Animals and animal care. Eleven cynomolgus monkeys (Macaca fascicularis) were supplied by Orient Bio Co. Ltd (Seongnam, Korea). The monkeys were isolated in individual cages. Water was given ad libitum, and biscuits were supplied twice a day (Certified Primate Diet 5048, LabDiet, St. Louis, MO, USA). Fresh fruits, vegetables, and nuts were also provided twice a day. The inhabited environment was maintained at a temperature of 25 ± 2°C, 40–60% humidity, 1–5 mmHg positive pressure air conditioning, and 300 lux illuminance alternating with darkness every 12 hours. All the procedures related to infection screening, housing, handling, care, and treatment in this study were performed as previously described33. This study was approved by the Institutional Animal Care and Use Committee of Orient Bio Laboratories (ORIENT–IACUC-14195), and the experiments were performed in accordance with the relevant guidelines and regulations. All methods were carried out in compliance with the ARRIVE guidelines.
Induction and management of type 1 diabetes mellitus. Pancreatectomy and the induction, confirmation, and maintenance of T1DM in cynomolgus monkeys were performed as previously described33. Briefly, the donor monkey’s pancreas was removed through subtotal (> 70% of the pancreas) or total pancreatectomy. The removed pancreas was used for islet isolation, and the donor monkey whose pancreas was removed became a recipient monkey after an injection of 60–80 mg/kg of streptozotocin (Sigma, St Louis, MO, USA). T1DM was diagnosed when the following criteria were satisfied: 1) sustained hyperglycemia (blood glucose level > 250 mg/dl), 2) fasting C-peptide level < 0.5 ng/ml, and 3) decrease in stimulated C-peptide response in the IVGTT. After the onset of diabetes and islet transplantation, the blood glucose level was monitored 2 to 4 times daily, and exogenous insulin (glargine: Lantus; Sanofi-Aventis, Bridgewater, NJ, USA, and glulisine: Apidra, Sanofi-Aventis) were used to maintain blood glucose levels < 200 mg/kg to protect the animals from hyperglycemia (Fig. 2).
Isolation and culture of islets. Resected partial or complete pancreases from the donor monkeys were processed as previously described to isolate the islets33. Briefly, the islets were isolated from the pancreas using the modified Ricordi method with collagenase MTF C/T (Roche, Indianapolis, IN). The discontinuous Ficoll density gradient method was used to purify the islets. The purified islets were cultured with CMRL-1066 supplemented medium (Corning, NY, USA) supplied with 10% fetal bovine serum (Gibco-Thermo Fisher Scientific, Waltham, MA, USA) and 1% antibiotics (Gibco-Thermo Fisher Scientific) in a humidified 5% CO2 atmosphere at 37°C.
Allogeneic islet transplantation and perioperative management. Initially, we planned for the follow-up time after transplantation to be 3 months (90 days). In fact, we stopped the experiment early for some monkeys in accordance with humanitarian treatment because the affected animals endured the following conditions for two continuous months after transplantation: 1) blood glucose level > 200mg/dL, 2) exogenous insulin usage > 3 IU/kg/day, and 3) serum C-peptide levels < 0.5 ng/ml. The transplantation of cultured islets and perioperative management were performed as previously described18,33. Briefly, at least 30 days after confirmation of T1DM, 11 monkeys underwent islet transplantation via the intraportal injection of cultured islets. According to the injected IEQ/kg, the monkeys were divided into three groups: group 1 (10,000 IEQ/kg, n = 2), group 2 (20,000 IEQ/kg, n = 5), and group 3 (> 25,000 IEQ/kg, n = 4) (Table 1). After inducing general anesthesia, we performed central venous access port insertion via the right internal jugular vein. Through this central line, all monkeys received ATG four times at 12-hour intervals to a cumulative dose of 20 mg/kg as induction immunosuppression. RTX injections at a dose of 375 mg/m2 were added in three monkeys (E, F, G) in group 2 as combination induction immunosuppression. Laparotomy began with an upper midline incision. After a self-retractor was applied, the portal vein was isolated. Islet-mixed heparin (75 IU/kg) was infused through an 18-gauge angiocatheter inserted into the portal vein. After islet infusion was finished, the angiocatheter was removed, and the puncture site was closed with 6 − 0 Prolene sutures. After each monkey awakened from anesthesia, it was returned to its cage. The immunosuppression schedule for pre- and post-transplantation is summarized in Supplementary Figure S1.
