of March 18, 2018.This information is current as

by Inducing Regulatory T Cells in MiceToleranceProtein Establish Robust Localized

Pancreatic Islets Engineered with SA-FasL

Haval ShirwanChantale Lacelle, Kyle B. Woodward, Nadir Askenasy and Esma S. Yolcu, Hong Zhao, Laura Bandura-Morgan,

http://www.jimmunol.org/content/187/11/5901doi: 10.4049/jimmunol.1003266November 2011;

2011; 187:5901-5909; Prepublished online 7J Immunol 

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6.DC1http://www.jimmunol.org/content/suppl/2011/11/09/jimmunol.100326

Referenceshttp://www.jimmunol.org/content/187/11/5901.full#ref-list-1

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is published twice each month byThe Journal of Immunology

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The Journal of Immunology

Pancreatic Islets Engineered with SA-FasL Protein EstablishRobust Localized Tolerance by Inducing Regulatory T Cellsin Mice

Esma S. Yolcu,*,†,1 Hong Zhao,*,†,1 Laura Bandura-Morgan,*,† Chantale Lacelle,*,†

Kyle B. Woodward,*,† Nadir Askenasy,‡ and Haval Shirwan*,†

Allogeneic islet transplantation is an important therapeutic approach for the treatment of type 1 diabetes. Clinical application of

this approach, however, is severely curtailed by allograft rejection primarily initiated by pathogenic effector T cells regardless of

chronic use of immunosuppression. Given the role of Fas-mediated signaling in regulating effector T cell responses, we tested if

pancreatic islets can be engineered ex vivo to display on their surface an apoptotic form of Fas ligand protein chimeric with

streptavidin (SA-FasL) and whether such engineered islets induce tolerance in allogeneic hosts. Islets were modified with biotin

following efficient engineering with SA-FasL protein that persisted on the surface of islets for >1 wk in vitro. SA-FasL–engineered

islet grafts established euglycemia in chemically diabetic syngeneic mice indefinitely, demonstrating functionality and lack of acute

toxicity. Most importantly, the transplantation of SA-FasL–engineered BALB/c islet grafts in conjunction with a short course of

rapamycin treatment resulted in robust localized tolerance in 100% of C57BL/6 recipients. Tolerance was initiated and main-

tained by CD4+CD25+Foxp3+ regulatory T (Treg) cells, as their depletion early during tolerance induction or late after established

tolerance resulted in prompt graft rejection. Furthermore, Treg cells sorted from graft-draining lymph nodes, but not spleen, of

long-term graft recipients prevented the rejection of unmodified allogeneic islets in an adoptive transfer model, further confirming

the Treg role in established tolerance. Engineering islets ex vivo in a rapid and efficient manner to display on their surface

immunomodulatory proteins represents a novel, safe, and clinically applicable approach with important implications for the

treatment of type 1 diabetes. The Journal of Immunology, 2011, 187: 5901–5909.

Type 1 diabetes (T1D) is an autoimmune disease caused bythe destruction of insulin-producing b cells by a complexset of immunological events initiated and coordinated by

CD4+ T cells responding to a set of b cell-specific Ags (1–3).Restoration of insulin-secreting b cell mass using allogeneic islettransplantation has been viewed as a preferred treatment modality,and its efficacy in restoring physiological glycemic control hasbeen demonstrated in clinical trials (4). However, the success ofallogeneic islet transplantation is compromised by immunologicalrejection and secondary graft failure due to the continuous useof immunosuppressive drugs to control rejection (5). Therefore,

novel approaches that specifically target and control destructiveauto- and alloimmune responses without continuous immuno-suppression remain to be developed for the successful applicationof allogeneic islet transplantation in the clinic.Inasmuch as T cells play a critical role in the initiation of islet-

destructive auto- and alloreactive immune responses (6), specificelimination of these cells or control of their function throughactive regulatory mechanisms may prove effective in achievinglong-term islet allograft survival without the continuous use ofimmunosuppression (7). In this context, immunomodulation withFasL presents an attractive approach due to the critical role playedby Fas/FasL-mediated apoptosis in activation-induced cell death(8), an important homeostatic molecular mechanism that controlsT cell responses to self-Ags (9). The immunomodulatory functionof FasL has been extensively exploited for the induction of tol-erance to auto- and alloantigens using gene therapy (10–15). How-ever, although gene therapy showed efficacy in some settings (10,12–15), the controlled ectopic expression of FasL in transfectedcells and tissues is not only technically challenging, but also posessafety concerns.We recently generated a chimeric form of FasL protein, strep-

tavidin (SA)-FasL, in which the extracellular domain of FasLlacking potential metalloproteinase sites was cloned C terminus tothe core SA (16). This molecule exists as tetramers and oligomerswith potent apoptotic activity and can be displayed on the sur-face of biotinylated cells in an efficient and rapid manner (16).Most importantly, systemic immunomodulation with SA-FasL–engineered donor splenocytes resulted in tolerance to cardiacallografts (17). However, the application of this novel approach toengineering tissues remains to be demonstrated. In this study, wetested if pancreatic islets, instead of isolated cells, can be engi-neered with SA-FasL protein and whether the engineered islets

*Institute for Cellular Therapeutics, University of Louisville, Louisville, KY 40202;†Department of Microbiology and Immunology, University of Louisville, Louisville,KY 40202; and ‡Frankel Laboratory of Experimental Bone Marrow Transplantation,Department of Pediatric Hematology/Oncology, Schneider Children’s Medical Centerof Israel, Petach Tikvah 49202, Israel

1E.S.Y. and H.Z. contributed equally to this work.

Received for publication October 1, 2010. Accepted for publication October 4, 2011.

