Abstract Introduction Durable transplant tolerance remains a major challenge. Prior studies have linked amphiregulin (Areg) to Treg cell stability, anti-inflammatory function, and regulation of T-cell differentiation and tumor immune evasion. As a mitogenic and cell differentiation ligand of EGFR, Areg may enable Tregs to restrain allospecific immune responses by activating exhaustion-associated transcriptional programs such as TOX and Nur77. We hypothesized that Tregs enforce long-term tolerance by inducing controlled Teff cell exhaustion through Areg—EGFR signaling. Methods Using a nonhuman primate model of allogeneic islet transplantation, we integrated high-dimensional CyTOF, donor MHC-II tetramer tracking, and ex vivo coculture assays to define Areg+Treg—EGFR+Teff cell interactions. Functional perturbations included Areg silencing in Tregs and EGFR neutralization/silencing in responder T cells to dissect causal signaling. Results Tolerant recipients displayed enrichment of Areg+ST2+FoxP3+ Treg cells, and HelioshiEOMEShiCCR2+ and EGFR+TOX+Nur77+ exhausted CD8+ T cells within splenic allospecific clusters. Trajectory and pseudotime analyses showed that activated naïve/memory CD8+ Teff cells progressively acquired exhaustion features under Areg+Treg influence. EGFR blockade or Areg knockdown abrogated Treg-mediated suppression, restored cytokine and perforin expression, and diminished TOX+Nur77+TIGIT+ exhaustion. Conclusion These findings establish the Areg+Treg—EGFR+Teff axis as a critical immunoregulatory mechanism that consolidates transplant tolerance by linking Treg-derived Areg signaling to Teff exhaustion. Mechanistically, Areg—EGFR engagement drives TOX—Nur77 transcriptional and metabolic reprogramming, restraining effector activation and sustaining immune quiescence. Funding Source NIH R21 Topic Categories Immune Response Regulation: Cellular Mechanisms (IRC)

OBJECTIVE Islet transplantation was recently US Food and Drug Administration approved for adults with type 1 diabetes complicated by recurrent severe hypoglycemia events (SHEs). We sought to understand the long-term benefit for glycemic control and risk of immunosuppression to kidney function associated with islet transplantation compared with ongoing standard of care. RESEARCH DESIGN AND METHODS We performed a case-control analysis of prospectively collected data from patients in the Collaborative Islet Transplant Registry (CITR) with at least one SHE in the year (2000–2014) before transplantation (cases) and compared them with data from patients in the T1D Exchange (T1DX) Registry with at least one SHE in the year (2010–2012) before enrollment (controls), with both cohorts observed over 5 years. SHEs were restricted to those resulting in seizure or loss of consciousness. RESULTS Cases from CITR (n = 71) compared with controls from T1DX (n = 213) more often achieved the primary outcome of HbA1c <7.0% and absence of an SHE (71–80 vs. 21–33% over 5 years; P < 0.001) and the outcome of HbA1c ≤6.5% and absence of an SHE (60–75% vs. 10–20%; P < 0.001) while requiring significantly less insulin (majority in CITR were insulin independent). Kidney function, measured by estimated glomerular filtration rate, declined from baseline to a greater extent in CITR than in T1DX (−8.8 to −20 vs. −1.3 to −6.5 mL ⋅ min−1 ⋅ 1.73 m−2 over 5 years; P < 0.001). CONCLUSIONS Islet transplantation for adults with type 1 diabetes complicated by SHEs results in near-normal glycemic control in the absence of SHEs more often than observed with standard of care, but at the cost of greater reduction in kidney function.

Objective Islet transplantation is recently FDA-approved for adults with type 1 diabetes complicated by recurrent severe hypoglycemia events (SHE). We sought to understand the long-term benefit for glycemic control and risk of immunosuppression to kidney function associated with islet transplantation compared to ongoing standard-of-care. Research Design and Methods We performed a case-control analysis of prospectively collected data from patients in the Collaborative Islet Transplant Registry (CITR) with at least one SHE in the year (2000-2014) prior to transplant (cases) and compared them to patients in the T1D Exchange (T1DX) Registry with at least one SHE in the year (2010-2012) prior to enrollment (controls), with both cohorts followed over 5-years. SHEs were restricted to those resulting in seizure or loss-of-consciousness. Results Cases from CITR (n=71) compared to controls from T1DX (n=213) more often achieved the primary outcome of HbA1c <7.0% and absence of SHE (71–80 vs. 21–33% over 5-years; P <0.001), and the outcome of HbA1c ≤6.5% and absence of SHE (60–75% vs. 10–20%; P <0.001) while requiring significantly less insulin (majority in CITR insulin-independent). Kidney function, measured by estimated glomerular filtration rate, declined from baseline to a greater extent in CITR than in T1DX (-8.8 to -20 vs. -1.3 to -6.5 mL·min-1·1.73 m-2 over 5-years; P <0.001). Conclusions Islet transplantation for adults with type 1 diabetes complicated by SHE results in near-normal glycemic control in the absence of SHE more often than that observed with standard-of-care, but at the cost of greater reduction in kidney function.

