doi:10.1182/blood-2012-08-452037Prepublished online April 11, 2013;2013 121: 4303-4310   

 Lise Pasquet, Jean-Yves Douet, Tim Sparwasser, Paola Romagnoli and Joost P. M. van Meerwijk immunotherapy involves host Foxp3-expressing T cellsLong-term prevention of chronic allograft rejection by regulatory T-cell

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Regular Article

IMMUNOBIOLOGY

Long-term prevention of chronic allograft rejection by regulatory T-cellimmunotherapy involves host Foxp3-expressing T cellsLise Pasquet,1-3 Jean-Yves Douet,1-3 Tim Sparwasser,4 Paola Romagnoli,1-3 and Joost P. M. van Meerwijk1-3

1Institut National de la Sante et de la Recherche Medicale, U1043, Toulouse, France; 2Centre National de la Recherche Scientifique, U5282, Toulouse, France;3Universite de Toulouse, Universite Paul Sabatier, Centre de Physiopathologie de Toulouse Purpan, Toulouse, France; and 4Institut fur Infektionsimmunologie,

TWINCORE-Zentrum fur Experimentelle und Klinische Infektionsforschung GmbH, Hannover, Germany

Key Points

• Administration of donor-specific regulatory T cellsprevents chronic rejection ofBM and skin allografts in themouse.

• Injected regulatory T cellsinduce the emergence of hostregulatory T cells with similarspecificity thus ensuringpersistence of tolerance.

Despite the use of immunosuppressive drugs, chronic allograft rejection remains a

major hurdle in transplantation medicine. Induction of specific immunologic tolerance

to antigens expressed by the graft would avoid its eventual functional loss and the

severe side effects of paralyzing the immune system. We previously showed that donor-

specific regulatory T-lymphocytes prevent rejection of fully allogeneic bone marrow

(BM) grafts in mice. Thus generated hematopoietic chimeras then accepted skin and

heart allografts of the same donor. We noticed that injected regulatory T-cells (Tregs)

disappeared with time and investigated themechanisms involved in the nevertheless long-

term persistence of allograft tolerance. Using Tregs that can be depleted in vivo with

diphtheria toxin, we show that injected cells are required for induction but not for main-

tenance of tolerance to BM allografts. We observed progressive deletion of donor-

specific T-lymphocytes, accounting at least in part for maintenance of tolerance.

Toxin-induced depletion of administered as well as host Tregs did not affect hema-

topoietic chimerism but it led to rapid loss of skin allografts. Therefore, our data show

that newly generated host Tregs can prevent chronic allograft rejection. Long-lasting tolerance to allografts is thus achieved.

(Blood. 2013;121(21):4303-4310)

Introduction

Transplantation of allogeneic tissue and organs is severely hinderedby the vigorous reaction of the host’s immune system to the foreigngraft. Unless inhibited by immunosuppressive drugs, this allo-reactivity leads to rapid rejection. However, these medicaments donot prevent chronic allograft rejection. Moreover, the inducedparalysis of the immune system explains the increased incidence ofcancer and opportunistic infections. The lifelong use of drugs is alsoassociated with direct toxic side effects. Means to prevent chronicallograft rejection should therefore be developed.1,2

Despite the random nature of the somatic rearrangements thatdetermine the antigen-specificity of developing T lymphocytes, thesecells normally do not attack self-tissues. Several mechanisms areinvolved in this self-tolerance.3,4 For example, developing autospe-cific T cells die upon recognition of their peptide/major histo-compatibility complex (MHC) ligand expressed by thymic dendriticcells. Indeed, the T-cell repertoire developing in mice in whichthymic dendritic cells do not express MHC molecules is stronglyautoreactive in vitro and in vivo.5-8 Some autospecific T cellsnevertheless escape to the periphery.9,10 These cells are kept undercontrol by regulatory T-cell (Treg)-populations. The most exten-sively studied Treg express the co-receptor CD4 and Foxp3. Patientsandmice carryingmutations in the gene encoding this forkhead/wingedhelix transcription factor rapidly develop a lethal autoimmune

syndrome, best illustrating the central role of Treg in the control ofautospecific lymphocytes.11-13 Self-tolerance is therefore main-tained by at least 2 important mechanisms: deletion and regulation.Induction of genuine tolerance to donor antigens, presumably alsoneeds these 2 mechanisms.

