DOI: 10.1126/scitranslmed.3001809, 83ra41 (2011);3 Sci Transl Med

, et al.Keli L. Hippenwith Minimal Loss of in Vivo Functional Activity

)regsMassive ex Vivo Expansion of Human Natural Regulatory T Cells (T

 Editor's Summary

 Cross-Checking Graft-Versus-Host Disease

   establish donor banks that would keep human GVHD and autoimmunity in check.significantly reduced mortality resulting from GVHD. Such large numbers of functional nTregs could be used tofunctional cells ~50 million�fold. When injected into mice at the same time as human T cells, these expanded Tregs cells maintained suppressor function. Stimulation of the nTreg population up to four times expanded the numbers ofexpansion, Hippen et al. expanded nTregs 80-fold after only one stimulation; they then showed that these multiplied Using good manufacturing practice conditions and artificial antigen-presenting cells designed to stimulate T cellnTregs in cord blood is limited. Therefore, the authors used peripheral blood as a source of nTregs for expansion.

Umbilical cord blood can be used to expand functional natural Tregs (nTregs); however, the initial number ofvivo.than those previously achieved while maintaining their ability to selectively suppress self-attacking cytotoxic T cells in altered function after expansion in vitro. Hippen et al. now report a new way to expand Tregs to numbers much largerand differs from organ rejection. However, using Tregs to prevent GVHD has been limited by low Treg numbers and patient�s own tissues. This process, called graft-versus-host disease (GVHD), is one of the risks of transplantationcytotoxic T cells, from an overly exuberant response and, in the case of a bone marrow transplant, from attacking the brawl. Regulatory T cells (Tregs) are the referees of the adaptive immune system. They prevent the enforcers,Black-and-white striped referees serve to uphold this balance, breaking up fights and preventing the bench-clearing

melee.these aggressive athletes. Yet, a balance must be maintained between the occasional high stick and an all-out Fighting in hockey is a long-standing tradition: Stitches and gap-toothed smiles are badges of honor among

http://stm.sciencemag.org/content/3/83/83ra41.full.htmlcan be found at:

and other services, including high-resolution figures,A complete electronic version of this article

http://stm.sciencemag.org/content/suppl/2011/05/16/3.83.83ra41.DC1.html can be found in the online version of this article at: Supplementary Material

http://stm.sciencemag.org/content/scitransmed/3/83/83ra42.full.html http://stm.sciencemag.org/content/scitransmed/3/83/83ra40.full.html http://stm.sciencemag.org/content/scitransmed/3/83/83ps19.full.html

can be found online at:Related Resources for this article

http://www.sciencemag.org/about/permissions.dtl in whole or in part can be found at: article

permission to reproduce this of this article or about obtaining reprintsInformation about obtaining

is a registered trademark of AAAS. Science Translational Medicinerights reserved. The title NW, Washington, DC 20005. Copyright 2011 by the American Association for the Advancement of Science; alllast week in December, by the American Association for the Advancement of Science, 1200 New York Avenue

(print ISSN 1946-6234; online ISSN 1946-6242) is published weekly, except theScience Translational Medicine

on

May

19,

201

1st

m.s

cien

cem

ag.o

rgD

ownl

oade

d fr

om

R E S EARCH ART I C L E

GRAFT -VERSUS -HOST D I S EASE

Massive ex Vivo Expansion of Human NaturalRegulatory T Cells (Tregs) with Minimal Lossof in Vivo Functional ActivityKeli L. Hippen,1* Sarah C. Merkel,1 Dawn K. Schirm,1 Christine M. Sieben,1 Darin Sumstad,2

Diane M. Kadidlo,2 David H. McKenna,2 Jonathan S. Bromberg,3 Bruce L. Levine,4

James L. Riley,4 Carl H. June,4 Phillip Scheinberg,5 Daniel C. Douek,5 Jeffrey S. Miller,6

John E. Wagner,1 Bruce R. Blazar1*

on

May

19,

201

1em

ag.o

rg

Graft-versus-host disease (GVHD) is a frequent and severe complication after hematopoietic cell transplantation.Natural CD4+CD25+ regulatory T cells (nTregs) have proven highly effective in preventing GVHD and autoimmunityin murine models. Yet, clinical application of nTregs has been severely hampered by their low frequency and un-favorable ex vivo expansion properties. Previously, we demonstrated that umbilical cord blood (UCB) nTregs couldbe purified and expanded in vitro using good manufacturing practice (GMP) reagents; however, the initial numberof nTregs in UCB units is limited, and average yield after expansion was only 1 × 109 nTregs. Therefore, we askedwhether yield could be increased by using peripheral blood (PB), which contains far larger quantities of nTregs. PBnTregs were purified under GMP conditions and expanded 80-fold to yield 19 × 109 cells using anti-CD3 antibody–loaded, cell-based artificial antigen-presenting cells (aAPCs) that expressed the high-affinity Fc receptor and CD86. Asingle restimulation increased expansion to ~3000-fold and yield to >600 × 109 cells while maintaining Foxp3 ex-pression and suppressor function. nTreg expansion was ~50 million–fold when flow sort–purified nTregs were re-stimulated four times with aAPCs. Indeed, cryopreserved donor nTregs restimulated four times significantlyreduced GVHD lethality induced by the infusion of human T cells into immune-deficient mice. The capabilityto efficiently produce donor cell banks of functional nTregs could transform the treatment of GVHD and auto-immunity by providing an off-the-shelf, cost-effective, and proven cellular therapy.

enc

stm

.sci

Dow

nloa

ded

from

INTRODUCTION

Acute graft-versus-host disease (GVHD) is a major cause of morbidityand mortality after hematopoietic cell transplantation (1). Natural regu-latory T cells (nTregs) express the transcription factor Foxp3 and are re-quired for immune self-tolerance (2). Inmurinemodels, adoptive transferof nTregs prevents GVHD and donor bonemarrow graft rejection, as wellas speeds immune recovery inGVHD-prone animals (3–5), making Tregsan attractive therapeutic tool for preventing and/or treating disease inhumans (6–9). However, clinical testing has been hampered by low nTregfrequency (1 to 2%) in peripheral blood (PB) (10), contamination withnon-Tregs, such as CD25

+ T-effector or T-memory cells (7, 11), and thelack of availability of good manufacturing practice (GMP)–compatibleprocedures for nTreg purification. Maximizing yield is also critical, be-cause murine studies find that high Treg doses (~1:1 with donor T cells)are required to efficiently and reproducibly suppress GVHD (5).

Previously, we found that nTregs were more readily purified fromumbilical cord blood (UCB) than PB because of the relative paucityof CD25+ non-Tregs in UCB; these cells could be expanded several

1Division of Bone Marrow Transplantation, Department of Pediatrics, University of Min-nesota Cancer Center, Minneapolis, MN 55455, USA. 2Molecular and Cellular TherapeuticsFacility, University of Minnesota, Minneapolis, MN 55455, USA. 3Division of Transplantation,Department of Surgery, University of Maryland School of Medicine, Baltimore, MD 21201,USA. 4Abramson Family Cancer Center Research Institute, University of PennsylvaniaCancer Center, Philadelphia, PA 19104, USA. 5Human Immunology Section, VaccineResearch Center, National Institute of Allergy and Infectious Diseases, National Institutesof Health, Bethesda, MD 20892–3005, USA. 6Division of Bone Marrow Transplantation, De-partment of Medicine, University of Minnesota Cancer Center, Minneapolis, MN 55455, USA.*To whom correspondence should be addressed. E-mail: hippe002@umn.edu (K.L.H.);blaza001@umn.edu (B.R.B.)

www.S

hundred-fold ex vivo using anti-CD3/CD28 monoclonal antibody(mAb)–coated microbeads and interleukin-2 (IL-2) (11, 12). Thesestudies allowed us to initiate the world’s first clinical trial to study thesafety of ex vivo–expanded nTregs. Transferred nTregs remained Foxp3+

and could be tracked in blood for up to 14 days. No adverse effectswere observed, and a trend toward a lower incidence of acute grade IIto IV GVHD was observed, but the maximum cell dose was limited byinsufficient and variable nTreg expansion rates for some UCB units(13). In other studies, we have shown that stimulation of UCB nTregswith cell-based artificial antigen-presenting cells (aAPCs) increases ex-pansion (about fourfold) over bead-based aAPCs, but this increasealone would not have much effect on clinical nTreg dose. Because thenTreg number in UCB is limited and the dose-limiting toxicity was notreached, other nTreg sources need to be explored to determine the max-imal efficacy of single- or multiple-dose nTreg therapy.

