Localized Immunomodulation with PD-L1 Results in Sustained Survival and Function of Allogeneic Islets without Chronic Immunosuppression

Abstract

Allogeneic islet transplantation is limited by adverse effects of chronic immunosuppression used to control rejection. The programmed cell death 1 pathway as an important immune checkpoint has the potential to obviate the need for chronic immunosuppression. We generated an oligomeric form of programmed cell death 1 ligand chimeric with core streptavidin (SA-PDL1) that inhibited the T effector cell response to alloantigens and converted T conventional cells into CD4⁺Foxp3⁺ T regulatory cells. The SA-PDL1 protein was effectively displayed on the surface of biotinylated mouse islets without a negative impact islet viability and insulin secretion. Transplantation of SA-PDL1-engineered islet grafts with a short course of rapamycin regimen resulted in sustained graft survival and function in >90% of allogeneic recipients over a 100-d observation period. Long-term survival was associated with increased levels of intragraft transcripts for innate and adaptive immune regulatory factors, including IDO-1, arginase-1, Foxp3, TGF-β, IL-10, and decreased levels of proinflammatory T-bet, IL-1β, TNF-α, and IFN-γ as assessed on day 3 posttransplantation. T cells of long-term graft recipients generated a proliferative response to donor Ags at a similar magnitude to T cells of naive animals, suggestive of the localized nature of tolerance. Immunohistochemical analyses showed intense peri-islet infiltration of T regulatory cells in long-term grafts and systemic depletion of this cell population resulted in prompt rejection. The transient display of SA-PDL1 protein on the surface of islets serves as a practical means of localized immunomodulation that accomplishes sustained graft survival in the absence of chronic immunosuppression with potential clinical implications.

FIGURE 1.

Construction, expression, and characterization of the SA-PDL1 protein. (A) Schematic presentation of pMT-Bip-SA-PDL1 construct. A synthetic gene encoding the extracellular domain of mouse PD-L1 linked to a modified form of streptavidin and 6His tag was cloned under the CuSO₄-inducible metallothionein (MT) promoter and the Bip secretion signal in the Drosophila S2 pMT/Bip/V5-HisA expression vector. (B) SDS-PAGE analysis of the purified SA-PDL1 protein. SA-PDL1 was purified from culture supernatants of S2 stable transfectants using immobilized metal affinity chromatography. Protein samples in reducing and denaturing loading buffer were either left unheated or heated in boiling water for 5 minutes and run on a 12.5% SDS-PAGE gel. The SA-PDL1 protein migrates as a single monomeric band (~52 kDa) with heat and as an oligomeric band (> 250 kDa) without heat. (C) Western blot analysis of the SA-PDL1 protein probed with anti-streptavidin Ab.

FIGURE 2.

SA-PDL1 protein enhances TGF-β-mediated conversion of Tconv into Treg cells and blocks alloantigen-mediated proliferation of T effector cells. (A) SA-PDL1 mediated augmentation of Tconv conversion into CD4⁺FoxP3⁺ Treg cells. Tconv cells (CD4⁺hCD2⁻) were flow sorted from C57BL/6 mice transgenic for the human CD2 molecule under the control of mouse FoxP3 promotor and cultured in medium supplemented with agonistic Ab to CD3 (5 μg/ml) and CD28 (1 μg/ml) and TGF-β (1 ng/ml), IL-2 (20 U/ml), and the indicated amounts of SA-PDL1 protein for 3 days. Streptavidin (SA) was used at equimolar levels as a control protein. The frequency of Treg cells (hCD2⁺CD4⁺FoxP3⁺) was assessed using flow cytometry. (B) SA-PDL1 suppresses proliferation of alloreactive T cells ex vivo. Splenocytes from 4C mice (C57BL/6 transgenic for a TCR specific for I-A^b) were co-cultured with irradiated BALB/c splenocytes as stimulators in a mixed lymphocyte reaction assay for 72 hrs. At 48 hrs of incubation, cultures were supplemented with the indicated amounts of SA-PDL1 or equimolar concentrations of SA as control and incubated for additional 24 hrs. Cultures were pulsed with [³H]-thymidine for the last 16 hrs of incubation and harvested to assess the DNA associated radioactivity. Data was graphed as percent inhibition. Both sets of these studies were performed in triplicate and repeated at least three times. Each data point is indicative of Mean ± SEM. Statistical analysis was performed using one-way ANOVA with Bonferroni’s multiple comparison test. *p < 0.05. **p < 0.01, ***p < 0.001.

