doi:10.1182/blood-2006-01-0329Prepublished online March 7, 2006;2006 108: 390-399   

 Christopher H. Contag and Robert S. NegrinRobert Zeiser, Vu H. Nguyen, Andreas Beilhack, Martin Buess, Stephan Schulz, Jeanette Baker, calcineurin-dependent interleukin-2 production

regulatory T-cell function by+CD25+Inhibition of CD4

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TRANSPLANTATION

Inhibition of CD4�CD25� regulatory T-cell function by calcineurin-dependentinterleukin-2 productionRobert Zeiser, Vu H. Nguyen, Andreas Beilhack, Martin Buess, Stephan Schulz, Jeanette Baker, Christopher H. Contag,and Robert S. Negrin

CD4�CD25� regulatory T (Treg) cells con-trol immunologic tolerance and antitumorimmune responses. Therefore, in vivomodification of Treg function by immuno-suppressant drugs has broad implica-tions for transplantation biology, autoim-munity, and vaccination strategies. In vivobioluminescence imaging demonstratedreduced early proliferation of donor-derived luciferase-labeled conventional Tcells in animals treated with Treg cellsafter major histocompatibility complexmismatch bone marrow transplantation.

Combining Treg cells with cyclosporine A(CSA), but not rapamycin (RAPA) or myco-phenolate mofetil (MMF), suppressed Tregfunction assessed by increased T-cell pro-liferation, graft-versus-host disease(GVHD) severity, and reduced survival.Expansion of Treg and FoxP3 expressionwithin this population was lowest in con-junction with CSA, suggesting that cal-cineurin-dependent interleukin 2 (IL-2)production is critically required for Tregcells in vivo. The functional defect of Tregcells after CSA exposure could be re-

versed by exogenous IL-2. Further, theTreg plus RAPA combination preservedgraft-versus-tumor (GVT) effector func-tion against leukemia cells. Our data indi-cate that RAPA and MMF rather than CSApreserve function of Treg cells in patho-logic immune responses such as GVHDwithout weakening the GVT effect. (Blood.2006;108:390-399)

© 2006 by The American Society of Hematology

Introduction

Naturally occurring CD4�CD25� regulatory T (Treg) cells are critical inthe reduction of experimental acute graft-versus-host disease (aGVHD)1-3

but also provide immune privilege for malignant disease.4 Therefore,affecting Treg-cell function in vivo by different immunosuppressantdrugs has relevance for transplantation biology and may be useful toenhance antitumor vaccination strategies. We have previously shownthat Treg cells are capable of suppressing experimental aGVHD whilepreserving the beneficial graft-versus-tumor (GVT) effect.1 Thesefavorable effects and the observation that murine Treg cells sharefunctional characteristics with their human counterparts have fueledinterest in exploring cell-based immunotherapy in clinical trials tofurther investigate the capability of Treg cells to prevent aGVHD or treatautoimmunity; however, there is little information on the impact ofimmunosuppressant drugs on Treg-cell function.

Treg suppressor function has been demonstrated to be criticallydependent on the presence of interleukin 2 (IL-2)5-7 and activation byT-cell receptor (TCR) engagement.8,9 Recently, IL-2 signaling has beenshown to be critical for survival of FoxP3� Treg cells in vivo.10-12

We therefore compared the relevance of calcineurin-dependentIL-2 production13 versus mammalian target of rapamycin (mTOR)

pathway activity14 versus inosine monophosphate dehydrogenaseenzyme activity15 for Treg function in vivo, by using the commonlyapplied immunosuppressant drugs cyclosporine A (CSA), rapamy-cin (RAPA), and mycophenolate mofetil (MMF).

Using a major histocompatibility complex (MHC) class I and IImismatch bone marrow transplantation (BMT) model, we observedthat trafficking of luciferase (luc�)–expressing Treg cells was notaltered by the different immunosuppressant drugs and proliferationwas only slightly affected. However, CSA administration led tosignificantly reduced Treg function in vivo. Mechanistically wehave identified that CSA acts by reducing the production of IL-2because exogenous IL-2 can overcome reduced FoxP3� expressionand the functional defect in Treg cells induced by CSA.

Materials and methods

Mice

FVB/N (H-2kq, Thy-1.1), C57B/6 (H-2kb, Thy-1.1 or Thy-1.2), and Balb/cmice (H-2kd, Thy-1.2) were purchased from Jackson Laboratory (Bar

From the Division of Bone Marrow Transplantation, Department of Medicine,Stanford University, Stanford, CA; Division of Hematology and Oncology,Department of Medicine, Freiburg University Medical Center, Freiburg,Germany; Division of Biochemistry, Department of Medicine, StanfordUniversity, Stanford, CA; Department of Pathology, Technische UniversitatMunchen, Munchen, Germany; and Division of Pediatrics/Microbiology andImmunology, Department of Medicine, Stanford University, Stanford, CA.

Submitted January 25, 2005; accepted February 24, 2006. Prepublished onlineas Blood First Edition Paper, March 7, 2006; DOI 10.1182/blood-2006-01-0329.

Supported by grants from the National Institutes of Health (NIH; RO1CA0800065 and P01 HL075462). R.Z. is supported by the Dr Mildred-Scheel-Stiftung, Germany. V.N. is supported by 5 K08 AI060888 from the NIH. A.B. issupported by a Supergen Postdoctoral Fellowship from the AmyStrelzer-Manasevit Research Program (NMDP).

R.Z. designed and performed research, analyzed data, and wrote the paper;V.H.N. performed research and analyzed data; A.B. contributed new reagents,performed research, and analyzed data; M.B., S.S., and J.B. performedresearch and analyzed data; C.H.C. designed research and contributed vitalnew reagents and analytic tools; and R.S.N. designed research and helped towrite the paper.

Reprints: Robert S. Negrin, Center for Clinical Science Research, 269 WCampus Dr, Rm 2205, Division of Bone Marrow Transplantation, StanfordUniversity School of Medicine, Stanford, CA 94305; e-mail:negrs@stanford.edu.

The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is herebymarked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.

© 2006 by The American Society of Hematology

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Harbor, ME) or Charles River Laboratory (Wilmington, MA). Mice wereused between 6 and 12 weeks of age. Only sex-matched combinations wereused for transplantation experiments. The luciferase-expressing (luc�)transgenic FVB/N line was generated as previously described.16 Maleheterozygous luc� offspring of the transgenic founder line FVB-L2G85were used for all transplantation experiments. All animal protocols wereapproved by the University Committee on Use and Care of LaboratoryAnimals at Stanford University.