Despite non-Treg contaminants, isolation of PB nTregs offers severaladvantages over UCB nTregs, including increased nTreg number,continued donor availability for additional isolations, and use of au-tologous cells. PB nTregs can be successfully purified with cell sorting(14, 15) and expanded ~80-fold in vitro. However, cell sorting is achallenging GMP procedure, and overall nTreg yield from PB obtainedwith this isolation and expansion approach is not greatly increased overthat from UCB. Restimulation increased total expansion to ~1000-fold,but cultures frequently lost Foxp3 expression and suppressive functionconcomitant with the appearance of effector T cells secreting IL-2 andinterferon-g (IFN-g) (16, 17). Although nTregs can also be purified withmAb-coated magnetic beads, and ~30-fold more CD25high cells can beisolated from PB than from UCB (~150 × 106 compared with ~5 × 106,

cienceTranslationalMedicine.org 18 May 2011 Vol 3 Issue 83 83ra41 1

R E S EARCH ART I C L E

respectively), bead-purified nTregs contain higher numbers of CD25lo

cells and are less pure than those obtained by flow cytometry sorting(18, 19). Thus, rapamycin, which preferentially inhibits cytokine re-sponses in and survival of effector and memory T cells when comparedwith nTregs, is often added to bead-purified expansion cultures, albeit atthe expense of a 5- to 10-fold reduction in nTreg expansion (20–23).

Here, we show, using GMP-grade reagents, that repetitive nTreg stim-ulation with cell-based aAPCs massively increases nTreg yield whilemaintaining Foxp3 and suppressive function. Expanded cells expressednTreg-specific markers [Foxp3 and LAP (latency-associated peptide)],displayed Treg-specific demethylation in the Foxp3 gene, and containedvery few IL-2–, IFN-g–, or IL-17–secreting cells. Despite four restimula-tions and expansion of >50 million–fold, fresh and cryopreserved nTregseach were capable of suppressing lethality in a xenogeneic model ofGVHD. These findings advance the clinical utility of expanded nTregsfor the prevention and treatment of GVHD after blood and marrowtransplantation, solid organ rejection, and autoimmune disease.

on

May

19,

201

1.o

rg

RESULTS

PB nTregs can be purified and expanded with GMPreagents and protocolsAlthough our Phase I studies showed UCB nTreg cellular therapy to bewell tolerated, a dose-limiting toxicity of nTregs was not reached, pos-sibly because of limitations in nTreg expansion rates. Moreover, GVHD

www.S

was significantly reduced but not eliminated compared to historicalcontrols (13). To determine whether nTreg yield could be increased ifthe source was changed from UCB to PB, we purified cells from leu-kapheresis products with a two-step protocol using GMP antibody-coated magnetic beads, whereby CD4+ cells were enriched by depletingcells expressing CD8, CD14, and CD19, followed by positive selection ofCD25high cells (Fig. 1A). The starting purity of PB nTregs was assessedby flow cytometry for a phenotype that displays potent suppressivecapacity (CD4+127−Foxp3+; fig. S1A) (24) and was comparable to pre-vious observations for UCB nTregs (95 ± 1% CD4+, of which 66 ± 2%were CD127−Foxp3+). Of the non-CD4+ cells in either cellular prepa-ration, <1% were positive for CD8, CD14, and CD19 (fig. S1A). Theaverage yield of PB nTregs after expansion (233 ± 31 × 106 cells) was~40-fold higher than with UCB nTregs (13).

Purified cells were stimulated with clinical-grade anti-CD3/CD28mAb-coated beads or KT64/86 cells, a recently GMP-licensed, cell-based,aAPC-expressing CD86 (a CD28 ligand) and CD64 (the high-affinityFc receptor) because of lentiviral gene transfer. KT64/86 cells were loadedwith anti-CD3 mAb. IL-2 (300 U/ml) and rapamycin (109 nM) wereadded to all cultures (Fig. 1B). As reported (25), stimulation with eitherKT64/86 cells or anti-CD3/CD28 mAb-coated beads increased PB nTregexpansion by ~5-fold (82 ± 11–fold; 18 ± 5–fold, respectively) (Fig. 1,C and D). More robust expansion observed with KT64/86 cells and anti-CD3/CD28 mAb-coated beads was associated with increased overallviability (94.3 ± 0.5%; 90.0 ± 0.3%, respectively; P ≤ 0.001) and de-creased granzyme B production (P < 0.02) (fig. S1, B to D). nTreg cul-

stm

.sci

ence

mag

Dow

nloa

ded

from

Fig. 1. Restimulation greatly increases PB nTreg expansion, and cell-basedaAPCs are more effective than bead-based aAPCs. nT were purified

stimulated with PMA and ionomycin for 4 hours in the presence of brefeldinA, and the percentage of cells secreting IL-2 or IFN-g was determined by

regs

from PB leukapheresis products and expanded with GMP anti-CD3/CD28mAb-coated beads or an anti-CD3 mAb-loaded cell line (KT64/86). (A)GMP purification schema. (B) Schema showing time course of experimentand ranges for size-based restimulation (R0 = no restimulation, R1 = onerestimulation, etc.). (C and D) Fold nTreg expansion (average ± SEM); total(C) or after each stimulation (D). (E) Percentage of cultured cells (CD4-gated)that are CD127−Foxp3+ after each stimulation. (F) Percent suppression ofin vitro, anti-CD3–mediated CD8+ T cell proliferation at 1:4 (nTreg/PBMNCs) asdetermined by CFSE dye dilution. (G) nTregs from each stimulation were re-

flow cytometry. (H) Bead-purified PB nTregs restimulated three or four times(black and gray symbols, respectively) with anti-CD3 mAb-loaded KT64/86cells were harvested and genomic DNA was purified. Foxp3 TSDR demeth-ylation status was assessed with bisulfite sequencing and is comparedto nTreg purity (percentage of CD4+ cells that are CD127−Foxp3+) or per-cent suppression at a 1:4 ratio of nTregs/PBMNCs. Averages are for threeindependent experiments. Individual symbols in (E) and (F) representindependent experiments. Brackets indicate the range of days for eachstimulus. *P < 0.05.

cienceTranslationalMedicine.org 18 May 2011 Vol 3 Issue 83 83ra41 2

R E S EARCH ART I C L E

on

May

19,

201

1st

m.s

cien

cem

ag.o

rgD

ownl

oade

d fr

om

tures stimulated once with anti-CD3/CD28mAb-coated beads or KT64/86maintained an nTreg phenotype (97 ± 2% or 99.5 ± 0.3% CD4+, ofwhich 81 ± 5% or 84 ± 7% were CD127−Foxp3+, respectively) andin vitro function [84 ± 12% or 83 ± 6% inhibition of carboxyfluoresceindiacetate succinimidyl ester (CFSE)–labeled CD8+ T cell proliferation af-ter anti-CD3 stimulation at a 1:4 ratio of nTreg/PB mononuclear cells(PBMNCs)] (Fig. 1, E and F). These data demonstrate that suppressive,Foxp3+ nTregs can be expanded from PB with GMP procedures and showthat stimulation with cell-based aAPC is superior to anti-CD3/CD28mAb-coated beads. However, because PB nTreg expanded 5- to 10-foldless than UCB nTregs expanded without rapamycin, overall nTreg yieldwas not substantially increased.

