Transplantation of PEGylated islets enhances therapeutic efficacy in a diabetic nonhuman primate model

Abstract

Islet cell transplantation can lead to insulin independence, reduced hypoglycemia, and amelioration of diabetes complications in patients with type 1 diabetes. The systemic delivery of anti-inflammatory agents, while considered crucial to limit the early loss of islets associated with intrahepatic infusion, increases the burden of immunosuppression. In an effort to decrease the pharmaceutical load to the patient, we modified the pancreatic islet surface with long-chain poly(ethylene glycol) (PEG) to mitigate detrimental host-implant interactions. The effect of PEGylation on islet engraftment and long-term survival was examined in a robust nonhuman primate model via three paired transplants of dosages 4300, 8300, and 10 000 islet equivalents per kg body weight. A reduced immunosuppressive regimen of anti-thymocyte globulin induction plus tacrolimus in the first posttransplant month followed by maintenance with sirolimus monotherapy was employed. To limit transplant variability, two of the three pairs were closely MHC-matched recipients and received MHC-disparate PEGylated or untreated islets isolated from the same donors. Recipients of PEGylated islets exhibited significantly improved early c-peptide levels, reduced exogenous insulin requirements, and superior glycemic control, as compared to recipients of untreated islets. These results indicate that this simple islet modification procedure may improve islet engraftment and survival in the setting of reduced immunosuppression.

Keywords: animal models: nonhuman primate; basic (laboratory) research/science; immunosuppression/immune modulation; immunosuppressive regimens; islet transplantation; islets of Langerhans; translational research/science.

Figure 1.. PEGylation of NHP islets did not impact viability or function but decreased coagulation.

NHP islets were surface modified with long-chain PEG to generate a peripheral PEG coating, as visualized via rhodamine tag on PEG chain (A); inset shows PEG-free controls (B). Light microscope and Live/Dead (green: live; red: dead) confocal images of control (C & E) and PEGylated (D & F) islets reveal similar morphology and viability. Scale bars = 100 μm (A-D) and 50 μm (E-F). Dynamic glucose stimulated insulin release curves (G) illustrate identical stimulation profiles for control and PEGylated islets. Error bars = standard deviation from technical replicates for each group (n = 4). 3G = 3 mM glucose; 11 G = 11 mM Glucose; KCl = 25 mM KCl. Coagulation kinetics of plasma was significantly delayed after incubation with NHP PEGylated islets versus control islets (H), with a significant increase in half-max time for PEGylated islets. Plasma-only wells served as the negative control. Error bars = standard deviation from technical replicates for each group (n = 4). **P < 0.01; ***P< 0.0001 per one way ANOVA with Tukey post-hoc

Figure 2.. Transplantation of a marginal mass (<5,000 IEQ/kg) of PEGylated islets resulted in stable blood glucose control, with decreased exogenous insulin requirement (EIR), as compared to transplantation with untreated islets.

Schematic of islet isolation and transplant, which was independently conducted for PEGylated and untreated islets (top panel). MHC designation and color coding were included for clarity (74, 75). PEGylated islets were PEGylated on POD −1. Immunosuppression scheme is shown and was initiated on POD −1. The recipient of control (untreated) islets received a dosage of 4,600 IEQ/kg, while the recipient of PEGylated islets received 4,160 IEQ/kg. Blood glucose (FBG: fasting blood glucose; PBG: postprandial glucose) of recipients of control (A) or PEGylated islets (B). Exogenous insulin requirement per kg body weight (C; normalized by average EIR/kg from POD −14 to −4) and C-peptide (D) for control and PEGylated islets. For recipients of control islets, necropsy was performed on POD 90. For recipients of PEGylated islets, elective necropsy was performed on POD 272.

Fig 3.. Immunohistochemical analysis of explanted grafts, from recipients of marginal mass transplants, illustrates significant rejection for the recipient of control islets.