Immunosuppressive treatment

For in vivo studies, CSA (Bedford Laboratories, Bedford, OH) and MMF(Roche Laboratories, Nutley, NJ) were dissolved by suspension in phosphate-buffered saline (PBS). RAPA (Wyeth-Ayerst, Princeton, NJ) was dissolvedin carboxymethylcellulose sodium salt (C-5013; Sigma-Aldrich, St Louis,MO) and polysorbate 80 (P-8074; Sigma-Aldrich). RAPA stock solutionwas stored at 4°C in the dark in distilled water according to themanufacturer’s instructions. Injections were given intraperitoneally oncedaily and started on day 0. Dosage was adjusted to the body weight everyother day. Mycophenolic acid (MPA; Sigma-Aldrich), the active metaboliteof MMF, was used for in vitro experiments. Immunosuppressive treatmentwas continued until death or end of the observation period (day 100).

Flow cytometric cell purification and analysis

The following antibodies were used for flow cytometric analysis: unconju-gated anti-CD16/32 (2.4G2), CD4 (RM4-5), CD8� (53-6.7), CD25 (PC61),CD11c (M1/70), CD45R/B220 (RA3-6B2), H-2Kq (KH114), H-2Kd (34-2-12), Thy-1.1 (H1S51), Thy-1.2 (53-2.1), and Foxp3 (FJK-16). All reagentswere purchased from BD PharMingen (San Diego, CA) and eBiosciences(San Diego, CA). Staining was performed in the presence of purifiedanti-CD16/32 at saturation to block unspecific staining. Propidium iodide(Sigma, St Louis, MO) was added prior to analysis to exclude dead cells.Apoptosis was measured by annexin V staining. All analytic flow cytometrywas done on a modified dual laser LSRScan (BD ImmunocytometrySystems, San Diego, CA).

Cell isolation and sorting

Single-cell suspensions from cervical lymph nodes (cLNs), axillary lymphnodes (aLNs), inguinal lymph nodes (iLNs), mesenteric lymph nodes(mLNs), and spleens were enriched for CD25� cells after sequentialstaining with anti-CD25 PE (BD PharMingen) and anti-PE magnetic beadsusing the auto-magnetically activated cell sorting (autoMACS) system(POSSEL program, Miltenyi Biotec, Auburn, CA). CD25� cells were thenstained with anti-CD4 allophycocyanin and sorted on a MoFlow cell sorter(Becton Dickinson, Mountain View, CA) for the CD25high population(15%-20% of the enriched CD25� cells). This sorting strategy yielded 96%or more CD4�CD25�FoxP3� cells.

T-cell–depleted bone marrow (TCD-BM) was obtained through nega-tive depletion using anti-CD4 and anti-CD8 magnetic beads (MiltenyiBiotec). For transplantation of conventional and luc� T-cell subsets, splenicsingle-cell suspensions from FVB-L2G85 mice were enriched with CD4/CD8-conjugated magnetic beads using the autoMACS system (POS-SEL_D, Miltenyi Biotec). After enrichment, CD4� and CD8� T cellsreached more than 90% purity.

Real-time quantitative PCR for Foxp3

Total RNA was isolated from fresh cell pellets using the RNeasy MiniKit(Qiagen, Valencia, CA) and gDNA was eliminated by digestion with amodified proprietary DNase (DNA-free; Ambion, Austin, TX). Total RNA(500 ng) was mixed with dT16 primer in a volume of 11 �L, incubated at65°C for 10 minutes, and immediately put on ice. Following addition of100 U Superscript II reverse transcriptase (RT; Gibco, Carlsbad, CA)reverse transcription was performed for 2 hours at 42°C in 1 � RT reactionbuffer (Gibco), 10 �M DTT, 500 �M dNTP (Amersham Biosciences,Pittsburgh, PA) with 2.5 �M dT16 primer in a volume of 20 �L.Polymerase chain reactions (PCRs) were performed in a final volume of 20�L with cDNA prepared from 20 ng RNA and a final concentration of 1 �

SYBR Green PCR Master Mix (ABI, Foster City, CA) and 200 nM of eachprimer (sequences: FoxP3 forward, GGAGCCGCAAGCTAAAAGC; FoxP3reverse, TGCCTTCGTGCCCACTG; GAPDH forward, GTCCTGAAGTAT-GTCGTGGAGTCTAC; and GAPDH reverse, GGCCCCGGCCTTCTC).The reaction was run in an ABI 7700 Sequence Detection System with thefollowing cycling conditions: 50°C for 2 minutes, 94°C for 10 minutes, then40 cycles of 94°C for 15 seconds and 60°C for 60 seconds. For each gene astandard curve was prepared and triplicate measurements were performedfor each sample.

Immunofluorescence

Sorted cells were fixed for 10 minutes to slides (Sigma) with ice-coldacetone. Cells were incubated for 30 minutes in PBS containing 10% FCSand were incubated 30 minutes at 4°C with a monoclonal rat anti–mouse-Foxp3 antibody (FJK-16s, eBioscience) and after PBS washing with asecondary goat anti–rat Alexa Fluor 546 antibody (A11081, Invitrogen,Carlsbad, CA) for 30 minutes. Cells were washed with PBS and wereincubated for 5 minutes with 4,6-diamidino-2-phenylindole (DAPI; Molecu-lar Probes, Eugene, OR) for nuclei stain.

TNF-� and IFN-� ELISA

Serum was collected from Balb/c recipients on day 7 after transplantation.Enzyme-linked immunosorbent assays (ELISAs) were performed accord-ing to the manufacturer’s instructions (R&D Systems, Minneapolis, MN).Briefly, samples were diluted 1:2 to 1:5, and TNF-� or IFN-� was capturedby the specific primary monoclonal antibody (mAb) precoated on themicroplate and detected by horseradish peroxidase–labeled secondarymAbs. Plates were read at 450 nm using a microplate reader (model SpectraMax 190; Bio-Rad Labs, Hercules, CA). Recombinant murine TNF-� andmurine IFN-� (BD PharMingen) were used as standards. Samples andstandards were run in duplicate, and the sensitivity of the assays was 16 to20 pg/mL for each cytokine, depending on the sample dilution.

