Original Basic Science

Enhancement of Islet Engraftmentand Achievement of Long-Term Islet AllograftSurvival by Toll-Like Receptor 4 BlockadeLaurianne Giovannoni,1 Yannick D. Muller,1 Stéphanie Lacotte,1 Géraldine Parnaud,1 Sophie Borot,1,2

Raphaël P.H. Meier,1,3 Vanessa Lavallard,1 Benoît Bédat,1,3 Christian Toso,1,3 Bruno Daubeuf,4 Greg Elson,4

Limin Shang,4 Philippe Morel,3 Marie Kosco-Vilbois,4 Domenico Bosco,1 and Thierry Berney1,3

Background.Toll-like receptors are key players in sterile inflammation phenomena and can link the innate and adaptive immunesystems by enhancing graft immunogenicity. They are also consideredmediators of types 1 and 2 diabetes development. The aimof the present study was to assess the role of Toll-like receptor-4 (TLR4) in mediating the inflammatory and immune responses topancreatic islets, thereby promoting inflammatory destruction and immune rejection of islet grafts.Methods. Experiments wereconducted in murine and human in vitro systems and in vivo murine islet transplant models, using species-specific anti-TLR4monoclonal antibodies. In vitro, mixed lymphocyte-islet reaction experiments were performed to assess T-cell activation and pro-liferation. In vivo, both a syngeneic (B6-to-B6) marginal mass islet transplant model to assess the impact of TLR4 blockade on isletengraftment and an allogeneic (DBA1-to-B6) model were used.Results. In vitro TLR4 blockade decreased lipopolysaccharide-mediated β-cell apoptosis and T-cell activation and proliferation against allogeneic islets. In vivo, TLR4 blockade resulted in signif-icantly better syngeneic marginal mass islet engraftment and in indefinite allogeneic islet graft survival. Tolerance was not observedbecause donor-specific skin graft rechallenge in nonrejecting animals resulted in rejection of both skin and islets, but without ac-celerated rejection as compared to naive animals. Conclusion. Taken together, our data indicate that TLR4 blockade leads toa significant improvement of syngeneic islet engraftment and of allogeneic islet graft survival. A mechanism of graft accommoda-tion with concurrent inhibition of donor-specific immune memory is likely to be involved.

(Transplantation 2015;99: 29–35)

A lthough clinical results of islet of Langerhans trans-plantation have been steadily improving in the past de-

cade, attrition of islet graft function is still commonlyobserved, and multiple grafts are necessary to achieve insulinindependence.1 This is the result of the combination of com-plex inflammatory and immune reactions. The causes and

Received 27 May 2014. Revision requested 17 June 2014.

Accepted 26 August 2014.1 Cell Isolation and Transplantation Center, Department of Surgery, University ofGeneva School of Medicine, Geneva, Switzerland.

2Department of Endocrinology-Metabolisme andDiabetology-Nutrition, JeanMinjozUniversity Hospital, Besançon, France.

3 Division of Transplantation and Visceral Surgery, Department of Surgery, Universityof Geneva Hospitals, Geneva, Switzerland.

4 NovImmune, Plan-les-Ouates, Switzerland.

This work was supported by a grant from the Swiss National Science Foundation(310030-149798), a grant from the Juvenile Diabetes Research Foundation (31-2008-416; human islet for research distribution program), a grant from theInsuleman Foundation and the 2010 Award from the “Fondation Romande pour laRecherche sur le Diabète”. CT is supported by a Professorship from the Swiss Na-tional Science Foundation (PP00P3-139021).

The authors declare no conflicts of interest.

