Diabetes is an emerging global epidemic that affects more that 285 million people worldwide. Engineering of endocrine pancreas tissue holds great promise for the future of diabetes therapy. Here we demonstrate the feasibility of re-engineering decellularized organ scaffolds using regenerative cell source. We differentiated human pluripotent stem cells (hPSC) toward pancreatic progenitor (PP) lineage and repopulated decellularized organ scaffolds with these hPSC-PP cells. We observed that hPSCs cultured and differentiated as aggregates are more suitable for organ repopulation than isolated single cell suspension. However, recellularization with hPSC-PP aggregates require a more extensive vascular support, which was found to be superior in decellularized liver over the decellularized pancreas scaffolds. Upon continued culture for nine days with chemical induction in the bioreactor, the seeded hPSC-PP aggregates demonstrated extensive and uniform cellular repopulation and viability throughout the thickness of the liver scaffolds. Furthermore, the decellularized liver scaffolds was supportive of the endocrine cell fate of the engrafted cells. Our novel strategy to engineer endocrine pancreas construct is expected to find potential applications in preclinical testing, drug discovery and diabetes therapy.

Bromocriptine is approved as a diabetes therapy, yet its therapeutic mechanisms remain unclear. Though bromocriptine's actions have been mainly attributed to the stimulation of brain dopamine D2 receptors (D2R), bromocriptine also targets the pancreas. Here, we employ bromocriptine as a tool to elucidate the roles of catecholamine signaling in regulating pancreatic hormone secretion. In β-cells, bromocriptine acts on D2R and α2A-adrenergic receptor (α2A-AR) to reduce glucose-stimulated insulin secretion (GSIS). Moreover, in α-cells, bromocriptine acts via D2R to reduce glucagon secretion. α2A-AR activation by bromocriptine recruits an ensemble of G proteins with no β-arrestin2 recruitment. In contrast, D2R recruits G proteins and β-arrestin2 upon bromocriptine stimulation, demonstrating receptor-specific signaling. Docking studies reveal distinct bromocriptine binding to α2A-AR versus D2R, providing a structural basis for bromocriptine's dual actions on β-cell α2A-AR and D2R. Together, joint dopaminergic and adrenergic receptor actions on α-cell and β-cell hormone release provide a new therapeutic mechanism to improve dysglycemia.

Dopaminergic agonism effectively treats dysglycemia, with dopaminergic agonist bromocriptine approved as a type 2 diabetes therapy. Though bromocriptine’s actions have been mainly attributed to stimulation of dopamine D2 receptors (D2R) in the brain, we previously showed that bromocriptine also targets metabolically-relevant peripheral tissues including the endocrine pancreas. Here, we employ bromocriptine as a tool to elucidate roles of dopaminergic and adrenergic signaling in regulation of pancreatic hormone secretion. Using bromocriptine, we demonstrate a new mechanism for metabolic actions in pancreatic α-cells and β-cells via D2R and adrenergic α2A receptor (α2A-AR) signaling. In β-cells, bromocriptine acts jointly on D2R and α2A-AR to reduce glucose-stimulated insulin secretion (GSIS) , while in α-cells, bromocriptine acts via D2R to reduce glucagon secretion. Bromocriptine reduces cAMP in β-cells via concurrent actions on D2R and α2A-AR, further emphasizing shared roles of adrenergic and dopaminergic receptor agonism in GSIS regulation. At the receptor level, α2A-AR activation by bromocriptine leads to receptor recruitment of an ensemble of G proteins driven mainly via G protein signaling with no β-arrestin2 recruitment. In contrast, D2R recruits both G proteins and β-arrestin2 upon bromocriptine stimulation, demonstrating signaling unique to each receptor. Docking studies also reveal that bromocriptine binding to α2A-AR is distinct from bromocriptine-bound D2R, providing a structural basis for bromocriptine’s dual actions on β-cell α2A-AR and D2R. Together, joint dopaminergic and adrenergic receptor actions on α-cell and β-cell hormone release provide a new therapeutic mechanism to improve dysglycemia in diabetes. Disclosure S. Bertera: None. L. Friggeri: None. R. Logan: None. R. Free: None. R. Bottino: Employee; Imagine Pharma. Funding Department of Defense (PR141292) , National Institutes of Health (R01DK124219, R01DK097160, RDA046138) , the John F. and Nancy A. Emmerling Fund of The Pittsburgh Foundation, Intramural Research Program of the National Institute of Neurological Disorders and Stroke (ZIA-NS002263) , Veterans Affairs VA-ORD-BLR&D (I01BX002678) , the Deutsche Forschungsgemeinschaft (SFB1423, project number 421152132) .