Obesity is a state of chronic low-grade inflammation, where alterations in the immune landscape plays a central role. Cholinergic anti-inflammatory pathway (CAP), mediated by acetylcholine (ACh) and its receptor α7nAChR, is an effective counter regulatory mechanisms to suppress pro-inflammatory cytokines. Immune profiling of PBMCs from obese and non-obese individuals was performed using mass cytometry. Unpaired t test identified significant differences in CAP among immune cells. Our data reveal significantly elevated levels of α7nAChR+ monocytes and B cells, along with increased ACh-expressing CD8+/CD4+ T cells, and Tregs including monocytes and B cells in obese vs. non-obese individuals. For the first time, we define CAP heterogeneity, showing ACh dominance in activated Teff and Treg cells, while nAChRs are primarily expressed on classical and non-classical monocytes. Key metabolic markers: C-reactive protein, dyslipidemia, and HbA1c, were positively correlated with ACh-expressing T cells, α7nAChR+ monocytes, and B cells, Discussion: This study is the first to report elevated levels of ACh and its receptors across immune cells in human. Future studies should investigate CAP’s functional role and interaction with Tregs highlighting its therapeutic potential. This increase in CAP components may reflect an adaptive response to chronic inflammation or a dysfunction where heightened CAP activity fails to regulate inflammation effectively. University of Minneosta Medical School Immune Response Regulation: Cellular Mechanisms (IRC)

Islet cell replacement therapies have evolved as a viable treatment option for type 1 diabetes complicated by problematic hypoglycemia and glycemic lability. Refinements of islet manufacturing, islet transplantation procedures, peritransplant recipient management, and immunosuppressive protocols allowed most recipients to achieve favorable outcomes. Subsequent phase 3 trials of transplantation of deceased donor islets documented the effectiveness of transplanted islets in restoring near-normoglycemia, glycemic stability, and protection from severe hypoglycemia, with an acceptable safety profile for the enrolled high-risk population. Health authorities in several countries have approved deceased donor islet transplantation for treating patients with type 1 diabetes and recurrent severe hypoglycemia. These achievements amplified academic and industry efforts to generate pluripotent stem cell-derived β-cells through directed differentiation for β-cell replacement. Preliminary results of ongoing clinical trials suggest that the transplantation of stem cell-derived β-cells can consistently restore insulin independence in immunosuppressed recipients with type 1 diabetes, thus signaling the profound progress made in generating an unlimited and a uniform supply of cells for transplant. Avoiding the risks of chronic immunosuppression represents the next frontier. Several strategies have entered or are approaching clinical investigation, including immune-isolating islets, engineering immune-privileged islet implantation sites, rendering islets immune evasive, and inducing immune tolerance in transplanted islets. Capitalizing on high-dimensional, multiomic technologies for deep profiling of graft-directed immunity and the fate of the graft will provide new insights that promise to translate into sustaining functional graft survival long-term. Leveraging these parallel progression paths will facilitate the wider clinical adoption of cell replacement therapies in diabetes care. ARTICLE HIGHLIGHTS:Transplantation of deceased donor-derived primary human islets has restored near-normoglycemia and protection from severe hypoglycemia in immunosuppressed recipients with type 1 diabetes. Transplantation of embryonic stem cell-derived β-cells has restored insulin independence in immunosuppressed recipients with type 1 diabetes. Clinical trials are underway and planned to evaluate the safety and efficacy of transplantation of mature stem cell-derived β-cells with transient, local, minimal, and/or no-maintenance immunosuppression in recipients with type 1 diabetes. The high-dimensional, multiomic monitoring of immunity to transplanted islets and of the fate of the islet graft will faciliate the identification of determinants of sustained islet graft function and of patients most likely to benefit from cell replacement therapies.