L.G. participated in research design, performance of the research, data analysis, andwriting of the article. Y.D.M. participated in performance of the research, data anal-ysis, and review/edition of the article. S.L. participated in performance of the re-search, data analysis, and review/edition of the article. G.P. participated in

Transplantation ■ January 2015 ■ Volume 99 ■ Number 1

Copyright © 2014 Wolters Kluwer H

mechanisms of these reactions are starting to be character-ized.2 Importantly, the role of innate immunity in the immedi-ate loss of a sizeable proportion of transplanted islets and inthe subsequent activation of adaptive immunity is unfolding.3

Toll-like receptors (TLR) are transmembrane receptors andare part of the innate immune system critical for the responseto microbial pathogens.4 They recognize exogenous pathogen-associated molecular patterns (PAMPs) but are also able to

performance of the research, data analysis, and review/edition of the article. S.B.participated in performance of the research, data analysis, and review/edition ofthe article. R.P.H.M. participated in performance of the research, data analysis,and review/edition of the article. V.L. participated in performance of the re-search and review/edition of the article. B.B. participated in performance of the re-search and review/edition of the article. C.T. participated in research design,data analysis, and review/edition of the article. B.D. contributed new reagentsand participated in review/edition of the article. G.E. contributed new reagents and par-ticipated in review/edition of the article. L.S. contributed new reagents and par-ticipated in study design and review/edition of the article. P.M. participated inreview/edition of the article. M.K.V. contributed new reagents and participatedin research design, data analysis, and writing of the article. D.B. participated in re-search design, data analysis and writing of the article. T.B. participated in researchdesign, data analysis, and writing of the article.

Correspondence: Thierry Berney,MD,MSc,Divisions of Visceral Surgery and Trans-plantation, Department of Surgery, University of Geneva Hospitals, 4, rue Gabrielle-Perret-Gentil, 1211 Geneva 14, Switzerland. (thierry.berney@hcuge.ch).

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ISSN: 0041-1337/15/9901-29

DOI: 10.1097/TP.0000000000000468

www.transplantjournal.com 29

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FIGURE 1. LPS-induced apoptosis in human β cells. Islet cells werecultured 24 hr with LPS andwith orwithout anti-TLR4mAb or isotypecontrol mAb. Anti-TLR4 mAb was able to inhibit LPS-induced β-cellapoptosis (P=0.014 vs. vehicle; P=0.039 vs. isotype control mAb)assessed by TUNEL labeling. Results of three independent exper-iments are presented as mean±SEM. *P<0.05. TUNEL, terminaldeoxynucleotide tranferase–mediated dUTP nick-end labeling;mAb, monoclonal antibody; TLR4, Toll-like receptor-4.

30 Transplantation ■ January 2015 ■ Volume 99 ■ Number 1 www.transplantjournal.com

sense endogenous ligands, collectively known as damage-associated molecular patterns (DAMPs), such as key mole-cules in autoimmune pathologies5,6 or necrotic cell products,usually released during ischemia-reperfusion injury in organor cell transplantation.5,7 Additional inflammatory mole-cules, and especially chemokines, can be recognized by TLRs,making them important players in sterile inflammation phe-nomena.8 Moreover, toll-like receptor-4 (TLR4)-mediateddendritic cell (DC) activation can lead, by means of the nu-clear factor kappa B pathway, to DC maturation and Tregsuppression.9 In the setting of transplantation, ischemicinjury can activate donor DCs by means of heat-shockprotein70-TLR4 interaction. Donor-derived activated DCscan enhance immunogenicity of the graft, thus promotinggraft rejection.7

Toll-like receptor-4 is one of the better studied members ofthe TLR family. It is the key receptor for lipopolysaccharide(LPS) but has also been linked to autoimmune pathologiesand graft rejection. In islets, TLR4 has been associated withβ cell function and viability through CXCL10 signaling andis considered a player in the development of types 1 and2 diabetes.10-13

The aim of this study was to investigate the role of TLR4on islet graft damage in human and murine models of islettransplantation, to explore a potential protective role of TLR4blockade on islet grafts, using anti-TLR4 monoclonal anti-bodies (mAb).