This work was supported in part by Grants R21 AI057903 and R41 DK077242 fromthe National Institutes of Health and Juvenile Diabetes Research Foundation (1-2001-328); Kentucky Diabetes Research Board (KDR-PP09-23); the Commonwealth ofKentucky Research Challenge Trust Fund; the Keck Foundation; and an AmericanHeart Association Grant-in-Aid (09GRNT2380136) and National Institutes of HealthTraining Fellowship 5T32 HL076138-07 (to H.Z. and L.B.-M.).

Address correspondence and reprint requests to Dr. Haval Shirwan and Dr. Esma S.Yolcu, Institute for Cellular Therapeutics, Donald Baxter Biomedical Building, Uni-versity of Louisville, 570 South Preston Street, Suite 404E, Louisville, KY 40202.E-mail addresses: haval.shirwan@louisville.edu (H.S.) and esma.yolcu@louisville.edu (E.S.Y.)

The online version of this article contains supplemental material.

Abbreviations used in this article: KDLN, kidney-draining lymph node; LN, lymphnode; MST, mean survival time; SA, streptavidin; T1D, type 1 diabetes; Teff, effectorT; Treg, regulatory T.

Copyright� 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00

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overcome rejection and establish euglycemia following trans-plantation into chemically diabetic allogeneic hosts. Our datademonstrate for the first time, to our knowledge, that pancreaticislets can be engineered with SA-FasL in a rapid and efficientmanner, and such engineered islets under transient cover of rapa-mycin induce localized allotolerance that was initiated and main-tained by CD4+CD25+Foxp3+ regulatory T (Treg) cells homingto the graft and graft-draining lymph nodes (LNs).

Materials and MethodsMice and recombinant proteins

C57BL/6 (CD45.2; H-2b), C57BL/10 (CD45.2; H-2b), C57BL/6Foxp3EGFP, and BALB/c (H-2d) mice were purchased from The JacksonLaboratory. Congenic C57BL/6.SJL (CD45.1; H-2b) and TCR-transgenicOT-I (CD8+ T cell) mice on the rag22/2 background were purchased fromTaconic Farms (Germantown, NY) and bred in our specific pathogen-freeanimal housing facility at the University of Louisville using protocolsapproved by the Institutional Animal Care and Use Committee. Recom-binant SA, human SA-CD40L, and rat SA-FasL proteins were produced inour laboratory using the Drosophila DES expression system (Invitrogen) aspreviously described (16, 18).

Pancreatic islet isolation and engineering with SA-FasL

Pancreatic islets were harvested from 8–12-wk-old BALB/c mice underanesthesia using a standard protocol as previously described (16). Isletswere engineered by first incubating in 5 mM EZ-Link Sulfo-NHS-LC-Biotin solution (Thermo Scientific) in PBS at room temperature for 30min followed by extensive washing to remove free biotin. Biotinylatedislets were then incubated in PBS containing SA-FasL or SA-CD40Lproteins (200 ng protein/450–550 islets/200 ml PBS) or equal molar ofSA as a control at room temperature for 30 min.

After several washes, islets were cultured in vitro for various days,stained first with anti–SA-FITC Ab (Vector Laboratories) to detect SA-FasL and SA-CD40L, washed several times in PBS, and then stained withSA-allophycocyanin (BD Biosciences) to visualize biotin. Z-stack analysiswas performed to measure fluorescence intensity of SA-FasL on engi-neered islets using LAS AF software on Leica TCS SP5 confocal mi-croscopy (Leica Microsystems). Unmodified or SA-engineered islets wereused as controls for background staining.

Islet transplantation

Diabetes was induced in C57BL/6 mice by i.v. injection of streptozotocin(200 mg/kg) and confirmed by two consecutive blood glucose readings.300 mg/dl. Pancreatic islets were harvested, cultured overnight, and thenengineered with SA, SA-CD40L, or SA-FasL proteins. These islets werethen immediately transplanted under the kidney capsule of diabetic mice(450–550 islets/mouse). Unless otherwise indicated, graft recipients wereinjected i.p. with 0.2 mg/kg rapamycin (LC Company) starting on the dayof transplantation daily for 15 d. Animals were monitored for diabetes, andthose with two consecutive daily measurements of $250 mg/dl bloodglucose level were considered diabetic and confirmation of graft failure.

Assessing localized tolerance

To investigate the nature of observed tolerance, we performed two sets ofexperiments on long-term (.100 d) graft acceptors. In the first set, uni-lateral nephrectomy was performed to remove the SA-FasL–engineeredislet graft. After confirmation of hyperglycemia, a second set of unmodi-fied islet graft was transplanted under the contralateral kidney capsule. Toeliminate the effect of surgery associated inflammation on graft rejection,in a second model, unmodified donor islets were transplanted under thecontralateral kidney capsule 40 d prior to surgical removal of the kidneyharboring SA-FasL–engineered islet graft.

CD4+CD25+Foxp3+ Treg cell analysis

Lymphocytes from peripheral LNs, spleen, and kidney-draining LNs(KDLNs) of various treatment groups were harvested, stained with Abs tomouse CD4-allophycocyanin and CD25-PE (BD Pharmingen) molecules,washed with PBS, and permeabilized/fixed overnight at 4˚C using a Per-meabilization/Fixation kit from eBioscience. The FcgII/III receptors wereblocked using 2.4G2 Ab, followed by staining with anti–Foxp3-FITC Abaccording to the manufacturer’s protocol (eBioscience). The cells were runon the FACSCalibur (BD Biosciences), and the data were analyzed byFlowJo software (Tree Star).

Immunohistochemical analyses

Snap-frozen sections of islet grafts were incubated in a blocking solution(0.5% Triton X-100, 0.1% BSA, 5% goat serum, and 1:400 FcgII/III re-ceptor block). Guinea pig anti-insulin Ab (DakoCytomation) and a rat anti-mouse CD4 mAb (BD Pharmingen) were used to detect islets and CD4+

T cells, respectively. Staining of these Abs was visualized using secondaryAbs conjugated with Alexa Fluor-647 (CD4) and Alexa Fluor-555 (insulin)followed by direct staining with FITC-conjugated rat anti-Foxp3 Ab(eBioscience) to visualize Treg cells. Hoechst (Molecular Probes) wasused to stain the nucleus of the cell. Fluorescent images were obtainedusing Leica TCS SP5 confocal microscopy (Leica Microsystems) under320 original magnification. H&E staining was performed on formalin-fixed and paraffin-embedded kidney tissue blocks as previously de-scribed (17).

