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

Published April 6, 2020, doi:10.4049/jimmunol.2000055 The Journal of Immunology

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

Lalit Batra,*,†,1 Pradeep Shrestha,*,†,1 Hong Zhao,*,† Kyle B. Woodward,*,†,2 Alper Togay,*,3 Min Tan,*,† Orlando Grimany-Nuno,*,† Mohammad Tariq Malik,*,† Marı´a M. Coronel,‡ Andre´s J. Garcı´a,‡,x Haval Shirwan,*,†,{,4 and Esma S. Yolcu*,†,{,4

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-b, IL-10, and decreased levels of proinﬂammatory T-bet, IL-1b,TNF-a,andIFN-g 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. The Journal of Immunology, 2020, 204: 000–000.

he potential of pancreatic islet grafts as a treatment option Programmed cell death-1 (PD-1; CD279) is a member of the for type 1 diabetes (T1D) has been demonstrated in a CD28/B7 superfamily of costimulatory molecules that shows low recent multicenter phase 3 clinical trial (1). Two signifi- levels of expression on resting T cells and high levels of expression T + + cant limitations of clinical islet transplantation include the paucity on activated CD4 and CD8 T cells, B cells, NKT cells, and of cadaveric pancreata and the adverse effects of chronic immu- monocytes (4). PD-1 ligand (PD-L1), one of the two physiological nosuppression to control rejection. Significant effort has recently ligands, is constitutively expressed on a variety of hematopoietic been devoted to the generation of a replenishable supply of insulin- and nonhematopoietic cells (4, 5). PD-1 is an important immune producing cells, such as porcine pancreatic islets (2) or b cells checkpoint pathway that modulates innate, adaptive, and regu- derived from stem cells (3), as an alternative to cadaveric human latory immune responses and as such, plays a critical role in islets for transplantation. Regardless of the b cell source, the immune homeostasis and tolerance to self-antigens (6). Mice widespread use of insulin-producing cells as an effective treatment deficient for PD-1 exhibit a breakdown of peripheral tolerance and for T1D will require immunomodulatory approaches that obviate manifest multiple autoimmune features, such as lupus and car- or mitigate the need for chronic immunosuppression. diomyopathy (7, 8). The role of this immune inhibitory pathway in

*Institute for Cellular Therapeutics, School of Medicine, University of Louisville, Research Challenge Trust Fund, and a William Marvin Petty Gift for Type 1 Diabetes. Louisville, KY 40202; †Department of Microbiology and Immunology, School of M.T.M. is supported by Foundation for the National Institutes of Health T32 HL134664. Medicine, University of Louisville, Louisville, KY 40202; ‡Woodruff School of x Address correspondence and reprint requests to Dr. Haval Shirwan and Dr. Esma Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA 30332; Petit S. Yolcu, Institute for Cellular Therapeutics and Department of Microbiology and Institute for Bioengineering and Biosciences, Georgia Institute of Technology, { Immunology, School of Medicine, University of Louisville, 570 South Preston Street, Atlanta, GA 30332; and Department of Child Health, School of Medicine, Univer- Baxter I Building, Suite 404E, Louisville, KY 40202-1760. E-mail addresses: sity of Missouri, Columbia, MO 65211 [email protected] (H.S.) and [email protected] (E.S.Y.) 1L.B. and P.S. share ﬁrst authorship. The online version of this article contains supplemental material. 2Current address: Clinical Research Division, Fred Hutchinson Cancer Research Abbreviations used in this article: ARG-1, arginase-1; AUC, area under the curve; C , Center, Seattle, WA. T threshold cycle; DT, diphtheria toxin; GSIS, glucose-stimulated insulin secretion; 3Current address: Izmir_ Tepecik Training and Research Hospital, Izmir, Turkey. hCD2, human CD2; iNOS, inducible NO synthase; IPGTT, i.p. glucose tolerance test; MDSC, myeloid-derived suppressor cell; MST, median survival time; mTORC1, 4H.S. and E.S.Y. share senior authorship. mammalian target of rapamycin complex 1; NHS-LC, N-hydroxysulfosuccinimide– ORCIDs: 0000-0002-4375-9004 (L.B.); 0000-0002-5524-6011 (K.B.W.); 0000-0002- long chain; NOS-2, NO synthase-2; PD-1, programmed cell death 1; PD-L1, PD-1 5256-0064 (A.T.); 0000-0002-1986-8438 (M.T.); 0000-0001-9972-5850 (M.T.M.); ligand; PD-L1.Ig, PD-L1 and Ig fusion protein; SA, streptavidin; SA-FasL, Fas 0000-0001-6602-2518 (A.J.G.); 0000-0002-1657-9470 (H.S.). ligand chimeric with SA; SA-PDL1, PD-L1 chimeric with core SA; Tconv, T conventional; T1D, type 1 diabetes; Teff, T effector; Treg, regulatory T. Received for publication January 17, 2020. Accepted for publication March 17, 2020. This work was supported by grants from the Foundation for the National Institutes of Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 Health (R21 EB020107, R01AI121281, U01AI132817), the Kentucky Science and Technology Corporation (KSEF-2927-RDE-016), the Commonwealth of Kentucky