RESULTS

TLR4 Blockade Inhibits LPS-Mediated -Cell Apoptosis

Human islet cells were incubated for 24 hr in the presenceof LPS and anti-TLR4 mAb. Apoptosis of β cells was thenanalyzed by terminal deoxynucleotide tranferase-mediateddUTP nick-end labeling after immunostaining for insulin.LPS increased β-cell apoptosis by twofold compared to cellscultured under control conditions. Toll-like receptor-4 block-ade was able to significantly inhibit LPS-induced β-cell apo-ptosis (Fig. 1).

TLR4 Blockade Decreases IsletImmunogenicity In Vitro

Murine lymph node cells were coculturedwith intact mouseislets for 7 days in the presence or absence of antimouse TLR4mAb. Immune cell proliferation was then assessed by Ki67staining. Toll-like receptor-4 blockade significantly decreasedallogeneic islet-induced lymph node cell proliferation as com-pared to control conditions (Fig. 2A). Similar results were ob-served for human peripheral bloodmononuclear cells (PBMC)exposed to allogeneic islets for 7 days in the presence of anti-human TLR4 mAb (Fig. 2C).

Allogeneic islet-induced lymphocyte activation was alsoassessed by measuring secretion of the prototype proinflam-matory cytokine interferon (IFN)-γ using an enzyme-linkedimmunospot (ELISPOT) assay. Antimouse and antihumanTLR4 mAb both significantly decreased the number of IFN-γ-secreting cells in the murine and human experimentalmodels, respectively (Fig. 2B and D).

TLR4 Blockade Leads to Enhanced IsletEngraftment in Mice

We assessed the impact of TLR4 blockade on islet en-graftment, in the absence of the confounding factor of

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allorejection, with a marginal mass syngeneic islet transplan-tation model. In this model, transplantation induces diabetesreversal in a delayed fashion.14 Any manipulation that allowsshortening the time to diabetes reversal can be interpreted asbeneficial in engraftment.14 Transplantation of anti-TLR4mAb-treated islets into anti-TLR4 mAb-treated animalsachieved reversal of diabetes in 100% of recipients after a me-dian time of 7 days, as compared to 75% after 21.5 days incontrols (Fig. 3).

TLR4 Blockade Leads to Indefinite Islet AllograftSurvival in Mice

As shown in Figure 4, anti-TLR4 treatment of both isletsand recipients led to indefinite graft survival (>100 days)in 62.5% of animals (P=0.02 versus controls), whereas treat-ment of islets or recipient mice only was associated with me-dian graft survivals of 41.5 days (P=0.08 vs. controls) and14 days (p ns vs. controls), respectively. Median graft survi-val in controls was 12.5 days (isotype control mAb) and17 days (vehicle).

Immunohistochemistry showed essentially identical CD4+and CD8+ T-cell infiltration in islet grafts of nonrejectinganti-TLR4-treated islets and recipients at 100 days aftertransplantation and of control mice at the time of rejection.Foxp3+ T cells were rarely seen both in treated and controlanimals. This indicates that TLR4 blockade had no impacton effector or regulatory T-cell recruitment to the site of im-plantation, but that recruited cytotoxic T cells were not func-tional with respect to donor-specific graft rejection.

Graft Acceptance is Associated With Lack ofDonor-Specific Immune Memory

To test if treated B6 recipients developed immune memoryagainst DBA1 islets, we assessed the in vitro proliferativecapacity of their lymph node cells in mixed lymphocyte-isletreactions (MLIR) or mixed lymphocyte reactions (MLR)

