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Am J Transplant. 2020;20:689–700. amjtransplant.com  | 689© 2019 The American Society of Transplantation and the American Society of Transplant Surgeons

1  | INTRODUC TION

For patients with type 1 diabetes (T1D), clinical islet transplanta-tion (CIT) can lead to improved glycemic control, elimination of

severe hypoglycemia, decreased complications, and insulin indepen-dence.1-4 A barrier to broad application of CIT is the substantial cell loss observed during the early posttransplant period, as this reduced functional islet mass limits the metabolic capacity and durability of

Received:17July2019  |  Revised:19September2019  |  Accepted:29September2019DOI: 10.1111/ajt.15643

O R I G I N A L A R T I C L E

Transplantation of PEGylated islets enhances therapeutic efficacy in a diabetic nonhuman primate model

Cherie L. Stabler1,2  | Jaime A. Giraldo2 | Dora M. Berman2,3  | Kerim M. Gattás‐Asfura1,2 | Melissa A. Willman2 | Alexander Rabassa2 | James Geary2 | Waldo Diaz2 | Norman M. Kenyon2 | Norma S. Kenyon2,3,4,5

Abbreviations:ALT,alanineaminotransferase;ALKP,alkalinephosphatase;AST,aspartateaminotransferase;CIT,clinicalislettransplantation;CBC,completebloodcount;EIR,exogenousinsulinrequirement;FBG,fastingBG;FN,Fibrinogen;GGT,gamma-glutamyltransferase;IHC,immunohistochemical;IBMIR,instantbloodmediatedinflammatoryresponse;IEQ,isletequivalents;MHC,majorhistocompatibilitycomplex;NHS,N-hydroxysuccinimide;NHP,nonhumanprimate;PTT,partialthromboplastintime;PEG,poly(ethyleneglycol);POD,post-operativeday;PBG,post-prandialbloodglucose;PT,prothrombintime;RBC,redbloodcellcount;SI,stimulationindex;TAT,thrombin-antithrombinIIIcomplex;TBIL,totalbilirubin;T1D,type1diabetes;WBC,whitebloodcellcount.

1J. Crayton Pruitt Family Department of BiomedicalEngineering,UniversityofFlorida,Gainesville,Florida2DiabetesResearchInstitute,UniversityofMiami,Miami,Florida3Surgery,MillerSchoolofMedicine,UniversityofMiami,Miami,Florida 4MicrobiologyandImmunology,MillerSchoolofMedicine,UniversityofMiami,Miami,Florida5BiomedicalEngineering,UniversityofMiami,Miami,Florida

CorrespondenceCherieL.StablerEmail:cstabler@bme.ufl.edu

NormaS.KenyonEmail:nkenyon@miami.edu

Present addressJaimeA.Giraldo,JDRF,NewYork,NY

Funding informationDiabetes Research Institute Foundation; NationalInstitutesofHealth,Grant/AwardNumber:R01DK100654,U01AI102456andP01A1089556

Islet cell transplantation can lead to insulin independence, reduced hypoglycemia, and amelioration of diabetes complications in patients with type 1 diabetes. The sys-temic delivery of anti-inflammatory agents, while considered crucial to limit the early loss of islets associated with intrahepatic infusion, increases the burden of immu-nosuppression. In an effort to decrease the pharmaceutical load to the patient, we modifiedthepancreaticisletsurfacewithlong-chainpoly(ethyleneglycol)(PEG)tomitigatedetrimentalhost-implantinteractions.TheeffectofPEGylationonisleten-graftment and long-term survival was examined in a robust nonhuman primate model via three paired transplants of dosages 4300, 8300, and 10 000 islet equivalents per kg body weight. A reduced immunosuppressive regimen of anti-thymocyte globulin induction plus tacrolimus in the first posttransplant month followed by maintenance with sirolimus monotherapy was employed. To limit transplant variability, two of thethreepairswerecloselyMHC-matchedrecipientsandreceivedMHC-disparatePEGylatedoruntreatedisletsisolatedfromthesamedonors.RecipientsofPEGylatedislets exhibited significantly improved early c-peptide levels, reduced exogenous in-sulin requirements, and superior glycemic control, as compared to recipients of un-treated islets. These results indicate that this simple islet modification procedure may improve islet engraftment and survival in the setting of reduced immunosuppression.

K E Y W O R D S

animal models: nonhuman primate, basic (laboratory) research/science, immunosuppression/immune modulation, immunosuppressive regimens, islet transplantation, islets of Langerhans, translational research/science

[CorrectionaddedonFebruary11,2020,afterfirstonlinepublication:Theauthorname,NormaS.KenyonhasbeencorrectedtoNormanM.Kenyon].

www.amjtransplant.commailto:https://orcid.org/0000-0003-0573-0646https://orcid.org/0000-0001-7817-9387mailto:https://orcid.org/0000-0003-3157-339Xmailto:cstabler@bme.ufl.edumailto:nkenyon@miami.edu

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the graft, while also increasing the required efficacious islet dos-age.5-9 Furthermore, the requirement for chronic immune suppres-sion and its associated risks limits the procedure to patients with severe hypoglycemia.10 Thus, research in the field is focused on the identification of approaches that limit early posttransplant inflam-mation and minimize immune suppression.