FIGURE 3.

Islets are effectively engineered with SA-PDL1 protein without a significant impact on their function. (A) Assessing cell surface engineering conditions with the SA-PDL1 protein. Mouse splenocytes were surface modified with 5 μM EZ-Link sulfo-NHS-LC-Biotin and engineered with the indicated amounts of SA-PDL1 protein (ng/10⁶ cells). The level of SA-PDL1 on the cell surface was assessed using an anti-SA Ab in flow cytometry. Biotinylated splenocytes without SA-PDL1 engineering served as control. (B) Mean fluorescence intensity (MFI) plotted against varying concentrations of SA-PDL1 protein. Data were tabulated from five independent experiments. (C) Engineering mouse pancreatic islets with the SA-PDL1 protein. Mouse islets were biotinylated (5 μM) followed by engineering with the SA-PDL1 protein (400 ng/500 islets). Biotinylation and the presence of SA-PDL1 on the islet surface were assessed using the streptavidin protein conjugated with APC (SA-APC, red) and anti-SA Ab (Anti-SA-FITC, green), respectively, in confocal microscopy. Islets positive for both molecules appear as yellow. Original magnification X 20. Staining patterns were consistent for samples across independent runs. (D) Engineering islets with SA-PDL1 protein does not impact insulin secretion. SA-PDL1-engineered and unmodified islets as control were stimulated with low (3 mM) and high (11 mM) glucose concentrations in a glucose stimulated insulin secretion assay. (E) Stimulation indices of studies conducted in D. Stimulation index was calculated by dividing the mean DNA normalized insulin value measured from high glucose samples by the low glucose samples. No significant difference (p = 0.73) was observed between the two groups using unpaired student t test.

FIGURE 4.

SA-PDL1-engineered pancreatic islets show sustained long-term survival and function in allogeneic graft recipients. (A) Experimental scheme showing islet engineering, transplantation, and the transient use of immunosuppression. (B) Survival of SA-PDL1-engineered islet grafts in allogeneic recipients. SA-PDL1-engineered or unmodified islets were transplanted under the kidney capsule of chemically diabetic recipients without or with a short course of rapamycin (0.2 mg kg/daily for 15 doses). Animals were monitored for blood glucose levels, and those with two consecutive daily readings of ≥ 250 mg/dl were considered diabetic. Graft survival was assessed using the log-rank (Mantel-cox) test, ***p < 0.001. (C) Intraperitoneal glucose tolerance test (IPGTT) showing sufficient mass and function of transplanted islets. Recipients with long-term (≥ 150 days) graft survival were subjected to IPGTT with naïve mice serving as controls. (D) Area-under-the-curve for each animal glucose clearance was computed using the trapezoid rule in GraphPad Prism and compared using the student two-tailed t test. (E) Long-term euglycemia is maintained by the transplanted SA-PDL1-enginered islet grafts. Blood glucose levels of mice transplanted with SA-PDL1-engineered islets with or without rapamycin. Surgical removal of the kidney harboring the long-term SA-PDL1-engineered islets results in prompt hyperglycemia, confirming graft function.

FIGURE 5.