CD25+ T cell depletion

Selected groups of mice were injected i.p. with 300 mg purified anti-CD25Ab/animal (PC61; Bio X Cell) to deplete CD25+ T cells on days 14 or 100posttransplantation of SA-FasL–engineered allogeneic islets. Depletionwas confirmed using the 7D4 Ab recognizing a different epitope of CD25than PC61 Ab (19) to stain PBLs harvested at various times post-PC61treatment. Groups of mice were also injected i.p. with 300 mg/animalisotype Ab or NK1.1 Ab (clone PK136; Bio X Cell) on day 14 post-transplantation as controls.

CD4+CD25+Foxp3+ Treg cell adoptive transfer assay

CD4+CD25+ T cells were sorted from KDLNs or spleens of long-term SA-FasL–engineered islet graft acceptors, SA-engineered islet graft rejectors,and naive C57BL/6 mice using Abs against CD4-FITC and CD25-PE inflow cytometry. CD4+CD252 effector T (Teff) cells from naive C57BL/6mice spleen were sorted using flow cytometry. The purity of cells was.95%. One million Teff cells were adoptively transferred i.v. into chem-ically diabetic OT-I mice 1 d prior to the transplantation of unmodifiedBALB/c islets either alone or with 2,500–10,000 CD4+CD25+ Treg cells.Mice were monitored for graft survival by assessing blood glucose levelson a regular basis.

Assessing apoptosis of islet-infiltrating T cells

BALB/c islets engineered with SA-FasL or SA proteins were transplantedunder the kidney capsule of streptozotocin diabetic C57BL/6 Foxp3EGFP

mice subjected to daily rapamycin (0.2 mg/kg) treatment starting on theday of transplantation. Three days posttransplantation, mice were eutha-nized, and grafted islets were dissected with fine forceps from the kidneycapsule and mechanically dispersed into single cells. Cells were stainedwith fluorochrome-conjugated Abs against CD3, CD4, CD8, CD25, Gr-1,and CD11b molecules and Annexin V, run on a BD LSR II flow cytometer(BD Biosciences), and analyzed using Diva software (BD Biosciences).

Assessing chemotactic function of SA-FasL for neutrophils

C57BL/6.SJL (CD45.1+) splenocytes were engineered with SA-FasL (40ng SA-FasL protein/106 cells) or equal molar of SA protein (20 ng SAprotein/106 cells), and 5–10 3 106 of the engineered cells were injectedi.p. into C57BL/6 (CD45.2+) mice. LPS (Sigma-Aldrich) injected i.p. at 10mg/mouse served as positive control. Animals were terminated at varioustimes (17–44 h) postinjection, and peritoneal lavage was aspirated underaseptic conditions. Lavage cells were stained with Abs against CD3,CD19, Gr-1, CD11b, CD45.1, CD45.2, and F4/80 molecules. Percentagesof lavage neutrophils (CD11b+Gr-1high) were assessed by gating on re-cipient (CD45.2+) cells using an LSR II and Diva software (BD Bio-sciences).

Statistical analyses

The t test and ANOVA were used to determine significance between twoand multiple groups, respectively. Graft survival was assessed using theKaplan-Meier method and the log-rank test. Data are expressed as mean 6SD. The p values ,0.05 were considered significant. Statistical analysiswas performed using SPSS 13.0 software (SPSS).

ResultsPancreatic islets engineered with SA-FasL protein ex vivoestablish euglycemia in diabetic syngeneic host

Various conditions were used to optimize the rapid and efficientdisplay of SA-FasL on the surface of mouse pancreatic islets

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ex vivo without compromising their survival and function. Aftertesting various doses of biotin and SA-FasL, concentrations of 5mM EZ-Link Sulfo-NHS-LC-Biotin and 200 ng SA-FasL/450–550 islets were found to be optimum for islet engineering (Fig.1A). Z-stack analysis demonstrated intense staining for both biotinand SA-FasL on the external surface of islets, with the inner coreshowing only moderate staining (Fig. 1B). Flow cytometric anal-ysis of mechanically dispersed SA-FasL–engineered pancreaticislets further confirmed the presence of both biotin and SA-FasLon the majority of islet cells (Fig. 1C). Under these conditions,SA-FasL did not have any toxic effect on islets, and the proteinpersisted on the surface of islets for .1 wk ex vivo (Fig. 1D).Importantly, transplantation of SA-FasL–engineered islet graftsinto chemically diabetic syngeneic C57BL/10 mice resulted innormalized blood glucose levels and survival in all mice over a100-d observation period (Fig. 1E). Collectively, these results dem-onstrate that pancreatic islets can be engineered with SA-FasLprotein in a rapid and efficient manner without compromisingtheir function for establishing glucose homeostasis in diabetichosts in the absence of detectable acute toxicity.