Hematopoietic humanized (hu) mice are powerful tools for modeling the action of human immune system and are widely used for preclinical studies and drug discovery. However, generating a functional human T cell compartment in hu mice remains challenging, primarily due to the species-related differences between human and mouse thymus. While engrafting human fetal thymic tissues can support robust T cell development in hu mice, tissue scarcity and ethical concerns limit their wide use. Here, we describe the tissue engineering of human thymus organoids from inducible pluripotent stem cells (iPSC-thymus) that can support the de novo generation of a diverse population of functional human T cells. T cells of iPSC-thymus-engrafted hu mice could mediate both cellular and humoral immune responses, including mounting robust proinflammatory responses on T cell receptor engagement, inhibiting allogeneic tumor graft growth and facilitating efficient Ig class switching. Our findings indicate that hu mice engrafted with iPSC-thymus can serve as a new animal model to study human T cell-mediated immunity and accelerate the translation of findings from animal studies into the clinic.

BACKGROUND SARS-CoV-2 infection or COVID-19 disease has been linked to the onset of diabetes and metabolic dysregulation because it has been suggested that viral entry proteins, specifically ACE2 and TMPRSS2, are expressed in the exocrine cells and ductal epithelium of the pancreas. Because of the unknown effect this can have on islet function, there can be doubt that patients with previous SARS-CoV-2 infections are good candidates for autologous islet transplantation after total pancreatectomy (TPAIT). CASE REPORT A patient with a history of chronic pancreatitis and previous non-surgical interventions was presented as a viable candidate for TPAIT at our institution. Approximately 1 month later, the patient contracted a SARS-CoV-2 infection, resulting in a mild case of COVID-19. The infection resolved without the need for hospitalization. At the time of this occurrence, COVID-19 was primarily considered a respiratory ailment, and little was known of the potential association between metabolic dysfunction and SARS-CoV-2. Islet isolation and surgery proceeded in a textbook manner with no surgical complications. The patient was weaned off exogenous insulin within 3 months after transplantation. CONCLUSIONS Favorable outcomes after surgery included pain reduction, islet function, and improved quality of life for the patient in the first 6 months after the procedure. These successful results demonstrate that SARS-CoV-2 infection did not prevent the patient from achieving good glucose regulation after auto-islet transplantation. This outcome suggests that, at least in this instance of mild infection, there were no long-lasting negative COVID-19-associated effects on the transplanted islets that might impact islet function.

Clinical islet transplantation was first realized over four decades ago at the University of Minnesota. Autologous islet transplantation is now widely recognized as a treatment to prevent diabetes in patients after pancreas excision and is offered at major transplant centers throughout the United States and the world. Type 1 diabetes represents a much larger demographic in which islet transplantation may benefit patients. Allogeneic islet transplantation can now offer similar outcomes to pancreas transplantation in a subset of patients with labile type 1 diabetes with less risk than whole organ transplantation. It is recognized as a standard of care in nations around the world but not in the United States, despite the important developmental role US scientists and physicians have played. Early reports of islet transplantation focused on insulin independence that proved to diminish over time. However, regardless of insulin status, islet transplantation provides benefits ranging from improved quality of life to reduction in diabetic complications. A National Institutes of Health sponsored multi-center Phase 3 Clinical Trial (CIT-07) demonstrated safety and efficacy, although the Food and Drug Administration chose to consider islets as a biologic that requires licensure, which makes offering the procedure in the clinic very challenging. Until regulations can be brought into communion with international standards, allogeneic islet transplantation in the United States is unlikely to match international levels of success and once promising programs are left to wither on the vine. Food and Drug Administration approval would open the door for third party medical reimbursement and allow many patients the opportunity to enjoy better health and quality of life. Establishment of clinical islet transplantation for type 1 diabetes would lead to optimizations in procedures making it more efficacious and cost effective while offering support for ongoing islet xenotransplantation studies that could bring islet transplantation to even more patients.

