FasL microgels induce immune acceptance of islet allografts in nonhuman primates

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.

Fig. 1.. Local presentation of SA-FasL via synthetic microgels for islet transplantation.

(A) Synthetic biotin-PEG microgels, generated by microfluidic polymerization, capture, and present SA-FasL. SA-FasL–presenting microgels and islets are immobilized on the surface of the omentum of NHPs using autologous plasma and thrombin, where they induce immune acceptance by potentially eliminating T effector cells and generating T regulatory cells. (B) Schema describing donor and diabetic MHC mismatched recipient NHPs and treatment protocol. SA-FasL–presenting microgels with transient rapamycin (RAPA) monotherapy (SA-FasL Microgels, n = 4); control microgels with transient rapamycin monotherapy (Microgels, n = 3).

Fig. 2.. SA-FasL–presenting microgels induce islet allograft acceptance.

(A) Kaplan-Meier survival curves of islet allografts in SA-FasL Microgel (n = 4) and Microgel (n = 3); Mantel-Cox test, P = 0.010. Mean graft survival times: SA-FasL Microgel, >180 days; Microgel, 27.7 days. (B) Nonfasting blood glucose levels (mean, blue line; SEM, gray shadow, left axis) and daily total EIR (mean, red bars; lower SEM, dark red bars, right axis) for SA-FasL Microgel subjects. Animals exhibited high blood glucose levels and external insulin demand after STZ induction but before transplant (defined as post-STZ). After cotransplantation of islets and SA-FasL microgels (Tx), animals rapidly became normoglycemic and had significantly reduced EIR. Animals reverted to hyperglycemic state after graft removal (blood glucose levels, purple lines, left axis; total EIR, tan bars, right axis). (C) Blood glucose levels for SA-FasL Microgel subjects after intravenous infusion of glucose before (pre-STZ) and after diabetes induction (post-STZ), at 3 and 6 months after transplantation, and after graft removal. (D) Insulin (left axis) and C-peptide (right axis) levels in serum for SA-FasL Microgel animals under fasting (F) and post-stimulation (S) before (naïve) and after diabetes induction, at 3 and 6 months after transplantation, and post-graft removal (PGR). (E) Nonfasting blood glucose levels (blue line; SEM gray shadow, left axis) and daily total EIR (mean, red bars; lower SEM, dark red bars, right axis) for Microgel subjects. After restoring normoglycemia following transplantation (Tx), animals became hyperglycemic and required higher external insulin around 1 month after transplantation. (F) Blood glucose after intravenous infusion in Microgel during prediabetic state (Pre-STZ), after diabetes induction (Post-STZ), and at 1 month after transplantation. (G) Insulin (left axis) and C-peptide (right axis) levels in serum for Microgel animals under fasting (F) and post-stimulation (S) before and after diabetes induction and at 1 month after transplantation.

Fig. 3.. Histological analyses of grafts showing increased FoxP3⁺ cells in recipients of SA-FasL–presenting microgels.

(A) Hematoxylin and eosin (H&E) staining of excised omentum biopsies at takedown demonstrates the presence of islet-like clusters (outlined in white dashed line) in SA-FasL Microgel subject with minimal immune cell infiltration (left image: bar, 20 μm; middle image: bar, 50 μm). Representative image from Microgel (right image: bar, 50 μm) shows islet-like cluster with significant cell infiltration. (B) Immunostaining for FoxP3 [green: FoxP3; blue: 4′,6-diamidino-2-phenylindole (DAPI); bar: 50 μm], a marker of T_regs, showing increased numbers of FoxP3⁺ at the graft site of animals receiving SA-FasL microgels compared to those receiving microgels. FoxP3⁺ cells were in closed apposition to microgels (dashed white lines). (C and D) Quantification of FoxP3⁺ cells over total cell counts and mean intensity in sections from Microgel and SA-FasL Microgel animals demonstrating increased frequency of T_regs in SA-FasL Microgel subjects (nested two-tailed t test). Plots show data points for each animal in a different color, with a minimum of three representative images analyzed per animal.

Fig. 4.. Local delivery of SA-FasL microgels does not alter peripheral blood lymphocyte populations.

(A to J) Numbers of circulating (A) CD3⁺, (B) CD20⁺, (C) CD4⁺, and (D) CD8⁺ cells, as well as naïve (Tn) (E) CD4⁺ and (F) CD8⁺, effector memory (EM) (G) CD4⁺ and (H) CD8⁺, and central memory (CM) (I) CD4⁺ and (J) CD8⁺ lymphocytes in SA-FasL Microgel–treated subjects (dashed blue lines, mean, SEM, n = 3 to 4) and Microgel-receiving recipient (red solid lines, mean, SEM, n = 3) animals.

Fig. 5.. SA-FasL microgel recipients display no significant changes in pre- to posttransplant anti-donor MHC antibodies, CD4⁺ and CD8⁺ T cell proliferative responses to donor and third-party stimulators, and IFN-γ–secreting cell numbers.

(A and B) IgG responses (mean, individual points) to donor-specific MHC (A) class I and (B) class II epitopes for SA-FasL Microgel (blue, n = 3 to 4) and Microgel (red, n = 3) subjects, demonstrating no donor-specific activation in treated subjects (class I: P = 0.2904, class II: P = 0.0754). No positive donor-specific antibodies to MHC I were detected in Microgel NHPs (P = 0.3292). Nonetheless, these control subjects generated antibodies against donor MHC II (P = 0.0326). (C to F) Mixed lymphocyte reaction (mean, individual points) to CD4⁺ (C) donor and (D) third-party stimulators and CD8⁺ (E) donor and (F) third-party antigens for SA-FasL Microgel (blue, n = 3 to 4) and Microgel (red, n = 3) subjects showing no differences in responses for the latter (CD4⁺ donor: P = 0.4200; CD4⁺ third-party: P = 0.6949; CD8⁺ donor: P = 0.4145; CD8⁺ third-party: P = 0.7858). Microgel animals exhibited no responses against donor (P = 0.6082) or third-party (P = 0.0555) antigens in CD4⁺ compartment, but CD8⁺ T cell responses were elevated against both donor (P = 0.0057) and third-party (P = 0.0216) stimulators. (G and H) ELISpot IFN-γ counts (mean, individual points) for (G) donor and (H) third-party stimulation for the SA-FasL Microgel (blue, n = 3 to 4) and Microgel (red, n = 3) subjects. No differences in frequency of IFN-γ–secreting cells in circulation between pre- and posttransplant time points for SA-FasL Microgel (donor: P = 0.2485; third-party: P = 0.1445) or Microgel subjects (donor: P = 0.0824; third-party: P = 0.1413). Repeated-measures ANOVA was used with pairwise comparisons to pretransplant/day 0 values using Dunnett’s test.