Seminal work by Billingham et al14 showed that congenitalhematopoietic chimerism strongly favors acceptance of skin grafts ofthe same donor. Deletion of autospecific T cells presumably playeda major role in this phenomenon. However, unless chimerisminduced Treg, which has never been shown, it appears unlikely that itwill lead to full tolerance to the skin grafts. Indeed, despite thesubstantial delay in acute rejection, chronic rejection is not preventedby hematopoietic chimerism (see review by Pasquet et al15). We andothers, therefore, assessed the capacity of Treg to induce long-lastingtransplantation tolerance in the mouse. Donor-specific Treg wereadministered to sublethally irradiated host mice at the time of bonemarrow (BM) transplantation. Thus, the hosts accepted the fullyallogeneic BM grafts.16,17 When these hematopoietic chimeras weregrafted with skin or heart from the same donor, transplants survivedfor at least 100 days and chronic rejection was fully prevented.The combination of the induction of hematopoietic chimerismand the administration of Treg of appropriate specificity, therefore,appeared to ensure full tolerance to the grafts.18 Separately, these 2

Submitted August 23, 2012; accepted April 5, 2013. Prepublished online as

Blood First Edition paper, April 11, 2013; DOI 10.1182/blood-2012-08-452037.

The online version of this article contains a data supplement.

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approaches also strongly favor acceptance of allografts,19,20 butbest results were obtained by associating them.

Whereas, in absence of rejection, hematopoietic chimerism ismaintained by self-renewing stem cells, little is known about the lifeexpectancy of administered Treg. When injected into mutant animalsin which production of Treg is impaired, Treg can persist overprolonged periods.21 In our earlier work we showed that injectedTreg survived substantially longer in the presence of hematopoieticcells for which they were specific than in their absence.18 However, 9weeks after injection, their representation in the blood had droppedsubstantially and it was unclear if these cells would be home totissues or entirely disappear. Therefore, it was important to assess ifpersistence of administered Treg is required for long-term mainte-nance of tolerance to the graft. Here we address this issue by firstquantifying the persistence of injected Treg in different tissues. Wethen actively eliminated these cells in vivo and monitored survival ofBM and skin allografts. Given we observed that injected Tregbecome dispensable for persistent tolerance to the grafts, we alsoinvestigated mechanisms involved in the long-term prevention ofchronic rejection.

Methods

Mice

Mice were purchased from the Centre de Recherche et d’Elevage Janvier (LeGenest St. Isle, France). C57BL/6 (B6) Thy1.1 and DEREGmice22 were bredin our animal facility. All experiments involving animals were performed incompliance with relevant laws (authorization #31 09 555 45), and institutionalguidelines were approved by the ethics committee (MP/01/40/11/06).

Antibodies

Anti (a)H-2Kb-FITC or -biotin (AF6-88.5); aH-2Kd-PE (SF1-1.1), aH-2Kk-PE(36-7-5); aCD4-Pacific Blue (RM4-5); aCD8-Pacific Blue (53.6.7);aCD3-Pacific Orange (500A2); aCD25-APC (PC61); aVb3, Vb4, Vb5,Vb6, and Vb14-biotin (BD Biosciences, Heidelberg, Germany); aNK-FITC (DX5); aCD4-PECy7 (GK1.5); aThy1.1-APC (HiS51) and aB220-APC (RA3-6B2); aCD25-PE (PC61.5); purified F4/80 (BM8) and aFoxp3mAb (FJK-16s); Streptavidin-eFluor710 (eBioscience, San Diego, CA).Hybridoma supernatants of aFcgRII/III (2.4G2), aCD8 (53.6.7), aMHCclass II (M5/114.15.2), and aThy1.2 (AT83).

Purification and in vitro culture of CD41CD251 Treg

CD41CD251 splenic T cells were purified and cultured with g-irradiatedsplenocytes as previously described.16 Cell purity was checked by flowcytometry on a FACSCalibur (BD Bioscience, San Jose, CA) and wasroutinely >95%.

BM and skin transplantation

Skins and T-cell depleted BM were transplanted as previously described.18

Skins were considered rejected if >70% of the surface was necrotic.

In vivo depletion of Treg

Chimeras were intraperitoneally injected with 1.5 mg Diphtheria Toxin(Sigma-Aldrich, St. Quentin Fallavier, France) diluted in PBS on 2 consecutivedays.