Following intraportal islet infusion, interactions between the islet surface and the recipient's blood result in activation of an in-stantblood-mediated inflammatory response (IBMIR).6 This innate response leads to substantial cell damage and loss during engraft-ment, with resulting stress signals and cellular debris potentiating adaptive immune response. The addition of systemic anti-inflamma-toryagents,suchasTNF-α inhibitors (eg, etanercept and infliximab), IL-1 receptor antagonists (eg, anakinra), and others, improves islet engraftment and survival11-19; however, increasing the drug regimen in CIT patients is not desirable.

Cell surface modification is an appealing method for masking cell features that may instigate inflammatory cascades. Polymer chains can be embedded or linked onto the cell surface via mul-tiple methods, typically using the polymer poly(ethylene gly-col) (PEG).20-27 PEG grafting, termed PEGylation, is commonlyachievedusingaheterofunctional linearPEG,forexample,NHS-PEG-CH3 (NHS-mPEG), where the N-hydroxysuccinimide (NHS)group permits spontaneous reactivity to free amines and the methyl group (CH3) serves as an inert terminal end. This simple and efficient polymeric grafting approach has been applied to drugs to increase circulation half-life and cell surfaces to reduce immunogenicity.28-31

ApplyingPEGylationto isletsdelays,butdoesnotcompletelyprevent, the rejection of allogeneic and xenogeneic grafts in rodent models,most likelyduethe limitedcapacityofsinglePEGchainsto provide durable protection against aggressive allo- and xeno-geneic immune responses.23-25,32-35 Combining PEG-based cellsurface grafting with low-dose or local immunosuppression results in augmented benefits,23,32-38 providing evidence that islet PEGgrafting complements immunosuppressive regimens to improve graft efficacy and/or decrease the overall drug load. Validation of these results in larger animal models is critical for the translation of promising approaches into the clinic; however, there is a paucity of published reports in nonhuman primate models.39 In this study, we explored the impact of combining reduced immunosuppression, including rapamycin monotherapy after the first posttransplant month, with islet surface PEGylation, using our well-establishedislet PEGylation protocol,23 on allogeneic islet engraftment and long-term survival in a diabetic nonhuman primate model.

2  | MATERIAL S AND METHODS

2.1 | Reagents and polymer fabrication

All chemical reagents were purchased from Sigma-Aldrich, unlessotherwise noted. All culture media was sourced fromMediatech.NHS-PEG-CH3wasfabricated,asdetailedinAppendixS1.

23

2.2 | Nonhuman primate donor islet isolation, recipient management, and immunosuppression

AllanimalprotocolswereapprovedbyIACUCofTheUniversityofMiami.Major histocompatibility complex (MHC)-typed donor andrecipientSPFMauritiancynomolgusmonkeyswereobtained fromthe Mannheimer Foundation (Homestead, FL) or Charles RiverLaboratories(Houston,TX),seeTablesS1andS2,andDataS1.Isletswere isolated (day0),PEGylated (day1), andcharacterizedand/ortransplanted(day2),asfullydescribedinAppendixS1.7,23,40,41

Full details on animal management and transplantation proce-dure are provided in Appendix S1.7 All recipients received induc-tion therapy consisting of four IV 10 mg/kg doses of thymoglobulin (Sanofi Genzyme) on postoperative days (PODs) −1, 0, 2, and 4.Intramuscular (IM) FK506 (Astellas Pharma Inc) was initiated onPOD−1through30,withatargettroughlevelof8-10ng/mL,andIMrapamycin (LC Labs) was initiated on POD 28, allowing for a 3 days overlap with FK506, with a targeted trough level of 8-12 ng/mL. Drug trough levels were measured weekly. For method details on coagulation panels, immunomonitoring of anti-donor alloantibodies, andhistopathologicanalysisofexplantedgrafts,seeAppendixS1.7

2.3 | Statistical analysis

Dataareexpressedas themean±SDofgroup replicates (n=3for each group), unless otherwise specified. Exogenous insulinrequirement (EIR) was reported as change from pretransplantbaselinelevels(averagefromPOD−14to−4)topermiteaseincom-parisonofEIRchangesacrossalltransplantrecipients.Statisticalanalysis was performed using unpaired t test for equal variances or unpaired t test with Welch's correction to account for unequal variances, as indicated. For multiple comparisons, one-way or two-way ANOVAwith Tukey's orWelch’s multiple comparisonswereemployed.All analyseswereperformedusingGraphPad (v8.0) and P < .05 was considered statistically significant.