SA-FasL–engineered islet grafts survive indefinitely inallogeneic hosts treated with a short course of rapamycin

We next tested if the SA-FasL protein on islet grafts is effective inpreventing rejection in allogeneic hosts in the absence of immu-nosuppression. SA-FasL–engineered BALB/c islets, although theyshowed significantly (p = 0.001) prolonged survival in the ab-sence of any immunosuppression in chemically diabetic allogeneicC57BL/6 mice as compared with unmodified or SA protein-engi-neered islets, only a moderate percentage (∼18%) of grafts sur-vived over the 100-d observation period (Fig. 2A). To improve

long-term survival, SA-FasL–engineered islets were transplantedin conjunction with a short course of rapamycin treatment. Therationale for using rapamycin was 2-fold. First, rapamycin hasbeen shown to cause apoptosis of alloreactive T cells (20, 21) andas such may serve to physically eliminate circulating alloreactiveT cells evading apoptosis induced by SA-FasL displayed on isletgrafts. Second, rapamycin has also been shown in various settingsto induce CD4+CD25+Foxp3+ Treg cells (22, 23). All SA-FasL–engineered islet grafts (n = 45) survived indefinitely and nor-malized blood glucose levels in chemically diabetic allogeneicC57BL/6 mice treated with 0.2 mg/kg rapamycin daily for 15doses starting on the day of transplant (Fig. 2B). In marked con-trast, all control islets engineered with SA (n = 10) or human SA-CD40L (n = 5) protein that does not interact with mouse CD40receptor (18) underwent acute rejection within 30 d. Taken to-gether, these data demonstrate that SA-FasL alone is effective inprolonging allogeneic islet graft survival with a modest effect onlong-term survival and that, in combination with a short course ofrapamycin treatment, SA-FasL is effective in inducing tolerance inall islet graft recipients. Therefore, we used rapamycin as a com-ponent of our immunomodulation protocol for the rest of thestudies.

Robust tolerance induced by SA-FasL–engineered islet grafts isislet specific and localized

To test if euglycemia is maintained by the transplanted SA-FasL–engineered allogeneic islet grafts, the kidney harboring the graftedislets was surgically removed 100 d posttransplantation. All hostsdeveloped hyperglycemia within 3 d (n = 5). These mice were thentransplanted with a second set of unmanipulated donor allogeneicislet grafts under the remaining kidney capsule. All islet graftswere rejected in acute fashion (n = 5; mean survival time [MST] =18 6 7.3 d; Fig. 3A). To test that surgery-associated trauma/inflammation or temporary lack of donor Ags in the mice withunilateral nephrectomy was not the cause of secondary islet graftrejection, another group of long-term survivors (n = 4) were firsttransplanted with a second set of unmanipulated allogeneic isletsunder the contralateral kidney capsule and then nephrectomized40 d later to remove the kidney harboring the primary graft. Allmice maintained euglycemia until the removal of the kidney har-boring the primary islet grafts, which then resulted in the devel-opment of hyperglycemia within 3 d (Fig. 3A). These datademonstrate that the secondary unmanipulated grafts are rejectedwithout an effect on the survival of the primary grafts, therebydemonstrating the localized nature of tolerance.Lack of systemic tolerance was further tested by performing

BALB/c skin allografts into C57BL/6 mice with long-term (.100d) surviving islet allografts. All skin grafts rejected at a similartempo (n = 4; MST = 12.756 1.26 d) to skin grafts (n = 6; MST =11.3 6 1.21 d) transplanted onto naive C57BL/6 mice (Fig. 3B).Importantly, unlike islet grafts (Fig. 3A), skin graft rejection re-sulted in acute rejection of long-term islet grafts within 21 d(Fig. 3B). Taken together, these data demonstrate that the transientdisplay of SA-FasL on pancreatic islet grafts is effective in induc-ing localized tolerance, which can be overcome by alloreactiveresponses against donor skin, but not islet, grafts.

Localized tolerance is induced and maintained by Treg cells

Although SA-FasL–engineered islet grafts may induce localizedtolerance by clonal deletion of alloreactive T cells (17), this mech-anism is expected not to operate when the levels of SA-FasL onpancreatic islet grafts decline with time. Therefore, newly arisingalloreactive T cells late posttransplantation need to be controlledby ongoing immunoregulatory mechanisms, such as Treg cells. To

FIGURE 1. Pancreatic islets can effectively be engineered to display

SA-FasL protein on their surface, and such engineered islets established

long-term euglycemia in chemically diabetic syngeneic recipients. A,

Confocal picture of SA-FasL–engineered pancreatic islets stained first with

anti–SA-FITC Ab (green) and then with SA-allophycocyanin (red) to vi-

sualize SA-FasL and biotin, respectively. Cells positive for both molecules

appear as yellow. Original magnification 320. B, Z-stack analysis of

pancreatic islets across the line shown in A. C, The FACS profile of

mechanically dispersed total engineered islet cells stained with SA-allo-

phycocyanin and anti–SA-FITC Ab to detect biotin and SA-FasL, re-

spectively. D, In vitro persistence of biotin (red) and SA-FasL protein

(green) on engineered pancreatic islets as a function of time shown in days.

Original magnification 320. E, Transplantation of 450–550 SA-FasL–

engineered islets under the kidney capsule of chemically diabetic synge-

neic host results in long-term euglycemia. Unmodified islets are used as

positive controls. Data shown for A–D are representative of two to three

independent experiments.

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investigate the role of Treg cells in localized tolerance, lymphoidorgans from long-term syngeneic and allogeneic SA-FasL–engi-neered allogeneic islet graft acceptors and SA-engineered isletgraft rejectors were analyzed at various times posttransplantation.There were no detectable differences in the percentages of Tregcells in the spleen, mesenteric LNs, or KDLNs of all three groups(Fig. 4A, 4B, and data not shown).In view of several recent reports providing evidence for in situ

intrapancreatic immune reactions playing a critical role in the de-velopment of T1D in NOD mice (24, 25), we analyzed islet graftsusing immunohistochemistry and confocal microscopy for thepresence of Treg cells. On day 5 posttransplant, SA-FasL–engi-neered islet grafts had significantly higher numbers of Treg cells thatwere localized to the grafts as comparedwith SA-engineered controlgrafts (Fig. 4C, 4D). Importantly, long-term (.100 d) SA-FasL–engineered allogeneic islet grafts also had higher numbers of Tregcells residing in the periphery of islet grafts as compared withsyngeneic grafts (Fig. 5A, 5B) and otherwise looked similar to