Flow cytometry

Cell suspensions were stained using saturating concentrations of antibody.Acquisition was performed on an LSRII or a FORTESSA cytometer (Becton

Dickinson, Franklin Lakes, NJ) and data were analyzed using FlowJosoftware (Tree Star, Ashland OR).

Histological analysis

Skin biopsies were fixed in 10% buffered formalin and embedded inparaffin. Sections were stained as indicated. F4/80 and aFoxp3 antibodybinding was revealed using arat Ig-biotin (Dako, Glostrup, Denmark) andstreptavidine-HRP (Vector Laboratories, Berlingame, CA).

Statistical analysis

Statistical significance was determined by using Student t test with Bonferronicorrection.

Results

Administered Treg wane away with time

We previously showed that donor-specific Treg durably preventedrejection of semi and fully allogeneic BM allografts.16 To assess ifadministered Treg need to persist to maintain tolerance to the donor,we set up an experimental system in which Treg can be deleted invivo. DEREG mice carry a transgenic bacterial artificial chromo-some construct in which theFoxp3-promoter drives expression of thediphtheria toxin receptor and enhanced green fluorescent protein(eGFP).22 Practically all CD41CD25high Treg in C57BL/6 (B6)DEREG mice express eGFP (Figure 1A). We isolated CD41

CD25high cells from spleens of transgenic animals (Figure 1B, leftpanel) and cultured them with (donor x host)F1 (ie, [CBA3 B6]F1)mice-derived–irradiated splenocytes as stimulators in presence ofhigh levels of interleukin-2, required for the proliferation of Treg.Semi-allogeneic F1 splenocytes were used because they presentalloantigens via the direct and indirect pathways. This allows for theexpansion of Treg specific for indirectly presented alloantigensrequired for prevention of chronic rejection of skin and heartallografts.18 After a 2-week culture period, Foxp3-expression, asassessed by eGFP fluorescence, was maintained (Figure 1B, rightpanel). We then used these donor-specific Treg to protect a BMallograft. Recipient B6 mice were sublethally irradiated (5 Gy g),reconstituted with T cell-depleted CBA BM, and simultaneouslyinjected with the in vitro cultured DEREG Treg. Thus, the fullyallogeneic BM graft was protected from rejection by the recipient’simmune system for more than 100 days (Figure 1C). Wild-type(WT) and DEREG Treg were equally efficient in inducing toleranceto the CBABM allograft (ie, similar levels of hematopoietic chimerismwere observed). In protected mice administered DEREG Treg wereclearly visible among blood CD41 T lymphocytes (Figure 1D).However, DEREG Treg waned away with time and after 70 daysthey were no longer visible in peripheral blood mononuclearcells (PBMC) of the chimeras (Figure 1D-E). At this time, also inspleen, lymph nodes, and BM, administered Treg had almostcompletely disappeared (Figure 1F).

Diphtheria toxin-mediated in vivo depletion of Treg

These data suggested that persistence of injected donor-specific Tregis not required for maintenance of tolerance to the BM allograft.However, at 70 days after injection, a very small number ofDEREG Treg still remained detectable, especially in BM(Figure 1F). Therefore, we cannot formally conclude that adminis-tered Treg no longer play a role in protection of the graft. To directlyaddress this question, next we depleted administered DEREG Treg

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by injection of diphtheria toxin. Preconditioned WT B6 mice weregrafted with CBA BM and simultaneously injected with in vitrocultured donor-specific WT or DEREG Treg. Diphtheria toxin wasinjected 4 weeks after BM transplantation. Two and 6 weeks aftertoxin injection, administered DEREG (ie, Foxp3-eGFP expressing)Treg were no longer detected in the spleen, BM, lymph nodes,and PBMC (Figure 1G). These results were confirmed using anexperimental setup in which administered DEREG Treg could betraced using the allelic marker Thy1.2. Again, upon administration ofdiphtheria toxin, we observed a very efficient depletion of admin-istered Treg (supplemental Figure 1). Therefore, the injection ofdiphtheria toxin very efficiently depleted administered DEREGTreg.