Immunomodulation with SA-PDL1 results in islet graft-localized tolerance. (A) T cells of long-term islet graft survivors generates a normal response to donor antigens. Splenocytes from naïve (Naïve), rejecting (SA-PDL1-islet), and long-term survivors (SA-PDL1-islet + rapa) were used as responders against irradiated donor-matched BALB/c or C3H third party splenocytes in a standard CFSE-based in vitro mixed lymphocyte reaction assay. Dilution of CFSE in CD4⁺ and CD8⁺ T cells was assessed using Abs to CD4 and CD8 molecules in flow cytometry and plotted as the percent division for each cell population. (B) Intracellular cytokine response. Splenocytes from groups in (A) were stimulated with PMA and Ionomycin for 6 hrs, stained with fluorescence-labelled Abs to CD4, CD8, CD44 (to define activated cells) along with IFN-γ, TNF-α, and IL-2 and analyzed using flow cytometry. Data was tabulated as percentage of the indicated cell population gated on total CD4⁺ T or CD8⁺ T cells. The experiments were performed in duplicates and each data point is indicative of Mean ± SEM. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparison test.

FIGURE 6.

Long-term survival of SA-PDL1-engineered islet grafts is associated with upregulated expression of immunoregulatory factors. Total RNA from unmodified islet grafts, unmodified islet grafts plus rapamycin (Rapa), SA-PDL1-engineered islet grafts (SA-PDL1), and SA-PDL1-engineered islet grafts plus rapamycin (SA-PDL1 + rapa) was extracted 3 days post-transplantation. The RNA was subjected to a TaqMan probe (FAM/VIC) based quantitative real-time RT-PCR using TaqMan primers to the indicated cytokines and transcriptional factors. Data were analyzed using DataAssist Software and plotted as fold change (2^−ΔΔCT) relative to GAPDH and unmodified islet graft only group. The experiments were performed in triplicate and repeated two times. Each data point is indicative of Mean ± SEM. *p < 0.05, **p <0.01, ***p <0.001 as assessed by one-way ANOVA with Tukey’s multiple comparison test.

FIGURE 7.

CD4⁺FoxP3⁺ Treg cells contribute to the maintenance of localized tolerance. T cells isolated from graft-draining lymph nodes of the indicated groups were analyzed on day 3 (A) or 7 (B) post-transplantation using flow cytometry with Abs to various cell surface markers. Absolute cell numbers for the indicated T cell populations are graphed. The ratio of CD4⁺CD25⁺FoxP3⁺ Treg to activated CD4⁺PD1⁺CD44^hi CD62L⁻ Teff cells on day 7 post-transplantation for SA-PDL1-engineered islets is significant when compared with the rapamycin control group, **p < 0.01 using unpaired t-test with Welch’s correction. Data (mean ± SEM) is representative of two independent experiments. (C) Long-term surviving SA-PDL1-engineered allogeneic islets show increased numbers of CD4⁺FoxP3⁺ Treg cells localized at the periphery of the graft. Tissue sections of kidney harboring long-term (> 100 days) islet grafts were stained with Abs against CD4 (red), Foxp3 (green), and insulin (blue) and analyzed using confocal microscopy (lower panel). Tissues stained with the same Abs, except an isotype Ab to Foxp3, served as control for Treg staining (upper panel). Circles indicate Treg cells in patches at the periphery of islet grafts. Upper right two panels are higher magnification of the different areas shown by circles. (D) Depletion of Treg cells results in rejection of long-term islet allografts. Streptozotocin diabetic C57BL/6.FoxP3^EGFP/DTR mice (n = 6) were transplanted with SA-PDL1-engineered islet grafts under the cover of a 15-day rapamycin regimen. Two out of 6 mice rejected within 33 days of transplantation (red solid lines). Four mice with sustained euglycemia were treated with diphtheria toxin (DT; 50 ng/gm) for two consecutive days on day 60 (n = 2, upward green arrows) and 80 (n = 2, upward blue arrows) post-transplantation. One animal in each treatment rejected (shown in respective broken lines) following DT treatment. One of the two euglycemic mice was treated again with DT (25 ng/gm two consecutive days; blue broken line, injection shown as downward grey arrows) 32 days post first treatment that resulted in graft rejection. The remaining one recipient (green unbroken line) maintained euglycemia for 140 days experimental end-point.