Postoperative management. Postoperative management were performed as previously described18. Briefly, the monkeys received oral FK506 (Tacrolimus, Prograf, Astellas Pharma Europe Ltd, Addlestone, UK) and mycophenolate mofetil (Cellcept, Roche Pharmaceuticals AG, Basel, Switzerland) as maintenance immunosuppressive drugs. To prevent inflammatory events, etanercept was given on the day of the transplant (day 0) and on days 3 and 6. A subcutaneous injection of anakinra (Kineret™, Swedish Orphan Biovitrum, Stockholm, Sweden) was also given daily from days 0 to 7 (Supplementary Figure S1). One month after transplantation, IVGTT was performed after 12 hours of fasting. After sedation with ketamine, two blood samples were drawn for C-peptide and blood glucose measurements. Then, 0.5 g/kg of dextrose was given intravenously, and blood samples were drawn 1, 3, 5, 7, and 10 min thereafter. Blood samples were also drawn at 15, 20, 25, 30, and 60 min to measure the glucose disappearance rate. Serum C-peptide was measured using a radioimmunoassay kit developed for human plasma (C-Peptide IRMA kit; IMMUNOTHECH, Beckman Coulter Inc., Prague, Czech Republic), which shows 90% cross-reactivity with plasma from cynomolgus monkeys. The acute C-peptide response was calculated as the difference between the mean C-peptide after glucose infusion and C-peptide at baseline (Figs. 2 and 3).
Liver biopsy and immunohistochemical staining. Liver biopsies and the immunohistochemical analyses were conducted following our previously published criteria32. Briefly, to observe and investigate the environment of the grafted islets after transplantation, a protocol liver biopsy was performed one month (30 days) and two months (60 days) after transplantation, dependent on each monkey’s health condition. Monkey K of group 3 received a left lateral lobectomy 2 months after islet transplantation to assess the continuation of insulin independence after the removal of a considerable volume of islets from the grafted liver. The monkeys underwent a laparotomy to expose the liver, and then segment 1 or 2 of the liver was excised. The biopsied liver tissues were fixed in 10% neutral buffered formalin for 24h and then embedded in paraffin. Each paraffin block was sliced into 30 paraffin slides that were 4 µm in thickness in a total of three sections, as previously described32 to produce 10 slides/section (Fig. 1). The slides produced in each section were stained for target proteins in the following order: hematoxylin and eosin (1st slide), anti-insulin from abcam (ab6995) (2nd slide), anti-CD3 from DAKO (A0452) (3rd slide), anti-CD20 from DAKO (M0755) (4th slide), anti-amyloid oligomers from abcam (ab126892) (5th slide), and anti-glucagon from abcam (ab92517) (6th slide). After deparaffinization and heat retrieval of the epitope, each slide was stained with the target protein for the 1st antibody. To visualize of each target protein as brown, 3, 3'-diaminobenzidine tetrahydrochloride staining was performed using a DAKO EnVision system (DAKO, Santa Clara, CA, USA) according to the manufacturer’s instructions (Fig. 4).
Analysis of immunohistochemically stained images. The immunohistochemistry slide images were analyzed as previously described32. Briefly, an image file of each stained slide was acquired using a ScanScope AT slide scanner (Leica Biosystems, Wetzlar, Germany) and Aperio ScanScope software (Leica Biosystems). Using the Aperio Positive Pixel Count algorithm (version 9.1) in the Aperio ImageScope program (version 184.108.40.20629; Leica Biosystems), we obtained the islet diameter (µm), islet area (µm2), and insulin-, glucagon-, CD3-, CD20-, and IAPP-positive areas within the total islet area (%) (Fig. 5). Because of the non-uniform shape and size of the islets and the change in the position of the islets on the slides from a continuous paraffin section, the number of islets for which we could analyze all the characteristics was small. To compensate for that, we analyzed the statistical significance of the characteristics between islets containing all the characteristics (complete data set) and islets containing only some of the characteristics (incomplete date set). As a result, we confirmed that there were no statistically significance differences in the remaining characteristics except for the size of the islets and the beta-cell expression rate within the islet area of group 2 at 2 months. Therefore, we used the incomplete data set (Supplementary Table S1).
CD3+T cell infiltration rate grade criteria. The CD3+ T cell infiltration rate into the islets was graded using the following criteria: 1) Grade 1 — CD3+ T cells infiltrated into < 1% of insulin-expressing islets, 2) Grade 2 — CD3+ T cells infiltrated into 1–5% of the insulin-expressing islets, 3) Grade 3 — CD3+ T cells infiltrated into > 5% of the insulin-expressing islets, 4) Grade 4 — Massive infiltration of CD3+ T cells into islets rarely expressing insulin (< 1% insulin), 5) Grade 5 — Massive infiltration of CD3+ T cells into islets not expressing insulin (Supplementary Figure S2).
Statistical analysis. The IVGTT results were analyzed using one-way ANOVA with Bonferroni`s multiple comparison post hoc test in GraphPad Prism version 5.00 (GraphPad Software, San Diego CA, USA) (Fig. 3A and B). The characteristics of the biopsied islets were analyzed using unpaired t testing with GraphPad Prism version 5.00 (GraphPad Software) (Fig. 5, Fig. 6B, and Supplementary Table S1). All data are presented as means ± standard error of the mean (SEM).