The instant blood-mediated inflammatory response (IBMIR) causes islet loss and compromises diabetes outcomes after total pancreatectomy with islet autotransplant (TPIAT). We previously reported a possible benefit of etanercept in maintaining insulin secretion 3 months post-TPIAT. Here, we report 2-year diabetes outcomes and peri-operative inflammatory profiles from a randomized trial of etanercept and alpha-1 antitrypsin (A1AT) in TPIAT. We randomized 43 TPIAT recipients to A1AT (90 mg/kg IV x6 doses, n = 13), etanercept (50 mg then 25 mg SQ x 5 doses, n = 14), or standard care ( n = 16). Inflammatory cytokines, serum A1AT and unmethylated insulin DNA were drawn multiple times in the perioperative period. Islet function was assessed 2 years after TPIAT with mixed meal tolerance test, intravenous glucose tolerance test and glucose-potentiated arginine induced insulin secretion. Cytokines, especially IL-6, IL-8, IL-10, and MCP-1, were elevated during and after TPIAT. However, only TNFα differed significantly between groups, with highest levels in the etanercept group ( p = 0.027). A1AT increased after IAT in all groups ( p < 0.001), suggesting endogenous upregulation. Unmethylated insulin DNA ratios (a marker of islet loss) and 2 years islet function testing were similar in the three groups. To conclude, we found no sustained benefit from administering etanercept or A1AT in the perioperative period.

Type 1 diabetes recipients of intrahepatic islet transplantation exhibit glucose-dependent suppression of insulin and activation of glucagon secretion in response to insulin-induced hypoglycemia associated with clinical protection from hypoglycemia. Whether sympathetic activation of adrenergic receptors on transplanted islets is required for these responses in defense against hypoglycemia is not known. To evaluate the adrenergic contribution to posttransplant glucose counterregulation, we performed a randomized, double-blind crossover study of responses during a hyperinsulinemic euglycemic-hypoglycemic clamp under phentolamine (α-adrenergic blockage), propranolol (β-adrenergic blockage), or placebo infusion. Characteristics of participants (5 females/4 males) were as follows: median (range) age 53 (34-63) yr, diabetes duration 29 (18-56) yr, posttransplant 7.0 (1.9-8.4) yr, HbA1c 5.8 (4.5-6.8)%, insulin in-/dependent 5/4, all on tacrolimus-based immunosuppression. During the clamp, blood pressure was lower with phentolamine and heart rate was lower with propranolol versus placebo (P < 0.05). There was no difference in the suppression of endogenous insulin secretion (derived from C-peptide measurements) during the euglycemic or hypoglycemic phases, and although levels of glucagon were similar with phentolamine or propranolol vs. placebo, the increase in glucagon from eu- to hypoglycemia was greater with propranolol vs. placebo (P < 0.05). Pancreatic polypeptide was greater with phentolamine versus placebo during the euglycemic phase (P < 0.05), and free fatty acids were lower and the glucose infusion rate was higher with propranolol versus placebo during the hypoglycemic phase (P < 0.05 for both). These results indicate that neither physiological α- nor β-adrenergic blockade attenuates transplanted islet responses to hypoglycemia, suggesting sympathetic reinnervation of the islet graft is not necessary for posttransplant glucose counterregulation.NEW & NOTEWORTHY Whether adrenergic input to islets is necessary for glucose homeostasis in humans is debated. Here, the adrenergic contribution to intrahepatically transplanted islet cell responses to hypoglycemia in individuals with type 1 diabetes was investigated through α- or β-adrenergic receptor blockade during hyperinsulinemic euglycemic-hypoglycemic clamps. Neither α- nor β-adrenergic blockage affected the suppression of endogenous insulin or activation of glucagon secretion, suggesting that sympathetic reinnervation of islet grafts is not required for posttransplant defense against hypoglycemia.

IntroductionConventional culture conditions, such as in T-flasks, require that oxygen diffuse through the medium to reach the islets; in turn, islet surface area density is limited by oxygen availability. To culture a typical clinical islet preparation may require more than 20 T-175 flasks at the standard surface area density of 200 IE/cm2. To circumvent this logistical constraint, we tested islets cultured on top of silicon gas-permeable (GP) membranes which place islets in close proximity to ambient oxygen.MethodsOxygenation of individual islets under three culture conditions, standard low-density, non-GP high density, and GP high density, were first modeled with finite element simulations. Porcine islets from 30 preparations were cultured for 2 days in devices with GP membrane bottoms or in paired cultures under conventional conditions. Islets were seeded at high density (HD, ∼4000 IE/cm2, as measured by DNA) in both GP and non-GP devices.ResultsIn simulations, individual islets under standard culture conditions and high density cultures on GP membranes were both well oxygenated whereas non-GP high density cultured islets were anoxic. Similarly, compared to the non-GP paired controls, islet viability and recovery were significantly increased in HD GP cultures. The diabetes reversal rate in nude diabetic mice was similar for HD GP devices and standard cultures but was minimal with non-GP HD cultures.DiscussionCulturing islets in GP devices allows for a 20-fold increase of islet surface area density, greatly simplifying the culture process while maintaining islet viability and metabolism.