Dopamine (DA) and norepinephrine (NE) are catecholamines primarily studied in the central nervous system that also act in the pancreas as peripheral regulators of metabolism. Pancreatic catecholamine signaling has also been increasingly implicated as a mechanism responsible for the metabolic disturbances produced by antipsychotic drugs (APDs). Critically, however, the mechanisms by which catecholamines modulate pancreatic hormone release are not completely understood. We show that human and mouse pancreatic α- and β-cells express the catecholamine biosynthetic and signaling machinery, and that α-cells synthesize DA de novo. This locally-produced pancreatic DA signals via both α- and β-cell adrenergic and dopaminergic receptors with different affinities to regulate glucagon and insulin release. Significantly, we show DA functions as a biased agonist at α 2A -adrenergic receptors, preferentially signaling via the canonical G protein-mediated pathway. Our findings highlight the interplay between DA and NE signaling as a novel form of regulation to modulate pancreatic hormone release. Lastly, pharmacological blockade of DA D 2 -like receptors in human islets with APDs significantly raises insulin and glucagon release. This offers a new mechanism where APDs act directly on islet α- and β-cell targets to produce metabolic disturbances.

Islet transplantation can restore glycemic control in patients with type 1 diabetes. Using this procedure, the early stages of engraftment are often crucial to long-term islet function, and outcomes are not always successful. Numerous studies have shown that mesenchymal stem cells (MSCs) facilitate islet graft function. However, experimental data can be inconsistent due to variables associated with MSC generation (including donor characteristics and tissue source), thus, demonstrating the need for a well-characterized and uniform cell product before translation to the clinic. Unlike bone marrow- or adipose tissue-derived MSCs, human embryonic stem cell-derived-MSCs (hESC-MSCs) offer an unlimited source of stable and highly-characterized cells that are easily scalable. Here, we studied the effects of human hemangioblast-derived mesenchymal cells (HMCs), (i.e., MSCs differentiated from hESCs using a hemangioblast intermediate), on islet cell transplantation using a minimal islet mass model. The co-transplantation of the HMCs allowed a mass of islets that was insufficient to correct diabetes on its own to restore glycemic control in all recipients. Our in vitro studies help to elucidate the mechanisms including reduction of cytokine stress by which the HMCs support islet graft protection in vivo . Derivation, stability, and scalability of the HMC source may offer unique advantages for clinical applications, including fewer islets needed for successful islet transplantation.

It is well known that some of the agents commonly used during immunosuppressive (IS) therapy after solid organ transplantation can contribute to beta cell dysfunction and result in diabetes mellitus in the transplant recipient. Some of the risks associated with post-transplant diabetes mellitus (PTDM) include cardiovascular disease (CVD), graft failure and mortality. Since this significance was recognized, many studies are ongoing to refine the IS therapy regimen to reduce or discontinue corticosteroids and calcineurin inhibitors (CNI). A promising addition to the immunosuppression treatment regimen to treat PTDM is glucagon-like peptide-1 receptor (GLP-1R) agonists or incretin mimetics normally used in the treatment of type 2 diabetes (T2D). Studies show that adding GLP-1R agonists to the immunosuppression regimen after solid organ transplant is beneficial not only for the health of the islet beta cells but also positively affects immune function in metabolic disorders by suppressing the activation of CD4+ T lymphocyte cytokine expression. Additional benefits include decreased cardiac graft vasculopathy, improvement of hepatic steatosis, preservation of kidney function, enhanced graft survival and improved all-cause mortality rates for solid organ transplant recipients.

The advancement toward a clinical application for porcine islets to cure diabetes in humans must include reproducible long-term successes in non-human primate (NHP) models. Many dedicated researchers around the world are continuing to work toward this goal. In this chapter, we describe procedures for islet isolation of pancreatic islets from adult and neonatal/fetal pigs. We further include procedures for the induction of diabetes in non-human primates and subsequent insulin therapy, islet transplantation, immunosuppression, and also the daily maintenance of xenotransplanted NHPs. The procedures that we outline in this chapter are ones that we have successfully utilized in pig-to-NHP islet transplantation models. However, where appropriate, alternative methods will also be identified.