3  | RESULTS

3.1 | In vitro effect of PEGylation on nonhuman primate (NHP) islets

NHPisletsweresurfacemodifiedwithlinearPEGchainsusingapro-tocol optimized and validated in rodent islets.23UniformconjugationofPEGtotheisletperipherywasobserved(Figure1A)withnovisuallydistinct differences in morphology or viability when compared to un-treatedcontrolislets(Figure1C-F).PEGylationdidnotalterisletfunc-tion, as measured by dynamic glucose stimulated insulin release42,43 and intracellular insulin content via membrane depolarization with KCl (Figure1G).Asshown,PEGylatedisletsexhibitedsimilarglucosesens-ing and insulin release dynamics under high glucose and KCl stimula-tion.Globalcomparisonofinsulinstimulationindex(SI)foruntreatedandPEGylatedisletsforalltestedisletisolationsmeasurednosignifi-cantdifferences(FigureS1;P=.63;pairedt test).

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The impactof isletPEGylationon invitroactivationof coagu-lation cascades was characterized by adding islets to platelet poor plasma and measuring the time to clot.41 As shown in the coagulation kineticcurves (Figure1H),PEGylated isletsexhibiteddelayedacti-vation of coagulation, with a significant decrease in kinetic half-max time when compared to untreated islets (P < .001; post hoc Tukey).

3.2 | Transplantation of PEGylated islets into diabetic NHP model

Transplant studies were initiated to determine if the protective effectofisletPEGylationobservedinrodentstudies23,35,44 would translatetotheNHP.Immunosuppressionconsistedofinduction

F I G U R E 1  PEGylationofnonhumanprimate(NHP)isletsdidnotimpactviabilityorfunctionbutdecreasedcoagulation.NHPisletsweresurfacemodifiedwithlong-chainpoly(ethyleneglycol)(PEG)togenerateaperipheralPEGcoating,asvisualizedviarhodaminetagonPEGchain(A);insetshowsPEG-freecontrols(B).LightmicroscopeandLive/Dead(green:live;red:dead)confocalimagesofcontrol(C&E)andPEGylated(D&F)isletsrevealsimilarmorphologyandviability.Scalebars=100μm (A-D) and 50 μm(E-F).Dynamicglucosestimulatedinsulinreleasecurves(G)illustrateidenticalstimulationprofilesforcontrolandPEGylatedislets.Errorbars=standarddeviationfromtechnicalreplicatesforeachgroup(n=4).3G=3mmol/Lglucose;11G=11mmol/LGlucose;KCl=25mmol/LKCl.CoagulationkineticsofplasmawassignificantlydelayedafterincubationwithNHPPEGylatedisletsvscontrolislets(H),withasignificantincreaseinhalf-maxtimeforPEGylatedislets.Plasma-onlywellsservedasthenegativecontrol.Errorbars=standarddeviationfromtechnicalreplicatesforeachgroup(n=4).**P<.01;***P<.0001perone-wayANOVAwithTukeyposthoc

A C

E F

D

B

G H

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with thymoglobulin and maintenance with tacrolimus for the first month, followed by a transition to rapamycin monotherapy 28 days after transplant (see Figure 2 top schematic). As rapa-mycin monotherapy was inadequate for prolongation of islet sur-vival,45 graft rejection was typically observed within 45-90 days.

Islets were isolated from a single donor, cultured overnight, PEGylated, and transplanted the following day into anMHC-mis-matched (partial or full) diabetic recipient (see Figure 2, top sche-matic). Due to limited islet yield, a subtherapeutic islet dosage of4160 islet equivalents (IEQ)per kgbodyweightwasdelivered,correlating to42%ofa standardNHPtherapeuticdose.7 Figure 2 comparesresultsobtainedfromtherecipientofPEGylatedisletstoa control animal transplanted with a similar mass of untreated islets (4600 IEQ/kg) and the same immunosuppression. Retrospectiveanalysis of islet quality revealed similar stimulation profiles for untreated vs PEGylated islets (SI = 4.92 and 3.78, respectively).Elevatedweight anddecreasedEIR for the control recipientwerenoted; however, pretransplant fasting blood glucose levels for both animalswerestatisticallyequivalent(FiguresS2andS8).

Posttransplantation, the recipient of control islets did not expe-rience a reduction in exogenous insulin requirement, demonstrated poor glycemic control, and fully rejected the graft with undetect-able c-peptide after POD 81. In striking contrast, the recipient of PEGylatedisletsexhibitedexcellentgraftfunction,withrobustfast-ingandpostprandialglycemiccontrol (Figure2B).EIR,reportedas

change in EIR from pretransplant levels, precipitously dropped tozero on POD 47 and the animal remained insulin-free for 84 days (Figure 2C). Although the animal returned to insulin supplementa-tiononPOD131,theEIRaveraged24.0%±8.8%ofitsinitialinsulinrequirement until elective necropsy on POD 272. Fasting c-peptide levels, while undetectable pretransplant, were robust through-out the transplant period, peaking at 4.18 ng/mL and averaging 2.23 ± 0.64 ng/mL throughout the experiment (Figure 2D).