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FIGURE 2. Mixed lymphocyte-islet reaction. Murine lymph node cells or human PBMC were cultured with allogeneic islets for 7 days for de-termination of cell proliferation by Ki67 staining or for 3 days for detection of IFN-γ–secreting cells by ELISPOT. A, proliferation of murine cellswas decreased in the presence of anti-TLR4mAb (P=0.004 vs. vehicle or isotype control mAb). B, proliferation of human cells was decreased inthe presence of anti-TLR4 mAb (P=0.0001 vs. vehicle or isotype control mAb). C, numbers of murine IFN-γ–secreting cells decreased in thepresence of anti-TLR4 mAb (P=0.0001 vs. vehicle or isotype control mAb). D, numbers of human IFN-γ–secreting cells decreased in the pres-ence of anti-TLR4 mAb (P=0.049 vs. vehicle; p ns vs. isotype control mAb). Results are presented as mean±SEM. *P<0.05, **P<0.005,***P<0.001. PBMC, peripheral blood mononuclear cells; IFN, interferon; mAb, monoclonal antibody; TLR4, Toll-like receptor-4.

© 2014 Wolters Kluwer Giovannoni et al 31

100 days after transplantation. There was no difference incell proliferation against DBA1 spleen cells or islets, whencomparing anti-TLR4-treated nonrejecting and naive mice,contrasting with a significantly higher proliferation againstdonor-specific spleen cells and islets in rejecting control mice(Fig. 5A).

DBA1 Skin Graft Rechallenge Leads toIslet Graft Rejection

To test if the B6 nonrejecting recipient mice developed atrue tolerance to DBA1 islets, we assessed the rejection of is-lets consecutive to skin graft. A hundred days after islet trans-plantation, graft-accepting animals received a donor-specific(DBA1) or third party (C3H) ear skin graft.WhenDBA1 skinwas transplanted, skin rejection occurred between days 8 and12 after transplantation (as for naive mice), and animalsbecame hyperglycemic, indicating simultaneous induction

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of islet graft rejection. When third party (C3H) skin wastransplanted, skin rejection also occurred between days 8 and12, but animals remained normoglycemic (Fig. 5B).

DISCUSSION

In this study, we have demonstrated that TLR4 plays a keyrole as a mediator of inflammatory and immune responses inthe setting of islet of Langerhans transplantation and thatTLR4 blockade can inhibit allogeneic immune responses invitro and lead to indefinite graft survival in vivo in a signifi-cant proportion of recipients. Second, we have demonstratedthat TLR4 blockade facilitates islet engraftment in the ab-sence of rejection. Finally, in this model, indefinite graft sur-vival is not a result of donor-specific immune tolerance, butrather of efficient inhibition of the immune response at the timeof initial cognate interaction, leading to graft accommodation.

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FIGURE 3. Diabetes reversal after syngeneic marginal mass islettransplantation. B6 islets were cultured with anti-TLR4 mAb or vehicle(PBS) before syngeneic transplantation. Diabetic B6 mice weretransplanted under the left kidney capsule with 150 IEQ and treatedby twice weekly intraperitoneal injections, from day 0 to day 28, withvehicle (n=8), or anti-TLR4 mAb (n=8). Anti-TLR4 treatment of bothislets and recipients led to enhanced islet engraftment demon-strated by reversal of diabetes in 100% of animals after a mediantime of 7 days, as compared to 75% in a median time of 20.5(P=0.022). *P=0.07, **P<0.05. PBS, phosphate-buffered saline;mAb, monoclonal antibody; TLR4, Toll-like receptor-4.

FIGURE 5. A, cell reactivity against spleen cells or islets after 35 orlonger than 100 days of graft acceptance. Lymph node cells were ex-tracted from rejecting (35 days) or indefinitely accepting (>100 days)B6 islet recipients or naive B6mice and tested for proliferation againstdonor-specific DBA1 irradiated spleen cells (MLR) or islets (MLIR) byKi67 staining. No significant difference in proliferation was seen be-tween naive and transplanted nonrejecting animals, contrasting witha significantly higher proliferation of lymph node cells from rejecting