CFSE-based cell proliferation

For cell-proliferation analysis, T cells (1 � 107/mL) were resuspended inplain PBS and stained with Vybrant carboxyfluorescein diacetate, succinimi-dyl ester (CFDA SE) tracer kit (Molecular Probes) at a final concentrationof 5 �M for exactly 6 minutes at 37°C. Immediately after staining, cellswere washed in 5 volumes of ice-cold RPMI plus 10% fetal bovine serum(Gibco, Life Sciences, Grand Island, NY) twice, resuspended in PBS, andcounted before use in an in vitro assay or intravenous injection. Theproportion of CFSE� cells proliferating in vitro was calculated as previ-ously described.17

Mixed leukocyte reaction for Treg suppressor function

Treg cells (CD4�CD25high�H-2kb�Thy-1.1�) were incubated for 96 hourswith �-irradiated (30 Gy) antigen-presenting cells (APCs; CD11c�H-2kd�)and CFSE�-labeled conventional T (TCONV) cells (CD4�CD25�H-k2b�Thy-1.2�), with each population 2 � 105 cells. Cell division was monitored bylevels of CFSE dilution. Cultures were evaluated in triplicate in 96-wellflat-bottom plates in a total volume of 200 �L. Cells were cultured in RPMI1640 medium with 10% FCS, 10 mM N-2-hydroxyethylpiperazine-N�-2-ethanesulfonic acid (HEPES), 1% nonessential amino acids, 2 mML-glutamine, 100 U/mL penicillin, 100 �g/mL streptomycin (all fromGibco), and 5 �g/mL 2-mercaptoethanol (Sigma-Aldrich).

GVHD model

To induce aGVHD, Balb/c recipients received 5 � 106 TCD-BM cells onday 0 plus 1.6 � 106 T cells from FVB/N donors on day 2 after lethalirradiation with 800 cGy as described previously.18 Treg cells (8 � 105

CD4�CD25high) were coinjected with TCD-BM on day 0 resulting in a 1:2ratio (Treg/TCONV). Mice were kept on antibiotic water (sulfamethoxazole-trimethoprim; Schein Pharmaceutical, Danbury, CT) for the first 30 days.For conventional histology, mice were killed 5 and 15 days after celltransfer. Hematoxylin and eosin (H&E) staining of paraffin-embedded

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tissue sections was performed according to standard protocols. HistologicGVHD scoring was performed according to a previously publishedsystem19 by a pathologist (S.S.) who received coded paraffin-embeddedtissue samples of liver and small and large bowel and was blinded to thetreatment groups.

In vivo and ex vivo bioluminescence imaging

In vivo bioluminescence imaging (BLI) was performed as previouslydescribed.18 Briefly, mice were injected intraperitoneally with luciferin(10 �g/g body weight). Ten minutes later mice were imaged using anIVIS200 charge-coupled device (CCD) imaging system (Xenogen, Alameda,CA) for 5 minutes. Imaging data were analyzed and quantified with LivingImage Software (Xenogen) and IgorProCarbon (WaveMetrics, LakeOswego, OR).

Tumor model

To investigate GVT activity of transferred donor T cells, we usedA20-luc/yfp B-cell leukemia cells that have been previously demonstratedto migrate primarily to the bone marrow with secondary infiltration ofspleen and other lymphoid organs when injected intravenously at the timeof BMT.1,20 Animals were given injections with A20-luc/yfp-leukemia cells2 days prior to administration of the TCONV cells to allow the tumor to homeand establish. Tumor growth was analyzed by BLI, quantification ofyfp-expressing cells by fluorescence-activated cell sorting (FACS), andH&E histology.

Statistical analysis

Differences in animal survival (Kaplan-Meier survival curves) wereanalyzed by log-rank test. Differences in proliferation of TCONV cells,FoxP3 expression, GVHD histopathology score, and serum cytokine levelswere analyzed using the 2-tailed Student t test.

Results

In vitro effect of CSA, RAPA, and MPA on Tregsuppressor function

Alloantigen-activated Treg cells significantly reduced alloantigen-driven proliferation of CD4�CD25� TCONV cells (Figure 1A leftcolumn), consistent with previous reports of ex vivo activatedmurine Treg cells.21 Importantly, RAPA, MPA, the active metabo-lite of MMF, and CSA-exposed murine Treg cells maintaineddifferential degrees of suppressor function in vitro (Figure 1A rightcolumn). Treg cells derived from cultures containing CSA wereless suppressive as compared to Treg cells exposed to RAPA orMPA (Figure 1A right column). Importantly, the decreased level ofsuppression correlated with reduced FoxP3 mRNA levels in Tregcells exposed to CSA (Figure 1B). Our finding that allostimulationincreases FoxP3 expression is in line with results reported byFontenot et al, who showed an increase of FoxP3 expression inCD4�CD25� T cells after TCR ligation.11

Interestingly, reduced Treg function in the presence of CSA, asmeasured by percentage of undivided TCONV cells, could bereversed from 24% � 2.7% to 50% � 3.4% by the addition of IL-2to the primary stimulation culture (‡ in Figure 1A right column),which again correlated with FoxP3 mRNA expression (Figure 1B).To examine whether the Treg stimulation culture was depleted ofIL-2 in the presence of different immunosuppressants, the cytokinewas measured in the culture supernatants at various dosages.Whereas RAPA and MPA had only a minor impact on the IL-2 levelin the primary Treg activation culture, the cytokine was reduced