IntroductionSuccessful diabetes reversal using pancreatic islet transplantation by various groups illustrates the significant achievements made in cell-based diabetes therapy. While clinically, intraportal islet delivery is almost exclusively used, it is not without obstacles, including instant blood-mediated inflammatory reaction (IBMIR), relative hypoxia, and loss of function over time, therefore hindering long-term success. Here we demonstrate the perihepatic surface of non-human primates (NHPs) as a potential islet delivery site maximizing favorable characteristics, including proximity to a dense vascular network for adequate oxygenation while avoiding IBMIR exposure, maintenance of portal insulin delivery, and relative ease of accessibility through minimally invasive surgery or percutaneous means. In addition, we demonstrate a targeted mapping technique of the perihepatic surface, allowing for the testing of multiple experimental conditions, including a semi-synthetic hydrogel as a possible three-dimensional framework to improve islet viability.MethodsPerihepatic allo-islet cell transplants were performed in immunosuppressed cynomolgus macaques using a targeted mapping technique to test multiple conditions for biocompatibility. Transplant conditions included islets or carriers (including hydrogel, autologous plasma, and media) alone or in various combinations. Necropsy was performed at day 30, and histopathology was performed to assess biocompatibility, immune response, and islet viability. Subsequently, single-injection perihepatic allo-islet transplant was performed in immunosuppressed diabetic cynomolgus macaques. Metabolic assessments were measured frequently (i.e., blood glucose, insulin, C-peptide) until final graft retrieval for histopathology.ResultsTargeted mapping biocompatibility studies demonstrated mild inflammatory changes with islet-plasma constructs; however, significant inflammatory cell infiltration and fibrosis were seen surrounding sites with the hydrogel carrier affecting islet viability. In diabetic NHPs, perihepatic islet transplant using an autologous plasma carrier demonstrated prolonged function up to 6 months with improvements in blood glucose, exogenous insulin requirements, and HbA1c. Histopathology of these islets was associated with mild peri-islet mononuclear cell infiltration without evidence of rejection.DiscussionThe perihepatic surface serves as a viable site for islet cell transplantation demonstrating sustained islet function through 6 months. The targeted mapping approach allows for the testing of multiple conditions simultaneously to evaluate immune response to biomaterials at this site. Compared to traditional intraportal injection, the perihepatic site is a minimally invasive approach that allows the possibility for graft recovery and avoids IBMIR.

Introduction: In total pancreatectomy with islet autotransplant (TP-IAT) for chronic pancreatitis, early islet damage from the instant blood-mediated inflammatory response (IBMIR) compromises diabetes outcomes. We previously reported that in our randomized trial with etanercept or alpha-1-antitrypsin (A1AT) to block IBMIR, etanercept-treated patients had higher insulin secretion at 3 months, but this was not sustained 1 year after surgery. Herein, we report 2 year diabetes outcomes and mechanistic data. Method: We randomized 43 TPIAT recipients to A1AT (90 mg/kg IV x6 doses, n=13), etanercept (50 mg then 25 mg SQ x 5 doses, n=14), or standard care (n=16). Inflammatory cytokines were drawn at pre-operative screening (x2), pre-islet infusion, and 1,3, 6, 12, 24h, and 3 and 7 days after IAT, including: IP-10, IL-1α, IL-1β, IL-6, IL-8, IL-10, MCP-1, TNF-α. Alpha 1 antitrypsin (A1AT) levels were measured pre-surgery and 7, 14, 21 and 28 days post islet infusion. Islet function was assessed 2 years after TPIAT with mixed meal tolerance test (MMTT), intravenous glucose tolerance test (IVGTT) and glucose potentiated arginine induced insulin secretion (GPAIS). Results: Participants were adults average 38.5 (SD 12) years old, 17 (40%) males. Cytokine measures, especially IL-6, IL-8, IL-10, and MCP-1, were elevated during and after TPIAT. However, only TNFa differed significantly by treatment, with highest levels in the etanercept group (p=0.027). A1AT levels increased significantly above pre-TPIAT baseline in all 3 groups (p <0.001, Figure 1), with the highest levels in the A1AT-treated group. At 2 years, insulin dependence and measures of insulin secretion by MMTT, IVGTT and GPAIS were similar in all groups (Table1).Conclusion: Diabetes outcomes and cytokine profiles (except TNFa) were similar in all groups. Notably, A1AT increased significantly from pre-surgery in all 3 groups, suggesting endogenous upregulation, which may explain the lack of therapeutic benefit from pharmacologic treatment with A1AT. This study was funded by the National Institute of Health. Grant number R01DK109914 (PI Bellin). References: 1. Naziruddin B, Iwahashi S, Kanak MA, Takita M, Itoh T, Levy MF. Evidence for instant blood-mediated inflammatory reaction in clinical autologous islet transplantation. Am J Transplant. Feb 2014;14(2):428-37. doi:10.1111/ajt.12558 2. Abdel-Karim TR, Hodges JS, Pruett TL, et al. A randomized controlled pilot trial of etanercept and alpha-1 antitrypsin to improve autologous islet engraftment. Pancreatology. Jan 2023;23(1):57-64. doi:10.1016/j.pan.2022.11.006