32 Transplantation ■ January 2015 ■ Volume 99 ■ Number 1 www.transplantjournal.com

Toll-like receptors provide a well-recognized link betweenthe innate and adaptive immune systems. In transplantation,they are a key link between ischemia-reperfusion injury andgraft rejection.7,15,16 They play this role by enhancing antigenpresentation, upregulating costimulatory molecule expressionand stimulating proinflammatory cytokine and chemokineproduction. Toll-like receptors can be engaged by exogenousligands (PAMPs, such as LPS) in infection, and by endoge-nous ligands (DAMPs), which are the products of inflamma-tory processes, such as in ischemia-perfusion.15-17 AlthoughTLR4 was initially described at the surface of cells of the in-nate immune system from the granulocyte and macrophagelineages, including antigen-presenting cells, it has been shownmore recently that theywere also expressed by other cell types,revealing a markedly broadened range of activity. First, TLRexpression by Tcells was demonstrated, suggesting that Tcellscan also be directly stimulated by TLR ligands.16 Second,islet cells were shown to express TLR4, and its engagementat the islet cell surface to mediate islet damage, for example,by means of the CXCL10 chemokine.13 Among DAMPs,

FIGURE 4. Allogeneic islet graft survival. Fully major histocompatibil-ity complex-mismatched allogeneic DBA1 islets were cultured withanti-TLR4 mAb or vehicle before transplantation. Diabetic B6 micewere transplanted under the left kidney capsule with 600 IEQ andtreated by twice weekly intraperitoneal injection, from day 0 to day28, with anti-TLR4mAb, isotype control mAb, or vehicle. Five groupsof animals (N=8 in each group) were transplanted: anti-TLR4 mAb-treated islets and recipients; anti-TLR4 mAb-treated islets only;anti-TLR4 mAb-treated recipients only; isotype control mAb-treatedrecipients only; vehicle-treated recipients only. *P<0.05. mAb,mono-clonal antibody; TLR4, Toll-like receptor-4.

mice against donor-specific spleen cells (P=0.0005) and islets (P=0.04).Results are presented as mean±SEM. *P<0.05, ** P<0.001. B,skin graft rechallenge in animals that indefinitely accepted is-let allograft. Anti-TLR4-treated mice with intact islet function longerthan 100 days after transplantation (N=6) were transplanted withdonor-specific DBA1 (N=3) or third party C3H (N=3) ear skin. MLIR,mixed lymphocyte-islet reaction; MLR, mixed lymphocyte reaction;TLR4, Toll-like receptor-4.

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high mobility group box 1 (HMGB1) is another example ofa TLR4 ligand that mediates inflammatory responses.18 In-deed, although it is a matter of debate, HMGB1 expressionby islet cells has been identified as an important determinantof islet graft failure or success.19,20

The lower graft survivals achieved when only islets or onlygraft recipients were treated with anti-TLR4 mAb, as com-pared to treatment of both, suggest that TLR4 signaling inboth islet and host immune cells are involved in mediatinggraft rejection. Thus, target cells for the anti-TLR4 mAb arelikely to comprise donor and host antigen-presenting cells, is-let cells and host T-cells. Further, we have shown that LPS-induced islet cell apoptosis could be prevented by TLR4

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© 2014 Wolters Kluwer Giovannoni et al 33

blockade, suggesting that TLR4 expressed at the islet cell sur-face also has a major implication in PAMP-associated isletcell damage.

It should also be noted that approximately 40% of recipi-ents escape from protection by TLR4 blockade and rejecttheir graft at the same time as controls, indicating that inter-individual variability may lead to a possible threshold effect.

Indefinite graft survival was probably achieved through amechanism of graft accommodation, a condition that couldbe defined as graft acceptance in the absence of donor-specific tolerance,21 as demonstrated by the rejection of theislet graft after donor-specific rechallenge by skin grafts. Itis noteworthy that rechallenge led to skin rejection simulta-neously with the ongoing islet graft. Interestingly, islet graftsurvival was maintained after third party skin transplanta-tion, indicating a quite robust accommodation. Our find-ing that timing of rejection in vivo occurred not faster fordonor-specific than for third party skins also indicates lackof immune memory toward donor antigens.