Restimulation greatly increases nTreg expansionTo maximize yield, we restimulated GMP bead-purified nTregs grownin rapamycin after they had returned to resting size (≤8.5 mm; fig.S1E), which we have shown maximizes CD4+ T cell expansion (26).nTregs stimulated with KT64/86 cells were found to have a higher peakcell size compared to anti-CD3/CD28 mAb-coated beads (fig. S1F). Re-stimulation with anti-CD3/CD28 mAb-coated beads or KT64/86cells increased expansion 18- and 36-fold, respectively, to a total of 330-or 3000-fold over input cell number (Fig. 1, C and D). Cultures re-mained >65% Foxp3+ and suppressed in vitro T cell proliferation>50% at a ratio of 1:4 (nTregs/PBMNCs) (Fig. 1, E and F). Of the non-CD4+ cells expanded with either stimulus, <1% were positive for CD8,CD14, CD19, or CD56 (fig. S1G).

Others have shown that nTreg restimulation in the absence ofrapamycin results in up to 30% of cells that secrete IL-2 and/or IFN-g,two cytokines that could potentially exacerbate GVHD (16, 17). There-fore, we quantified the number of IL-2– and IFN-g–secreting cells byintracellular cytokine staining after phorbol 12-myristate 13-acetate(PMA)/ionomycin stimulation of bead-purified nTreg cultured withanti-CD3/CD28 mAb-coated beads or KT64/86 cells and rapamycin.As shown in Fig. 1G, <1% of cells expanded with either anti-CD3/CD28mAb-coated beads or KT64/86 cells secreted IL-2. Although less than 6%of cells in any culture expanded with either aAPCs or rapamycin wereIFN-g+, significantly fewer IFN-g+ cells were found in nTreg culturesexpanded with KT64/86 than CD3/CD28 beads (1% for KT64/86 re-stimulation; 4% for R0 bead restimulation; 6% for R1 bead restimulation).

Multiple restimulations lead to reduced suppressivefunction despite the presence of Foxp3To determine whether bead-purified nTregs could be expanded evenfurther, we stimulated the above cultures another three times (fourrestimulations total) (Fig. 1C). In contrast to anti-CD3/CD28 mAb-coated bead-expanded cultures, whose peak size declined after eachstimulation and was <9.0 mm after the fourth restimulation, peak sizeafter KT64/86 cell stimulation remained high at ~9.5 mm (fig. S1, E andF). In addition, the fold expansion induced by successive stimulationswith anti-CD3/CD28 mAb-coated beads decreased more rapidly thanwith KT64/86 cells, ultimately resulting in 200-fold lower total expan-sion than with KT64/86 cells (25,000-fold versus ~5 million–fold, re-spectively) (Fig. 1, C and D). nTregs restimulated with anti-CD3/CD28mAb-coated beads remained >80% Foxp3+, and although expressiongradually decreased in KT64/86 cell–expanded nTregs, Foxp3 remainedin >60% of cells after the fourth restimulation (Fig. 1E). nTregs ex-panded with anti-CD3/CD28mAb-coated beads also had higher amountsof Foxp3 on a per cell basis than those expanded with KT64/86 cells

www.S

(fig. S1, H and I). Despite achieving Foxp3 levels previously associatedwith significant suppressive function by expanded UCB nTregs (13),<50% suppression of anti-CD3 mAb-driven CD8+ T cell proliferationat a 1:4 nTreg/PBMNC ratio was observed in two of two and one ofthree cultures restimulated three or four times with anti-CD3/CD28mAb-coated beads, respectively, as well as in two of three cultures re-stimulated with KT64/86 cells either three or four times (Fig. 1F).

Stable expression of Foxp3 is a trait of natural, but not induced, Tregsand is conferred through epigenetic modification of the Foxp3 geneat the Treg-specific demethylated region (TSDR) (27). To assess themethylation status of the Foxp3 gene in restimulated nTreg, we purified,bisulfite-modified, and sequenced DNA from cultures receiving three orfour restimulations, and we determined the average percent methylationof 11 CpG sites contained in the TSDR. Because Foxp3 is on the Xchromosome and becomes hypermethylated during X-inactivation,the data shown are restricted to male samples. An evaluation of two in-formative samples, Fig. 1H suggests that TSDR demethylation status isproportional to Foxp3 and slightly decreases between the third and thefourth restimulation, although the effects were not significant (r = 0.65,P = 0.35). As observed for Foxp3, TSDR demethylation is not directlyproportional to suppressive function (r = 0.75, P = 0.25).

Sort-purified nTregs maintain Foxp3 and suppressivefunction after multiple stimulationsDecreased suppressive function could be caused by contaminatingcells that become amplified after restimulation and acquire Foxp3 dur-ing the process of massive cell expansion. Therefore, PB nTregs werepurified by flow cytometry sorting and restimulated with KT64/86cells in the presence or absence of rapamycin (Fig. 2A). To enable moremeaningful comparisons of the various restimulations, we also devel-oped freeze/thaw conditions that allow expanded nTregs to maintainphenotype and suppressive function so that all samples are assayedsimultaneously (fig. S2). The most common strategy for sorting nTreg

is to first purify CD4+ cells and then gate on the 2% of cells with thehighest expression of CD25. Although cells purified in this manner areregularly >90% Foxp3+; this method is relatively inefficient and onlycaptures ~20% of the total Foxp3+ cells. To maximize yield, we per-formed an initial purification with magnetic anti-CD25 mAb-coatedbeads and then sorted for CD4+25hi127− cells, which allowed >25% ofsorted cells to be positively selected. This method increased initialnTreg purity from 66 ± 2% CD127−Foxp3+ for bead-based purificationto 84 ± 3 (P≤ 0.003), and resulted in a routine yield of 15 × 106 to 30 ×106 nTregs from 2 × 109 PBMNCs (fig. S3, A and B).

nTregs stimulated with KT64/86 cells in the absence of rapamycin,which is known to affect size and proliferation (28), had both a largerpeak size (Fig. 2B; 10.4 without rapamycin; 9.9 mMwith rapamycin; P <0.01) and increased expansion (Fig. 2, C andD; 290- and 55-fold, respec-tively). nTregs cultured in the presence or absence of rapamycin were re-stimulated at 8.5 mm (day 13 ± 1), and after 4 days of expansion and sizeincrease, cultures started to decrease in size and stop expanding. How-ever, after day 25,without additional stimulation, cultures grownwithoutrapamycin increased in size to ~9.3 mMand started proliferating, impres-sively expanding>5×1011–fold throughout the 55-day observation period.Additional restimulationdidnot increase either cell size ormaximal expan-sion. Day 55 cultures contained few CD127−Foxp3+ nTregs and were notsuppressive, whereas those harvested on day 25 had expanded 11,000 ±2000–fold, were≥60% CD127−Foxp3+, and conferred≥60% suppres-sion of CD8+ T cell proliferation (Fig. 2, E and F).

cienceTranslationalMedicine.org 18 May 2011 Vol 3 Issue 83 83ra41 3

R E S EARCH ART I C L E

In contrast to cultures established in the absence of rapamycin,sort-purified nTregs expanded with KT64/86 cells in the presence ofrapamycin returned to resting size and ceased proliferating after eachrestimulation (Fig. 2, B and C). Cumulative expansion after restimula-tion of sort-purified nTregs + rapamycin was >6-fold higher than mAb-coated bead-purified nTregs (31 ± 14 × 106–fold and 4.7 ± 0.7 × 106–foldexpansion, respectively, P < 0.05), due mainly to the fact that the foldexpansion did not decline after each restimulation (fig. S3C). Re-peated stimulation caused a gradual decrease in Foxp3 such that afterthe fourth restimulation, 63 ± 12% of cells were CD127−Foxp3+ (Fig.2G). However, unlike bead-purified nTregs, sort-purified nTregs ex-panded after four repetitive stimulations maintained >50% suppressionof T cell responses for all restimulations (Fig. 2H). Table S1 summarizes

www.S

the in vitro expansion characteristics (yield, Foxp3 expression, T-effectorcell contamination, and suppressive function) for nTreg derived fromUCB and PB with varying culture and restimulation conditions.