Immunohistochemical (IHC) analysis of tissues retrieved fromthe control animal on POD 90 revealed no insulin positive islets and significant residual lymphocytic infiltration (Figure 3, top row), with aggregated CD3+ T and CD20+Bcellsobserved invariouspocketsthroughouttheliver.Instarkcontrast,IHCsectionsobtainedfromtis-suestakenduringelectivenecropsyoftherecipientofPEGylatedis-lets on POD 272, revealed robust insulin-positive islets, with minimal CD3+TandCD20+BcellsandnoCD68+cells(Figure3,bottomrow).

3.3 | Characterization of the impact of islet PEGylation using a paired NHP transplant model

To permit a more rigorous and direct evaluation of the effect of PEGylation on islet survival, a paired transplant model was em-ployed.7 For these transplants, a pair ofMHC-matched recipientsreceivedanequivalentisletdosageofPEGylatedoruntreatedisletssourcedfromthesameMHC-mismatchedisletpool (seetoppanel

F I G U R E 2   Transplantation of 4160 IEQ/kgPEGylatedisletsresultedinstableblood glucose control, with decreased exogenousinsulinrequirement(EIR),ascompared to transplantation of 4600 IEQ/kguntreatedislets.Schematicofislet isolation and transplant, which was independentlyconductedforPEGylatedanduntreatedislets(toppanel).MHCdesignation and color coding were included for clarity.70,71PEGylatedisletswerePEGylatedonpostoperativeday(POD)−1.TheimmunosuppressionschemeshownwasinitiatedonPOD−1.Summaryofbloodglucose(FBG:fastingbloodglucose;PBG:postprandialglucose)ofrecipientsofcontrol(A)orPEGylatedislets(B).ChangeinEIRperkgbodyweight(C;normalizedbyaverageEIR/kgfromPOD−14to−4)andC-peptide(D)forcontrolandPEGylatedislets.Forrecipients of control islets, necropsy was performed on POD 90. For recipients of PEGylatedislets,electivenecropsywasperformed on POD 272

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in Figures 4 and 5). This approach reduces the variability associated with (a) donor pancreas quality and islet isolation variability, as recip-ients receive islets from the same preparation; and (b) alloreactivity, asrecipientsshareMHCandreceiveisletsfromthesameMHC-mis-matched preparation. A therapeutic islet dosage was targeted for these transplants, although the final transplant dosage depended on isolation yields.

For the pair shown in Figure 4, 8300 IEQ/kg untreated orPEGylatedisletsweretransplanted.Bothanimalsexperiencedare-duction inEIRandwerec-peptidepositive in theearlyposttrans-plantperiod,supportingthequalityoftheislettransplant.However,the control animal exhibited destabilization of postprandial blood glucose(PBG)byPOD20,withsubsequentincreasesinfastingBG(FBG) and EIR; c-peptide levels dropped substantially by POD 28(the last day with c-peptide >1 ng/mL) and the last positive c-peptide measurement on POD 39 was minimal (0.37 ng/mL). In contrast, the recipientof anequivalentdosageofPEGylated islets showed sta-ble graft function with gradual increases in c-peptide after the first month posttransplant and a c-peptide >1 ng/mL through POD 60. Although c-peptide levels began declining after that time point, c-peptide was 0.58 ng/mL at the time of elective necropsy on POD 96.Overall,c-peptide levelsforPEGylatedisletswereconsistentlyhigher than untreated islets and ranged from 0.51 to 4.44 ng/mL. After POD 20, the stability of glycemic control in the recipient of PEGylated islets was notably superior to its matched control re-cipient (Figure S3). EIR for the PEGylated islet recipient was also35% ± 12% lower than the control recipient after POD 30.

ThematchedpairshowninFigure5eachreceived10000IEQ/kg.ThePEGrecipientbecameinsulinfreeonPOD100andremainedso until the elective termination of the experiment on POD 187. In contrast,thecontrolanimalrequired61.8%±0.1%ofitsoriginalEIRposttransplant. Although fasting c-peptide levels in the control trans-plantwere>1ng/mLuntilPOD143, the recipientofPEGylated is-lets consistently maintained higher c-peptide levels, with an average c-peptide 2.32 ± 0.73 vs 1.34 ± 0.58 ng/mL for the control (P < .001, t testwithWelch'scorrection;POD7-187).GlycemiccontrolwasmorestablefortherecipientofPEGylatedislets,withprecipitousreductioninEIR,no insulin requiredafterPOD100,decreasedaverageFBG,and minimal glycemic excursions as compared to the matched control recipient(FigureS4).PBGstabilityforthePEGgraftrecipientwasalsoenhancedoverthetransplantperiod,whereasPBGincontrolanimalsretained its variability throughout the transplant period (187 days).