In the clinical setting, in contrast to this study, islet ofLangerhans transplantation is performed into the liver throughthe intraportal route. This is quite a unique situation in thefield of clinical transplantation, inasmuch as it generates, inaddition to ischemia-reperfusion injury, an intense inflamma-tory reaction at the site of implantation.2 The intraportalgeneration of an “instant blood-mediated inflammatory re-action” and the release of reactive oxygen species and othernoxious inflammatory mediators by the Kuppfer cell-richmicroenvironment have been extensively studied.22-24 It iswidely accepted that the inflammatory phenomena describedabove translate into poor islet engraftment. Islet transplanta-tion could therefore represent a situation in which TLR4 en-gagement is especially active, and in turn, TLR4 blockadeespecially relevant for the facilitation of graft survival. Thisis exemplified in this study by the observation that engraft-ment is significantly improved by TLR4 blockade in theabsence of immune rejection, in a model of marginal masssyngeneic islet transplantation.14 This strongly suggests amajor impact of TLR4 blockade on the activation of cellsof the innate immune system at the islet implantation site.

Survival of islet grafts in the absence of TLR4 signalinghas only been reported so far in genetically deficient mice,as opposed to through direct blockade by pharmacologicalagents.25-29 Studies on TLR4−/− syngeneic or allogeneic is-let transplantation are contradictory, with some positiveand some negative reports of their impact on graft implanta-tion and survival. The different mouse strain combinationsused may partly explain these discrepancies. They also exem-plify the limitations of genetic manipulation. Our observa-tions, indicating the superior results of pharmacologicalblockade, and especially the in vitro data obtained by targetingTLR4 in human islets with a specific mAb that works in aligand-independent mechanism, open the way for therapeuticstrategies in clinical islet transplantation.

Taken together, our data indicate that TLR4 blockadeleads to indefinite islet graft survival in a significant proportionof recipients, through a strong mechanism of graft accom-modation, with concurrent inhibition of donor-specific im-mune memory, but in the absence of donor-specific tolerance.These data show that inhibition of TLR4 signaling seems tobe as critical on the donor’s islets as on the donor’s or host’simmune cells.

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MATERIALS AND METHODS

Antibodies

Antihuman TLR4 (15C1), antimouse TLR4 (5E3) and anirrelevant control isotype antimouse mAb were produced byNovImmune (Geneva, Switzerland). Briefly, a humanized ver-sion of 15C130 on a human immunoglobulin (Ig)G1 κ back-bone and a chimeric version of 5E331 on a mouse IgG2a κbackbone were produced in CHOK1 cells. The mouse IgG2a

κ isotype matched control, W6/32, was produced from amouse hybridoma (ATCC, No. HB-95). Antibodies were pu-rified by Protein A affinity chromatography (GE Healthcare,MabSelect SuRe, No. 17-5438-03). Human IgG1 was pur-chased from Sigma (Buchs, Switzerland) and used asisotype-matched control. Anti-insulin and antiglucagon anti-bodies were purchased from Dako (Lausen, Switzerland);fluorescein isothiocyanate anti-Ki-67 Set, anti-CD4 andanti-CD8a from BD Pharmingen (Allschwil, Switzerland).For Western blotting, mouse antihuman TLR4 antibodywas purchased from Sigma.

Human Islets

The use of human islets for research was approved by ourlocal institutional ethical committee. Islets were isolated asdescribed, using the automated method described by Ricordiet al.32,33 with local modifications. Before in vitro experiments,islets were incubated overnight in nonadherent 60-mm Petridishes containing 5 mL Connaught Medical Research Labo-ratories medium-10% fetal calf serum, 11.2 mM glucose,110 μg/mL sodium pyruvate and supplemented with 110units/mL penicillin, 110 μg/mL streptomycin, and 50 μg/mLgentamycin, with or without 1.5 nM anti-TLR4 mAb.