To determine whether restimulation affects the Treg phenotype, weassessed the level of several Treg-associated markers (including LAP,CD62L, CD27, CCR7, and CD45RA) on cells receiving either singleor multiple stimulations. Although LAP, derived from the N-terminalregion of transforming growth factor–b (TGFb), was expressed onFoxp3+ cells after all four restimulations (fig. S4A), CD62L and CD27staining was lost after two and four restimulations, respectively (fig.S4B). CCR7 behaved like an activation marker; it was more highlyexpressed at day 7 than at resting size (fig. S4C). However, if nTregs weremaintained in culture after returning to basal size, a subpopulation of

on

May

19,

201

1st

m.s

cien

cem

ag.o

rgD

ownl

oade

d fr

om

Fig. 2. Sort-purified nTregs maintain Foxp3 and suppressive function aftermultiple stimulations. PB nTregs were sort-purified (CD4+25hi127−) and

examples (C) and average (D) expansion of nTregs ± rapamycin, respectively.Arrows in (C) on days 25 and 55mark twodistinct phases (plateauandgrowth

expanded with anti-CD3 mAb-loaded KT64/86 in the presence or absenceof rapamycin using four or two restimulations, respectively. (A) Schemashowing time course of experiment and time ranges for size-based restim-ulation (R0 = no restimulation, R1 = one restimulation, etc.). Brackets indicatethe range of days for each stimulus. (B) Average cell size (±SEM) over time forPB nTreg cultures restimulated ± rapamycin. (C and D) Representative

phase) seen after first restimulation of nTreg cultures grownwithout rapamycin.(E to H) Average percent CD127−Foxp3+ (CD4-gated) or percent suppressionof in vitro T cell proliferation at a 1:4 ratio of nTregs/PBMNCs for culturesexpanded in the absence (E and F) or presence (G and H) of rapamycin,respectively. Bars represent average; other symbols represent individualexperiments.

cienceTranslationalMedicine.org 18 May 2011 Vol 3 Issue 83 83ra41 4

R E S EARCH ART I C L E

ay 1

9, 2

011

nTregs spontaneously regained CCR7 staining (fig. S4D). Although itis not surprising that restimulation decreased CD45RA expressed onnaïve, resting T cells and Tregs, the finding that cells regained stainingafter returning to resting size was unanticipated (fig. S4E, especiallyrestimulations 2 and 3).

We next examined changes in surface phenotype and T cell re-ceptor (TCR) repertoire usage of the Tregs expanded with one (R0)or a total of five stimulations (R4). After multiple rounds of stimula-tion, the nTreg phenotype changed from CD27+CD45RA−CD57− toCD27−CD45RA−CD57−, suggesting that these cells were undergoing dif-ferentiation to a more mature state (fig. S5). However, an increase inCD57 expression was not noted after expansion, suggesting that the cellsdid not become terminally differentiated or senescent (29). Finally, TCRVb usage was essentially unchanged between R0 and R4, suggesting thatparticular TCR Vb families were not preferentially expanded despite amassive increase in nTreg number during the course of cell culture (fig. S6).

nTreg cultures restimulated with KT64/86 cells in the presenceof rapamycin do not secrete IL-2 or effector cytokinesRepetitive stimulation of TH cells in the absence of rapamycin generateseffector cells, which secrete cytokines that could exacerbate GVHD. Todetermine the extent of effector T cell contamination in our cultures,

www.S

we stimulated samples of each restimulation from KT64/86-expandedcultures grown with or without rapamycin with PMA/ionomycin andassayed them for IL-2, IL-4, IL-17, and IFN-g (Fig. 3A) using intra-cellular cytokine staining. To make comparisons between various re-stimulations more valid, we assayed frozen nTregs representing allconditions simultaneously and co-stained them for Foxp3 to differentiatesecretion by nTregs and non-Treg cells. Adding rapamycin suppressedeffector cell generation such that ≤3% of PMA/ionomycin-stimulatedcells secreted IL-2 and ≤2% secreted IFN-g, compared to ≥17% and≥6% for cultures without rapamycin (Fig. 3, B and C). In contrast,rapamycin was less effective at inhibiting IL-4 production in Foxp3+

or Foxp3− cells, and the percentage of IL-4+ cells increased with eachsuccessive restimulation from 8 ± 2% to 58 ± 17% (Fig. 3D). Finally,the total number of IL-17–secreting cells present in cultures of sortednTregs was consistently low (<3.1%) for all stimulations with or with-out rapamycin (Fig. 3E).

nTregs expanded with multiple rounds of stimulationameliorate disease in a xenogeneic model of GVHDSeveral groups have reported that nTregs are not terminally differen-tiated and can be reprogrammed into helper T cells (30) and T-effectorcells (31, 32), which are capable of inducing proinflammatory responses

on

Mst

m.s

cien

cem

ag.o

rgD

ownl

oade

d fr

om

Fig. 3. nTreg cultures restimulated with KT64/86 cells in the presence ofrapamycin do not secrete IL-2 or effector cytokines. PB nTregs were sort-purified

mycin sample corresponds to the day 25 time point with high Foxp3 staining.(A) Representative example of cytokine production by Foxp3+ and Foxp3−

and expanded with multiple rounds of stimulation with anti-CD3mAb-loadedKT64/86 cells in the presence or absence of rapamycin. The R1 without rapa-

cells (CD4-gated). (B to E) Average (±SEM) percent of cells secreting IL-2 (B),IFN-g (C), IL-4 (D), or IL-17 (E). Averages are for three independent experiments.

cienceTranslationalMedicine.org 18 May 2011 Vol 3 Issue 83 83ra41 5

R E S EARCH ART I C L E

www.ScienceTranslationalMedicine.or

on

May

19,

201

1st

m.s

cien

cem

ag.o

rgD

ownl

oade

d fr

om

and disease (32). Therefore, we used axenogeneic model of GVHD in whichnTregs are co-transferred at a 1:1 ratiowith allogeneic PBMNCs (30 × 106 each)into NOD/Scid/gc

−/− recipients to com-pare the stability and safety of in vitro–expanded nTregs and CD4+CD25− cellsthat were restimulated four times culturedin the absence or presence of TGFb, whichwas used to induce Foxp3 (Fig. 4A). Adopt-ive transfer of nTregs increased mediansurvival from 39 to 55 days (P < 0.01).Transfer of non-Tregs appeared to exacer-bate GVHD, even if Foxp3 was inducedwith TGFb (Fig. 4B), whereas Foxp3−

cells present in nTreg cultures did not ex-pand or persist long-term and, in contrastto cultures expanded fromCD4+CD25− cells,did not exacerbate GVHD. We also testedthe in vivo potency, stability, and safety ofnTregs expanded 50 million–fold with fourrestimulations using KT64/86 cells. Al-though recipients of PBMNCs rapidlyand uniformly succumbed to GVHD, micegiven nTregs had a significantly prolongedsurvival, with 25% of mice surviving today 55 (Fig. 4D; n = 8 to 10 per group;P < 0.05). GVHD amelioration was alsoindicated by a significant decrease in weightloss between days 14 and 21 (Fig. 4E). Thepartial protection seen using nTregs in thisxenogeneic GVHD model has also beenobserved using UCB nTregs obtained aftera single stimulation with anti-CD3/CD28mAb-coated beads, which we have shownto rescue 50% of macrophage-depleted,sublethally irradiated Rag2−/− gc−/− re-cipients when infused at a 1:1 ratio withPBMNCs (12).