Administered Treg are required for induction, but not

maintenance, of tolerance to MHC mismatched BM allografts

We next assessed if persistence of administered Treg is required formaintenance of tolerance. WT B6 mice were preconditioned, graftedwith CBA BM, and simultaneously injected with donor-specificDEREG Treg. Mice injected with diphtheria toxin at 1 week afterBM grafting did not develop hematopoietic chimerism (Figure 2A).This effect was due neither to a nonspecific effect of the toxin (miceinjected with WT Treg and treated with toxin developed chimerism,not shown) nor to an intrinsic defect of DEREG Treg (mice injectedwith DEREG Treg but not treated with toxin developed chimerism)(Figure 2A). Therefore, administered Treg are required during theinitial phases of tolerance induction. These data also confirmed thatinjection of diphtheria toxin efficiently depleted injected DEREGTreg.

Next we performed similar experiments but injected diphtheriatoxin at 2 or 4 weeks after BM grafting and Treg injection. In bothcases, hematopoietic chimerism developed normally and persistedfor at least 100 days (Figure 2B-C). No significant difference inchimerism was observed between the groups injected with WT orDEREG Treg, treated or not with diphtheria toxin. Therefore, it

appears that 2 weeks after transplantation, administered Treg are nolonger required for maintenance of tolerance to the BM allograft.

After depletion of administered Treg, tolerance to BM allografts

appears maintained by deletion of donor-specific T cells, not by

host Treg

We then assessed which mechanisms were involved in thetolerance to BM allografts that persisted despite depletion ofadministered Treg. First, we wished to investigate what happens to

Figure 1. Administered Treg wane away with time. (A) DEREG splenocytes were analyzed by flow cytometry. CD25 vs Foxp3-eGFP expression on electronically gated

CD41 cells. (B) Expression of CD25 vs Foxp3-eGFP by purified DEREG Treg was analyzed before (left) and after (right) in vitro culture with donor-type irradiated splenocytes

as stimulators. Dot plots are representative of 4 experiments. Numbers indicate percentages within indicated electronic gates. Sublethally irradiated B6 WT mice were grafted

with CBA BM under cover of 2.106 DEREG Treg in vitro cultured with (CBA 3 B6)F1 antigen-presenting cells. (C) Hematopoietic chimerism in PBMC and percentage of

Foxp3-eGFP1 cells among CD41 T lymphocytes in (D-E) blood and (F) spleen, lymph node (LN), and BM was assessed at the indicated time points. Indicated are mean

values 6 SD (n $ 3, 4 independent experiments). (G) Four weeks after BM grafting, chimeric mice were left untreated (white bars) or received (black bars) 2 consecutive

injections of diphtheria toxin (DTx). Percentage of Foxp3-eGFP1 cells among CD41 T lymphocytes was analyzed in spleen, lymph node (LN), BM, and PBMC. Indicated are

mean values 6 SD (n 5 5, 2 independent experiments).

Figure 2. Administered Treg are dispensable for persistence of tolerance to

BM allografts. Sublethally irradiated B6 WT mice were grafted with CBA BM

coinjected with 2.106 DEREG or WT Treg in vitro cultured with (CBA 3 B6)F1

antigen-presenting cells. Treg depletion by diphtheria toxin (DTx) injection was

performed 1 (A), 2 (B), or 4 (C) weeks after transplantation. Allograft survival was

followed up to 100 days. Shown is the percentage of donor cells among PBMC, as

assessed by flow cytometry, mean values6 SD (n$ 5, 4 independent experiments).

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donor-specific T lymphocytes. To track such cells, we chose to useDBA/2 donors. These mice present a large number of mammarytumor virus-encoded superantigens by their I-Ed MHC class IImolecules. T cells expressing several T cell receptor (TCR) Vbsegments, among which Vb3, Vb5, Vb6, but not Vb4 and Vb14,recognize these superantigens.23 Preconditioned B6 (Thy1.2) micewere grafted with T cell-depleted DBA/2 BM and injected withdonor-specific B6 Thy1.1 Treg. The TCR Vb repertoire of host(ie, Thy1.21 H-2Kb1) T cells was analyzed in spleen andthymus of these chimeras. In sublethally irradiated B6 mice thathad received a DBA/2 BM in absence of Treg, and therefore thatrapidly rejected the allograft, the TCR Vb repertoire in the spleendid not change much during the 28-day follow-up period and wassimilar to that observed in B6 → B6 control chimeras (Figure 3A).In contrast, in B6 mice that had received a DBA/2 BM allograftand donor-specific Treg, and that accepted the allograft and becamechimeric, superantigen-specific TCR Vb3, 5, and 6-expressingCD41 T cells gradually disappeared and reached levels found inDBA/2 → DBA/2 control chimeras by 28 days (Figure 3A).Similar results were obtained for the thymus (Figure 3B). Therepresentation of CD41 T cells expressing Vb4 (Figure 3A-B)and Vb14 (not shown), which do not recognize donor-encodedsuperantigens, was similar in spleen and thymus of all 4 types of