Immunohistochemical analysis was performed at the time of necropsy.For8300IEQ/kgtransplants,significantlymphocyticin-filtration(bothCD3+TandCD20+Bcells)wasobservedintissuesfrom both animals (Figure 6). Insulin was not detected in control animal explants (POD 69), but clusters of insulin-positive cells were stilldetectableforthePEGylatedisletrecipient(POD96).MinimalCD68+ cells were distinguished for both animals, denoting a lim-ited macrophage presence at the graft site. For the higher dosage transplants (10000IEQ/kg),amorebenign immuneresponsewasobserved (Figure 7), but greater lymphocytic infiltration and fewer insulin-positive cells were visualized for the control animal in the POD 187 necropsy tissues.

F I G U R E 3   Immunohistochemical analysis of explanted grafts, from recipients of marginal mass transplants, illustrates significant rejectionfortherecipientofcontrolislets.ExplantedgraftswerestainedviaH/E(A&E),CD3/insulin(B&F);CD20/insulin(C&G),orCD68/insulin(D&H).Toprow:Explantsfromrecipientofcontrolislets(ID:H10C21)atnecropsyonpostoperativeday(POD)90.Bottomrow:ExplantsfromrecipientofPEGylatedislets(ID:10C59)atelectivenecropsyonPOD272.Insulinantibody=red;CD3,CD20,orCD68antibody=green;DAPInucleistaining=blue.Scalebar=50µm

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To examine the impact of the islet transplant on systemic inflammation following infusion, blood was collected pre- and up to 24 hours posttransplant on the matched pair recipients. Prothrombin time (PT) and partial thromboplastin time (PTT) were measuredandcirculatingfibrinogen(FN),d-dimer,andthrombin-antithrombin III complex (TAT) levels were quantified. In previ-ous reports, attenuation of IBMIR results in decreased TAT andD-Dimer levels and increased fibrinogen.7 The recipient of 8500 PEGylated IEQ/kg exhibited decreased d-dimer and TAT and in-creased circulating fibrinogen in the early transplant period, when compared to its paired control recipient; however, these trends were not observed in the higher islet dosage pair (Figures S5andS6).Ofnote,PTlevelsforrecipientsof10000IEQ/kgislets(FigureS6A)weresignificantlyhigherthanhistoricalvalues,indi-cating potential technical challenges with the blood draw.

Circulating class I and II alloantibodies within matched pair re-cipients were globally higher for recipients of control islets, as com-pared to recipients of PEGylated islets (Figure S7). The pair thatreceived8300IEQ/kghadagreaterdegreeofMHCmismatchthanthe10000IEQ/kgpair,whichcorrespondedtogenerallyelevatedanti-Class II alloantibodies (note differences in scale between the pairsforFigureS7).Withtheexceptionofdonor4C105,higheranti-class I and II alloantibody (as compared to baseline for each recipient) wasobservedbeginningonPOD14-28forthe8300IEQ/kgcontrolandwasminimaltonegativeforthePEGrecipientuntilPOD74.For

donor 4C105, anti-class II alloantibodies were very high for both the controlandthePEGanimalsonPOD14,whichmayhavecontrib-utedtoearliergraftlossinthispair.Forrecipientsof10000IEQ/kg,levels of anti-class I alloantibodies for 2 of the 3 donors were neg-ativetominimalforboththecontrolandPEGrecipients,withonedonor eliciting a higher anti-class I response for the control recipient atalltimepoints.Foranti-classIIalloantibodies,thePEGrecipientwas negative to all donors at all time points, while the control recip-ient had positive levels against all 3 donors.

3.4 | Evaluation of overall impact of PEGylated islets on NHP transplant outcomes

Forthethreedosagesevaluated(~4300,8300,and10000IEQ/kg), examination of EIR (Figure 8A,B) and glycemic control(Figure 8C,D) during early (POD 10-25) and late (POD 45-60) stage islet engraftment revealed global impacts. The most notable dif-ferenceinEIRwasatthelowestisletdosage;however,PEGylatedislet recipients required significantly decreased exogenous insulin at late stage engraftment for all dosages (P < .0001; t test with Welch's correction). The average fasting glucose was significantly lower for the 4300 and 10 000 IEQ/kg dosages, for both earlyandlatestageengraftment.Althoughrecipientsofthe8300IEQ/kg dosagewere not significantly different, the variance of FBGfor the control animal during those time periods was substantially