Animals

Two-month-old male C57BL/6 (B6), DBA1, and C3Hmice were purchased from Janvier (Le Genest-St-Isle,France). All animals were kept in the animal facilities at theUniversity of Geneva with free access to food and water. Allexperiments were conducted under protocols reviewed andapproved by our institutional animal care and use committee.

Mouse Islets

Islets of Langerhans were isolated by collagenase (SigmaType XI; Sigma), digestion of pancreases from male DBA1or B6 mice, followed by Ficoll purification using a methodroutinely used in our laboratory.34 Before in vitro and in vivoexperiments, islets were incubated overnight in nonadherent60-mm diameter Petri dishes containing 5 mL of RoswellPark Memorial Institute 1640 complete medium (10% fetalcalf serum, 11.2 mM glucose, 110 μg/mL sodium pyruvateand supplemented with 110 units/mL penicillin, 110 μg/mLstreptomycin, and 50 μg/mL gentamycin) with or without1.5 nM anti-TLR4.

Cell Apoptosis

Aliquots of 50,000 islet cells were cultured 1 hr at 37°Cin CMLR 10% fetal calf serum in Cunningham chambersprecoated 1 hr with 0.1 mg/mL poly-L-lysine, followed by24 hr in the presence or absence of LPS (0.5 μg/mL; Sigma)or anti-TLR4 mAb, and fixation in 4% paraformaldehyde.

Terminal deoxynucleotide tranferase-mediated dUTP nick-end labeling staining was performed with the In Situ CellDeath Detection Kit (Roche, Rotkreuz, Switzerland). Cells

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34 Transplantation ■ January 2015 ■ Volume 99 ■ Number 1 www.transplantjournal.com

were stained by immunofluorescence for insulin and withHoechst 33342 (Sigma). Quantification of mononucleosomesand oligonucleosomes present in the cytoplasm of apoptoticcells was performed using the cell death detection ELISAPLUSkit (Roche).

Cell Extraction, Mixed Lymphocytes Reactions andMixed Lymphocyte-Islet Reactions

Human blood was taken from healthy volunteers and PBMCwere obtained by centrifugation for 20 min at 2000 rpm onHistopaque-1077 (Sigma).

Cells from mesenteric lymph nodes or spleens were extrac-ted by manual structural destruction and red cell lysis usingACK lysing buffer.

For MLIR, aliquots of 25 IEQ were seeded with 500,000human PBMC or mouse lymph node cells, in MilliporeMultiscreen-IP 96 wells plate, precoated with captured IFN-γantibody (human and mouse IFN-γ ELISPOT Ready-SET-Goassay kits, eBiosciences), in a total volume of 200 μL RoswellPark Memorial Institute 1640 complete medium supple-mented with 0.5 mM β-mercaptoethanol and MEM nones-sential amino acid solution 1× (Sigma), in the presence ofanti-TLR4 or isotype control mAb. As a control, cells wereseeded without islets.

After 3 days, cells and islets were transferred to non-adherent 96wells plates. ELISPOTmembranes were revealedaccording to manufacturer recommendations. Spots werecounted using an automated immunospot analyzer (CellularTechnology Ltd, Bonn, Germany).

Reaction between cells and islets was continued duringfour additional days before cell proliferation measurement.Cells were separated from islets by centrifugation for 1 minat 1000 rpm, and permeabilized using the Foxp3 StainingBuffer Set (eBiosciences). Cells were then labelled with fluo-rescein isothiocyanate anti-Ki-67 antibody. Data acquisitionand analysis was done on a FACSCalibur flow cytometer(BDBiosciences, Allschwil, Switzerland).

For MLR, 500,000 lymph node cells were seeded with500,000 irradiated (3500 Rad) splenocytes from B6, DBA1,or C3H. Cell culture, staining and analysis were conductedas for MLIR.