chimeras studied. Peripheral and central deletion of donor-specificCD41 T cells, therefore, was gradually established in the chimerasand most surely contributed to the maintenance of tolerance afterdepletion of administered Treg.

We also investigated if host Treg played a role in the maintenanceof tolerance to the BM allograft after depletion of administered Treg.To this end, we preconditioned B6 DEREG hosts and grafted themwith DBA/2 BM coadministered with donor-specific DEREGTreg. Injection of diphtheria toxin into these mice would depleteadministered, as well as host, Treg. Rapid reconstitution of newlydeveloping Treg avoids autoimmune pathology.22 As expected,depletion of administered plus host Treg (but not host Tregonly, supplemental Figure 2) at 1 week after reconstitution led torejection of the BM allograft (Figure 4A). Injection of diphtheriatoxin at 2 or 4 weeks did not lead to rejection of the allografts(Figure 4B-C). These results strongly suggest that host Treg are notinvolved in the maintenance of tolerance to the BM allografts afterdepletion of administered Treg.

Persistence of administered Treg is not required for tolerance

to skin allografts

We next investigated if persistence of injected Treg was requiredfor maintenance of tolerance to fully allogeneic skin grafts. WT B6

Figure 3. BM allografts induce deletion of donor-specific T cells. Sublethally

irradiatedB6micewere graftedwithDBA/2BMcoinjected (or not) with 2.106 Thy1.11Treg

in vitro cultured in presence of DBA/2 antigen-presenting cells. Control syngeneic

chimeras were realized by reconstituting sublethally irradiated B6 and DBA/2 mice with

syngeneic BM. TCR-Vb3, Vb5, Vb6-expressing CD41 T cells are specific for super-

antigens presented in DBA/2 (but not B6) mice. The percentage of Vb1 cells among host

CD41 T cells was assessed by flow cytometry in the spleen (A) and thymus (B) at the

indicated time points after reconstitution. Vb4-expressing T cells do not recognize

superantigens in DBA/2 mice. Indicated are mean values 6 SD (n $ 4, 4 independent

experiments). *P , .05, **P , .01, ***P , .001. ns, not significant, Student t test.

Figure 4. Host Treg are not required for persistence of BM tolerance.

Sublethally irradiated DEREG or WT mice were grafted with DBA/2 BM under cover

of 2.106 DEREG Treg in vitro cultured in presence of (B6 3 DBA/2)F1 antigen-

presenting cells. Treg depletion, by diphtheria toxin (DTx) injection, was performed

1 (A), 2 (B), or 4 (C) weeks after transplantation. BM allograft survival was assessed

by flow cytometry (illustrated in left panels) up to 10 weeks after grafting by analyzing

the percentage of allogeneic cells in PBMC (quantitative assessment in right panels).

Indicated are mean values 6 SD (n $ 4, 4 independent experiments).

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hosts were preconditioned and reconstituted with DBA/2 BM.DEREG Treg in vitro expanded using (donor3 host)F1 (ie, [DBA/2 3 B6]F1) antigen-presenting cells, and therefore specific fordirectly and indirectly presented alloantigens, were injected withthe BM allograft. Thus generated hematopoietic chimeras weregrafted with skin from the same donor strain 6 weeks after BMreconstitution. Grafts were monitored for macroscopic signs ofrejection and, if no rejection was observed at the end of the 100-day observation period, they were taken for histologic analysis ofchronic rejection.

In a first experiment, we depleted administered DEREG Tregat 10 weeks after BM reconstitution (ie, at 4 weeks after skingrafting). During the 100-day observation period, we did notobserve any macroscopic signs of rejection of the skins(Figure 5A-B, upper panels). Histologic analysis of the graftstaken at 100 days after skin grafting did not reveal any signs of(chronic) rejection either (Figure 5C, upper panels). Mostimportantly, we did not see infiltration by mononuclear cells(eg, macrophages) or by eosinophils, previously observed inchronic rejection of skin allografts.18,24 Therefore, it appears

that once the skin allograft is in place, administered Treg aredispensable for the maintenance of tolerance.