syngeneic grafts with respect to insulin expression and lack of sig-nificant levels of inflammatory infiltrates (Fig. 5C, 5D).The critical role of Treg cells in the localized tolerance was

further confirmed by in vivo elimination of these cells using an Ab(PC61) against the CD25 molecule. Consistent with a previousreport (26), i.p. treatment of mice with PC61 Ab (300 mg/mouse)partially depleted Treg cells because a significant percentage(∼6%) of CD4+ T cells in the blood remained Foxp3 positive withdownregulated expression of CD25, as assessed by a second Ab(7D4) to a different CD25 epitope (Fig. 6A). Partial depletion ofTreg cells either early (day 14 posttransplant) during the estab-lishment of tolerance or late (day 100 posttransplant), when tol-erance has already been established, resulted in prompt islet graftrejection (Fig. 6B). There was a direct correlation between thedepletion of CD4+Foxp3+ Treg cells and the tempo of graft re-jection. Mice that rejected islet grafts at a rapid tempo had fewerCD4+Foxp3+ Treg cells remaining in the periphery as comparedwith mice that rejected at slower tempo (data not shown). Indeed,one out of five mice that did not reject the allograft had inefficientdepletion of CD4+Foxp3+ Treg cells (29 versus 39–66% depletionin rejecting mice). Treatment with an isotype Ab or NK1.1-depleting Ab did not prompt graft rejection in either setting (Fig.6B), demonstrating that it is not mere depletion of a lymphocytepopulation that sets off the rejection reaction. Collectively, thesedata demonstrate that Treg cells play a critical role in the main-tenance of localized tolerance.

Treg cells sorted from kidney draining LNs, but not spleens, oflong-term tolerant graft recipients prevent rejection ofunmodified donor islets in an adoptive transfer model

Although the partial depletion of Treg cells using anti-CD25 Abdemonstrates the importance of these cells in the induced tolerance,it does not assess if they are the primary mechanism of tolerance.We, therefore, tested the function of these cells in an adoptivetransfer allogeneic islet model. Given that the established tolerancewas not systemic and localized to the graft, we also tested if therewas a functional difference between Treg cells residing in the graftdraining LNs and spleens of tolerant mice. Treg cells were sortedfrom the KDLNs or spleens of long-term (.100 d) SA-FasL–engineered islet graft acceptors, SA-engineered islet graft rejec-tors, and unmanipulated naive C57BL/6 mice. The sorted cellswere then adoptively transferred into chemically diabetic rag22/2

OT-I mice on C57BL/6 background 1 d prior to BALB/c alloge-neic islet transplantation. Animals transplanted with unmodifiedBALB/c allogeneic islets did not reject their grafts (n = 5;.100 d)due to the specificity of the TCR in OT-I mice for the OVA Ag(Fig. 7). However, adoptive transfer of 1 3 106 CD4+CD252

FIGURE 2. SA-FasL–engineered pancreatic islets establish robust allotolerance when transplanted under the transient cover of rapamycin. A, Prolonged

survival of SA-FasL–engineered BALB/c islet grafts in C57BL/6 mice in the absence of rapamycin as compared with unmodified or islet-SA groups

(p = 0.001 versus others). B, SA-FasL–engineered islet grafts induce robust tolerance when transplanted under the transient cover of rapamycin as

compared with SA, nonfunctional human SA-CD40L–engineered, or unmodified control islet grafts (p , 0.0001). Because some animals were euthanized

for mechanistic studies at various time points, the survival curve represents: n = 45, 100 d; n = 23, 300 d; n = 18, 400 d; and n = 13, 500 d post-

transplantation.

FIGURE 3. Tolerance induced by SA-FasL–engineered allogeneic islets

is localized to the graft. A, Long-term tolerant mice reject unmodified

secondary islet grafts from the donor. C57BL/6 recipients of primary SA-

FasL–engineered islet grafts underwent unilateral nephrectomy to remove

the kidney harboring primary SA-FasL–engineered grafts 100 d post-

transplantation. After the confirmation of hyperglycemia within 3 d, these

mice were transplanted with a second set of unmodified BALB/c alloge-

neic islets under the contralateral kidney capsule. All of the secondary

grafts were rejected in a normal tempo (MST = 18 6 7.3 d; 2nd islet post-

nephrectomy). To ensure that the rejection of the second set of islets is not

due to nephrectomy-associated inflammation, a second group of long-term

SA-FasL–engineered islet graft recipients was transplanted with unma-

nipulated BALB/c islets under the contralateral kidney capsule. Removal

of the kidney harboring primary islets 40 d later resulted in hyperglycemia

in all animals (2nd islet pre-nephrectomy). B, Long-term allogeneic islet

graft survivors reject allogeneic skins from BALB/c mice in a normal

tempo (MST = 12.756 1.26 d) as compared with naive mice (MST = 11.361.21 d). Allogeneic skin graft rejection also results in the rejection of long-

term (100 d) allogeneic islet grafts (MST = 19.25 6 6.90 d) in a similar

tempo to naive islets (MST = 14.5 6 1.76 d).

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T cells alone from naive C57BL/6 mice resulted in acute rejectionof allogeneic islets (n = 5; MST = 29.0 6 9.2 d). Cotransfer of2,500–10,000 Treg cells sorted from graft-draining LNs of SA-FasL–islet graft acceptors prevented islet graft rejection inducedby 1 3 106 CD4+CD252 T cells (n = 6; MST .100 d). In markedcontrast, cotransfer of the same number of Treg cells sorted fromeither SA-islet graft rejectors or naive C57BL/6 mice did notprevent rejection mediated by CD4+CD252 T cells (n = 4; MST =36 6 2.3 d and n = 5; MST = 34.4 6 3.5 d, respectively; Fig. 7).Importantly, splenic Treg cells sorted from SA-FasL–islet graftacceptors had significantly reduced efficacy in preventing the re-jection of unmanipulated donor islet allografts in the adoptivetransfer model as compared with Treg cells sorted from graft-draining LNs. Indeed, only one out of six mice, which receivedthe highest number of Treg cells, in this group accepted the donorgraft, whereas all of the rest had rejection, but in a delayed fashion(MST = 48 6 3.8 d) as compared with controls (Fig. 7). These

studies provide direct evidence for the role of Treg cells in theinduced tolerance and further demonstrate that graft-protectiveTreg cells preferentially home to the graft-draining LNs, but notdistant lymphoid organs, such as spleen.