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tolerance to self-antigens was further substantiated by the performed in accordance to approved protocols by Institutional Animal demonstration that PD-L1 knockout NOD mice develop a rapid Care and Use Committee, University of Louisville. onset of diabetes as compared with wild-type NOD (9, 10). PD-1 Construction, expression, and characterization of signaling is also extensively exploited by chronic infections and SA-PDL1 protein tumors for immune evasion, providing additional evidence for the importance of this pathway in negative regulation of immune A synthetic gene was constructed to include the extracellular domain of mouse PD-L1 (68–728 bp, GI: AF233517.1) N terminus to a modified form responses (11, 12). The PD-1 pathway as an important thera- of core SA and a 63His tag for purification. The synthetic gene was then peutic target in immuno-oncology has been verified by the subcloned into the pMT/BiP/V5-His A CuSO4-inducible expression vector reported remarkable clinical efficacy of blocking Abs for various (Invitrogen, San Diego, CA) for stable expression in Drosophila S2 cells, tumor types (13). following published protocols (20, 21). SA-PDL1 protein was purified The PD-1 pathway also regulates alloreactive immune responses. using metal affinity chromatography (GE Healthcare Life Sciences, Marlborough, MA) and assessed for structure and purity using SDS-PAGE PD-L1 blockade was shown to result in enhanced alloreactive T cell and Western blots. Purified protein was tested for concentration and en- proliferation, Th1 cell differentiation, and accelerated MHC class dotoxin using the bicinchoninic acid and limulus amebocyte lysate tests, II–mismatched skin graft rejection in mice (14). A dimeric form of respectively, and aliquoted and frozen in 280˚C until use. PD-L1 and Ig fusion protein (PD-L1.Ig) blocked T cell prolifer- SA-PDL1–mediated Tconv cell conversion into Treg cells ation in vitro and prevented cardiac allograft rejection in combination with anti-CD154 blockade, providing direct evidence for Splenocytes harvested from C57BL/6.hCD2 mice transgenic for human the potential of this pathway to induce allograft tolerance (15). CD2 (hCD2) were expressed under the control of Foxp3 (22), stained with anti-mouse CD4-allophycocyanin and anti–hCD2-PE Abs, and suspended The combination of PD-L1.Ig and anti-CD154 Ab was shown to in cell sorting media (HBSS with 2% FBS). Tconv (CD4+hCD22) cells have robust efficacy in an allogeneic islet transplantation setting in were sorted using FACSAria (.99% purity) and cultured in 96-well which tolerance, rather than prolonged survival, was achieved U-bottom plates (0.2 3 106 cells per well) coated with anti-CD3 Ab (5 mg/ml). (16). Treatment with another form of PD-L1.Ig fusion protein Cultures were supplemented with anti-CD28 Ab (1 mg/ml), various concentrations of SA-PDL1 protein, human TGF-b1 (1 ng/ml; R&D Systems), alone was shown to significantly prolong the survival of orthotopic and 20 U/ml recombinant human IL-2 (PeproTech) in complete MLR corneal allografts in mice (17). Thus, the PD-1 pathway has sig- medium (20). Cultures were incubated for 72 h at 37˚C in a 5% CO2 in- nificant potential for modulating alloreactive responses to over- cubator. Cells were harvested, stained with anti-mouse CD4-Alexa 700 and come graft rejection. anti–hCD2-PE Abs, and run on a BD LSR II Flow Cytometer. Data were We have previously reported an approach that allows the tran- analyzed using FlowJo (Tree Star, San Carlos, CA) software. sient display of immunological ligands on biologic and nonbiologic SA-PDL1–mediated inhibition of alloreactive surfaces for localized immunomodulation (18–20). This concept T cell proliferation relies on the generation of recombinant proteins that constitute Splenocytes from 4C mice (C57BL/6 transgenic for a TCR recognizing the functional domains of immunological ligands fused to a modified BALB/c MHC class I-Ad) (23) were used as responders against irradiated form of core streptavidin (SA) and the positional display of these BALB/c splenocytes as stimulators in a standard MLR assay (19). Briefly, molecules on biotinylated surfaces by taking advantage of the high 4C splenocytes were cultured in a petri dish for 45 min at 37˚C to enrich affinity interaction between biotin and SA (21). The transient for T cells. Nonadherent cells were collected, washed, and incubated with 2000 cGy irradiated BALB/c splenocytes in 96-well U-bottom plates display of a modified form of ligand for the Fas death receptor, Fas 5 (10 cells per well) in complete MLR medium (19). After 48 h of incubation, ligand chimeric with SA (SA-FasL), on pancreatic islets resulted cultures were supplemented with varying concentrations of SA-PDL1 proin indefinite graft survival in allogeneic hosts in the absence of tein or equimolar concentrations of control SA protein. Cultures were in- chronic immunosuppression (20). The importance of the PD-1 cubated for a total of 72 h, with the last 16 h pulsed with [3H] thymidine checkpoint pathway in self-tolerance, taken together with the (1 mCi per well). Cultures were then harvested with Tomtec cell harvester to assess DNA-associated radioactivity (cpm) as the measure of cell prolifer- clinical success of blocking Abs to PD-L1 in cancer immuno- ation using a b plate counter. The percentage inhibition of T cell prolifer- therapy, served as the impetus to generate a recombinant form of ation was calculated using the formula of 1 2 (cpm in test proliferation/cpm PD-L1 chimeric with core SA (SA-PDL1), and test its immuno- in control proliferation) 3 100. modulatory function in a preclinical model of allogeneic islet MLR assay transplantation. SA-PDL1 blocked the proliferation of T cells in response to alloantigens and facilitated the conversion of naive T Splenocytes from various C57BL/6 graft recipients were labeled with conventional (Tconv) cells into CD4+Foxp3+ regulatory T (Treg) CFSE dye and used as responders against irradiated (2000 cGy) allogeneic BALB/c donor or third-party C3H splenocytes in a standard MLR assay cells. The transient display of SA-PDL1 on allogeneic islet grafts (20). The cultures were harvested 4 d later and run on a BD LSR II Flow resulted in sustained survival and function in the absence of Cytometer to assess the proliferation of CD4+ and CD8+ T cells by gating chronic immunosuppression. Immune protection was localized to on live cells. Data were analyzed using FACS Diva software and graphed the graft and associated with intragraft expression of various in- using GraphPad Prism. nate and adaptive immunoregulatory factors and accumulation of Modification of cell membrane with biotin and engineering + + CD4 Foxp3 Treg cells. Depletion of Treg cells resulted in prompt with the SA-PDL1 protein rejection of long-term islet grafts. Thus, the transient display of immunomodulatory molecules on the surface of islets serves as an Splenocytes were prepared from theBALB/cmiceandincubatedin5mM EZ-Link-Sulfo–N-hydroxysulfosuccinimide–long chain (NHS-LC)-biotin so- effective approach to prevent rejection in the absence of chronic lution (Thermo Fisher Scientific) in PBS for 30 min at room temperature in immunosuppression with significant translational potential. the dark, per a published protocol (21). After washing to remove free biotin, cells were incubated in PBS supplemented with various amounts of SA-PDL1 protein (10–1280 ng per 106 cells) for 30 min at 4˚C. Cells were then washed, Materials and Methods stained with anti–SA-FITC Ab or SA-allophycocyanin protein, and run on a Animals BD LSR II Flow Cytometer, and data were analyzed using FlowJo. C57BL/6, BALB/c, and C3H animals were purchased from the Jackson Islet isolation, engineering with the SA-PDL1 protein, hCD2 Laboratory. C57BL/6.Foxp3 (22) and B6.SJL-4C.TCR-tg (23) animals and transplantation were generously provided by Drs. H. Waldmann (Oxford University) and T.V. Brennan (Duke University). All animals were maintained in our specific Pancreatic islets were harvested from 8– to 12–wk-old BALB/c mice and pathogen-free vivarium at the University of Louisville. All experiments were engineered with SA-PDL1 protein, per published protocols (20). Briefly, Downloaded by guest on October 1, 2021 The Journal of Immunology 3