Figure 1. In vitro effects of CSA, RAPA, and MPA on Treg cells. In vitro effects of CSA, RAPA, and MPA on allostimulated Treg cells with respect to suppressor function(A), FoxP3 expression (B,D), and IL-2 levels in the primary coculture (C). (A) Fresh isolated C57B/6 Treg cells (CD4�CD25highH-2kb�) were incubated with Balb/c APCs (CD11cH-2kd) with CSA (100 ng/mL), RAPA (10 ng/mL), or MPA (50 ng/mL). Treg cells were reisolated by sorting for CD4�CD25highH-2kb� cells after 72 hours of primary coculture.Reisolated Treg cells were then incubated for 96 hours with �-irradiated (30 Gy) APCs (CD11c�H-2kd�) and CFSE-labeled TCONV cells (CD4�CD25�H-2kb�Thy-1.2�), witheach population 2 � 105 cells. Cell division was monitored by levels of CFSE dilution. The congenic markers Thy-1.1 and Thy-1.2 were used to distinguish between Treg andTCONV cells, respectively. Histograms show the FACS profile of CFSE� TCONV cells. Numbers of events in each cell division (n) are indicated below the respective peak. Thepercentage of undivided CD4�CFSE� cells in each culture condition is indicated next to the right upper corner of the respective histogram. One representative experiment of 4is presented. Allostimulated Treg cells suppress alloantigen-driven expansion of CFSE� TCONV (*) significantly more strongly than autostimulated Treg cells (�; P .027).CSA-exposed Treg cells are still suppressive, but significantly less as compared to RAPA (#P .031) and addition of IL-2 (50 IU/mL) to the activation culture restores Tregfunction partially (‡; P .037). (B) Relative FoxP3 mRNA expression level in Treg cells (H-2kb�) exposed to allogeneic APCs (H-2kd�) alone or in conjunction with RAPA (10ng/mL), MPA (50 ng/mL), CSA (100 ng/mL), or CSA (100 ng/mL) plus IL-2 or exposed to autologous APCs (H-2kd). The increase of FoxP3 expression in Treg cells duringallostimulation is reduced by CSA addition and restored by IL-2. Data represent means � SD of triplicates (**P .004; ***P .008; ****P .007). (C) IL-2 concentrationsmeasured by ELISA in supernatants from the following cocultures are depicted (y-axis): Treg cells (H-2kb) incubated with APCs (H-2kd) and CSA (f), RAPA (‚), or MPA (F) atdifferent concentrations (x-axis) after 72 hours. CSA but not RAPA or MPA reduces IL-2 levels in the Treg activation culture in a dose-dependent manner. (D) Histograms depictthe intracellular expression profile of FoxP3 in live-gated Treg cells exposed to medium, CSA (100 ng/mL), RAPA (10 ng/mL), or MPA (50 ng/mL) for 72 hours (filled histogram,unstained cells; open histogram, FoxP3 staining). The mean fluorescence intensity (MFI) for FoxP3 expression is significantly lower in Treg cells reisolated from CSA(213 � 24) as compared to cultures containing RAPA (412 � 42) or MPA (392 � 31; P .05).

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dose dependently in the presence of CSA (Figure 1C). In contrast toIL-2 the cytokines IL-7 and IL-15 could not reverse the CSA effect(data not shown). Intracellular FoxP3 staining confirmed quantita-tive real-time PCR results and indicated that CSA exposureinduced a shift toward lower FoxP3 expression and not 2 distinctFoxP3high versus FoxP3low Treg populations (Figure 1D). Theexperiments were reproducible in an eGFP-based Treg reisolationsystem (data not shown).

CSA, RAPA, and MMF reduce aGVHDin a dose-dependent manner

To define the impact of different immunosuppressant drug doseson the expansion of donor-derived TCONV cells and aGVHDlethality, CSA, RAPA, and MMF were investigated in a recentlydescribed GVHD model18 where CD4� and CD8� T cells fromluciferase transgenic FVB/N animals (H-2kq) are transferredinto MHC class I and class II mismatched BALB/c recipients(H-2kd). Doses of the immunosuppressants were chosen accord-ing to previous publications on murine GVHD models forCSA,22-24 RAPA,22,25 and MMF.26

Higher doses of CSA (50 mg/kg) and RAPA (1.5 mg/kg) wereassociated with a significantly improved survival as compared toanimals receiving TCONV and PBS or the lower dose of therespective immunosuppressant (Figure 2A-B). Administration ofthe higher MMF dose (200 mg/kg) did not result in improvedsurvival as compared to PBS injection (Figure 2C). Lower doses ofCSA, RAPA, and MMF were associated with aGVHD-relatedlethality within the first 30 days, indicating the modest impact ofthe immunosuppressants at these dosages on aGVHD, and weretherefore chosen for further adoptive Treg transfer studies. Thesuppressive impact of CSA, RAPA, and MMF on the proliferation

of luc� CD4� and CD8� donor T cells was visualized by BLI(Figure 2D). Interestingly, the homing of TCONV cells to the cLNs,spleen (SPL), and gastrointestinal tract (GIT) was conserved in thepresence of the different immunosuppressants, whereas TCONV

expansion was gradually reduced according to dosing (Figure 2D).To exclude that the BLI signal only reflects the impact of theimmunosuppressants on TCONV-cell expansion, we performed atitration study with different doses of the immunosuppressants withrespect to signal intensity. We found that the immunosuppressantdrugs at the following doses, CSA 10 mg/kg, RAPA 0.5 mg/kg,MMF 90 mg/kg, had no significant impact on the BLI signal ascompared to PBS (data not shown).

Impact of immunosuppressants on adoptively transferred Tregcells with respect to GVHD-related morbidity, mortality,and TCONV expansion

We tested the effect of the immunosuppressants CSA, RAPA, andMMF on Treg function in our lethal aGVHD model,18 where Tregtransfer is essential for survival. We have found that Treg cells aremost effective when given prior to TCONV (V.H.N., unpublisheddata, May 2005). Therefore, we administered Treg cells togetherwith TCD-BM on day 0, whereas TCONV cells were consistentlygiven on day 2 after transplantation.

Mice given TCD-BM alone appeared healthy, and more than90% of the animals survived for at least 70 days. Mice that receivedTCONV cells along with TCD-BM developed severe GVHD signsincluding reduced activity, hunched posture, ruffled fur, diarrhea,and weight loss and all animals died within 30 days aftertransplantation. Treg-cell cotransfer reduced GVHD-related deathafter BMT and more than 75% survived longer than 70 days. These

Figure 2. Effects of different doses of CSA, RAPA,and MMF on GVHD-related mortality. Data from 3independent experiments are combined. (A) Percentagesurvival of animals receiving CSA 10 mg/kg (�, n 12),CSA 50 mg/kg (f, n 12), or PBS (�, n 12). Thehigher CSA dose improves survival (f versus �,P 0.032). (B) Percentage survival of animals receivingRAPA 0.5 mg/kg (E, n 12), RAPA 1.5 mg/kg (F,n 12), or PBS (�, n 12). The higher RAPA doseimproves survival (F versus �, P 0.029). (C) Percent-age survival of animals receiving MMF 90 mg/kg (‚,n 12), MMF 200 mg/kg (Œ, n 12), or PBS (�,n 12), not significant (NS). Control animals receivedTCD-BM only (*). (D) Distribution of donor derived luc�

TCONV cells in Balb/c recipients at day 5 after BMT withcoadministration of different doses of CSA (top row),RAPA (middle row), and MMFl (bottom row). At this timepoint the major TCONV expansion is found in cLNs, GIT,and SPL. Although the localization pattern of TCONV isconserved, proliferation is gradually reduced when differ-ent doses of immunosuppressants are administered.