Because there was a modest decrementin %CD127−Foxp3+ and in vitro suppres-sion of nTregs in the GVHD model, weused a suboptimal ratio of nTregs/PBMNCs(1:2) to help uncover potential differencesbetween nTreg restimulated three or fourtimes. Figure 4F shows that nTregs restimu-lated three or four times maintained theirphenotype and in vitro–suppressive func-tion after cryopreservation and thawing.Both expanded nTreg preparations signifi-cantly reduced GVHD-induced lethalitywhen compared with PBMNC controls,and therewas nodifference in their relativepotency (Fig. 4G; P < 0.003 and P < 0.001compared with PBMNC controls for threeor four restimulations, respectively). Ex-pansion of PB-derived CD4+ and CD8+

T cells is predictive of GVHD severity,

Fig. 4. PB nTregs expanded >50 million–fold can still ameliorate disease in a xenogeneic model of GVHDeven after freezing and thawing. (A) Summary of purity (percentage of CD4+ cells that are CD127−Foxp3+)and in vitro–suppressive function for in vitro–expanded nTregs or CD4

+CD25− cells (grown ± TGFb) after asingle stimulation with KT64/86 cells. (B) Kaplan-Meier survival curve comparing NOD/Scid/gc−/− mice thatreceived human PBMNCs only or co-transferredwith nTregs, CD4

+CD25− cells, or CD4+CD25− cells expanded inTGFb co-transferred at 1:1 (for example, 30 × 106 PBMNCs and 30 × 106 nTregs). (C) Summary of fold expansion,purity (percentageofCD4+ cells that areCD127−Foxp3+), and in vitro–suppressive function for nTregs expandedwith four restimulations (R4). (D) Kaplan-Meier survival curve comparing NOD/Scid/gc−/− mice receiving hu-man PBMNCs ± fresh nTregs restimulated four times (R4) co-transferred at 1:1. (E) Average weight (percentageof initial) formice survivingonagivenday fordifferentgroupsofmice.P≤0.05 for freshnTregs fromdays14 to21.(F) Summaryof foldexpansion, purity (percentageofCD4+cells that areCD127−Foxp3+), and invitro–suppressivefunction for expanded nTregs restimulated three or four times (R3 and R4, respectively). (G) Kaplan-Meiersurvival curve showing survival of mice receiving human PBMNCs ± cryopreserved and thawed R3 or R4nTregs (HLA-A2

+) co-transferred at 1:2 (that is, 15 × 106 nTregs and 30× 106 PBMNCs).n=10, 8, and 7 for groupsPBMNCs, R3 nTregs, and R4 nTregs, respectively. (H) Average number (±SEM) of human CD4+HLA-A2−,CD8+HLA-A2−, or total CD4+/CD8+/HLA-A2− cells per microliter of blood on day 30 for animals in (G).

g 18 May 2011 Vol 3 Issue 83 83ra41 6

R E S EARCH ART I C L E

and Fig. 4H shows that, like UCB nTregs, co-transfer of restimulated PBnTregs significantly reduced the number of GVHD-causing T cells onday 30 after transfer.

on

May

19,

201

1st

m.s

cien

cem

ag.o

rgD

ownl

oade

d fr

om

DISCUSSION

The therapeutic potential of nTregs to prevent or cure multiple auto-immune diseases or GVHD in murine or xenogeneic models has beenwell documented (3–5). Two critical obstacles to overcome before im-plementing this therapy in humans are generating sufficient cellnumbers and demonstrating their in vivo safety and stability. Here, weshow that sort-purified nTregs could be expanded at least 50 million–fold by repetitive stimulation with cell-based aAPCs while maintainingsuppressive function in vitro and in vivo. Addition of rapamycin mini-mized contamination with T helper 1 (TH1) inflammatory cytokine-secreting cells, but not TH2 cells, which skew immunity away frominflammatory responses. Restimulated nTregs differentiated from aCD27+ memory phenotype to CD27− memory phenotype but, impor-tantly, did not adopt a senescent (CD57+) phenotype (33, 34). The lackof Vb skewing in the TCR repertoire indicates that massively expandednTregs retain a broad spectrum of reactivities and are not transformed.

Maximizing nTreg expansion, while minimizing loss of suppressivefunction and contamination with non-Tregs, is critical for establishingan nTreg cellular therapy. Three studies have shown that nTregs can beexpanded >1000-fold if restimulated in the absence of rapamycin, butin each case, cultures contained high numbers of IL-2– and IFN-g–secreting cells that were both Foxp3− and Foxp3+ (16, 17, 35). Weconfirmed these data and found that nTreg cultures eventually lost Foxp3and suppressive function in the absence of rapamycin. Loss of Foxp3correlated with an increased ratio of cycling (that is, Ki-67+) Foxp3− cells(fig. S7), suggesting that loss of purity is due to the outgrowth ofFoxp3− cells as opposed to conversion of Foxp3+ cells as suggestedby one report (16).

We previously demonstrated that the increased stimulatory capacityof cell-based aAPCs allowed PB nTregs to be expanded 1000-fold with asingle restimulation, even in the presence of rapamycin, and nTregs

expanded with aAPCs were equal to anti-CD3/CD28 mAb-coatedbead-expanded cells at suppressing xenogeneic GVHD [fig. S1K and(25)]. For these initial studies, restimulation was performed at thegrowth plateau phase, but the high variability (days 8 to 12) and diffi-culty of determining this time point are not conducive to clinical pro-duction. Restimulation on a specific day is optimal for clinical trials.However, although studies without rapamycin showed that restimula-tion on day 7 increased expansion, we observed no increase in expan-sion of day 7 restimulation (n = 3) using bead-purified nTreg stimulatedwith anti-CD3/CD28 mAb-coated beads (25- and 18-fold for with orwithout day 7 restimulation, respectively). Although restimulation basedon cell size resulted in more variability in the day of optimal re-stimulation than would be the case at a single time point, such an ap-proach identified a time range (day 13 ± 1) more suitable for clinicalrestimulation.

All nTreg cultures contain some number of Foxp3− cells, which havethe potential, especially after restimulation, to become effector T cellsand exacerbate disease. Although nTreg cultures restimulated in theabsence of rapamycin contained high numbers of IL-2– and IFN-g–secreting cells, the number of these cells did not increase with restim-ulation in the presence of rapamycin. Furthermore, when transferred

www.S

in vivo, Foxp3− cells present in nTreg cultures did not expand or persistlong-term and, in contrast to cultures expanded from CD4+CD25− cells,did not exacerbate GVHD. In addition, studies show that rapa-mycin temporally imparts Foxp3 expression and Treg-like activity toeffector T cells, which can reacquire T-effector cell function if rapa-mycin is removed (36). LAP expression differentiates activated nTregs

from stimulated CD4+CD25− T cells expressing Foxp3 spontaneouslyor after exposure to TGFb or rapamycin (17). Even after four restimu-lations, most Foxp3+ cells expressed LAP even 7 days after restimula-tion, showing that the cultures remain primarily nTregs. Furthermore,cultures expanded >1 million–fold maintained nTreg-specific de-methylation in the Foxp3 gene. Murine T cells expanded in rapamycinare TH2-skewed, secrete IL-4 and IL-10, and, after adoptive transfer,decrease allospecific IFN-g secretion and ameliorate disease in amurinemodel of GVHD (37). Although rapamycin almost completely inhib-ited the differentiation of IL-2– and IFN-g–secreting cells in our cul-tures of human cells, the effect on IL-4 was not complete, and >50%of cells secreted IL-4 (both Foxp3+ and Foxp3− cells) after the fourthrestimulation.