the pancreas was perfused with cold Liberase TL (0.18 mg/ml; Roche secondary Ab mixture of Alexa Fluor 647–conjugated (CD4) goat Diagnostics) and digested for 17–18 min at 37˚C. Islets were isolated anti-rabbit polyclonal Ab (1:100; Life Technologies) and Alexa Fluor on a discontinuous Ficoll (Sigma-Aldrich) gradient and maintained 555–conjugated (insulin) anti–guinea pig Ab (1:300; Invitrogen) for 1 h at overnight in RPMI-1640 medium supplemented with 10% FBS and room temperature. Slides were then washed with PBS and incubated for penicillin/streptomycin (100 U/ml and 100 mg/ml) in a 5% CO2 incubator 1 h at room temperature with rat anti-Foxp3 Ab conjugated with FITC (1:20; at 37˚C. Islets were collected in a 15-ml polypropylene round-bottom tube, eBioscience) to visualize the Treg cells. Fluorescent images were obtained washed with PBS, and incubated in 5 mM EZ-Link sulfo-NHS-LC-biotin using a Leica TCS SP5 Confocal Microscope under 203 magnification. solution at room temperature for 30 min. Biotinylated islets were then engineered with SA-PDL1 protein (∼400 ng per 500–550 islets) for 30 min RNA isolation and quantitative real-time RT-PCR at room temperature. Engineered islets were washed and then transplanted Islet grafts under the kidney capsule were surgically removed on day 3 under the kidney capsule of streptozotocin-diabetic C57BL/6 recipients, posttransplantation and preserved in the RNAlater solution for stabilization per published protocols (19, 20). Selected groups of graft recipients were and immediate RNase protection. RNA was extracted with TRIzol reagent i.p. injected with rapamycin (0.2 mg/kg; LC Laboratories) starting on the (Thermo Fisher Scientific), and cDNA was synthesized from 4 mgof day of transplantation for 15 d. Animals were monitored for blood glucose the total RNA using SuperScript IV VILO Master Mix (Thermo Fisher $ levels, and those with two consecutive daily measurements of 250 mg/dl Scientific). Quantitative real-time RT-PCR was performed for different were considered diabetic and confirmation of graft rejection. genes using the TaqMan probe assay on QuantStudio 3 Real-Time PCR To assess the role of Treg cells in the maintenance of long-term grafts, system (Applied Biosystems). Each PCR consisted of 5 ml of TaqMan Fast SA-PDL1–engineered islets were transplanted into streptozotocin-diabetic EGFP/DTR Advanced Master Mix, 1 ml of TaqMan primer and FAM/VIC–labeled C57BL/6.Foxp3 mice (Jackson Laboratory) transgenic for human minor groove binder probes, 1 ml of cDNA sample (from 1:10 dilution), diphtheria toxin (DT) receptor expressed under the control of Foxp3 promoter. and 3 ml of nuclease-free water in a final volume of 10 ml. The thermal Graft recipients were treated i.p. with DT (50 ng/g) for 2 consecutive d at the cycling conditions were as follows: one cycle of 95˚C for 20 s, followed by indicated days posttransplantation. Treg cell depletion was confirmed in pe- 40 cycles of 95˚C for 1 s and 60˚C for 20 s. The relative quantitation was ripheral blood using flow cytometry. analyzed using comparative threshold cycle (CT) protocol based on the 2DDCT Flow cytometry 2 method with DataAssist Software (Thermo Fisher Scientific). Briefly, differences in the CT for the target gene and the CT for the GAPDH Abs against mouse CD4 (RM4-5), CD3 (500A2), CD8 (53-6.7), CD62L gene control were calculated as DCT, to normalize for differences in the (MEL-14), CD44 (IM7), CD25 (PC61.5), PD-1 (RMP1-30), Foxp3 amount of total nucleic acid added to each reaction and the efficiency of (FJK-16s), IFN-g (XMG1.2), TNF-a (MP6-XT22), and IL-2 (JES6-5H4) the quantitative real-time RT-PCR step. Finally, the DCT values were 2 were purchased from BD Biosciences or eBioscience. Optimal concen- normalized to unmodified islet only group and expressed as 2 DDCT. Thus, trations for all fluorochrome-conjugated Abs were determined by titration. all the experimental samples were expressed as n-fold change relative to For surface staining, cells were incubated with respective Abs for 30 min the control (unmodified islet only group) as published (25). at 4˚C. For intranuclear Foxp3 staining, fixation-permeabilization buffer (eBioscience) was used, as per manufacturer’s instruction, followed by Statistical analysis incubation with anti-Foxp3 Ab for 30 min at 4˚C. For intracellular cyto- Student t test (two-tailed) was used to assess differences between two kine analysis, spleen cells were incubated for 6 h at 37˚C in complete MLR groups, whereas one-way ANOVA with Bonferroni multiple comparisons media with PMA (50 ng/ml), ionomycin (1 mg/ml) and Golgiplug (for last or Tukey multiple comparisons were used to determine differences among 4 h; BD Biosciences). After surface staining, Cytofix (BD Biosciences) three or more groups. Graft survival was assessed using the log-rank was used, according to manufacturer’s instruction, to fix and home-made (Mantel–Cox) test. Data were expressed as mean 6 SEM where indi- perm buffer (0.1% saponin in PBS) for permeabilization followed by in- cated, and the p values , 0.05 were considered statistically significant. cubation with Abs against cytokines and analysis using flow cytometry, as Statistical analyses were performed using GraphPad Prism 9 software. reported (19). Glucose-stimulated insulin secretion assay Results Glucose-stimulated insulin secretion (GSIS) was performed for naive and Construction and production of the SA-PDL1 SA-PDL1 engineered BALB/c islets using a static incubation protocol (24). recombinant protein Briefly, low (3 mM) and high (11 mM) glucose solutions were prepared in Krebs-Ringer Bicarbonate buffer. Islets were handpicked and equilibrated A synthetic gene containing the coding sequences for the extra- at low glucose (100 islets per milliliter) in a millicell cell culture insert cellular domain of mouse PD-L1 and a modified form of core SA (Merck) for 1 h at 37˚C in a 5% CO2 incubator. Islets were then challenged was subcloned in frame with the BiP secretion signal in the pMT/ with high glucose solution for stimulation by incubating the inserts holding BiP/V5-HisA expression vector (Fig. 1A). Drosophila S2 cells islets in the well containing high glucose solution for 1 h at 37˚C in a 5% stably transfected with this construct secreted the SA-PDL1 protein CO2 incubator. The supernatant was collected from each well at different time points and stored at 280˚C until insulin content analysis by ELISA following stimulation with CuSO4. The protein was successfully (Mercodia). DNA quantification (Qiagen) was done from each pelleted purified from the culture supernatants using metal affinity chro- islet mass for normalization against individual insulin content. The stim- matography taking advantage of a 63His tag engineered into the ulation index was calculated as the ratio of insulin secreted in high-glucose construct. SA-PDL1 ran as oligomers in a reducing SDS-PAGE gel stimulation to low-glucose stimulation. without heat (Fig. 1B). Heating the protein at 100˚C resulted in i.p. glucose tolerance test the dissociation of oligomers into monomers, a structural feature An i.p. glucose tolerance test (IPGTT) was performed on mice fasted for dictated by the native SA molecule that forms oligomers in solution 6 h. Blood samples were obtained at 0, 10, 20, 30, 45, 60, 90, and 120 min (21, 26). Western blot analysis using polyclonal anti-SA Abs con- postintraportal glucose injection (2 g/kg body weight). Blood glucose levels firmed the identity and integrity of the protein (Fig. 1C). were measured using an Accu-Check Nano Glucometer and Smart View Test Strips (Roche Diagnostics). IPGTT was calculated by estimating the SA-PDL1 protein enhances the conversion of Tconv into Treg total area under the curve (AUC) using GraphPad trapezoid rule. cells and blocks proliferation of alloreactive T cells Immunohistochemical analysis The interaction of PD-L1 with PD-1 receptor on CD4+ Tconv cells was shown to augment TGF-b–mediated in vitro conversion of Kidneys harboring islet grafts frozen in Tissue-Tek O.C.T. compound + + + (Sakura FineTek) were cut into 5–8 mm sections and fixed with ice-cold these cells into CD4 CD25 Foxp3 –induced Treg cells (27). To acetone or 4% paraformaldehyde on frosted slides. Slides were incubated assess if SA-PDL1 protein has a similar function, Tconv cells in blocking solution (0.5% Triton X-100, 1% BSA, 5% goat serum and rat from a transgenic C57BL/6 mouse expressing the hCD2 under the anti-mouse CD16/CD32 FcgII/III receptor block) for 30 min at room mouse Foxp3 promoter (22) were sorted by flow cytometry gating temperature. Sections were then incubated for 1 h at room temperature in a + + 2 mixture of rat anti-mouse CD4 Ab (1:40; BD Pharmingen) and guinea pig on CD3 CD4 hCD2 cells. The sorted Tconv cells were then cul- anti-insulin polyclonal Ab (1:100; Dako) in PBS supplemented with 1% tured in the presence of agonistic Abs to CD3, CD28, and a mixture BSA. After washing twice with PBS, tissue sections were incubated in a of human TGF-b1 and IL-2 cytokines. There was a significant Downloaded by guest on October 1, 2021 4 PD-L1–ENGINEERED ALLOGENEIC ISLETS ACHIEVE SUSTAINED SURVIVAL

