A hydrogel platform for co-delivery of immunomodulatory proteins for pancreatic islet allografts

Abstract

Type 1 diabetes (T1D), an autoimmune disorder in which the insulin-producing β-cells in the islets of Langerhans in the pancreas are destroyed, afflicts over 1.6 million Americans. Although pancreatic islet transplantation has shown promise in treating T1D, continuous use of required immunosuppression regimens limits clinical islet transplantation as it poses significant adverse effects on graft recipients and does not achieve consistent long-term graft survival with 50%-70% of recipients maintaining insulin independence at 5 years. T cells play a key role in graft rejection, and rebalancing pathogenic T effector and protective T regulatory cells can regulate autoimmune disorders and transplant rejection. The synergy of the interleukin-2 (IL-2) and Fas immunomodulatory pathways presents an avenue for eliminating the need for systemic immune suppression by exploiting IL-2's role in expanding regulatory T cells and leveraging Fas ligand (FasL) activity on antigen-induced cell death of effector T cells. Herein, we developed a hydrogel platform for co-delivering an analog of IL-2, IL-2D, and FasL-presenting microgels to achieve localized immunotolerance to pancreatic islets by targeting the upregulation of regulatory T cells and effector T cells simultaneously. Although this hydrogel provided for sustained, local delivery of active immunomodulatory proteins, indefinite allograft survival was not achieved. Immune profiling analysis revealed upregulation of target regulatory T cells but also increases in Granzyme B-expressing CD8⁺ T cells at the graft site. We attribute the failed establishment of allograft survival to these Granzyme B-expressing T cells. This study underscores the delicate balance of immunomodulatory components important for allograft survival - whose outcome can be dependent on timing, duration, modality of delivery, and disease model.

Keywords: cell transplantation; immunomodulation; type 1 diabetes.

Figure 1:

Protease-degradable PEG-4MAL hydrogels for the controlled release of covalently-tethered IL-2D. (A) Schematic detailing the synthesis of IL-2D-encapsulating PEG-4MAL hydrogels and protease-dependent degradation. (B) SDS PAGE gel of IL-2D and PEG-4MAL + IL-2D showing an increase in MW, demonstrating successful covalent tethering of IL-2D to PEG-4MAL macromers. (C) IL-2D in vitro release profile from PEG-4MAL hydrogels treated in PBS or collagenase as measured by fluorescence (mean ± SE, N=4). (D) IL-2D loading efficiency at 3 different doses as measured by fluorescence on hydrogels encapsulating tagged IL-2D (mean ± SE N=4).

Figure 2:

Bioactivity of IL-2D is not negatively affected after being PEGylated with PEG-4MAL. (A) Dose-dependent proliferation of IL-2-sensitive CTLL-2 cells by unPEGylated and PEGylated IL-2D (mean ± SD, N=3). (B) Bioactivity of stored IL-2D and PEGylated IL-2D over time as measured by CTLL-2 proliferation via EdU proliferation kits and flow cytometry (mean ± SD, N=3).

Figure 3:

Sustained and localized in vivo release of IL-2D from degradable PEG-4MAL hydrogels. (A) Signal from IL-2D encapsulated in PEG-4MAL hydrogels is sustained, in contrast with rapid clearance of free IL-2D. (B) Normalized signal is presented for both conditions; ****P<0.0001; ***P<0.001; **P<0.01, multiple t tests (N=7 mice per group). (C) IL-2D signal from explanted (EFPs) at D19 (n=14); ***P<0.001, unpaired t test.

Figure 4:

Islet viability and SA-FasL microgel bioactivity are unaffected by IL-2D-delivering PEG-4MAL hydrogels. (A) Islets co-cultured with or without IL-2D-delivering hydrogels for 48 h and there were no differences in secretion of danger associated molecular patterns (DAMPs) HSP70 and uric acid (mean ± SE, N≥3; unpaired t-test). (B) Metabolic activity of islets was unaffected by presence of IL-2D-delivering PEG-4MAL hydrogels (mean ± SE, N=3; unpaired t-test). (C) No differences were detected in the apoptotic activity of SA-FasL microgels in the presence of IL-2D PEG-4MAL hydrogels (mean ± SE, N=3; one-way ANOVA). (D) The combination therapy does not have detrimental effects on graft function in a syngeneic transplant model (shadowed area represents SE for each group, N=3).

Figure 5:

The combination therapy of SA-FasL microgels with an IL-2D-delivering PEG-4MAL hydrogel induces significant changes to T cell subpopulations at the graft site. (A) The combination therapy group exhibited a significant upregulation of Tregs (CD4⁺CD25⁺FoxP3⁺) at D3 that was further heightened at D6. (B) There were statistically significant differences in CD4⁺Teffs (CD4⁺CD44^hiCD62L^lo) across groups, stemming from the decrease in CD4⁺ Teffs at D6 exhibited by the combination therapy group. (C) This decrease in CD4⁺ Teffs is paralleled by a lower presence of CD8⁺ Teffs (CD8⁺CD44^hiCD62L^lo) at D3 and D6 in the combination therapy group. (D) The combination therapy group also exhibited a significant upregulation of GranzymeB⁺CD8⁺ T cells at D3 that was sustained at D6 (mean ± SE, N≥4; two-way ANOVA).

Figure 6:

(A) The combination therapy induces an IL-2D dose-dependent upregulation of Tregs that is further heightened at D6. (B, C) This dose dependence is not observed in the presence of CD4+ and CD8+ Teffs. (D, E) A time-dependent decrease in CD4+ Teffs was observed, an indication that SA-FasL-induced Teff apoptosis may be occurring. However, (F) a dose-dependent increase of Granzyme B-expressing CD8+ T cells was observed, hinting at a broader activation of T cells caused by IL-2D (mean ± SE, N≥4; two-way ANOVA).

Figure 7:

(A) Metabolic allograft function, based on non-fasting blood glucose levels (shadowed area represents SE; N≥4). (B) Graft function and IL-2D dose are inversely correlated is prolonged with a lowered dose of IL-2D (simple linear regression, R² =0.3074, P<0.05).