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data confirm the potential of Treg cells to suppress aGVHD aftermajor mismatch BMT.

To examine whether donor-derived Treg cells would preventexpansion of TCONV cells in living animals in the presence ofdifferent immunosuppressants, we used luc� TCONV cells. Mice thatreceived TCD-BM plus TCONV cells displayed vigorous TCONV

expansion in contrast to animals that received additional Treg cells(Figure 3A). By day 8, TCONV cells were distributed throughout theanimal and accumulated in skin, lymph nodes, and GIT (Figure 3Afirst column from left). Animals receiving Treg cells and CSAshowed a reduced expansion of TCONV cells as compared to thegroup that received TCONV cells alone but significantly higher signalintensity as compared to groups receiving Treg cells plus RAPA orTreg cells plus MMF in serial whole-body BLI (Figure 3A-B).Animals receiving CSA along with Treg cells had significantly

reduced overall survival as compared to Treg plus PBS, Treg plusRAPA, and Treg plus MMF as shown in Figure 3C.

To more precisely localize the abdominal BLI signal, ex vivoimaging of the GIT as a GVHD target organ was performed. Theadoptive transfer of Treg cells reduced the ex vivo signal of luc�

TCONV cells in the GIT and the mLNs, an effect that was maintainedin the presence of RAPA and MMF and reduced when Treg cellswere given in conjunction with CSA (Figure 3D).

Proinflammatory cytokine production and histopathologicGVHD scoring

IL-2 serum levels were below the detectable level in any of theanimals given transplants, but there was a significant increase inserum IFN-� in animals that received TCONV cells and developedGVHD. IFN-� levels were significantly lower in animals that

Figure 3. CSA but not RAPA or MMF interferes with Treg suppressor function in vivo. Expansion of luc� donor TCONV cells in animals receiving TCONV cells alone or TCONV

and Treg cells or TCONV and Treg cells in conjunction with CSA (10 mg/kg), RAPA (0.5 mg/kg), or MMF (90 mg/kg) as shown for 3 representative animals at days 4, 6, 8, and 14after BMT (A) and as quantified by emitted photons over total body area at serial time points after BMT (B). GVHD was induced as described in “Materials and methods.” Datafrom 3 independent experiments are combined. (A) Proliferation of luc� TCONV (first column from left) is reduced by cotransfer of Treg cells (second column from left). Addition ofCSA reduces the suppressive effect of Treg cells (third column from left). In contrast RAPA allows for Treg function (fourth column from left) and MMF has minimal impact (fifthcolumn from left). (B) TCD-BM (Œ, n 15), with TCONV cells (F, n 15), with TCONV and Treg cells (�, n 10), with TCONV and Treg cells and CSA (‚, n 10), with TCONV andTreg cells and RAPA (�, n 10), or with TCONV and Treg cells and MMF (E, n 10). Signal intensity is significantly higher in animals receiving TCONV and Treg cells and CSA ascompared to RAPA (‚ versus �, P .001, day 14) and MMF (‚ versus E, P .009, day 14). (C) Percentage survival of Balb/c recipients is significantly lower when combiningTreg cells with CSA as compared to RAPA (‚ versus �, P .001) or MMF (‚ versus E, P .007). (D) Expansion of luc� TCONV cells at day 10 after BMT in the GIT. (Di)Background signal in the GIT of animals having received only TCD-BM without any luc� cells. Proliferation of TCONV cells in mLNs and the GIT (Dii) is reduced by cotransfer ofTreg cells (Diii). Addition of CSA reduces the protective effect of Treg cells (Div). RAPA (Dv) and MMF (Dvi) do not abrogate Treg suppression of TCONV proliferation in vivo.

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received Treg cells and TCONV cells plus RAPA as compared to Tregcells and TCONV cells plus CSA (Figure 4A). The same trend wasobserved for serum TNF-� although this difference did not reachstatistical significance (Figure 4B). These data indicate that Tregcells alone or in combination with RAPA reduce proliferation andaccumulation of proinflammatory cytokines after BMT. Histopatho-logic scoring of a total of 36 animals (6 from each group)demonstrated a significantly higher GVHD score (*P .05) in thegroup that received Treg cells along with CSA as compared to PBS,RAPA, or MMF (Figure 4C).

Expansion of adoptively transferred FoxP3�CD4�CD25� Tregcells in the irradiated recipient

To investigate the impact of the different immunosuppressants onTreg expansion, we used Treg cells derived from luc� animals,which allows for monitoring of in vivo expansion. Serial BLIshowed that none of the immunosuppressants completely abro-gated the expansion of the adoptively transferred Treg cells (Figure5A-B). Administration of CSA was associated with the lowestsignal intensity indicating reduced Treg expansion; however, thisdifference did not reach statistical significance.

To correlate these results with the level of FoxP3 expression, wequantified donor-derived CD4�FoxP3� T cells from spleen, cLNs,and mLNs by FACS analysis at various time points after transplan-tation. Interestingly, the frequency of donor derivedH-2kq�CD4�FoxP3� cells in Balb/c recipients having receivedTCONV and Treg cells in conjunction with CSA was significantlylower as compared to TCONV and Treg cells alone (P .009) orwith RAPA (P .008) or MMF (P .02) as shown in Figure 5C.

The observation made with BLI was reproducible in anotherBMT model (C57B/63Balb/c) that we previously reported1 andthat allowed us to use the congenic Thy-1 markers to distinguishbetween donor-derived TCONV versus Treg cells for CFSE-basedproliferation analysis. These studies demonstrated reduced Tregexpansion in animals receiving CSA (Figure 6A). To obtain a more

detailed understanding of the in vivo effect of CSA on FoxP3�

expression at a single-cell level, we quantified FoxP3� cells withinthe sorted Thy-1.1�CD4�CD25� cell population by immunofluo-rescence. In contrast to RAPA and MMF, CSA-treated micedisplayed significantly decreased numbers of FoxP3� cells withinthe transferred CD4�CD25� cell population (Figure 6B). Thedecreased FoxP3� Treg/TCONV ratio in the presence of CSA (Table1) supports the conclusion that FoxP3� expression and survival ofTreg cells within the CD4�CD25� cell population may requireintact early IL-2 signaling, which is altered by CSA.