SA-FasL–engineered pancreatic islets or splenocytes lackchemotactic activity for neutrophils

It has been shown that FasL can cause tissue destruction by servingas a chemotactic factor for neutrophils (27–29). We, therefore,conducted two sets of studies to directly test if SA-FasL has sucha function. In the first set of studies, SA-FasL–engineered syn-geneic splenocytes were injected i.p. into mice, and peritonealexudate cells were harvested at various time points to assess therecruitment of neutrophils using flow cytometry. Splenocytes en-gineered with SA served as negative control, whereas LPS wasused as positive control. We observed about the same percentages(∼12%) of neutrophils among peritoneal exudate cells harvested

FIGURE 4. Localized tolerance is associated with

increased numbers of CD4+CD25+Foxp3+ Treg cells

within the islet grafts. A, Analysis of CD4+Foxp3+

Treg cells gated on total lymphocytes harvested from

spleen, mesenteric LN, and KDLN of SA-allogeneic

islet graft rejectors and long-term (.100 d) SA-FasL–

allogeneic or syngeneic islet graft survivors. B, Tabu-

lation of the data presented in A (n = 3–5 animals/

group). C, Representative confocal images of Treg

cells in day 5 posttransplant SA-FasL– or SA-engi-

neered allogeneic islet grafts. Nuclear staining is

shown in blue. Scale bars, 50 mm. D, Tabulation of

Treg cells shown in C. The data are shown as average

of Treg cells per section and a minimum of three sec-

tions per animal with three animals per group.

FIGURE 5. Long-term SA-FasL–engineered allogeneic islet grafts maintain increased numbers of CD4+CD25+Foxp3+ Treg cells localized within the

periphery of the graft in the absence of significant inflammatory infiltrates. A and B, Kidney harboring islet grafts were surgically removed from recipients

of syngeneic or SA-FasL–engineered allogeneic islets on day 100 posttransplantation. Tissues were stained with Abs against CD4 (red), Foxp3 (green), and

counterstained with Hoechst (blue). Arrows indicate islet grafts. Scale bars, 100 mm. Right panels are higher magnification of areas shown by rectangles in

A and B. Scale bar, 25 mm. C and D, Tissues in A and B were stained with Abs against insulin (red) and counterstained with Hoechst (blue). Scale bar, 100

mm. H&E staining pattern for syngeneic and allogeneic islet grafts is depicted in insets. Original magnification 320.

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from mice injected with SA-FasL– and SA-engineered spleno-cytes (Fig. 8A). In marked contrast, ∼60% of peritoneal exudatecells were neutrophils in the LPS-positive group. In a second set ofstudies, we assessed the presence of neutrophils among recipientcells infiltrating into SA- and SA-FasL–engineered BALB/c isletgrafts transplanted into C57BL/6 mice under the cover of rapa-mycin. SA-FasL–islet grafts had low levels of neutrophils ascompared with SA-engineered islets (Fig. 8B). Taken together,these data demonstrate that SA-FasL displayed on splenocytes orpancreatic islets lacks chemotactic activity for neutrophils.

DiscussionWe demonstrate for the first time in this study, to our knowledge,that pancreatic islets can be engineered ex vivo in a rapid andefficient manner to display on their surface an apoptotic form ofFasL protein, SA-FasL, and that such islets induce robust local-ized, rather than systemic, tolerance when transplanted into fullyallogeneic hosts under transient cover of rapamycin without de-tectable toxicity to the graft or host. Tolerance by this protocol isinitiated and sustained by Treg cells, as physical depletion of thesecells in vivo early (14 d) or late (.100 d) posttransplantation

resulted in acute graft rejection. Furthermore, sorted Treg cellsfrom long-term, but not rejecting, graft recipients prevented therejection of unmanipulated donor allogeneic islets in an adoptivetransfer model, further confirming their role in the induced tol-erance.Tolerance in this model is completely dependent on SA-FasL

protein and accentuated by a short course of rapamycin treat-ment. This observation is consistent with the demonstrated roleof Fas-meditated apoptosis in lymphocytes for the regulation ofchronic immune responses and control of autoimmunity (9). Uponactivation, T cells upregulate both Fas and FasL and become sen-sitive to autocrine and paracrine apoptosis following repeatedengagement with the challenge Ag (9, 30). Therefore, the pre-sentation of alloantigens in the context of FasL may specificallyeliminate alloreactive T cells that upregulate the Fas receptorand thereby become sensitive to Fas/FasL-mediated apoptosis.Indeed, analysis of T cells infiltrating into the grafts 3 d post-transplantation revealed higher percentages of CD4+ Teff andCD8+ Teff cells undergoing apoptosis in SA-FasL–islet graftsas compared with SA-islet grafts (Supplemental Fig. 1). Thesefindings are also consistent with our recent studies demonstratingthat systemic immunomodulation with SA-FasL–engineered do-nor splenocytes induces apoptosis in alloreactive T cells, resultingin the inhibition of primary and secondary alloreactive immuneresponses (16) and induction of tolerance to cardiac allografts(17). Our findings are also consistent with several studies by others

FIGURE 6. Treg cells are critical for the induction and maintenance of localized tolerance. A, Depletion of Treg cells using PC61 (anti-CD25) Ab. PBLs

were collected from SA-FasL–engineered islet graft recipients before (D0) or 3 d (D3) after treatment with the PC61 Ab and typed using the 7D4 Ab

recognizing a different CD25 epitope than PC61 (n = 3). A significant percentage (∼6%) of CD4+ T cells expressing Foxp3 remains after PC61 Ab

treatment. B, Depletion of Treg cells in C57BL/6 mice transplanted with SA-FasL–engineered allogeneic BALB/c islets early (day 14) or late (day 100)

posttransplantation using PC61 anti-CD25 Ab. An isotype Ab and an anti-NK1.1 Ab are used on day 14 as controls. *, **p values for NK 1.1-depleted

group or isotype control versus groups depleted with PC61 on days 14 and 100 posttransplantation, respectively.