Islet Transplantation

Single-injection streptozotocin (200 mg/kg intraperitone-ally) was used to induce diabetes in B6 recipient mice beforeislet transplantation. Diabetes was defined as nonfastingblood glucose levels greater than 18.0mM for 2 ormore con-secutive days. Islets were transplanted under the kidney cap-sule after overnight culture at 37°C/5% CO2. For syngeneicmarginal mass transplantation experiments, 150 syngeneicIEQ were transplanted as described.22 For allogeneic trans-plantation experiments, 600 IEQ isolated fromDBA1donorswere transplanted as described.34 In syngeneic marginal massexperiments,micewere injected intraperitoneally twiceweeklywith 500 μg anti-TLR4 or vehicle (phosphate-buffered saline[PBS]), from days 0 to 28 after transplantation. Graft functionwas defined as nonfasting blood glucose less than 16 mMfor longer than 5 days. In allogeneic experiments, micewere injected intraperitoneally twice weekly with 500 μganti-TLR4 or control mAb, or vehicle, from days 0 to 28after transplantation. Graft rejection was defined as blood

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glucose greater than 18 mM. Three days after graft rejectionor after 100 days graft function, graft-bearing kidneyswere harvested, and fixed in formalin for 24 hr and embed-ded in paraffin or snap-frozen and embedded in optimal cut-ting temperature compound (Sakura Finetek Europe B. V.,Zoeterwoude, The Netherlands) prior to being sectionedfor histology. Lymph node and bloodmononuclear cells wereextracted and used immediately in MLR or MLIR.

Histology and Immunohistochemistry

For insulin and glucagon costaining, paraffin-embedded kid-neys were sectioned at 5 mm, and sections were deparaffinized,rehydrated with a series of alcohol solutions of decreasingconcentrations, and washed with PBS. Then, sections werepermeabilized (0.1% triton-X100, 20 min), preincubated(30 min, room temperature) in 0.5% BSA in PBS and ex-posed 2 hr to guinea pig anti-insulin (1:500) and rabbitantiglucagon (1:150) antibodies. After rinsing, sections wereexposed 1 hr to Alexa 488-conjugated anti-guinea pig(1:500) and Alexa 555-conjugated antirabbit antibodies(1:500; Invitrogen, Basel, Switzerland). Nuclei were stainedwith Hoechst 33342 (Sigma).

For CD4, CD8, or FoxP3 and insulin costaining, optimalcutting temperature-frozen kidneys were cryosectioned at5 mm, and sections treated 10 min with ethanol at −20°C.Sections were rinsed with PBS, preincubated (30 min, roomtemperature) in 0.5% BSA in PBS and exposed 2 hr toa guinea pig anti-insulin antibody (1:500) mixed with ratanti-CD4 (1:20, Pharmingen, Allschwil, Switzerland), ratanti-CD8 (1:20; AbDSerotec, Kidlington, UK), or rat anti-Foxp3 (1:10, eBioscience, Vienna, Austria) antibodies. Afterrinsing, sections were exposed 1 hr to Alexa 488-conjugatedanti-guinea pig (1:500) and Alexa 555-conjugated antirat an-tibodies (1:300; Invitrogen, Basel, Switzerland). Nuclei werestained with Hoechst 33342 (Sigma).

Skin Grafts

Ventral side skin was harvested from DBA1 or C3H earsand transplanted as described35 on a dorsal graft bed in B6recipients. Rejection was monitored by necrotic area mea-surement. Rejection was defined when 80% or higher necro-sis was seen.

Presentation of Data and Statistical Analysis

Data are presented as mean±SEM. Levels of significancefor differences between groups were assessed by two-tailedStudent t test or analysis of variance test for unpaired groups.Graft survival and diabetes reversal curves were generated byKaplan-Meyer analysis and differences assessed using theMantel-Cox log-rank test. All statistical analyses were doneusing the GraphPad PRISM software (Graphpad Softwares,La Jolla, CA). Significance level was set at P less than 0.05.

ACKNOWLEDGMENTSThe authors thank Florentina Naville, Solange Masson,Nadine Pernin and David Matthey-Doret for their excellenttechnical assistance.

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