FasL microgels induce immune acceptance of islet allografts in nonhuman primates
Data files
Jan 15, 2023 version files 15.50 KB
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Combined_control.csv
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Combined_SA-FasLmicrogel.csv
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README.txt
Abstract
Islet transplantation to treat insulin-dependent diabetes is greatly limited by the need for maintenance immunosuppression. We report a strategy through which cotransplantation of allogeneic islets and streptavidin (SA)–FasL–presenting microgels to the omentum under transient rapamycin monotherapy resulted in robust glycemic control, sustained C-peptide levels, and graft survival in diabetic nonhuman primates for >6 months. Surgical extraction of the graft resulted in prompt hyperglycemia. In contrast, animals receiving microgels without SA-FasL under the same rapamycin regimen rejected islet grafts acutely. Graft survival was associated with increased number of FoxP3 + cells in the graft site with no significant changes in T cell systemic frequencies or responses to donor and third-party antigens, indicating localized tolerance. Recipients of SA-FasL microgels exhibited normal liver and kidney metabolic function, demonstrating safety. This localized immunomodulatory strategy succeeded with unmodified islets and does not require long-term immunosuppression, showing translational potential in β cell replacement for treating type 1 diabetes.
Methods
NHPs were fasted overnight prior to administration of streptozotocin (STZ) at dose of 75 mg/kg. Starting the next day, blood glucose levels were monitored twice daily (8:00-9:30 AM, 3:30-5:00 PM) via tail pricking (Accu-check Aviva, Roche Diagnostics). STZ-induced diabetes was defined as three consecutive fasting glucose level readings >300 mg/dL and C-peptide levels < 0.5 ng/mL (C-peptide ELISA, Mercodia AB). Post-STZ period is defined from the time of STZ injection until the day of islet transplantation. Post-transplant graft rejection was defined as three consecutive fasting blood glucose readings >180 mg/dL or non-fasting blood glucose >250 mg/dL. Exogenous insulin (Humulin R, Lilly) and Lantus (Lilly) were administered on a sliding scale regimen to achieve a postprandial blood glucose level <250 mg/dL pre-transplant or after graft rejection was defined. Animals having induced diabetes for at least 12 days were used as recipients of islet transplantation.
For all recipient animals, the day of allogeneic islet transplant is defined as day 0. Animals received rapamycin (LC Laboratories) at a dose of 0.2 mg/kg I.M. daily for total 3 months starting on day -3 with target blood trough level of 40 ng/mL for the first two weeks, then 20 ng/mL until the 3-month time point post-transplant, when rapamycin was discontinued without weaning. Subjects received islets and SA-FasL-presenting PEG microgels or control microgels at a 1 IEQ:2 microgel ratio. Post-transplantation, daily low dose exogenous insulin (2-4 units) was administrated to all recipients for the first 28 days to promote islet engraftment by allowing islet “rest”. Graft recipients were also subjected to prophylactic Ganciclovir treatment for CMV.
Donor pancreatectomy was performed on the same day as islet transplantation. In brief, a median sternotomy with a midline abdominal incision was performed. Once a 12-gauge cannula connected to infusion set of hypothermic UW organ preservation solution was placed into the aorta at the renal artery level, I.V. 2000 U of heparin (SAGENT Pharmaceuticals) was given. Subsequently, the abdominal aorta was cross clamped at the sub-diaphragmatic level. Lesser sac was packed with iced saline while 1000 mL of UW solution was delivered from the aorta cannula for perfusion. The inferior vena cava was transected to provide proper venous drainage during perfusion. The entire pancreas was mobilized and excised subsequently.
The protocol of islet isolation was based on a modified human islet isolation protocol as we previously reported [Ji et al., Am J Transplant 15, 2739-2749 (2015)]. Briefly, the pancreas was enzymatically digested using purified Thermolysin and Collagenase blend (Vitacyte). Islets were purified from the digestion using continuous Optiprep gradient (densities 1.11 to 1.06) and a COBE 2991 blood cell processor (Gambro BCT) to separate islet from exocrine tissue. Samples taken from different fractions after purification were used to assess the purity of each cell fraction. Only fractions with >50% purity by dithizone stain were combined for transplantation. Final islet preparations were enumerated by manual counting, sizing, and converting islet particle number to islet equivalents (IEQ) based on a 150-mm diameter. Islets were then put into 15 mL CMRL 1066 transplant media (Cellgro), supplemented with 10% human serum albumin (Grifols Therapeutics) and heparin 70 units/kg recipient body weight ready for transplantation.
Under general anesthesia, a small midline abdominal incision was performed. The omentum was mobilized and draped over on a sterile moist towel with as minimal manipulation as possible. Islets were resuspended with PEG microgels with or without SA-FasL protein at a 1:2 ratio of IEQ to microgel in a minimal volume of NHP autologous plasma and carefully dripped onto the omentum surface. The islets were immobilized on the omentum by topical recombinant thrombin (Recothrom, Zymogenetics) layered over the islet slurry, followed by another layer of autologous plasma to create a degradable biologic fibrin matrix. The omentum was then folded onto itself and held in place by the thrombin-induced fibrin glue to form a microenvironment (omental pouch) to host the islets and SA-FasL microgels. Two separate spreadsheets (SA-FasL microgel, control) are provided for daily blood glucose (BG) and external insulin requirement (EIR) for non-human primates receiving allogeneic islets and either SA-FasL microgel or control microgels.