DOI: 10.5061/dryad.83bk3j9tg
title: FasL microgels induce immune acceptance of islet allografts in nonhuman primates

description: 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
 &gt;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.
description: 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
 &gt;300 mg/dL and C-peptide levels &lt; 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
 &gt;180 mg/dL or non-fasting blood glucose &gt;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
 &lt;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 &gt;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.