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 tran- scripts 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 immunosup- pression 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 con- ventional; 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 www.jimmunol.org/cgi/doi/10.4049/jimmunol.2000055 Downloaded by guest on October 1, 2021 2 PD-L1–ENGINEERED ALLOGENEIC ISLETS ACHIEVE SUSTAINED SURVIVAL 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 combi- nation 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 con- centrations 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.