We next investigated if administered Treg can be depleted beforeskin grafting. Diphtheria toxin was injected 4 weeks after BMgrafting. Skin from the same donor strain was transplanted 2 weekslater. During the 100-day observation period, no macroscopic signsof rejection were observed in 9 of 11 thus treated mice (Figure 5A-B,middle panels) and histologic analysis of the grafts did not reveal anysigns of chronic rejection (Figure 5C, middle panels).

Host Treg are required for maintenance of skin

allograft tolerance

Central and peripheral deletion, as shown in Figure 3, may beinvolved in the continued protection from chronic rejection of theskin grafts after depletion of injected Treg. We wished to investigatethe possibility that host Treg also played a role. B6 DEREG micewere preconditioned and grafted with fully allogeneic DBA/2 BMunder cover of DEREG Treg specific for directly and indirectlypresented alloantigens. Four weeks later, administered and host

Figure 5. Upon depletion of administered Treg, host Treg are required for acceptance of skin allografts. (A-C) Sublethally irradiated WT B6 mice were grafted with

DBA/2 BM under cover of 2.106 DEREG or WT Treg in vitro cultured in presence of (B6 3 DBA/2)F1 antigen-presenting cells. Mice were left untreated or received diphtheria

toxin (DTx) 4 or 10 weeks after reconstitution, as indicated. Skin allografts were placed 6 weeks after BM grafting. Control mice were not injected with Treg at the time of BM

transplantation. (A) Skin allograft survival was monitored daily by assessment of macroscopic signs of rejection. Graft-survival curves for indicated conditions (4 independent

experiments). Representative macroscopic (B) and histologic (C) features of skins 100 days after transplantation. (HE, hematoxylin and eosin; F4/80, immunohistocheministry

with antibody to the macrophage marker F4/80; Luna, Luna’s eosinophil stain) Scales represent 200 mm for HE and F4/80 staining and 20 mm for Luna staining. (D-E)

Sublethally irradiated DEREG mice were grafted with DBA/2 BM under cover of DEREG Treg in vitro cultured in presence of (B6 3 DBA/2)F1 stimulator cells. Injected Treg

were depleted or not by diphtheria toxin (DTx) injection 4 weeks after BM grafting. DBA/2 Skin transplantation was performed 2 weeks later. Skin allograft survival was

monitored daily. (D, left panel) Skin allograft survival during the 100-day observation period. (D, right panels) In toxin-treated mice, rejection was macroscopically clearly

visible (2 independent experiments). (E) Chimerism was analyzed in PBMC 2 days before skin transplantation and immediately after rejection in toxin-treated mice.

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Foxp31 Treg were depleted by injection of the diphtheria toxin.Treg depletion did not change the hematopoietic chimerism(Figure 4C). Two weeks later, DBA/2 skins were transplanted onthese chimeras. Grafts were monitored for signs of rejection duringa 100-day observation period. Depletion of administered and hostTreg in DEREG hosts led to rapid rejection, which was clearly,macroscopically visible (Figure 5D right panel). All grafts onDEREG hosts (5 of 5 performed) were rejected by 23 days(Figure 5D, left panel). Importantly, in all these mice the BMallograft remained in place (Figure 5E), and the loss of tolerancewas therefore specific for the skin grafts. These data show that hostTreg were involved in the continued prevention of skin rejectionafter depletion of injected Treg. They also demonstrate that DEREGhost Treg are effectively depleted by injection of diphtheria toxin inour experimental model and thus confirm that host Treg are notrequired for maintenance of the allogeneic BM graft (Figure 4).Combined, our data demonstrate that administered Treg hadtransmitted their role in the protection of fully allogeneic skingrafts to host Treg that became sufficient for the prevention ofchronic rejection.