Background. Although diabetes after total pancreatectomy and islet autotransplantation (TP-IAT) is one of the biggest concerns for TP-IAT recipients and physicians, reliable prediction of post-TP-IAT glycemic control remains unestablished. This study was conducted to identify early predictors of insulin independence and goal glycemic control by hemoglobin A1c (HbA1c) ≤ 6.5% after TP-IAT. Methods. In this single-center, retrospective study, patients who underwent TP-IAT (n = 227) were reviewed for simple metabolic markers or surrogate indices of β-cell function obtained 3 mo after TP-IAT as part of standard clinical testing. Long-term metabolic success was defined as (1) insulin independence and (2) HbA1c ≤ 6.5% 1, 3, and 5 y after TP-IAT. Single- and multivariate modeling used 3-mo markers to predict successful outcomes. Results. Of the 227 recipients, median age 31 y, 30% male, 1 y after TP-IAT insulin independence, and HbA1c ≤ 6.5% were present in 39.6% and 72.5%, respectively. In single-predictor analyses, most of the metabolic markers successfully discriminated between those attaining and not attaining metabolic goals. Using the best model selected by random forests analysis, we accurately predicted 1-y insulin independence and goal HbA1c control in 77.3% and 86.4% of the patients, respectively. A simpler “clinically feasible” model using only transplanted islet dose and BETA-2 score allowed easier prediction at a small accuracy loss (74.1% and 82.9%, respectively). Conclusions. Metabolic testing measures performed 3 mo after TP-IAT were highly associated with later diabetes outcomes and provided a reliable prediction model, giving valuable prognostic insight early after TP-IAT and help to identify recipients who require early intervention.

Background: In total pancreatectomy with islet auto-transplantation, successful diabetes outcomes are limited by islet loss from the instant blood mediated inflammatory response. We hypothesized that blockade of the inflammatory response with either etanercept or alpha-1-antitrypsin would improve islet function and insulin independence. Methods: We randomized 43 participants to receive A1AT (90 mg/kg x 6 doses, n = 13), or etanercept (50 mg then 25 mg x 5 doses, n = 14), or standard care (n = 16), aiming to reduce detrimental effects of innate inflammation on early islet survival. Islet graft function was assessed using mixed meal tolerance testing, intravenous glucose tolerance testing, glucose-potentiated arginine-induced insulin secretion studies, HbA1c, and insulin dose 3 months and 1 year post-TPIAT. Results: We observed the most robust acute insulin response (AIRglu) and acute C-peptide response to glucose (ACRglu) at 3 months after TPIAT in the etanercept-treated group (p <= 0.02), but no differences in other efficacy measures. The groups did not differ overall at 1 year but when adjusted by sex, there was a trend towards a sex-specific treatment effect in females (AIRglu p = 0.05, ACRglu p = 0.06), with insulin secretion measures highest in A1AT-treated females. Conclusion: Our randomized trial supports a potential role for etanercept in optimizing early islet engraftment but it is unclear whether this benefit is sustained. Further studies are needed to evaluate possible sex-specific responses to either treatment. Clinical trial notation: This study was performed under an Investigational New Drug Application (IND #119828) from the Food and Drug Administration and was registered on clinicaltrials.gov (NCT#02713997).(c) 2022 IAP and EPC. Published by Elsevier B.V. All rights reserved.