Pluripotent stem cells are promising source of cells for tissue engineering, regenerative medicine and drug discovery applications. The process of stem cell differentiation is regulated by multi-parametric cues from the surrounding microenvironment, one of the critical one being cell interaction with extracellular matrix (ECM). The ECM is a complex tissue-specific structure which is an important physiological regulator of stem cell function and fate. Recapitulating this native ECM microenvironment niche is best facilitated by decellularized tissue/organ derived ECM, which can faithfully reproduce the physiological environment with high fidelity toin vivocondition and promote tissue-specific cellular development and maturation. Recognizing the need for organ specific ECM in a 3D culture environment in driving phenotypic differentiation and maturation of hPSCs, we fabricated an ECM array platform using native-mimicry ECM from decellularized organs (namely pancreas, liver and heart), which allows cell-ECM interactions in both 2D and 3D configuration. The ECM array was integrated with rapid quantitative imaging for a systematic investigation of matrix protein profiles and sensitive measurement of cell-ECM interaction during hPSC differentiation. We tested our platform by elucidating the role of the three different organ-specific ECM in supporting induced pancreatic differentiation of hPSCs. While the focus of this report is on pancreatic differentiation, the developed platform is versatile to be applied to characterize any lineage specific differentiation.

Islet transplantation has progressively become a safe alternative to pancreas transplantation for the treatment of type 1 diabetes. However, the long-term results of islet transplantation could be significantly increased by improving the quality of the islet isolation technique even exploring alternative islet transplantation sites to reduce the number of islets required to mitigate hyperglycemia. The goal of the study was to test the lymph node as a suitable anatomical location for islet engraftment in a rodent model. Forty Lewis rats, 6–8 weeks old, body weight 250–300 g, have been used as islet donors and recipients in syngeneic islet transplantation experiments. Ten rats were rendered diabetic by one injection of 65 mg/Kg of streptozotocin. After pancreas retrieval from non diabetic donors, islet were isolated and transplanted in the mesenteric lymph nodes of 7 diabetic rats. Rats were followed for 30 days after islet transplantation. A total of 7 islet transplantations in mesenteric lymph nodes have been performed. Two rats died 24 and 36 h after transplantation due to complications. No transplanted rat acquired normal glucose blood levels and insulin independence after the transplantation. However, the mean blood levels of glycemia were significantly lower in transplanted rats compared with diabetic rats (470.4 mg/dl vs 605 mg/dl, p 0.04). Interestingly, transplanted rats have a significant weight increase after transplantation compared to diabetic rats (mean value 295 g in transplanted rats vs 245 g in diabetic rats, p < 0.05), with an overall improvement of social activities and health. Immunohistochemical analysis of the 5 mesenteric lymph nodes of transplanted rats demonstrated the presence of living islets in one lymph node. Although islet engraftment in lymph nodes is possible, islet transplantation in lymph nodes in rats resulted in few improvements of glucose parameters.