although nonsigniﬁcant, plausibly due to nonspeciﬁc inhibition caused by SA protein or contaminants derived from insect cell proteins. PD-L1, as an immune checkpoint negative regulator, blocks T cell proliferative responses (28, 29). Thus, we tested the SA-PDL1 protein for blocking alloreactive T cell responses in vitro. Splenocytes from 4C mice transgenic for a TCR on the C57BL/6 background recognizing BALB/c H-2 I-Ad molecule (23) were used as responders to irradiated BALB/c splenocytes in the presence of various doses of SA-PDL1 protein in a 3-d proliferation assay. There was robust inhibition of 4C proliferation in cultures supplemented with higher doses of SA-PDL1 (10 and 20 mg/ml) as compared with cultures supplemented with equimolar amounts of SA control protein (Fig. 2B; p , 0.001). Taken together, these results demonstrate the immunomodulatory function of SA-PDL1 both on Treg and T effector (Teff) cells, consistent with published literature (27–29).

FIGURE 1. Construction, expression, and characterization of the SA-PDL1 Pancreatic islets are effectively engineered to display the protein. (A) Schematic presentation of pMT-BiP–SA-PDL1 construct. A SA-PDL1 protein on their surface without a major impact on synthetic gene encoding the extracellular domain of mouse PD-L1 linked their viability and function to a modified form of SA and 6His tag was cloned under the CuSO4- To assess if SA-PDL1 protein can be transiently displayed on the inducible metallothionein (MT) promoter and the BiP-secretion signal in surface of pancreatic islets, splenocytes were used as a rapid and the Drosophila S2 pMT/BiP/V5-HisA expression vector. (B) SDS-PAGE quantitative platform to establish engineering conditions. Our analysis of the purified SA-PDL1 protein. SA-PDL1 was purified from culture supernatants of S2 stable transfectants using immobilized metal previous studies have shown that surface modification of cells and affinity chromatography. Protein samples in reducing and denaturing islets with 5–15 mM biotin was optimum for the transient display loading buffer were either left unheated or heated in boiling water for of various immunomodulatory molecules at desired densities 5 min and run on a 12.5% SDS-PAGE gel. The SA-PDL1 protein migrates (21, 30–32). Thus, splenocytes modified with 5 mM EZ-Link as a single monomeric band (∼52 kDa) with heat and as an oligomeric sulfo-NHS-LC-biotin were incubated with various concentrations band (.250 kDa) without heat. (C) Western blot analysis of the SA-PDL1 of SA-PDL1 protein and cells were analyzed in flow cytometry. We protein probed with anti-SA Ab. observed a dose-dependent binding of SA-PDL1 to biotinylated splenocytes that plateaued at 640 ng of protein per 1 3 106 cells (p = 0.004) increase in the number of induced Treg cells in cul- (∼98.5% of targeted cells with mean fluorescence intensity values tures supplemented with the highest dose of SA-PDL1 as com- of .13,000; Fig. 3A, 3B). pared with equimolar SA used as a control protein (Fig. 2A). As Our previous studies with FasL as another immunomodulator compared with cultures without proteins, cultures supplemented used for islet engineering (20) and the broad range of SA-PDL1 with the SA had a lower percentage of Treg cell conversion, protein displayed on the surface of splenocytes led us to use 5 mM

FIGURE 2. SA-PDL1 protein enhances TGF-b–mediated conversion of Tconv cells into Treg cells and blocks alloantigen-mediated proliferation of Teff cells. (A) SA-PDL1–mediated augmentation of Tconv cell conversion into CD4+Foxp3+ Treg cells. Tconv cells (CD4+hCD22) were flow sorted from C57BL/6 mice transgenic for the hCD2 molecule under the control of mouse Foxp3 promotor and cultured in medium supplemented with agonistic Ab to CD3 (5 mg/ml), CD28 (1 mg/ml), TGF-b (1 ng/ml), IL-2 (20 U/ml), and the indicated amounts of SA-PDL1 protein for 3 d. 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-Ad) were cocultured with irradiated BALB/c splenocytes as stimulators in an MLR assay for 72 h. At 48 h of incubation, and cultures were supplemented with the indicated amounts of SA-PDL1 or equimolar concentrations of SA as control and incubated for additional 24 h. Cultures were pulsed with [3H]-thymidine for the last 16 h of incubation and harvested to assess the DNA-associated radioactivity. Data were 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 6 SEM. Statistical analysis was performed using one-way ANOVA with Bonferroni multiple comparison test. *p , 0.05, **p , 0.01, ***p , 0.001. Downloaded by guest on October 1, 2021 The Journal of Immunology 5

biotin and 800 ng of SA-PDL1 protein per 1000 islets for engi- treatment with a short course of rapamycin in a similar model neering. Confocal microscopy analysis demonstrated the intense using FasL as an immunomodulator resulted in tolerance to al- presence of both biotin and SA-PDL1 protein on the surface of logeneic islet grafts (20). Treatment of SA-PDL1–engineered islet islets (Fig. 3C) with moderate levels of SA-PDL1 in the inner core graft recipients with a 15 d course of rapamycin regimen starting as assessed by Z-stack analysis (Supplemental Fig. 1). To assess the day of transplantation resulted in sustained survival of .90% the impact of engineering on function, islets were tested in a GSIS of grafts over an observation period of 100 d. In marked contrast, secretion assay. Although there was a nonsignificant reduction, naive unmanipulated islet grafts under the same rapamycin regi- plausibly due to the engineering process, in insulin secretion from men were rejected in an acute fashion (MST = 19 d). the SA-PDL1–engineered islets as compared with unmodified is- IPGTT performed on long-term graft recipients revealed a lets (Fig. 3D), stimulation indices showed comparable functions similar pattern of blood glucose clearance as compared with naive (Fig. 3E). mice (Fig. 4C). The AUC of the blood glucose concentration during the IPGTT showed no differences between naive and long- SA-PDL1–engineered islets survive indefinitely in allogeneic term graft recipients (Fig. 4D; p = 0.23), demonstrating sufficient hosts under a short course of rapamycin regimen functional mass of transplanted islets. Importantly, surgical re- Pancreatic islets from mice genetically modified to lack PD-L1 moval of the kidney harboring long-term SA-PDL1–engineered were shown to undergo accelerated rejection in allogeneic recip- grafts resulted in prompt hyperglycemia, confirming the role of ients, emphasizing the importance of this immune checkpoint li- transplanted islets in normalizing the blood glucose levels (Fig. 4E). gand in modulating alloreactive responses (33). Thus, we assessed the impact of SA-PDL1 protein displayed on the surface of islet Recipients of long-term allogeneic grafts generate a systemic grafts on survival in allogeneic hosts (Fig. 4A). Chemically dia- response to donor Ags betic C57BL/6 mice receiving unmodified BALB/c islets under To assess if localized immunomodulation with the transient display the kidney capsule acutely rejected all grafts with a median of SA-PDL1 protein on allogeneic islets results in systemic un- survival time (MST) of 14 d (Fig. 4B). SA-PDL1–engineered responsiveness, splenocytes from long-term graft recipients were islet grafts showed significantly prolonged survival, but all grafts tested in vitro against donor Ags in a standard in vitro proliferation eventually rejected (MST = 28 d). We have previously shown that assay (18, 19). Both CD4+ and CD8+ T cells from long-term graft