Murine and human nTregs are not terminally differentiated and canbe reprogrammed to secrete IL-17 in vitro or in vivo when activated inthe presence of IL-6 (31, 32, 38). Adoptive transfer of reprogrammedmurine nTregs induced autoimmune diabetes but, unlike their humancounterparts, these cells also produced IFN-g and tumor necrosisfactor–a (TNFa). It is not known whether reprogrammed humannTregs will cause disease, because only ~5% of nTregs become IL-17+

in vitro (38), and these retain suppressive function (31). Several findingsfrom this study suggest that nTreg reprogramming may not be a graveissue in developing a cellular therapy for in vitro–expanded nTregs. First,IL-17 was undetectable in the supernatants of all restimulation samplescultured with rapamycin (limit of detection, 0.3 pg/ml). Second, thenumber of expanded cells that were IL-17+ cells was very low (<2%total and ≤0.5% Foxp3+IL-17+) and, even more important, did not in-crease significantly over the four restimulation cycles (fig. S1J). Althoughthe likelihood for in vivo reprogramming of nTregs and especiallyexpanded nTregs may be context-dependent, the high degree of TSDRdemethylation of these cells may provide some degree of resistance tothe reprogramming process.

In summary, the degree of nTreg expansion reported here couldlead to the widespread application of nTreg cellular therapy for GVHDand graft rejection through the creation of an off-the-shelf therapyusing nTreg banks generated from human leukocyte antigen (HLA)–typed donors with known safety and potency records. The massiveexpansion observed with repetitive polyclonal stimulation should alsoallow relatively rare, autoantigen-specific nTreg clones to be expandedto treat autoimmune diseases. Ultimately, this strategy could be ap-plied to expansion of antigen-specific nTregs, which are more effectivethan polyclonal Tregs at suppressing disease. This strategy is potentiallypreferable to using Tregs induced in vitro by Foxp3 gene transfer orother conditions that favor Foxp3 expression. Furthermore, if increasedpurity and/or suppressive function is required, nTregs could be re-isolated after expansion using a protocol described recently by Shevach’sgroup based upon LAP expression (17). Although GMP sorting can bechallenging for many institutions, restimulation-driven expansion couldproduce sufficient numbers of cells in a small number of sorts tosupport the creation of a master cell bank that would contain matchesfor multiple patients. Finally, an nTreg master cell bank would be aneffective treatment for multiple diseases because, as shown here, nTregs

cienceTranslationalMedicine.org 18 May 2011 Vol 3 Issue 83 83ra41 7

R E S EARCH ART I C L E

suppress third-party responses, are able to maintain suppressive functionafter freeze/thaw, and ameliorate disease without long-term persistence.

on

May

19,

201

1st

m.s

cien

cem

ag.o

rgD

ownl

oade

d fr

om

MATERIALS AND METHODS

Treg isolation and cultureFor all experiments, nonmobilized PB leukapheresis products werecollected from normal adult volunteers with Food and Drug Admin-istration (FDA)–approved/cleared apheresis instruments. Written in-formed consent was obtained from all subjects with approval from theUniversity of Minnesota Institutional Review Board. nTregs were puri-fied with GMP magnetic beads or by sorting and cultured as in theSupplementary Material. Where indicated, rapamycin (Rapammune,Wyeth-Ayerst) at 109 nM was added on day 0 and with subsequentmedia supplementation. Cell size and viability were determined byViCell (Beckman Coulter).

For mAb bead-based nTreg purification, CD4+ T cells were en-

riched by MACS (all beads from Miltenyi Biotec) by depleting non-CD4+ cells with GMP-grade mAb-coated microbeads (cocktail ofCD8, CD14, CD19 ± CD56, 7.5 ml each/apheresis product) in com-bination with a CliniMACS (Depletion 2.1, max TNC = 2.0 × 1010).Unbound cells were washed and CD25high Tregs were subsequentlypurified by positive selection with GMP-grade anti-CD25 mAb-coatedmicrobeads (7.5 ml/apheresis product) and CliniMACS (Enrichment3.2). CD8−/CD14−/CD19−/CD25− cells were subsequently enrichedfor CD3+ feeder cells with GMP-grade anti-CD3 microbeads. All beadincubations were performed as specified by the manufacturer (thatis, 30 min at room temperature for GMP-grade beads). All washeswere performed at 300g for 10 min at room temperature.

nTregs were sort-purified from PBMNCs (Ficoll-Hypaque, AmershamBiosciences) in a two-step procedure in which CD25+ cells were ini-tially enriched from PBMNCs by AutoMACS (PosselD2) with GMP-grade anti-CD25 microbeads (75 ml/2 × 108 cells). CD25high cells werestained with CD4, CD8, CD25, and CD127 and sorted via FACSAriaas CD4+, CD8−, CD25high, and CD127−. Note that the bead-boundand fluorochome-conjugated anti-CD25 antibodies recognize differ-ent epitopes.

Purified CD4+CD25+ cells were cultured either with GMP anti-CD3/CD28 mAb-coated Dynabeads (26) (3:1 bead/cell) or with K562cell lines engineered to express CD86 and the high-affinity Fc receptor(CD64) (37) (2:1 nTreg/KT), which had been irradiated with 10,000cGy and incubated with anti-CD3 (Orthoclone OKT3, Janssen-Cilag).In some experiments, nTregs were stimulated with KT64/86 cells thatwere preloaded, irradiated, and frozen (1:1 nTreg/KT). Irradiatedfeeder cells (26 Gy, CD8−/CD14−/CD19−/CD25−/CD3+) were addedto CD3/CD28 bead cultures at 1:1 feeder/Treg. nTregs were cultured inX-Vivo 15 media (BioWhittaker) supplemented with 10% human ABserum (Valley Biomedical), GlutaMAX (Gibco), and N-acetylcysteine(USP). Recombinant IL-2 (300 IU/ml, Chiron) was added on day 2and maintained for culture duration. Cultures were maintained at0.3 × 106 to 0.5 × 106 viable nucleated cells/ml every 2 to 3 days.

Intracellular cytokine stainingFresh or frozen nTregs were cultured in supplemented X-Vivo 15 for4 hours ± PMA (2 pg/ml) and ionomycin (1 mg/ml) in the presence ofbrefeldin A (100 ng/ml) (all Sigma). Frozen/thawed samples werecultured for 1 hour at 37°C before restimulation. Cells were then har-

www.S

vested and stained for CD4, CD25, Foxp3, and cytokine (IL-2, IL-4,IL-17, and IFN-g) or granzyme B with the standard Foxp3 intra-cellular staining kit.

Suppression assaysThe in vitro–suppressive capacity of expanded nTregs was assessed witha CFSE inhibition assay as previously published. Briefly, PBMNCs werepurified, labeled with CFSE (Invitrogen), and stimulated with anti-CD3mAb-coated beads (Dynal) ± culturednTreg (1:2 to 1:32 nTregs/PBMNCs).On day 4, cells were stained with antibodies to CD4 and CD8 and pro-liferation, data were analyzed with FlowJo (8.8.7), and suppressionwas determined from the Division Index (TreeStar). nTregs suppressedCD4+ and CD8+ T cell responses equivalently (fig. S8), and only CD8data are presented. Xenogeneic GVHD experiments were performedas in (25) and are described in the Supplementary Material.