Autologous human islet transplantation to mitigate or prevent surgically induced diabetes after total pancreatectomy for the relief of chronic pancreatitisinduced pain was pioneered in the late 1970s. Islet allotransplantation using islets from human donors to treat type 1 diabetes was first reported several years later. Over the last 40 years, methods for clinical islet transplantation have been methodically standardized to become the methods used today. Human islets are also isolated from donors with pathologies with the aim to conduct a broad range of investigational studies. Human islet isolation undertaken for research can provide the opportunity to further optimize and develop methods that may be helpful in the clinic. The variability of conditions associated with donor and pancreas makes consistent success in isolating islets a challenge, thus, we should re-evaluate the effectiveness of our methods of islet isolation. In order to do this, we must first consider that the intended outcomes for clinical islet transplantation and experimental investigations involving islet isolation are, in fact, quite different and that these differences impact the status and future considerations of islet isolation procedures. AbbreviAtions CIT = Consortium for Islet Transplantation, CP = Chronic Pancreatitis, IBMIR = Instant Blood-Mediated Inflammatory Reaction, IEQ = Islet Equivalent, QOL = Quality of Life, T1D = Type 1 Diabetes, TPAIT = Total Pancreatectomy and Autologous Islet Transplantation. introduction Based on several studies we estimate a normal adult human pancreas to weigh approximately 90 grams and have a volume of 80 ml1,2. Estimates to the number of islets in a normal human pancreas range from 1 million3, 3.2 million4, to 3.6 and to 14.8 million5 islets. Islet cells account for as much as 4.49% of the pancreas volume4. Korsgren et al6 estimate that a normal 70 grams pancreas contains 500,000 Islet Equivalents (IEQ). One IEQ is the volume of a standard islet, i.e. a sphere with a diameter of 150 μm. Further, they state that most centers with an active clinical islet transplantation program report that they obtain between 300,000 to 600,000 IEQ/pancreas (between 4 and 10 ml islets by volume). Based on these calculations it seems likely that most of the native islets are successfully isolated from the donor pancreas. However, since islet total is normally highly variable, it may be difficult to isolate sufficient islets for transplantation even with proven methods. HumAn islets for reseArcH Several programs around the world focus on islet isolation and distribution of human islets for clinical and research purposes. The integrated Islet Distribution Program (IIDP) in the USA, the Alberta Islet Distribution Program (AIDP) in Canada, the European Consortium for Islet transplantation (ECIT), the Oxford Consortium for Islet Transplantation (OXCIT) in Europe, and the Clinical Islet Transplantation (CIT) Consortium International are currently active. Our Allegheny Health Network (AHN) Islet Isolation Center works closely with researchers to provide islets from investigator-defined organ characteristics including donor age and type 1 diabetes (T1D) pathology. Within these and other protocols, flexibility is the key to success. Broad discretion is built into the protocols, which may *Based on a presentation at the 3rd Cleveland Clinic Beta Cell Therapy Symposium on Diabetes, Cleveland, OH, USA, November 9-10, 2018. 2 R. Bottino, S. Bertera, C. Knoll, M. Knoll, M. Trucco creatic ducts. These conditions require adjusting the procedure protocols, increasing efficiency in breaking the extra cellular matrix, which allows to better release the islets. Higher islet numbers are associated with better outcome following intraportal infusion. In clinical islet allotransplantation, at least one, usually two and sometimes more deceased organ donors are needed to achieve success8. More stringent conditions (when compared to islet isolations for autologous islet transplantation) define success. Isolation fails to produce a useable batch of islets for allotransplantation approximately 50% of the time9 most often due to the inability to provide an efficacious mass of islets consolidated into less than 10 ml of tissue required for patient safety. Approximately 44% of recipients have been reported to achieve insulin independence at one year10, which falls to about 10% after 5 years, although, most recipients retain partial function which provides some important benefits11, including an abatement of the risk of hypoglycemia unawareness and diabetes complications. Islet isolation (independent of clinical outcome) is generally considered successful if 5,000-10,000 IEQ are isolated for allotransplantation and half that amount for autotransplantation. The CIT clinical trial CIT-07 must be considered the most complete attempt yet to standardize and optimize the production of a purified human islet product for allotransplantation in the USA. However, even this clinical trial was plagued by inconsistency in isolation and transplantation outcomes that cannot be totally laid at the door of post-transplant islet loss. To begin, only 52.5% of the lots produced for transplantation met the qualifications for release and ultimately only 75 were transplanted9, one participating center reported only 24.3% of islet lots met release conditions. This inefficiency is not unusual although it is mainly exclusive to islet allotransplantation. The first step in the process of pancreas sourcing for islet isolation is donor selection, which is based on criteria designed to provide the best chance to isolate a large islet mass for transplantation. The inclusion criteria of the CIT-07 protocol are typical and includes (1) donor age between 15 and 65 years, (2) cause of death acceptable by transplant team, (3) maximum of 12-hour cold ischemia, (4) and an acceptable preservation fluid for transport9. TPAIT does not select donors and donors used for research are based on investigator-initiallow modifications to the key steps of pancreas digestion and islet purification. However, there are several general characteristics shared by most research isolations. Several thousand islets are often all that is needed for the various studies conducted by the investigators. Therefore, pre-isolation conditions that affect islet yield are not necessarily critical to the success or even the attempt to isolate islets. Conditions that would disqualify an organ donor from clinical islet allotransplantation such as surgical damage, cold ischemia time, and organ size, are minor factors to be considered. Protocols may be challenging to institute and not universally accepted; however, once found they can be effective in producing successful outcomes for the limited needs of research. islets for clinicAl trAnsplAntAtion While clinical islet procedures for Total Pancreatectomy and Autologous Islet Transplantation (TPAIT) and allogeneic islet transplantation are similar, they are not identical because they are based on the expectation of different outcomes. Autologous islet isolation is a procedure in which islets are isolated from the excised pancreas of a patient suffering from chronic pancreatitis (CP) and returned to the patient in order to mitigate the effects of surgical diabetes. This type of transplantation is performed in the absence of immunosuppression. Clinical islet allotransplantation, in contrast, refers to the infusion of islets isolated from one or more deceased organ donors to replace endogenous insulin production in patients with T1D, typically after islet culture and in the presence of immunosuppression, with the goal to improve diabetes management, reduce hypoglycemic unawareness, and long-term complications. Although the primary goal to perform total pancreatectomy in severe CP is to treat pain, autologous islet transplantation may provide a sufficient islet mass that allows to achieve in many patients (approximately 30%) insulin independency, and to better control glycaemia in the majority of the recipients for several years7. Islets are isolated from the recipient’s own pancreas, no immunosuppression is needed, and there is often less than 15 ml of tissue isolated which typically eliminates the need for purification. The challenge in isolating islets from pancreatic organs with CP is to process tissue with significant fibrotic infiltration, necrotic areas, blood remnants, and dilated panThe effectiveness of human islet isolation methods 3 stream. 25% of islets were determined to be lost in the first 19 min after infusion began based on radioactivity given off by labeled islets as part of clinical islet allotransplantation13. In vitro studies modeling IBMIR with human islets and syngeneic mouse islet transplantation suggest that the loss may be closer to 50-60% in just a few days14-16. Estimates of islet loss range up to 70%17 or even 90%11 one-month post-transplant. 10% islet survival after transplantation would roughly correspond to 7% of the originally isolated islets surviving to potentially engraft. Obviously, any improvement in islet isolation efficiency would be welcome, more especially towards rendering islets more resistant to the events that characterize early engraftment. looking forwArd What does the future of islet isolation hold? The effectiveness of the individual procedure is directly related to the expected outcome. Islets isolated for research and as part of TPAIT can provide successful outcomes on a consistent basis. The standardized methods of isolation for islet allotransplantation are effective to a large extent, however, having the most severe measure of success, many preparations cannot be utilized. Some costs must be borne, and incentives made available to encourage testing new approaches including organ provision and transport. Perhaps the biggest boost for allotransplantation would be a method of islet infusion that allows a greater volume of