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 mM EZ-Link sulfo-NHS-LC-biotin and engineered with the indicated amounts of SA-PDL1 protein (nanogram per 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 mM) followed by engineering with the SA-PDL1 protein (400 ng per 500 islets). Biotinylation and the presence of SA-PDL1 on the islet surface were assessed using the SA protein conjugated with allophycocyanin (SA-allophycocyanin, red) and anti-SA Ab (anti–SA-FITC, green), respectively, in confocal microscopy. Islets positive for both molecules appear as yellow. Original magnification 320. 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 GSIS 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. Downloaded by guest on October 1, 2021 6 PD-L1–ENGINEERED ALLOGENEIC ISLETS ACHIEVE SUSTAINED SURVIVAL

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) IPGTT showing sufficient mass and function of transplanted islets. Recipients with long-term ($150 d) graft survival were subjected to IPGTT with naive mice serving as controls. (D) AUC 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–engineered 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.

recipients responded to BALB/c donor and C3H third-party Ags at group as compared with the SA-PDL1 islets or unmodified islets levels comparable to T cells from naive mice or from rejecting plus rapamycin groups (Fig. 6). There was not a significant dif- recipients (SA-PDL1–engineered islet graft without rapamycin; ference in the expression of proinflammatory cytokines IL-6 Fig. 5A). T cells from long-term graft recipients also expressed among the groups, whereas NO synthase-2 (NOS-2) as a marker similar levels of IL-2 and proinflammatory TNF-a and IFN-g for M1-type macrophages and ROR-gT transcriptional factor for cytokines as compared with T cells isolated from naive or recip- Th17 cells showed 1.5- to 2-fold increased expression in both ients with graft rejection (Fig. 5B). Taken together, these data rapamycin and SA-PDL1 islets plus rapamycin groups over the demonstrate the lack of systemic tolerance in long-term recipients SA-PDL1 islet group. These results demonstrate that SA-PDL1 of surviving islet allografts, thereby revealing the graft-localized works in synergy with rapamycin immediately posttransplantation nature of immune protection. to change the balance of regulatory and effector responses in favor of sustained islet graft survival. Long-term graft survival is associated with increased intragraft levels of transcripts for immunoregulatory factors CD4+Foxp3+ Treg cells play an important role in sustained To provide further insight into the mechanistic basis of the observed graft survival localized immune protection, quantitative real-time RT-PCR was We next assessed whether SA-PDL1–engineered allogeneic islets performed on total RNA isolated from various groups on day 3 impact T cell frequencies in lymphoid organs early posttransplantation. posttransplantation to assess the intragraft expression of transcripts Flow analysis of various T cell populations isolated from islet for pro- and anti-inflammatory genes. We observed a significant graft-draining lymph nodes on days 3 and 7 posttransplantation increase in intragraft expression of the regulatory transcriptional did not reveal significant differences in the absolute cell numbers factors Foxp3 (Treg; p , 0.05) and GATA-3 (Th2; p , 0.001) and (Fig. 7A) or percentages (Supplemental Fig. 2), except that the ratio the regulatory cytokines TGF-b (p , 0.001), IL-10 (p , 0.001), of CD4+CD25+Foxp3+ Treg cells to CD4+PD-1+CD44hiCD62L2 and IL-4 (p , 0.001) in SA-PDL1–engineered islets plus rapa- activated Teff cells was higher in the SA-PDL1 islets plus rapamycin group as compared with SA-PDL1-islets or unmodified mycin group compared with the unmodified islets plus rapamycin islets plus rapamycin groups (Fig. 6). The levels of transcripts group (p , 0.01) (Fig. 7B). A similar profile was also observed for for the proinflammatory transcriptional factor T-bet (p , 0.01) splenic T cell subtypes, except that the increase in the ratio of and cytokines IFN-g (p , 0.01), TNF-a (p , 0.001), and IL-1b CD4+CD25+Foxp3+ Treg cells to CD4+PD-1+CD44hiCD62L2– (p , 0.0001) were significantly lower in the SA-PDL1 islet and activated Teff cells was nonsignificant on either day 3 or 7 SA-PDL1 islets plus rapamycin versus the islet plus rapamycin posttransplantation (Supplemental Fig. 3). Immunohistochemi- group. Importantly, we also observed increased expression of cal analysis of long-surviving (.100 d) grafts revealed dense regulatory IDO-1 (p , 0.01) and arginase-1 (ARG-1, p , 0.0001) as peri-islet presence of CD4+Foxp3+ Treg cells occurring in patches markers for M2 macrophages in the SA-PDL1 islets plus rapamycin (Fig. 7C). Downloaded by guest on October 1, 2021 The Journal of Immunology 7

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 Ags. Splenocytes from naive, rejecting (SA-PDL1 islet), and long-term survivors (SA-PDL1 islet + rapamycin [rapa]) were used as responders against irradiated donor-matched BALB/c or C3H third-party splenocytes in a standard CFSE-based in vitro MLR 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 h; stained with fluorescence-labeled Abs to CD4, CD8, CD44 (to define activated cells) along with IFN-g, TNF-a, and IL-2; and analyzed using flow cytometry. Data were tabulated as the 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 6 SEM. Statistical analysis was performed using one-way ANOVA with Tukey multiple comparison test.