Host Treg acquire graft-protective capacities similar to that of

administered Treg

We previously showed that Treg specific for directly presenteddonor antigens (ie, presented by donor MHC molecules), protectedskin, and heart allografts from acute but not chronic rejection. Bycontrast, Treg specific for indirectly presented donor antigens(ie, presented by host MHC molecules) fully prevented chronicrejection.18 We wished to investigate if the capacity to protect fromacute vs chronic rejection is transmitted from administered to hostTreg. When Treg were specific for directly and indirectly presenteddonor antigens, chronic rejection of the skin allografts was prevented,even after depletion of administered Treg (Figure 5). We thenperformed similar experiments but used Treg specific for directlypresented alloantigens only. These cells protected skin grafts fromacute rejection (supplemental Figure 3A) but failed to prevent chronicrejection, thus confirming our previously reported results (supple-mental Figure 3B-C). When administered DEREG Treg specific fordirectly presented donor antigens were depleted at 4 weeks post-BMgrafting, subsequently transplanted skin grafts did not undergo acuterejection but clear signs of chronic rejection were observed(supplemental Figure 3A-C). It therefore appears that host Treg thatprotected skin grafts from rejection had acquired the capacity toprotect from acute and/or chronic rejection similar to that ofadministered Treg. In both cases some sparse host Treg could befound inside the skin grafts, suggesting that they may act locally(supplemental Figure 4).

Discussion

Previously published data from our laboratory and those of othersdemonstrated the potential of Treg to induce tolerance to BM, skin,and heart allografts in mice.17-19,25 Since injected Treg vanish withtime, it was important to study if tolerance would persist in theirabsence. Here we show that injected Treg are essential for theinduction of tolerance to BM allografts. By contrast, once tolerance isestablished, its maintenance no longer requires injected Treg. Micethat had accepted the BM allograft were also tolerant to subsequentskin grafts from the same donor strain. Injected Treg were not

required for acceptance of the skin grafts. However, when injectedTreg were depleted, host Treg became critical for skin graft accep-tance. Therefore, it appears that the Treg-mediated immunotherapyagainst skin graft rejection leads to development of host Treg-mediated tolerance. This mechanism will ensure long-term toleranceto allografts.

Administration of in vitro expanded Treg specific for directlypresented donor antigens leads to permanent protection of BMallografts in mice.16 Treg-mediated immunotherapy may therefore beenvisaged for clinical protocols in which administration of donorstem cells or BM is used to promote tolerance to kidney allografts, forexample. Treg administration may allow for induction of stablehematopoietic chimerism, not detected in the clinical protocols, andthus further improve results.15,26,27 Importantly, our data demonstratethat once the administered Treg had induced tolerance, its main-tenance no longer required the presence of these cells. The long-termpersistence of donor chimerism also indicates that progenitor cells arenot rejected. Deletion of donor-specific T cells upon interaction withdonor hematopoietic cells, as also shown by others before, probablyplays an important role in this persistent, self-sustained, tolerance.28

Depletion of injected and host Treg at 2 weeks did not lead torejection of the BM allograft, despite the presence of some donor-specific T cells. Therefore, it appears that the latter cells must beanergic29 or kept under control by regulatory cells not expressingFoxp3. Whatever the explanation, host Foxp3-expressing Treg arenot involved.

In our experimental approach, skin grafts are placed after hostsaccepted BM grafts. The hematopoietic chimerism alone isinsufficient for full tolerance to the skin grafts, which is consistentwith the literature.15 Here we show that in chimeras the administeredTreg are not required for acceptance of skin grafts. However, afterdepletion of injected Treg, host Treg became critically involved inthe prevention of skin allograft rejection. Despite the fact that theyrejected the skin grafts, recipients in which administered and hostTreg were depleted did not reject the BM allograft. T cells specificfor tissue-specific antigens, which apparently are not deleted (orrendered anergic) by hematopoietic cells, therefore, presumablyrejected the skin grafts. These data confirm that hematopoieticchimerism is insufficient for maintenance of tolerance to allogeneicskin in this model, and that Treg are required. More importantly,our data demonstrate that injected Treg had transferred theirtolerogenic role to host Treg.

Transmission of tolerogenic activity from one T-cell population toanother one is known as “infectious tolerance” (see reviews by Kendaland Waldmann30 and Cobbold et al31). Allograft tolerance can beinduced by blockade of CD4 and CD8 coreceptors, for example.32,33

Treg from tolerant mice transferred tolerance to a secondary host.34

When the transferred donor T cells were subsequently depleted,tolerance persisted, and it is thought that host Foxp31 Treg areinvolved.35 Using a very different experimental system, we directlyshow here that administered Treg had transferred their tolerogenicactivity to host Treg. In this report, “host” Treg are defined as thosecells that can be depleted with diphtheria toxin. Even at later timepoints, donor BM-derived CD41 T cells represented only a smallminority of the total pool of CD41 T cells in our experimental model(supplemental Figure 5), potentially explaining their manifest in-capacity to protect the skin allografts.