F I G U R E 4  Recipientof8300IEQ/kgPEGylatedisletsinapairedtransplantmodel resulted in stable blood glucose control with decreased exogenous insulin requirement(EIR)ascomparedtotherecipient of the same dosage of untreated islets.Schematicofisletisolationandtransplant:Donors(n=3)wereMHCmatched and pooled during isolation. Isolated islets were split into control (untreated)andPEGylatedislets,withPEGylationconductedonpostoperativeday(POD)−1.Foreachgroup,isletsweretransplantedwithmatchedIEQ/kgdosageintoMHC-matchedpairs(fullMHCmismatchtoisolatedislets),with initiation of immunosuppression scheme (summarized in top schematic) on POD−1.Summaryofbloodglucose(FBG:fastingbloodglucose;PBG:postprandialglucose) of matched recipients of control (A;H12C4)orPEGylatedislets(B;H12C33).(C-D)ChangeinEIRperkgbodyweight(C;normalizedbyaverageEIR/kgfromPOD−14to−4)andC-peptide(D)forcontrolandPEGylatedislets.NecropsywasperformedonPOD69forH12C4andonPOD96forH12C33

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F I G U R E 5  Recipientof10000IEQ/kgPEGylatedisletsinapairedtransplantmodel resulted in stable blood glucose control with decreased exogenous insulin requirement(EIR)ascomparedtotherecipient of the same dosage of untreated islets.Schematicofisletisolationandtransplant:Donors(n=3)wereMHCmatched and pooled during isolation. Isolated islets were split into control (untreated)andPEGylatedisletswithPEGylationconductedonpostoperativeday(POD)−1.Foreachgroup,isletsweretransplantedwithmatchedIEQ/kgdosageintoMHCmatchedpairs(fullMHCmismatch to isolated islets), with initiation of immunosuppression (summarized in top schematic)onPOD−1.Summaryofbloodglucose(FBG:fastingbloodglucose;PBG:postprandialglucose)ofmatchedrecipientsofcontrol(A)orPEGylatedislets(B).ChangeinEIRperkgbodyweight(C;normalizedbyaverageEIR/kgfromPOD−14to−4)andC-peptide(D)forcontrolandPEGylatedislets.Necropsywas undertaken on POD 187 for both animals

F I G U R E 6   Immunohistochemicalanalysisofexplantedgraftsfrom8300IEQ/kgtransplantsillustratesignificantrejectioninbothgroups.ExplantedgraftswerestainedviaH/E(A&E),CD3/insulin(B&F),CD20/insulin(C&G),orCD68/insulin(D&H).Toprow:Explantsfromrecipientofcontrolislets(H12C4)69dposttransplant.Bottomrow:ExplantsfromrecipientofPEGylatedislets(H12C33)96dposttransplant.Insulinantibody=red;CD3,CD20,orCD68antibody=green;DAPInucleistaining=blue.Scalebar=50µm

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F I G U R E 7   Immunohistochemicalanalysisofexplantedgraftsfrom10000IEQ/kgtransplantsillustratedecreasedrejectioninPEGylatedisletgroup.ExplantedgraftswerestainedviaH/E(A&E),CD3/insulin(B&F);CD20/insulin(C&G),orCD68/insulin(D&H).Toprow:Explantsfromrecipientofcontrolislets(H12C105)187dposttransplant.Bottomrow:ExplantsfromexplantsfromrecipientofPEGylatedislets(13C40)187dposttransplant.Insulinantibody=red;CD3,CD20,orCD68antibody=green;DAPInucleistaining=blue.Scalebar=50µm

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F I G U R E 8  PEGylationresultedindecreased overall insulin requirements and blood glucose levels with concurrent improved glycemic stability. Comparison of average change in exogenous insulin requirement(EIR)(A&B)andfastingbloodglucose(C&D)forearly(A&C;POD10-25)andlate(B&D;POD45-60)stageengraftmentofcontrolorPEGylatedisletsatvariabledosages.(E)ComparisonofC-peptidelevels,normalizedtoIEQ/kgfor each recipient, 4 d posttransplantation forallrecipientsofcontrol(n=8)orPEGylated(n=4)islets.Unpairedt-test with Welch's correction for unequal variances**P<.01;****P < .0001; γ=F test P < .01, unequal variances between groups

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higherthanthatofthePEGylatedisletrecipient,indicatinginferiorglycemic control.

Poolingdatafromcontrol(n=8;3 concurrent and 5 historical) and PEGylatedislet(n=4)recipientsfurthersupportedelevatedearlyisletengraftment for the treated group. Specifically, circulating nonstim-ulated c-peptide levels, normalized to islet dosage, were significantly elevatedforPEGylatedisletgraftsonPOD4ascomparedtountreatedcontrols(Figure8E;P=.0042;unpairedt test). Data were pooled from transplantsreceivinganaverageIEQof7472±1911IEQ/kgforcon-troltransplants(n=8)and6421±3470IEQ/kg(n=4)forPEGylatedtransplants,anonsignificantdeviationinIEQ/kgloadingbetweenthetwo groups (P=.505;unpairedt test).