To assess the contribution of Treg cells to sustained graft sur- this scheme, we generated a functional form of mouse PDL1, vival, BALB/c allogeneic islets engineered with the SA-PDL1 SA-PDL1, and assessed its efficacy when displayed on the sur- protein were transplanted under the kidney capsule of chemi- face of allogeneic islets in establishing sustained euglycemia in cally diabetic C57BL/6.Foxp3EGFP/DTR recipients (n = 6) under the the absence of chronic immunosuppression in chemically diabetic same rapamycin regimen. Two out of six recipients rejected their mice. grafts on day 29 and 33, whereas the other four showed sustained SA-PDL1 exists as an oligomeric molecule with immunoregu- euglycemia and were treated with two consecutive injections of latory functions. SA-PDL1 augmented the TGF-b–mediated DT on day 60 or 80 posttransplantation to deplete Treg cells conversion of CD4+ Tconv cells into induced CD4+Foxp3+ Treg (Fig. 7D). Two of four recipients rejected islet grafts following cells and blocked the proliferative response of Teff cells to allo- the treatment with DT, whereas the other two remained eugly- antigens, consistent with the reported regulatory functions of the cemic despite significant depletion of Treg cells in the blood native ligand (27–29). SA-PDL1 was efficiently displayed on the (Supplemental Fig. 4). Treatment of one of these mice with DT surface of pancreatic islets modified with biotin without a sig- for a second time resulted in prompt graft rejection, indicating nificant negative impact on the viability and insulin secretion insufficient depletion of Treg cells with one treatment in the function of islets. This observation is also consistent with our target tissues, plausibly the islet graft. Taken together, these data previous studies, demonstrating that the transient display of a signify the contribution of Treg cells to the observed long-term chimeric form of FasL, SA-FasL, on the surface of various cells, islet graft survival. including hematopoietic stem cells, pancreatic islets, and cardiac graft vasculature, did not negatively impact their viability or Discussion function (21, 31, 35, 36, 38, 39). The PD-1 pathway is an important immune checkpoint that reg- Transplantation of SA-PDL1–engineered islets into chemically ulates self-reactive responses and is extensively exploited by diabetic allogeneic recipients resulted in sustained survival and chronic infections and tumors for immune evasion (11, 34). These function in the absence of chronic immunosuppression. This ob- traits of PD-1 led to intensive efforts to modulate this pathway for servation is consistent with several reports targeting the PD-1 therapeutic purposes. Indeed, PD-1 has been proven as an im- pathway for modulation in the transplantation setting (14–17, portant therapeutic target in oncology with impressive clinical 40). Pancreatic islet or solid organ grafts genetically modified to outcomes for various malignancies. However, the efficacy of this lack PD-L1 expression showed accelerated rejection following immune checkpoint for modulating alloreactive responses in transplantation into allogeneic recipients (33, 40). Hematopoietic transplantation has not been extensively explored, which is stem cells genetically or pharmacologically modulated to over- the subject of this manuscript. As a practical alternative to the express PD-L1, but not wild-type cells with low levels of ex- DNA-based ectopic expression of immunomodulatory proteins, pression, were effective in reversing T1D in the NOD mouse we previously reported the concept of generating recombinant model (41). Blockade of PD-L1 using an Ab resulted in the rejec- chimeric immune ligands with a modified form of SA and their tion of allogeneic fetus, implicating this pathway in fetomaternal transient display on biologic surfaces, including islets, for regu- tolerance (42). Our findings are also consistent with published lating immune responses with demonstrated efficacy in various studies reporting the efficacy of a dimeric form of PD-L1.Ig when transplantation and autoimmune settings (20, 21, 35–37). Using used in combination with CD40 blockade in prolonging cardiac Downloaded by guest on October 1, 2021 8 PD-L1–ENGINEERED ALLOGENEIC ISLETS ACHIEVE SUSTAINED SURVIVAL

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 d posttransplantation. 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 2 as fold change (2 DDCT) relative to GAPDH and unmodified islet graft only group. The experiments were performed in triplicates and repeated two times. Each data point is indicative of mean 6 SEM. Assessed by one-way ANOVA with Tukey multiple comparison test. *p , 0.05, **p , 0.01, ***p , 0.001.

allografts survival (15) and supporting sustained survival of islet Treg cells; it blocks Teff cell proliferation by inhibiting p34cdc2 allografts (16). Furthermore, infusions of ethylene carbodiimide– kinase (58) while facilitating the generation, expansion, and sus- treated donor-splenic APCs resulted in tolerance to allogeneic tained survival of CD4+CD25+Foxp3+ Treg cells (53–57). PDL1 islets, and the induction of tolerance required a synergistic action converts Teff cells into Treg cells, as shown in the current study of the PD-1 pathway and Treg cells (43). and reported by others (59). The depletion of Treg cells resulted in SA-PDL1–mediated long-term graft survival required a short acute rejection of SA-PDL1–engineered long-term islet grafts, course (15 d) of rapamycin, as SA-PDL1–engineered grafts without thus implicating the generation of Treg cells as the potential basis rapamycin were rejected, albeit in a delayed fashion as compared of synergy between SA-PDL1 and rapamycin. with untreated controls. This observation is consistent with our T cells from long-term graft recipients generated a proliferative reported findings using SA-FasL as an immunomodulator in dif- response to donor Ags that was comparable to the response gen- ferent islet transplantation settings (18–20) and with those of erated by naive animals, demonstrating the lack of systemic tol- others using a short course of rapamycin in conjunction with erance. This finding is not surprising, given the localized nature various immunomodulatory protocols in transplantation and au- of immunomodulation by presenting SA-PDL1 on the surface toimmunity settings (44–46). The mechanisms underlying the of transplanted islets. Furthermore, the localized nature of graft synergy between rapamycin and SA-PDL1 are presently unknown. protection is consistent with our previous studies with SA-FasL Rapamycin has pleiotropic immune regulatory functions on cells displayed on the surface of islets (20), poly(ethylene glycol) micro- of innate, adaptive, and regulatory immunity, and as such, may gels cotransplanted with unmodified islets (19), or poly(lactic-co contribute to the SA-PDL1–induced graft survival by various means glycolic acid) scaffolds housing unmodified islets (18). Quantitative (47–57). Particularly, rapamycin has opposite effects on Teff and real-time PCR performed on day 3 posttransplantation showed Downloaded by guest on October 1, 2021 The Journal of Immunology 9