The combination of Treg cells with RAPA does not interferewith GVT activity

To investigate whether the Treg plus RAPA combination would causegeneralized immunoparalysis or allow for GVT activity, we usedA20-luc/yfp-leukemia cells. This cell line has been shown to result inleukemia with primary migration to the bone marrow and secondaryinfiltration of spleen and other lymphoid organs when injected at thetime of BMT.1 Balb/c recipients (H-2kd) were given intravenously5 � 106 FVB/N TCD-BM cells (H-2kq) and 1 � 105 A20-luc/yfp-leukemia cells (H-2kd) after lethal irradiation with 800 cGy on day 0.

Figure 4. Serum IFN-� and TNF-� levels. Serum was collected on day 7 aftertransplantation of Balb/c recipients as indicated for each individual bar (each 5animals). Data are derived from one of 2 independent experiments. (A) Animalsreceiving Treg cells in conjunction with RAPA have a significantly lower IFN-� serumlevel as compared with Treg cells plus CSA, *P .02. (B) TNF-� levels are lowest inanimals receiving TCD-BM. Differences between the groups that receive TCONV/Tregcells in conjunction with different immunosuppressants are not statistically signifi-cantly different. (C) The histopathologic score for the different groups is shown.Analysis included the large bowel, small bowel, and liver from recipients on days 5and 15. Coded tissue samples were scored by a pathologist blinded to the treatmentgroups as described in “Materials and methods.”

Figure 5. CSA administration is associated with reduced numbers ofCD4�FoxP3� cells and has only minor impact on the expansion of donor-derived CD4�CD25� cells. Expansion of luc� donor Treg cells in Balb/c mice (H-2kd)receiving TCONV and Treg cells (both H-2kq) alone or TCONV and Treg cells along withCSA (10 mg/kg), RAPA (0.5 mg/kg), or MMF (90 mg/kg) as shown for representativeanimals on day 7 after BMT (A) and as quantified by emitted photons over total bodyarea at serial time points after BMT (B). (A) Expansion of luc� Treg (first column fromleft) is slightly reduced by conjunction of CSA (second column from left) and to alesser extent by MMF (first column from right). Addition of RAPA (third column fromleft) has no impact on Treg expansion. (B) TCD-BM (*, n 10), with TCONV and Tregcells (�, n 10), with TCONV and Treg cells and CSA (‚, n 10), with TCONV and Tregcells and RAPA (�, n 10), or with TCONV and Treg cells and MMF (E, n 10).Signal intensity is decreased in animals receiving TCONV and Treg cells and CSA orMMF as compared to RAPA (‚ versus �, and E versus �, NS). (C) Splenic CD4donor T cells (H-2kq) were investigated for FoxP3 expression in Balb/c recipients onday 7 after BMT, having received TCONV and Treg cells alone (first column from left) inconjunction with CSA (second column from left), RAPA (third column from left), orMMF (fourth column from left). The percentage of FoxP3� Treg cells is significantlyreduced in the presence of CSA as compared to PBS injection (P .005) or RAPA(P .004).

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When indicated 8 � 105 Treg cells (day 0) with or without RAPA plus1.6 � 106 CD4�/CD8� 1:4 T cells (day 2) were given (both H-2kq).

BLI demonstrated tumor-cell infiltration in the bone marrow ofthe humeri, the femora, and the sternum at day 5 after transplanta-tion in all animals, proving initial engraftment of the A20-luc/yfp-

leukemia with a tumor-cell distribution similar to that of theTCD-BM control group (Figure 7A upper row). Tumor progressionand additional infiltration of the spleen on day 15 were observed inanimals receiving TCD-BM only (Figure 7A first column from theleft). Animals that received TCD-BM and TCONV cells achievedtumor control (Figure 7A second column from the left); however,they died even earlier from aGVHD. In contrast, animals receivingTCONV with Treg cells rejected the tumor cells, as determined byloss of tumor signal independent of additional RAPA coinjection,indicating that this combination allows for effector function ofcytotoxic T cells against A20 tumor cells. These animals had thebest overall survival (Figure 7E).

To confirm that the observed signal loss was indeed associated withtumor clearance, FACS analysis and examination of histologic sectionsfrom spleen and bone marrow were performed. FACS analysis of bonemarrow cells at day 12 after transplantation indicated a distinct yfp� cellpopulation in the group that received TCD-BM alone, whereas therewas tumor clearance in animals receiving TCONV and TCONV plus Tregcells with or without RAPA (Figure 7B). H&E staining demonstrated ahomogeneous spleen infiltration withA20-luc/yfp-leukemia cells on day12 in TCD-BM animals (Figure 7Ci), whereas there was no evidence fora significant tumor infiltration in the other groups (Figure 7Cii-iv).Tumor growth or rejection was confirmed by the increase or loss of BLIsignal intensity over time (Figure 7D). BALB/c mice coinjected withTCD-BM and A20-luc/yfp-leukemia cells died before day 40 fromleukemia and animals receiving additionally TCONV cells died before day22 due to aGVHD (Figure 7E). Addition of RAPA in BM recipientscoinjected with A20-luc/yfp-leukemia cells did not lead to improvedsurvival nor reduced signal intensity (data not shown), indicating thatRAPA has no significant impact on the A20-luc/yfp tumor growth incontrast to other murine tumor models.27

Discussion

Preclinical studies indicate that it might be possible to promote thegeneration of Treg cells or enhance their activity by treatment with

Table 1. Donor-derived cells reisolated from different lymphoid organs of Balb/c recipients at day 7 after transplantationin absolute cell numbers