Statistical analysisData were analyzed by analysis of variance (ANOVA) or Student’s t test.Probability (P) values of ≤0.05 were considered statistically significant.

SUPPLEMENTARY MATERIAL

www.sciencetranslationalmedicine.org/cgi/content/full/3/83/83ra41//DC1Materials and MethodsFig. S1. nTregs stimulated with cell-based aAPCs have increased peak size but decreased Foxp3.Fig. S2. Cultured nTregs maintain Foxp3-suppressive function after cryopreservation and thawing.Fig. S3. Sort-purified nTregs expand more than bead-purified nTregs.Fig. S4. Phenotype of restimulated nTregs.Fig. S5. Massively expanded nTreg phenotype as memory T cells, but are not exhausted.Fig. S6. No T cell receptor (TCR) Vb skewing after massive expansion of Tregs.Fig. S7. Loss of Foxp3+ cells is due to increased cycling of Foxp3− cells.Fig. S8. nTregs stimulated once or a total of five times suppress CD4+ and CD8+ T cell responsesequivalently.Table S1. Summary of in vitro nTreg expansion comparing source, stimulation conditions, andrestimulation.

REFERENCES AND NOTES

1. L. A. Welniak, B. R. Blazar, W. J. Murphy, Immunobiology of allogeneic hematopoietic stemcell transplantation. Annu. Rev. Immunol. 25, 139–170 (2007).

2. R. S. Wildin, A. Freitas, IPEX and FOXP3: Clinical and research perspectives. J. Autoimmun.25, 56–62 (2005).

3. P. Hoffmann, J. Ermann, M. Edinger, C. G. Fathman, S. Strober, Donor-type CD4+CD25+

regulatory T cells suppress lethal acute graft-versus-host disease after allogeneic bonemarrow transplantation. J. Exp. Med. 196, 389–399 (2002).

4. E. M. Shevach, R. A. DiPaolo, J. Andersson, D. M. Zhao, G. L. Stephens, A. M. Thornton, Thelifestyle of naturally occurring CD4+CD25+Foxp3+ regulatory T cells. Immunol. Rev. 212,60–73 (2006).

5. P. A. Taylor, C. J. Lees, B. R. Blazar, The infusion of ex vivo activated and expanded CD4+CD25+

immune regulatory cells inhibits graft-versus-host disease lethality. Blood 99, 3493–3499 (2002).6. M. Gavin, A. Rudensky, Control of immune homeostasis by naturally arising regulatory CD4+

T cells. Curr. Opin. Immunol. 15, 690–696 (2003).7. C. H. June, B. R. Blazar, Clinical application of expanded CD4+25+ cells. Semin. Immunol. 18,

78–88 (2006).8. C. A. Piccirillo, E. M. Shevach, Naturally-occurring CD4+CD25+ immunoregulatory T cells:

Central players in the arena of peripheral tolerance. Semin. Immunol. 16, 81–88 (2004).9. M. G. Roncarolo, M. Battaglia, Regulatory T-cell immunotherapy for tolerance to self anti-

gens and alloantigens in humans. Nat. Rev. Immunol. 7, 585–598 (2007).10. C. Baecher-Allan, J. A. Brown, G. J. Freeman, D. A. Hafler, CD4+CD25high regulatory cells in

human peripheral blood. J. Immunol. 167, 1245–1253 (2001).11. W. R. Godfrey, Y. G. Ge, D. J. Spoden, B. L. Levine, C. H. June, B. R. Blazar, S. B. Porter, In vitro–

expanded human CD4+CD25+ T-regulatory cells can markedly inhibit allogeneic dendriticcell–stimulated MLR cultures. Blood 104, 453–461 (2004).

cienceTranslationalMedicine.org 18 May 2011 Vol 3 Issue 83 83ra41 8

R E S EARCH ART I C L E

on

May

19,

201

1st

m.s

cien

cem

ag.o

rgD

ownl

oade

d fr

om

12. K. L. Hippen, P. Harker-Murray, S. B. Porter, S. C. Merkel, A. Londer, D. K. Taylor, M. Bina,A. Panoskaltsis-Mortari, P. Rubinstein, N. Van Rooijen, T. N. Golovina, M. M. Suhoski, J. S. Miller,J. E. Wagner, C. H. June, J. L. Riley, B. R. Blazar, Umbilical cord blood regulatory T-cell expansionand functional effects of tumor necrosis factor receptor family members OX40 and 4-1BBexpressed on artificial antigen-presenting cells. Blood 112, 2847–2857 (2008).

13. C. G. Brunstein, J. S. Miller, Q. Cao, D. H. McKenna, K. L. Hippen, J. Curtsinger, T. Defor,B. L. Levine, C. H. June, P. Rubinstein, P. B. McGlave, B. R. Blazar, J. E. Wagner, Infusionof ex vivo expanded T regulatory cells in adults transplanted with umbilical cord blood:Safety profile and detection kinetics. Blood 117, 1061–1070 (2011).

14. P. Hoffmann, R. Eder, T. J. Boeld, K. Doser, B. Piseshka, R. Andreesen, M. Edinger, Only theCD45RA+ subpopulation of CD4+CD25high T cells gives rise to homogeneous regulatory T-celllines upon in vitro expansion. Blood 108, 4260–4267 (2006).

15. S. Karakhanova, M. Munder, M. Schneider, M. Bonyhadi, A. D. Ho, M. Goerner, Highly efficientexpansion of human CD4+CD25+ regulatory T cells for cellular immunotherapy in patientswith graft-versus-host disease. J. Immunother. 29, 336–349 (2006).

16. P. Hoffmann, T. J. Boeld, R. Eder, J. Huehn, S. Floess, G. Wieczorek, S. Olek, W. Dietmaier,R. Andreesen, M. Edinger, Loss of FOXP3 expression in natural human CD4+CD25+ regula-tory T cells upon repetitive in vitro stimulation. Eur. J. Immunol. 39, 1088–1097 (2009).

17. D. Q. Tran, J. Andersson, D. Hardwick, L. Bebris, G. G. Illei, E. M. Shevach, Selective expres-sion of latency-associated peptide (LAP) and IL-1 receptor type I/II (CD121a/CD121b) onactivated human FOXP3+ regulatory T cells allows for their purification from expansioncultures. Blood 113, 5125–5133 (2009).

18. P. Hoffmann, T. J. Boeld, R. Eder, J. Albrecht, K. Doser, B. Piseshka, A. Dada, C. Niemand,M. Assenmacher, E. Orsó, R. Andreesen, E. Holler, M. Edinger, Isolation of CD4+CD25+

regulatory T cells for clinical trials. Biol. Blood Marrow Transplant. 12, 267–274 (2006).19. J. H. Peters, L. B. Hilbrands, H. J. Koenen, I. Joosten, Ex vivo generation of human alloantigen-

specific regulatory T cells from CD4posCD25high T cells for immunotherapy. PLoS One 3, e2233(2008).

20. M. Battaglia, A. Stabilini, B. Migliavacca, J. Horejs-Hoeck, T. Kaupper, M. G. Roncarolo, Rapamycinpromotes expansion of functional CD4+CD25+FOXP3+ regulatory T cells of both healthysubjects and type 1 diabetic patients. J. Immunol. 177, 8338–8347 (2006).

21. M. Battaglia, A. Stabilini, M. G. Roncarolo, Rapamycin selectively expands CD4+CD25+FoxP3+

regulatory T cells. Blood 105, 4743–4748 (2005).22. J. J. Coenen, H. J. Koenen, E. van Rijssen, L. B. Hilbrands, I. Joosten, Rapamycin, and not

cyclosporin A, preserves the highly suppressive CD27+ subset of human CD4+CD25+ regula-tory T cells. Blood 107, 1018–1023 (2006).