Explanted grafts were stained via H/E (A & E), CD3/insulin (B & F); CD20/insulin (C & G), or CD68/insulin (D & H). Top row: Explants from recipient of control islets (ID: H10C21) at necropsy on POD 90. Bottom row: Explants from recipient of PEGylated islets (ID: 10C59) at elective necropsy on POD 272. Insulin antibody = red; CD3, CD20, or CD68 antibody = green; DAPI nuclei staining = blue. Scale bar = 50 μm.

Figure 4.. Recipient of 8,300 IEQ/kg PEGylated islets in a paired transplant model resulted in stable blood glucose control with decreased exogenous insulin requirement (EIR) as compared to the recipient of the same dosage of untreated islets.

Schematic of islet isolation and transplant: Donors (n = 3) were MHC matched and pooled during isolation. Isolated islets were split into control (untreated) and PEGylated islets with PEGylation conducted on POD −1. For each group, islets were transplanted with matched IEQ/kg dosage into MHC matched pairs (full MHC mismatch to isolated islets), with initiation of Immunosuppression scheme is shown and was initiated on POD −1. Blood glucose (FBG: fasting blood glucose; PBG: postprandial glucose) of matched recipients of control (A; H12C4) or PEGylated islets (B; H12C33). Exogenous insulin requirement per kg body weight (C; normalized by average EIR/kg from POD −14 to −4) and C-peptide (D) for control and PEGylated islets. Necropsy was performed on POD 69 for H12C4 and on POD 96 for H12C33.

Figure 5.. Recipient of 10,000 IEQ/kg PEGylated islets in a paired transplant model resulted in stable blood glucose control with decreased exogenous insulin requirement (EIR) as compared to the recipient of the same dosage of untreated islets.

Schematic of islet isolation and transplant: Donors (n = 3) were MHC matched and pooled during isolation. Isolated islets were split into control (untreated) and PEGylated islets with PEGylation conducted on POD −1. For each group, islets were transplanted with matched IEQ/kg dosage into MHC matched pairs (full MHC mismatch to isolated islets), with initiation of immunosuppression starting on POD −1. Blood glucose (FBG: fasting blood glucose; PBG: postprandial glucose) of matched recipients of control (A) or PEGylated islets (B). Exogenous insulin requirement per kg body weight (C; normalized by average EIR/kg from POD −14 to −4) and C-peptide (D) for control and PEGylated islets. Necropsy was undertaken on POD 187 for both animals.

Fig 6.. Immunohistochemical analysis of explanted grafts from 8,300 IEQ/kg transplants illustrate significant rejection in both groups.

Explanted grafts were stained via H/E (A & E), CD3/insulin (B & F); CD20/insulin (C & G), or CD68/insulin (D & H). Top row: Explants from recipient of control islets (H12C4) 69 days post-transplant. Bottom row: Explants from recipient of PEGylated islets (H12C33) 96 days post-transplant. Insulin antibody = red; CD3, CD20, or CD68 antibody = green; DAPI nuclei staining = blue. Scale bar = 50 μm.

Fig 7.. Immunohistochemical analysis of explanted grafts from 10,000 IEQ/kg transplants illustrate decreased rejection in PEGylated islet group.

Explanted grafts were stained via H/E (A & E), CD3/insulin (B & F); CD20/insulin (C & G), or CD68/insulin (D & H). Top row: Explants from recipient of control islets (H12C105) 187 days post-transplant. Bottom row: Explants from explants from recipient of PEGylated islets (13C40) 187 days post-transplant. Insulin antibody = red; CD3, CD20, or CD68 antibody = green; DAPI nuclei staining = blue. Scale bar = 50 μm.

Figure 8.. PEGylation resulted in decreased overall insulin requirements and blood glucose levels with concurrent improved glycemic stability.

Comparison of average exogenous insulin requirements (A & B) and fasting blood glucose (C &D) for early (A&C; POD 10–25) and late (B&D; POD 45–60) stage engraftment of control or PEGylated islets at variable dosages. E) Comparison of C-peptide levels, normalized to IEQ/kg for each recipient, 4 days post-transplantation for all recipients of control (n = 8) or PEGylated (n = 4) islets. Unpaired T-test with Welsh’s correction for unequal variances **P < 0.01; ****P< 0.0001; γ = F test P< 0.01, unequal variances between groups.