Lymphoid organ, group

Donor TCONV cells, �105 Donor Treg cells, �105

CD4�/Thy-1.2� CD8�/Thy-1.2� CD4�/FOXP3�/Thy-1.1�

Spleen

TCONV 7.3 � 0.8 4.2 � 0.9 —

TCONV � Treg 2.4 � 0.6 1.5 � 0.3 1.5 � 0.3

TCONV � Treg � CSA 4.8 � 1.2 2.8 � 0.9 0.4 � 0.2

TCONV � Treg � RAPA 2.7 � 0.9 1.9 � 0.4 1.6 � 0.5

TCONV � Treg � MMF 3.1 � 0.4 1.7 � 0.5 1.2 � 0.1

cLN

TCONV 2.6 � 0.4 1.6 � 0.4 —

TCONV � Treg 1.0 � 0.3 0.8 � 0.1 0.4 � 0.1

TCONV � Treg � CSA 2.1 � 0.4 1.0 � 0.3 0.1 � 0.1

TCONV � Treg � RAPA 0.9 � 0.1 0.6 � 0.3 0.5 � 0.1

TCONV � Treg � MMF 1.0 � 0.3 0.6 � 0.5 0.3 � 0.3

mLN

TCONV 3.9 � 0.9 1.6 � 0.4 —

TCONV � Treg 1.4 � 0.6 0.5 � 0.2 0.9 � 0.2

TCONV � Treg � CSA 2.7 � 0.2 1.1 � 0.3 0.3 � 0.1

TCONV � Treg � RAPA 1.7 � 0.3 0.4 � 0.1 0.8 � 0.2

TCONV � Treg � MMF 1.4 � 0.6 0.6 � 0.1 0.7 � 0.1

On day 7 after BMT, lymphoid organs were removed and total cells per organ were counted from a single-cell suspension. Subsequent analysis of the resulting cellsuspensions by FACS provided the percentages based on which the absolute cell numbers were calculated. Data are derived from 3 independent experiments.

— indicates below detection.

Figure 6. CFSE-based proliferation analysis. (A) In vivo expansion of CFSE-labeled C57B/6 Treg (H-2kb, Thy-1.1) in Balb/c recipients (H-2kd) on day 6 after BMTis depicted. The congenic markers Thy-1.1 and Thy-1.2 were used to distinguishbetween Treg and TCONV cells, respectively. All Balb/c recipients (H-2kd) were given5 � 106 C57B6 TCD-BM cells (H-2kb) after lethal irradiation with 800 cGy. Addition-ally 5 � 105 Treg (day 0) plus 1 � 106 CD4�/CD8� T cells (day 2) were given (bothH-2kb). Histograms show the FACS profile of CFSE� Treg cells (H-2kbThy1.1).Numbers of events in each cell division (n) are indicated below the respective peak.Percentages of cells having undergone either 0 or 1 cell division are indicated in eachhistogram. Proliferation of Treg cells (first column from left) is slightly reduced in thepresence of CSA (10 mg/kg; second column from left) and to a lesser extent by MMF(90 mg/kg; fourth column from left). Addition of RAPA (0.5 mg/kg; third column fromleft) has no impact on Treg expansion. Cells are gated on the donor marker H-2kb andthe congenic marker Thy 1.1. (B) Intracellular expression of Foxp3 in donor-derivedThy1.1�H-2kb�CD4�CD25high� T cells derived from Balb/c recipients having re-ceived TCONV (Thy-1.2) and Treg (Thy-1.1) cells alone (first column from left) inconjunction with CSA (second column from left), RAPA (third column from left), orMMF (fourth column from left). Cells were sorted, fixed onto glass slides, andanalyzed by immunofluorescence. FoxP3 (Alexa 546), red; DNA (DAPI), blue; doublepositive, purple; magnification � 200. Percentage of FoxP3� cells within the donorCD4�CD25� population (average of 3 independent experiments): 95% � 3% noimmunosuppressant, 57% � 3.2% CSA (second column from left), 94% � 3.5%RAPA (third column from left), 89% � 1.7% MMF (fourth column from left).

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various drugs, such as MMF28 or RAPA.29 However, studies onMMF combined the drug with 1�,25-dihydroxyvitamin D3 in adiabetes model.28 Therefore, no direct evidence that the expansionof CD4�CD25� cells was due to either MMF or 1�,25-dihydroxyvitamin D3 was provided. Furthermore, there was noinformation on the level of FoxP3 expression within this popula-tion. Recent in vitro studies showed that RAPA can serve as a toolfor murine Treg-cell expansion,30 whereas CSA, but not RAPA,reduces the highly suppressive subpopulation of humanCD4�CD25�CD27� Treg cells.31

Our study provides the first evidence that RAPA and MMFrather than CSA preserve murine Treg function and expansion andFoxP3 expression levels when administered after allogeneic BMT.Consistent with findings in human Treg cells,31 we observed thatCSA did not abrogate but significantly reduced the function ofallostimulated Treg cells in vitro. Reduced FoxP3 gene transcrip-tion of murine Treg cells when exposed to CSA, but not to RAPAand MPA, is consistent with a recent study showing that calcineurininhibitors reduce FoxP3 expression in human Treg cells, whereasmTOR inhibitors have little or no impact.32 Reduced IL-2 levels inthe presence of CSA and reversion of the CSA effect by IL-2addition to the Treg activation culture indicated that IL-2 depletionmay be involved in the interference of CSA with Treg function. Thecritical importance of signaling by IL-2 for Treg activation andsuppressive activity has been shown in previous studies.6,7,33

Furthermore, it has been demonstrated that RAPA and MPAprofoundly affect the phenotype and function of APCs by reducingtheir antigen-uptake capacity34 and the expression of costimulatory

molecules,35 thereby favoring the differentiation of tolerogenicAPCs. Thus, suppressor function of Treg cells exposed to RAPA orMPA could be enhanced by an indirect effect of RAPA and MPA onAPCs. APC-derived IL-2 has been demonstrated to be critical forthe proliferation of allogeneic T cells and was found to be enrichedat the APC/T-cell interface during in vitro cognate interaction,36,37

suggesting that IL-2 production by APCs may account for thepresence of the cytokine in the primary Treg activation culture thatwe applied. The observation that Treg cells reisolated fromCSA-containing cultures were less suppressive and had reducedlevels of FoxP3 expression could be in part due to increasingnumbers of TCONV cells within this culture. However, the primarilyapplied Treg population was highly purified (� 96%CD4�CD25�FoxP3�) and we did not detect a significant change inGITR and CTLA-4 expression during the coculture (data notshown), making a massive overgrowth of TCONV cells unlikely.