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)or7(B) posttransplantation 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 cells to activated CD4+PD-1+CD44hi CD62L2 Teff cells on day 7 posttransplantation for SA-PDL1–engineered islets is significant when compared with the rapamycin control group. Using unpaired t test with Welch correction; **p , 0.01. Data (mean 6 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 d) islet grafts were stained with Abs against CD4 (red), Foxp3 (green), and insulin (blue) and analyzed using confocal microscopy (lower panel; composedof 4 fields of view from the same tissue section spliced together). Tissues stained with the same Abs, except an isotype Ab to Foxp3, served as control for Treg cell staining (upper panel; composed of 3 fields of view from the same tissue section spliced together). 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. Images were taken with a 20X objective with magnification of 4.63 and 6.22 optical zooms for insets. (D) Depletion of Treg cells results in rejection of long-term islet allografts. Streptozotocin-diabetic C57BL/6.Foxp3EGFP/DTR mice (n = 6) were transplanted with SA-PDL1–engineered islet grafts under the cover of a 15 d rapamycin regimen. Two out of six mice rejected within 33 d of transplantation (red solid lines). Four mice with sustained euglycemia were treated with DT (50 ng/g) for 2 consecutivedon day 60 (n = 2; upward green arrows) and 80 (n = 2; upward blue arrows) posttransplantation. 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/g 2 consecutive d; blue broken line; injection shown as downward gray arrows) 32 d after the first treatment that resulted in graft rejection. The remaining one recipient (green unbroken line) maintained euglycemia for 140 d, which was the experimental end point.

significantly (p , 0.001) higher levels of intragraft transcripts for compared with the rapamycin group. Importantly, SA-PDL1 had Foxp3, TGF-b, and IL-10 in the SA-PDL1–engineered islet grafts a synergistic effect with rapamycin in significantly increasing plus rapamycin group as compared with SA-PDL1–engineered the levels of intragraft transcripts for IDO-1 and ARG-1, two islet grafts or unmodified islet grafts plus rapamycin. The TGF- canonical markers of M2 regulatory macrophages (62). Both b/Treg cell axis has been shown to play a dominant role in sys- SA-PDL1 and SA-PDL1 plus rapamycin groups had significantly temic tolerance to autoantigens. Treatment with an anti-CD3 Ab lower levels of transcripts for proinflammatory TNF-a and IL-1b, was shown to result in the prevention of experimental autoimmune two predominantly M1 cytokines (63). The predominant M2 re- encephalomyelitis through a TGF-b/Treg cell axis (60). Phago- sponse is also consistent with increased levels of transcripts for cytes engulfing apoptotic bodies produced by anti-CD3 Ab- IL-4 and its transcriptional regulator GATA-3 in the SA-PDL1 mediated apoptosis of T cells were shown to produce TGF-b plus rapamycin as compared with the other two groups. IL-4 that, in turn, facilitates the generation of Treg cells. Similarly, the induces the expression of ARG-1 and plays a critical role in treatment of NOD mice with the anti-CD3 Ab was shown to polarizing macrophages toward an M2 phenotype (64). Also, prevent and reverse diabetes through a TGF-b/Treg cell axis (61). cotransplantation of allogeneic islets with recipient autologous The direct display of SA-FasL on pancreatic islets or poly(ethylene mesenchymal stem cells under the kidney capsule resulted in glycol) microgels cotransplanted with unmodified islets were prolonged graft survival with immune responses skewed toward a also shown to results in localized tolerance maintained by Treg Th2 profile (65). In addition to playing a critical role in the sup- cells (19, 20). Immunohistochemical analysis of long-term pression of Teff cells and generation, homeostasis, and regulatory grafts revealed the peri-islet presence of CD4+Foxp3+ Treg function of Treg cells (27, 29, 34, 66), PD-L1 has also been shown cells occurring in patches. Depletion of Treg cells in long-term to polarize macrophages toward M2 regulatory phenotype both in graft recipients resulted in the rejection of islet grafts, con- mice and humans (67, 68) and block the generation and immune firming the dominant role of Treg cells in sustaining allograft stimulatory function of M1 macrophage (69, 70). M1 macrophages survival. have been shown to play a critical role in graft rejection by direct The SA-PDL1 group with or without rapamycin treatment on damage or through priming the adaptive immunity for a Th1 response day 3 posttransplantation also showed decreased levels of intragraft (50–52). In marked contrast, M2 macrophages expressing ARG-1 transcripts for IFN-g and its transcriptional regulator T-bet as and IDO-1 prevent graft rejection by generating anti-inflammatory Downloaded by guest on October 1, 2021 10 PD-L1–ENGINEERED ALLOGENEIC ISLETS ACHIEVE SUSTAINED SURVIVAL

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FIGURE S1. Distribution of biotin and the SA-PDL1 protein in engineered mouse islets. (A)

Confocal picture of SA-PDL1-engineered pancreatic islets stained with SA-allophycocyanin (red) and anti-SA-FITC Ab (green) to visualize islet engineered with biotin and SA-PDL1, respectively.

(B) Z-stack analysis of engineered islets across the lines shown in A to assess the distribution of biotin and SA-PDL1 within islets. Magnification X 20.

FIGURE S2. Frequency of T cells in graft-draining lymph nodes. Percentages of

CD4+PD1+CD44hiCD62L- activated Teff and CD4+CD25+FoxP3+ Treg cells in graft-draining

lymph nodes on day 3 (A) and 7 (B) post-transplantation. The experiments were performed in triplicates and each data point is indicative of Mean ± SEM. Statistical analysis was performed

using one-way ANOVA with Tukey’s multiple comparison test.

FIGURE S3. Frequency of T cells in the spleen of graft recipients. Absolute cell numbers, ratios, and frequency of CD4+PD1+CD44hiCD62L- activated Teff and CD4+CD25+FoxP3+ Treg cells in the spleen of the indicated groups on day 3 (A) and 7 (B) post-transplantation as determined by flow cytometry. The experiments were performed in triplicates and each data point is indicative of

Mean ± SEM. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparison test. *p < 0.05. **p < 0.01, ***p < 0.001.

FIGURE S4: Depletion of FoxP3 Treg cells using diphtheria toxin. Peripheral blood from diphtheria toxin (DT) treated graft recipients in Figure 7D (n = 4) were analyzed for CD4+FoxP3+

Treg cells pre- and post-DT treatment. (A) Representative flow plot showing depletion of Treg cells. (B) Depletion data tabulated for all 4 mice.