23. L. Strauss, T. L. Whiteside, A. Knights, C. Bergmann, A. Knuth, A. Zippelius, Selective survivalof naturally occurring human CD4+CD25+Foxp3+ regulatory T cells cultured with rapamycin.J. Immunol. 178, 320–329 (2007).

24. W. Liu, A. L. Putnam, Z. Xu-Yu, G. L. Szot, M. R. Lee, S. Zhu, P. A. Gottlieb, P. Kapranov,T. R. Gingeras, B. Fazekas de St Groth, C. Clayberger, D. M. Soper, S. F. Ziegler, J. A. Bluestone,CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+

T reg cells. J. Exp. Med. 203, 1701–1711 (2006).25. T. N. Golovina, T. Mikheeva, M. M. Suhoski, N. A. Aqui, V. C. Tai, X. Shan, R. Liu, R. R. Balcarcel,

N. Fisher, B. L. Levine, R. G. Carroll, N. Warner, B. R. Blazar, C. H. June, J. L. Riley, CD28 costimula-tion is essential for human T regulatory expansion and function. J. Immunol. 181, 2855–2868(2008).

26. B. L. Levine, W. B. Bernstein, M. Connors, N. Craighead, T. Lindsten, C. B. Thompson, C. H. June,Effects of CD28 costimulation on long-term proliferation of CD4+ T cells in the absence ofexogenous feeder cells. J. Immunol. 159, 5921–5930 (1997).

27. J. Huehn, J. K. Polansky, A. Hamann, Epigenetic control of FOXP3 expression: The key to astable regulatory T-cell lineage? Nat. Rev. Immunol. 9, 83–89 (2009).

28. J. C. Rathmell, E. A. Farkash, W. Gao, C. B. Thompson, IL-7 enhances the survival and maintainsthe size of naive T cells. J. Immunol. 167, 6869–6876 (2001).

29. J. M. Brenchley, N. J. Karandikar, M. R. Betts, D. R. Ambrozak, B. J. Hill, L. E. Crotty, J. P. Casazza,J. Kuruppu, S. A. Migueles, M. Connors, M. Roederer, D. C. Douek, R. A. Koup, Expression ofCD57 defines replicative senescence and antigen-induced apoptotic death of CD8+ T cells.Blood 101, 2711–2720 (2003).

www.S

30. M. D. Sharma, D. Y. Hou, B. Baban, P. A. Koni, Y. He, P. R. Chandler, B. R. Blazar, A. L. Mellor,D. H. Munn, Reprogrammed Foxp3+ regulatory T cells provide essential help to supportcross-presentation and CD8+ T cell priming in naive mice. Immunity 33, 942–954 (2010).

31. G. Beriou, C. M. Costantino, C. W. Ashley, L. Yang, V. K. Kuchroo, C. Baecher-Allan, D. A. Hafler,IL-17–producing human peripheral regulatory T cells retain suppressive function. Blood 113,4240–4249 (2009).

32. S. Radhakrishnan, R. Cabrera, E. L. Schenk, P. Nava-Parada, M. P. Bell, V. P. Van Keulen,R. J. Marler, S. J. Felts, L. R. Pease, Reprogrammed FoxP3+ T regulatory cells become IL-17+

antigen-specific autoimmune effectors in vitro and in vivo. J. Immunol. 181, 3137–3147 (2008).33. C. A. Klebanoff, L. Gattinoni, N. P. Restifo, CD8+ T-cell memory in tumor immunology and

immunotherapy. Immunol. Rev. 211, 214–224 (2006).34. P. Scheinberg, J. J. Melenhorst, J. M. Brenchley, B. J. Hill, N. F. Hensel, P. K. Chattopadhyay,

M. Roederer, L. J. Picker, D. A. Price, A. J. Barrett, D. C. Douek, The transfer of adaptiveimmunity to CMV during hematopoietic stem cell transplantation is dependent on thespecificity and phenotype of CMV-specific T cells in the donor. Blood 114, 5071–5080(2009).

35. A. L. Putnam, T. M. Brusko, M. R. Lee, W. Liu, G. L. Szot, T. Ghosh, M. A. Atkinson, J. A. Bluestone,Expansion of human regulatory T-cells from patients with type 1 diabetes. Diabetes 58,652–662 (2009).

36. D. Valmori, V. Tosello, N. E. Souleimanian, E. Godefroy, L. Scotto, Y. Wang, M. Ayyoub,Rapamycin-mediated enrichment of T cells with regulatory activity in stimulated CD4+ Tcell cultures is not due to the selective expansion of naturally occurring regulatory T cellsbut to the induction of regulatory functions in conventional CD4+ T cells. J. Immunol.177, 944–949 (2006).

37. J. E. Foley, J. Mariotti, K. Ryan, M. Eckhaus, D. H. Fowler, Th2 cell therapy of establishedacute graft-versus-host disease requires IL-4 and IL-10 and is abrogated by IL-2 or host-typeantigen-presenting cells. Biol. Blood Marrow Transplant. 14, 959–972 (2008).

38. H. J. Koenen, R. L. Smeets, P. M. Vink, E. van Rijssen, A. M. Boots, I. Joosten, HumanCD25highFoxp3pos regulatory T cells differentiate into IL-17–producing cells. Blood 112,2340–2352 (2008).

39. Acknowledgments: We would like to thank C. Nelson for assistance with animal husband-ry. Funding: This work was supported in part by research grants from the Children’s CancerResearch Fund and Blood and Marrow Transplant Research Fund to K.L.H.; Leukemia andLymphoma Translational Research (grant R6029-07), R37 HL56067, and P01 AI056299 toB.R.B., P01 CA067493 and N01HB037164 to B.R.B., J.E.W., and J.S.M.; support from MiltenyiBiotec to B.R.B. and J.E.W.; a grant from Becton Dickinson to J.E.W.; and support from theJDRF Collaborative Centers for Cell Therapy and the JDRF Center on Cord Blood Therapiesfor Type 1 Diabetes to J.L.R. and C.H.J. Author contributions: K.L.H. designed the research,performed the experiments, interpreted the data, and wrote the paper. S.C.M., D.K.S., C.M.S.,and P.S. performed the experiments, interpreted the data, and assisted with the paper. D.S.and D.M.K. performed the research. J.S.M., J.E.W., D.C.D., D.H.M., J.S.B., B.L.L., C.H.J., and J.L.R.designed the research and wrote the paper. B.R.B. designed the research, interpreted the data,and wrote the paper. Competing interests: J.L.R., C.H.J., and J.E.W. have research fundingfrom Becton Dickinson and C.H.J. and B.R.B. were previously scientific consultants for BectonDickinson, although this funding did not conflict with this manuscript. K.L.H., J.L.R., C.H.J., andB.R.B. are authors on U.S. provisional patent application number 61/322, 186, “Methods toexpand a T regulatory cell master cell bank.” The other authors declare that they have nocompeting interests.

Submitted 14 October 2010Accepted 25 March 2011Published 18 May 201110.1126/scitranslmed.3001809

Citation: K. L. Hippen, S. C. Merkel, D. K. Schirm, C. M. Sieben, D. Sumstad, D. M. Kadidlo,D. H. McKenna, J. S. Bromberg, B. L. Levine, J. L. Riley, C. H. June, P. Scheinberg, D. C. Douek,J. S. Miller, J. E. Wagner, B. R. Blazar, Massive ex vivo expansion of human natural regulatoryT cells (Tregs) with minimal loss of in vivo functional activity. Sci. Transl. Med. 3, 83ra41 (2011).

cienceTranslationalMedicine.org 18 May 2011 Vol 3 Issue 83 83ra41 9