To further evaluate the relevance of these findings in vivo, weused an established aGVHD model18 in which adoptive transfer ofdonor-type Treg cells can prevent GVHD lethality. Consistent withthe in vitro findings RAPA and to a lesser extent MMF did notinterfere with Treg function. The fact that MMF had some effect onTreg cells in vivo may be explained by its impact on lymphocyteproliferation in general and corresponds to the observation thatTreg proliferation in vivo was lower as compared to the RAPAgroup. The observation that CSA interfered with Treg-relatedprotection is in line with reports that CSA but not RAPA canantagonize the induction of tolerance.38-41 This finding has been sofar explained by the interference of CSA with activation-induced

Figure 7. GVT effector function of TCONV cells is preserved in the presence of Treg cells and RAPA. Tumor growth and elimination are visualized by bioluminescenceimaging (A), FACS analysis (B), histology (C), emitted photons over time (D), and survival (E). All Balb/c recipients (H-2kd) were given 1 � 105 A20 leukemia cells (H-2kd) andBMT was performed as described in “Materials and methods.” (A) luc�A20 tumor-cell localization at 5 days (top row), 10 days (middle row), and 15 days (bottom row) after BMTin representative animals given transplants with TCD-BM alone (first column from left), TCD-BM plus TCONV cells (second column from left), TCD-BM plus TCONV and Treg cells(third column from left), and TCD-BM plus TCONV and Treg cells and RAPA (fourth column from left). ST indicates sternum; HU, humerus; FE, femur; SPL, spleen. (B) Bonemarrow samples with yfp� A20 cells of recipients on day 12 after transplantation with TCD-BM alone (first column from left), TCD-BM plus TCONV cells (second column from left),TCD-BM plus TCONV and Treg cells (third column from left), and TCD-BM plus TCONV and Treg cells and rapamycin (fourth column from left). (C) H&E stains of representativespleen samples gathered on day 12 from animals receiving TCD-BM only (Ci), TCD-BM plus TCONV cells (Cii), TCD-BM plus TCONV and Treg cells (Ciii), and TCD-BM plus TCONV

and Treg cells and RAPA (Civ). A homogenous leukemic infiltration is found only in animals receiving TCD-BM only (Ci) but not in the other groups. Microscopic evaluation wasperformed on a Nikon Eclipse TE 300 microscope equipped with 20 �/0.45 and 40 �/0.60 objective lenses (Nikon, Melville, NY). Photographs were captured by using a Spotdigital camera (Diagnostic Instruments, Sterling Heights, MI). Digital images were saved as TIFF files, which were inserted into and processed with PowerPoint software(Microsoft, Redmond, WA). (D) Expansion of luc�A20 tumor cell as measured in photons over total body area (photons/second/mouse). Animals rejecting the A20 tumordemonstrate a rapid signal loss within the first 15 days after BMT. (E) Survival of BALB/c mice receiving TCD-BM alone (‚, n 15), TCD-BM plus A20 leukemia cells (�,n 15), TCD-BM plus A20 leukemia cells plus TCONV cells (F, n 15), TCD-BM plus A20 leukemia cells plus TCONV and Treg cells (�, n 12), or TCD-BM plus A20 leukemiacells plus TCONV and Treg cells and RAPA 0.5 mg/kg; E, n 12). Data from 3 independent experiments are combined.

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cell death of effector T cells, which is not seen when the mTORsignaling pathway is blocked with RAPA.42 By showing thereduced number of FoxP3� Treg cells in vivo when exposed toCSA, our data provide an additional explanation for these findings.The observation that in vivo exposure to CSA but not to RAPA andMMF reduced the level of intracellular expression of FoxP3, whichis critical for Treg function,43,44 is consistent with our in vitro dataand indicates that blockade of calcineurin-dependent IL-2 produc-tion prevents Treg-mediated tolerance. The fact that reducedexpansion of Treg cells in the presence of CSA was not statisticallysignificant is consistent with our in vitro data, where equal numbersof Treg cells were reisolated following CSA exposure, indicatingthat reduced expansion of Treg cells cannot alone account for theimpaired suppressor function of Treg cells in the presence of CSAin vivo. Furthermore, our in vivo findings with impaired IL-2 genetranscription in the presence of CSA are consistent with studies inmice deficient for IL-2 or the �-subunit of the IL-2 receptor.10,11 Apossible method to overcome the detrimental effect of CSA-dependent depletion of IL-2 while conserving its beneficial immu-nosuppressant effect may be the coadministration of IL-2.

Consistent with the concept of Treg-cell–mediated tolerance,depletion of Treg cells in mice with tumors improves immune-mediated tumor clearance45 and enhances the response to immune-based therapy.46 In light of these data our observation that CSA hasa significant impact on Treg function in vivo may be relevant in theattempt to break Treg-mediated tolerance to tumor-associatedantigens in humans with cancer and increased numbers of peripheral47

or intratumoral48 Treg cells.The observation that the Treg/RAPA combination demonstrated

a sustained suppression of proliferation of TCONV cells could be

potentially unfavorable in allogeneic transplantation attempting toexploit the GVT effect of donor T cells. Our results, however, showthat the Treg/RAPA combination did not cause generalized immuneparalysis because it did not interfere with bone marrow engraft-ment and did not abrogate the ability of alloreactive CD4� andCD8� TCONV cells to eradicate an established tumor. Previousstudies have demonstrated that Treg cells reduce expansion ofalloreactive T cells after BMT but not their activation, which iscritical for tumor clearance.1

We conclude that the RAPA/Treg combination was mostfavorable with respect to GVHD-related morbidity and mortality.Reduced suppressor function of CSA-exposed Treg cells was IL-2dependent and correlated with a decreased number of FoxP3� Tcells both in vitro and in vivo. Beside adequate GVHD control, thecombination of Treg cells with RAPA allowed for GVT activity,suggesting a novel scientifically justified immunosuppressive com-bination of RAPA and MMF, which will be studied in the clinic.Conversely, the inhibitory effects by CSA on Treg function mayhave important implications for boosting immune responses tovaccination protocols.

Acknowledgments

The authors are grateful to Ruby M. Wong for expert assistancewith statistical analysis; to Janelle Olson, Lena Ho, and Jing-ZhouHou for helpful discussion; and to Mobin Karimi for excellenttechnical support.

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CALCINEURIN INHIBITION AFFECTS Treg-CELL FUNCTION 399BLOOD, 1 JULY 2006 � VOLUME 108, NUMBER 1

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