Due to intensive and individualized management of exog-enous insulin in response to measured blood glucose levels, all transplantrecipientsexhibitedstableweight(FigureS8)andnor-malkidney(perBUN,creatine,TP,Na,K,andCl)andliverfunc-tion (per AST, ALT, ALKP, CGT, and TBIL) during the course ofthestudy(datanotshown).Completebloodcounts(CBCs;WBC,RBC, lymphocytes, and platelets) also did not indicate any sig-nificant deviations in trends between groups (data not shown). Circulating levels of FK506 or rapamycin generally remained within targeted trough levels (Figure S9); recipients of eitheruntreated or PEGylated islets exhibited statistically equivalentFK506troughlevelsandvariance(FigureS10;P=.22;unpairedt test; P=.63f test).

4  | DISCUSSION

The conjugation of poly(ethylene glycol) (or PEG) to various bio-molecules and implants is a well-established clinical approach. The PEGylationofproteinandpeptidedrugssubstantiallyincreasestheirpharmacodynamic and pharmacokinetic profiles,46,47 while PEGlinkage to biomedical implants prevents protein adsorption and cell adhesion.48,49Forcellsurfaceengineering,PEGylationresultsinde-creased recognition of cell surface markers, such as integrins and MHC,which decreases cell adhesion and immunorecognition.26,28 ThesedesirablepropertiesofPEGareattributedtoitsstrongasso-ciation with water, which forms a hydration zone that shields under-lying agents from host interactions.50

Inthisstudy,weusedouroptimizedPEGylationproceduretoconjugatePEGchainstothesurfaceofnonhumanprimateislets.This protocol uses an easily fabricated polymer and significantly reduced manipulation and incubation periods, when compared to other published reports.39,51 Resulting PEGylated islets werehighly coated, with no notable changes in morphology. In vitro, dynamic glucose stimulated insulin release kinetics were identi-calforPEGylatedandcontrol islets,afeaturelostwhenemploy-ing micro-scale encapsulation strategies.43 Furthermore, in vitro studies measured decreased coagulation kinetics in response to PEGylated islets,whencompared to control islets.As inflamma-tory islet surface proteins (eg, tissue factor and thrombin) play key rolesininstigatingIBMIR,9,52PEGgraftingmaymasktheseagents,

resultingindelayedactivationofcoagulation.ThecapacityofPEGchain grafting to impair inflammatory pathways has not been re-portedpreviouslyforNHPislets,indicatingauniquebenefitofourPEGylationprocedure.

ToevaluatetheeffectofisletPEGylationontransplantoutcomesin the rigorous nonhuman primate model, a reduced immunosup-pression(IS)regimenwasemployed,withaninductionwindowthatsupported islet engraftment in the first posttransplant month and subsequent tapering to monotherapy maintenance.45 The infusion ofPEGylatedisletsintotheliverresultedinconsistentimprovementin graft outcomes. For the lowest islet dosage tested (4160 IEQ/kg),PEGylationimparteddramaticimprovementsinglycemicfunc-tion and complete independence from exogenous insulin, in stark comparison to untreated controls. Robust insulin staining and min-imal lymphocytic presence for tissues obtained at necropsy from thePEGylated islet recipient indicates long-termgraftacceptance.Although these results were highly encouraging, potential variables relatedtoMHCdisparitymayhavecontributedtothedisparateout-comes,independentoftheimpactofPEGylation.

To delineate the effects, two additional matched-pair model studieswereconductedusingMHC-matchedrecipientsanddonors.This approach eliminated the variables of pancreas quality, islet iso-lation, and alloreactivity. For both studies, a marked improvement inisletgraftfunctionandstabilitywasobservedforNHPrecipientsofPEGylated islets,with improvedglycemic stability (both fastingand postprandial), decreased exogenous insulin requirement, and increased fasting c-peptide levels, when compared to control recipi-ents.ExplantanalysisfurthersupportedthebenefitsofPEGylation.Although all three control animals exhibited substantial lymphocytic infiltration,sectionsfromrecipientsofPEGylatedgraftsrevealedin-creased insulin positive islets and decreased lymphocyte infiltrate. Circulating class I and II alloantibodies within matched-pair recipi-ents also indicated reduced adaptive responses for recipients of PEGylatedislets,whencomparedtotheirmatchedcontrolcounter-parts. Reliable evidence of decreased coagulation markers following intrahepatic islet infusion were not measured for these two pairs, indicating that further transplant studies and analyses are needed to fullydelineatetheroleofPEGylationinthesebeneficialoutcomes.