Islet transplantation has been proposed to be a potential treatment for type 1 diabetes. Recent compelling evidence indicates that intravascular islet infusion is far from ideal and therefore, the omentum is re-emerging as a potentially valuable site for islet transplantation. This experiment requires the isolation of high quality islets and the implantation of the islets to the diabetic recipients. Transplantation to the omentum requires surgical steps that can be better demonstrated visually. Here, the detailed steps for this procedure are presented. Two methods of mixing the isolated islets with hydrogel before placing the mixture into the omental pouch of diabetic mice are described here. Different hydrogels are used for the different conditions. Blood glucose levels of diabetic mouse recipients of syngeneic islets in the omentum were monitored for up to 35 days. Some animals were sacrificed after 14 days to perform immuno-histochemical analysis. This pre-clinical transplantation approach can be used as preliminary data leading up to translation to clinical transplantation.

Thymic epithelial cells (TECs), the primary stromal population in the thymus, are critical to establish the microenvironments required for generating self-tolerant, pathogen-responsive T cells. However, due to the inaccessibility of the thoracic cavity and the fragility of TECs, manipulating the thymus to modulate adaptive immune responses remains challenging. We developed a novel surgical procedure that allowed access to thymus glands for intrathymic injection (ITI), without the need to cut the manubrium of the sternum bone. Horizontal cuts of the muscle layers between the first and second bones of the rib cage expose the thymus with minimal side effects. This novel ITI procedure enabled us to implant 16.5-day embryonic TECs into an adult syngeneic recipients’ thymus. Long-term survival and colonization of the implanted TECs was observed via live imaging, flow cytometry, and immunohistochemistry. We further investigated if introduction of allogeneic TECs in the thymus promote negative selection against allo-reactive T cells and induce donor-specific immune tolerance. We engrafted B6 islets under the kidney capsules of chemically induced diabetic Balb/C mice, preconditioned with T-cell depletion sera and intrathymic injection of B6 16.5-day embryonic TECs. Prolonged survival of the transplanted islets was observed compared to sham-operated controls. Mixed lymphocyte reaction experiments further showed dampened T cell proliferation responses against donor B6 antigens. Moreover, increased levels of Foxp3+ T-regulatory cells were detected in Balb/C recipients with ITI of B6 TECs, suggesting that implanted TECs could also promote the generation of alloantigen-specific T regulatory cells in addition to facilitating negative selection of donor antigen-reactive T cells. Our results suggest that intrathymic transplantation of TECs could be an effective way to induce donor-specific immune tolerance with limited general immune suppression. Disclosure I. Pradhan: None. S. Wong-Noonan: None. S. Bertera: None. R.J. Lakomy: None. H. Cohen: None. D. Wells: None. M. Trucco: None. Y. Fan: None.