Treg specific for directly presented donor-antigens protected skingrafts from acute but not from chronic rejection. This observation isconsistent with a role for donor-specific antibody (the generation ofwhich requires indirect recognition of donor antigens) in chronicorgan-graft rejection.36 When administered Treg were depleted, the

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host Treg involved in protection of the skin grafts from acuterejection still failed to prevent chronic rejection. Injection of Tregwith indirect donor-specificity protected from chronic rejection.When these Treg were depleted, the host Treg involved in protectionof the skin grafts still prevented chronic rejection. These observationsstrongly suggested that initially administered and emerging host Treghad similar specificities. This conclusion is consistent with datashowing that hemagglutinin-specific Treg can induce Treg with thesame specificity from responder T cells in vivo.37 We postulate thatinteraction of administered Treg with host cells presenting donorantigens generates a tolerogenic microenvironment in which locallyactivated conventional T cells differentiate into Treg. This may occurunder influence of TGF-b or depletion of essential amino acids, bothknown to induce Foxp3-expression.37-40 Formally, we cannot excludethe possibility that host Treg of appropriate specificity mayexpand or develop in the host thymus under influence of themixed hematopoietic chimerism. However, hematopoietic chime-rism alone fails to prevent chronic rejection, and the latter possibilityappears rather unlikely. These issues merit further investigation.

To our knowledge, this is the very first demonstration that Tregcan transfer their tolerogenic capacity to other Treg in hematopoieticchimeras in absence of other donor tissue. It strongly suggests theinvolvement of hematopoietic cells, probably dendritic cells, actingin secondary lymphoid organs. In any case, solid donor tissue is notrequired.

Finally, in previous reports it was shown that CD41CD251

Foxp31 Treg could mediate infectious transplantation tolerance.34,35

This conclusion was based on adoptive transfer experiments, andthe potential implication of other regulatory populations was notassessed. By in vivo depleting only Foxp31 Treg, we abolisheddevelopment of tolerance to skin grafts. Potential other regulatorypopulations were clearly insufficient for prevention of skin allograftrejection in our experimental model.

In conclusion, experimental Treg-mediated immunotherapyagainst acute and chronic rejection of skin allografts does not requirepersistence of administered Treg. A newly emerging host Tregpopulation fully prevents chronic rejection. This mechanism allowsfor long-term maintenance of tolerance. Administration of in vitro

manipulated Treg for the treatment of graft rejection or graft-versus-host disease for example, should turn out to be an attainable goal,41

and our results indicate that thus induced tolerance should be self-perpetuating.

Acknowledgments

The authors thank Drs Sylvie Guerder, Jean-Charles Guery, andOlivier Joffre for critical reading of the manuscript, and Drs Fatima-Ezzahra L’Faqihi-Olive and Valerie Duplan-Eche (Inserm U1043flow cytometry facility), the personnel of the Inserm US006Animalerie Exploration Fonctionnelle/histology facility (Dr TalalAl Saati, Florence Capilla, and Alain Marrot) and the personnel ofthe Inserm US006 Animalerie Exploration Fonctionnelle/CentreRegional d’Exploration Fonctionnelle et Ressources Experimentalesanimal facility, in particular, Maryline Calise, for expert technicalassistance.

This work was supported by institutional funds, the AgenceNationale pour la Recherche (ANR-08-BLAN-0187), the RegionMidi-Pyrenees (08004389), and the Agence de la Biomedecine(2007).

Authorship

Contribution: L.P., P.R., and J.P.M.v.M. designed experiments;L.P. and J.-Y.D. performed experiments and analyzed results; T.S.contributed transgenic mice; L.P., J.-Y.D., P.R. and J.P.M.v.M.analyzed the data; and L.P., P.R., and J.P.M.v.M. wrote themanuscript.

Conflict-of-interest disclosure: The authors declare no compet-ing financial interests.

Correspondence: Joost P. M. van Meerwijk, Inserm U1043, BP3028, 31024 Toulouse Cedex 3, France; e-mail: joost.van-meerwijk@inserm.fr.

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