase reporter, RNAi, EMSA, and CHIP assays. Results A negative correlation was observed between the serum levels of TSH and the serum BA levels in healthy volunteers. TSH administration led to a decrease in BA content and CYP7A1 activity in thyroidectomized rats supplemented with thyroxine. When Tshr was silenced in mice, the BA pool size, fecal BA excretion rate, and serum BA levels all increased. Additionally, we found that HNF-4α acts as a critical molecule through which TSH represses CYP7A1 activity. We further confirmed that the accumulation of mature SREBP-2 protein could impair the capacity of nuclear HNF-4α to bind to the CYP7A1 promoter, a mechanism that appears to mediate the effects of TSH. Conclusions TSH represses hepatic BA synthesis via a SREBP-2/HNF-4α/CYP7A1 signaling pathway. This finding strongly supports the notion that TSH is an important pathophysiological regulator of liver BA homeostasis independently of thyroid hormones. [ABSTRACT FROM AUTHOR]