FIGURE 7. Treg cells sorted from LNs of long-term islet grafts prevent

rejection of unmanipulated donor islets in an adoptive transfer model.

Survival of allogeneic unmodified BALB/c islet grafts in rag22/2 OT-I

mice that did not receive T cell transfer (No CD4+ T cells) or adoptively

transferred with 13 106 flow-sorted conventional CD4+ T cells from naive

C57BL/6 mice alone (Naive CD4+ T cells) or in combination with flow-

sorted Treg cells from KDLN (n = 3, 2.53 103; n = 2, 53 103; n = 1, 103103 cells) or spleens (n = 2, 2.5 3 103; n = 2, 5 3 103; n = 2 10 3 103

cells) of long-term SA-FasL–islet graft survivors (tolerant CD4+ Tregs),

KDLN of SA-islet rejectors (rejector CD4+ Tregs; n = 4, 5 3 103 cells), or

naive C57BL/6 mice (naive CD4+ Tregs; n = 1, 2.5 3 103; n = 2, 5 3 103;

n = 2, 10 3 103 cells). p = 0.002, KDLN tolerant CD4+ Treg versus all the

other adoptive T cell transfer groups.

FIGURE 8. SA-FasL lacks chemotactic activity for neutrophils. A,

C57BL/6 (CD45.2) mice were injected i.p. with LPS or C57BL/6.SJL

(CD45.1) splenocytes engineered with SA-FasL or SA. Peritoneal lavage

cells were harvested at various times postinjection, stained with Abs to

various cell surface markers, and analyzed using flow cytometry by gating

on recipient cells (CD45.2). Data are shown for 20 h postinjection (n = 3/

group). B, Graft-infiltrating cells were harvested at 3 d posttransplantation

from SA or SA-FasL–engineered BALB/c islets transplanted under the

kidney capsule of STZ diabetic C57BL/6 mice treated with rapamycin.

Cells were stained with various cell-surface markers and analyzed using

flow cytometry (n = 3/group). Results are expressed as mean 6 SD.

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using cells (13), tissues (15), or organs (31) genetically manipu-lated to express FasL for immunomodulation.Contradictory data demonstrating that immunomodulation with

FasL does not prevent graft rejection have also been reported. Forexample, islets and heart grafts genetically modified to expressFasL lacked protection in allogeneic hosts (11, 32). These negativeresults have been attributed to the use of a cleavable isoform ofFasL and diverse functions associated with the membranous andsoluble forms of this molecule. FasL is a type II membranousprotein that is converted into a soluble form via cleavage bymatrix metalloproteinases in response to various physiologicstimuli (33). The membranous form is noted for its ability to in-duce apoptosis in autoreactive and alloreactive T cells, thus pro-moting tolerance. In contrast, the soluble form may inhibit ap-optosis, initiate inflammatory responses, and promote the activerecruitment of neutrophils, thereby accelerating disease or allo-graft rejection (29, 34, 35). Although the separation of thesedistinct functions of the soluble versus membranous forms of FasLhas been the source of great controversy, a recent study usingtransgenic mice expressing either membranous or secreted formsof FasL demonstrated that the membranous form only has apo-ptotic activity and is responsible for the elimination of self-reactive T cells and prevention of systemic autoimmunity (9). Inmarked contrast, the soluble form had no apoptotic activity andappeared to promote autoimmunity and tumorogenesis via non-apoptotic functions.The SA-FasL molecule used in this study lacks potential met-

alloproteinase sites and as such is not cleaved into a trimeric solubleform, but rather forms stable tetramers or oligomers (16) withoutany detectable chemotactic activity for neutrophils as demon-strated in this study (Fig. 8). Given that oligomerization of the Fasreceptor on the surface of T cells is critical for the deliveryof apoptotic signals (36), SA-FasL has potent apoptotic activityin soluble form (16) or when displayed on the cell surface (17).Therefore, SA-FasL–engineered allogeneic cells or tissues havethe potential to effectively eliminate alloreactive T cells via apo-ptosis, thereby initiating a cascade of regulatory events resultingin either systemic tolerance (17) or localized tolerance as shownin this study, with CD4+CD25+Foxp3+ Treg cells serving as thecommon denominator. Treg cells were shown to be relatively re-sistant to Fas-induced apoptosis under selected experimental con-ditions (37) and are spared by rapamycin (22). Consequently, weobserved high numbers of Treg cells homing to the islet graft.Physical depletion of these cells either early or late posttrans-plantation resulted in the abrogation of tolerance. The critical roleof these cells in the observed tolerance was further demonstratedin an adoptive transfer model in which Treg cells sorted fromgraft-draining LNs of tolerant, but not those from naive or SA-islet, graft rejectors prevented the rejection of unmanipulateddonor islet grafts. Importantly, splenic Treg cells isolated fromtolerant SA-FasL–islet graft recipients were inferior to Treg cellsfrom graft-draining LNs in preventing rejection in the adoptivetransfer model. Taken together, these data demonstrate that graft-protective Treg cells preferentially home to graft-draining LNsand potentially to the graft and play a critical role in keeping incheck the pathogenic Teff cells. Although we cannot totally ex-clude other regulatory mechanisms in addition to Treg cells in-volved in the observed tolerance, our data are consistent with arecent study by Kendal et al. (38), demonstrating that Treg cellsplay a critical role in tolerance to skin allografts induced bynondepleting anti-T cell Abs by preferentially residing in the graftand keeping in check the pathogenic function of Teff cells.SA-FasL may contribute to the generation/expansion of Treg