Evaluation of the impact of islet dosage on PEGylated graftoutcomes is confounded by multiple factors, including islet quality, MHCmismatchbetweentheisletdonorandrecipient,andtheallo-immune response to the transplant. As such, global trends of the role of islet dosage cannot be made from the experiments conducted to date and further study is needed. When all transplants were consol-idated to examine global trends, a significant improvement in early c-peptide levels was measured for PEGylated grafts. Because wehave previously established a positive correlation between islet quality/potency and c-peptide within the first week of transplant, these data support the hypothesis that PEGylation enhances isletsurvival posttransplantation.7 Despite the lack of a clear mechanism ofactionfortheroleofPEGylationinvivo,thisearlyelevatedpo-tency appears to impart measurable and substantial improvements in islet graft outcomes within this robust preclinical animal model.

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ThemechanismbywhichPEGylationprolongsisletgraftsurvivaland improves islet transplant outcomes is likely due to the masking of inflammatory islet surface markers and a delay of protein and cellu-lar adhesion to the islet. The correlation between inflammation and early graft loss, both in allogeneic and autologous clinical trials, is well known.12,53-56 Furthermore, it is established that early inflam-matory events accelerate detrimental host responses and fibrotic re-actions,57,58 thus modulation of local inflammatory processes during early engraftment should lead to decreased adaptive responses. When compared to soluble anti-inflammatory approaches, such as TNF-α inhibitors, IL-1 receptor antagonists, and anti-tissue factor, the use of polymeric grafting is appealing, as this strategy imparts similar trends in positive islet engraftment but avoids the side effects associ-ated with the systemic delivery of anti-inflammatory agents.7,11-19,59,60

AnotherpotentialimpactofPEGylationisinrestrictingtheinfil-tration of immunosuppressive agents into the cellular graft. Due to the highly lipophilic nature of FK506 and rapamycin, these agents typically readily absorb through the beta cell membrane, where they can impart detrimental functional and proliferative effects.61 The graftingoflong-chainPEGtothesurfaceofthepancreaticisletmayserve to mitigate drug infiltration, as the zone of hydration generated bythePEGchainsnotonlymasksthecellmembranebutimpairsthemobility of lipophilic agents. Future studies will seek to further eval-uatecorrelationsbetweenthePEGcoating,inflammation,anddrugtoxicity via more detailed characterization of engraftment, drug dos-ageeffects,andthepersistenceofthePEGcoatinginvivo.

Withthesepromisingresults,theintegrationofisletPEGylationintocurrent CIT protocols appears highly feasible. The procedure and time-line are easily adaptable to current islet isolation and transplant proto-cols and the capacity to continue use of the CIT intraportal infusion site provides high ease in translation, a feature not practical in traditional islet microencapsulation.62-64 Future work is focused on improving the PEGylationprotocoltopermiteaseinclinicaltranslationandtopreventislet lossduring theprocedure.PEGgraftingcouldalsobe leveragedto tether and present bioactive agents to the host. For example, oth-ers have linked anti-inflammatory motifs such as heparin, alpha1-anti-trypsin, urokinase, thrombomodulin, and human soluble complement receptor 1 (sCR1) with observed benefits in vitro and in rodent mod-els.41,65-69InoneNHPtrial,ParketalobservedimprovedengraftmentinfullyimmunosuppressedNHPrecipientswhenheparinwaslinkedtoPEGylatedallogenicislets.39 Thus, bioactivity can be incorporated onto PEGylatedisletsinfutureworktoimpartadditionalbenefits.

DISCL AIMER

DrGiraldoiscurrentlyemployedatJDRF,NewYork,NY.Theopin-ions expressed in this article are the author's own and do not neces-sarily reflect the views of the JDRF.

ACKNOWLEDG MENTS

ThisworkwassupportedbytheNationalInstitutesofHealthgrantsR01DK100654, U01AI102456, and P01A1089556, as well as the

Diabetes Research Institute Foundation. We thank Kevin Johnson intheDRIHistologicalCoreforhishistologicalprowessandtheDRIAnalyticalImagingandHistologyCoresforuseoftheirfacilities.

DISCLOSURE

The authors of this manuscript have no conflicts of interest to dis-close as described by the American Journal of Transplantation.

DATA AVAIL ABILIT Y S TATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.

ORCID

Cherie L. Stabler https://orcid.org/0000-0003-0573-0646

Dora M. Berman https://orcid.org/0000-0001-7817-9387

Norma S. Kenyon https://orcid.org/0000-0003-3157-339X

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SUPPORTING INFORMATION

Additional supporting information may be found online in the SupportingInformationsection.

How to cite this article:StablerCL,GiraldoJA,BermanDM, etal.TransplantationofPEGylatedisletsenhancestherapeuticefficacy in a diabetic nonhuman primate model. Am J Transplant. 2020;20:689–700. https ://doi.org/10.1111/ajt.15643

https://doi.org/10.1111/ajt.15643https://doi.org/10.1111/ajt.15643