cells by two distinct mechanisms: 1) preferential elimination of

activated alloreactive Teff cells due to their enhanced sensitivity toFasL-mediated apoptosis and as such tilting the balance towardTreg cells (37); or 2) generation of induced Treg cells throughmechanisms involving apoptosis (39). Unlike the human Treg cellsthat are not only resistant to Fas/FasL-mediated apoptosis, butalso use FasL as an effector molecule to induce apoptosis in Teffcells as a means of immune suppression (40, 41), the preferentialsensitivity of mouse Treg cells over the Teff cells to Fas/FasL-mediated apoptosis has been the subject of significant controversy.A series of studies in the autoimmunity and cancer settings hasdemonstrated that Treg cells are more sensitive than Teff cells toFas/FasL-mediated apoptosis (42–44). For example, immunomo-dulation with FasL protein was recently reported to selectivelydeplete Treg cells from tumor (43). Similarly, the efficacy ofimmunomodulation with IL-12 was shown to be dependent onCD8+ T cells expressing FasL and eliminating Treg cells withinthe tumor via Fas/FasL-mediated apoptosis (42). In marked con-trast, freshly isolated CD4+CD25+ Treg cells were shown to beless sensitive to Fas-mediated apoptosis as compared with CD4+

CD252 T cells in response to CD3 stimulation in vitro (45).However, this differential sensitivity to Fas/FasL-mediated apo-ptosis could be reversed in coculture experiments depending on theTreg/Teff cell ratios that were regulated by IL-2. Consistent withthis study, we recently demonstrated relative resistance of NODTreg cells to SA-FasL–mediated apoptosis under inflammatoryconditions as compared with Teff cells (37, 46, 47). Indeed, thedirect display of SA-FasL on the surface of Treg cells endowedthem with better regulatory activity by inducing apoptosis in Teffcells via SA-FasL/Fas interaction (46). In marked contrast, thedirect display of SA-FasL on NOD Teff cells resulted in theiraccelerated apoptosis and reduced onset and incidence of diabetesin an adoptive transfer model (47). Consistent with these studies,we observed higher percentages of CD4+ Teff and CD8+ Teff cellsundergoing apoptosis in SA-FasL–islet grafts as compared withSA-islet group. In marked contrast, CD4+ Treg cells appeared tobe resistant to SA-FasL–induced apoptosis (Supplemental Fig. 1).Although the molecular nature of these opposing observations isnot known and remains to be elucidated, they indicate the complexnature of Fas/FasL-mediated homeostasis of Treg and Teff cellsunder normal physiological conditions and disease settings.Alternative and/or additive to the sparing of Treg cells from Fas-

mediated death, SA-FasL–engineered islet grafts may facilitate denovo generation of induced Treg cells via a cascade of immuno-regulatory mechanism orchestrated by apoptosis of Teff cells. Con-sistent with notion is a recent study demonstrating that immu-nomodulation with anti-CD3 Ab results in apoptosis of T cells,digestion of apoptotic bodies by phagocytes, and their secretionof TGF-b that converts Ag-specific Teff cells into Treg cells (39).Importantly, Treg cells harvested from SA-FasL–engineered, butnot SA-control, islet graft recipients prevented rejection of un-modified second set donor islet grafts in an adoptive transfermodel, suggesting that these cells may represent the induced Tregcells that show Ag specificity.The tolerogenic effect of SA-FasL was greatly enhanced by

a short course of rapamycin treatment. Rapamycin may work insynergy with SA-FasL to enhance tolerance by eliminating Teffcells (20) and contributing to the generation of Treg cells eitherby sparing natural Treg cells (22) or actively contributing to theconversion of Teff cells into induced Treg cells through the reg-ulation of the mammalian target of rapamycin (23). Important inthis context is a recent study demonstrating that in the presenceof rapamycin TCR/IL-2 signaling resulted in the upregulation ofantiapoptotic Bcl-2 family members in Treg cells and their relativeresistance to apoptosis (48). In marked contrast, under the same

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conditions, rapamycin downregulated the expression of Bcl-2 fam-ily members while increasing the expression of proapoptotic Baxin T conventional cells, leading to their increased sensitivity to ap-optosis.Genetic manipulation of pancreatic islets ex vivo to express

immunoregulatory molecules for long-term survival in allogeneichosts represents an attractive immunomodulatory approach thatmay spare the recipient from harmful immunosuppressive treat-ments. However, introduction of foreign DNA into the graft is notonly technically challenging but also has safety concerns, andcontinuous expression of potent immunomodulatory molecules,such as FasL, on the graft may have long-term complications.Therefore, our approach of engineering donor grafts ex vivo todisplay on their surface immunomodulatory molecules, such as SA-FasL, in a rapid, effective, and transient manner represents a novelmeans of immunomodulation with therapeutic implications incancer, autoimmune disease, and transplantation.

AcknowledgmentsWe thank O. Grimany for technical help with SA-FasL, SA-CD40L, and SA

protein expression and purification and Drs. Suzanne T. Ildstad, Michele

Kosiewicz, and Huang-Ge Zhang for critical reading of the manuscript.

DisclosuresThe SA-FasL protein and ProtEx technology described in this article are

licensed from the University of Louisville by ApoVax, Inc., Louisville,

KY, for which H.S. serves as a Member of the Board and Chief Scientific

Officer, and H.S. and E.S.Y. have significant equity interest in the com-

pany. The other authors have no financial conflicts of interest.

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