of July 6, 2018.This information is current as

βof TGF-Lymphocytes by Stimulation in the Presence

PeripheralRegulatory Function from Human Differentiation and Expansion of T Cells with

Patricia E. Rao, Andria L. Petrone and Paul D. Ponath

http://www.jimmunol.org/content/174/3/1446doi: 10.4049/jimmunol.174.3.1446

2005; 174:1446-1455; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/174/3/1446.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2005 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Differentiation and Expansion of T Cells with RegulatoryFunction from Human Peripheral Lymphocytes by Stimulationin the Presence of TGF-�

Patricia E. Rao,1 Andria L. Petrone, and Paul D. Ponath

T cells with immunoregulatory function have been described in human and mouse systems. In both systems these cells can bedifferentiated either in the thymus or from peripheral T cells. To date, more progress has been made in the study of murineregulatory T cells, because it has been very difficult to isolate human regulatory T cells of sufficient purity and in sufficient numbersto permit detailed examinations of their biochemistry. We report in this study that human T cells with regulatory function can bedifferentiated in vitro from naive (CD4�CD45RA�) cord blood or peripheral T cells by stimulation with anti-CD3 and anti-CD28in the presence of TGF-�. Cells derived in this manner express a surface phenotype (CD25�, CD122�, HLA-DR�, glucocorticoid-induced TNF receptor-related gene�, CD103�, CTLA-4�) described for human and mouse regulatory T cells and express proteinand message for the transcription factor forkhead/winged helix transcription factor (FOXP3). They produce primarily TGF-� andIL-10, with lesser amounts of IFN-� and IL-13, when stimulated through their TCRs and are capable of inhibiting cytokineproduction and proliferation by stimulated naive T cells. Unlike Th1 and Th2 cells, these TGF-�-derived regulatory T cells do notappear to be dependent on the protein kinase C� pathway of NF-�B activation for Ag-induced responses. The Journal ofImmunology, 2005, 174: 1446–1455.

W hen initially described, regulatory T cells were iden-tified from the CD4�CD25� subset of murine periph-eral lymphocytes (1–8). These cells were shown to be

directly thymus derived (9–11) and capable of inhibiting prolifer-ation in vitro of murine CD4�CD25�cells (12), and CD8� cells(13). Additionally, transfer of CD4�CD25� cells in various dis-ease models has demonstrated their suppressive ability in vivo(14–19). Association of regulatory function with a similar humanpopulation was also described several years later (20–24). Al-though obtaining populations of murine lymphocytes sufficientlyenriched in a regulatory population to permit studying these cellshas been possible, it has proved more difficult to obtain sufficientlypure human regulatory T cells. Human CD4�CD25� cells withregulatory function have been obtained from peripheral blood insufficient purity for study by using only a subpopulation ofCD4�CD25� cells expressing high levels of CD25 (25, 26).

The possibility that regulatory cell populations might also bederived from naive peripheral lymphocytes, distinct from the thy-mus-derived CD4�CD25� population, has also been recognized inthe description of TR1 cells (27, 28), which are capable of inhib-iting colitis in mice, and Th3 cells (29, 30), which are capable ofmediating tolerance to orally administered Ags in mice. Additionalevidence for the ability of peripheral lymphocytes to differentiateinto regulatory T cells was recently presented in the murine systemthrough cultivation of murine CD4�CD25�peripheral lympho-cytes in vitro in the presence of TGF-� (31). Regulatory T cellsdifferentiated under these conditions expressed forkhead/winged

helix transcription factor (FOXP3),2 secreted TGF-�, suppressedproliferation of normal T cells in vitro, and inhibited Ag-specificresponses of TCR transgenic animals in vivo.

We report in this study that human regulatory T cells can bedifferentiated in vitro by cultivation of naive human cord blood orPBL in the presence of exogenous IL-2, TGF-�, and IL-15. Ourdata strongly suggest that exposure of peripheral blood CD4 cellsto TGF-� can up-regulate the expression of FOXP3, which mayhave physiological significance during healing when TGF-� ishighly expressed. Cells differentiated in this manner express a sur-face phenotype associated with human and murine regulatory Tcells (CD4�, CD25�, CD69�, glucocorticoid-induced TNF recep-tor-related gene (GITR)�), express cytokines appropriate to regu-latory cells, up-regulate the transcription factor FOXP3, and sup-press division and cytokine production when cocultured withstimulated naive lymphocytes. These culture conditions permit theexpansion of cells with a regulatory phenotype to numbers usefulfor larger biological studies or screening assays.

Materials and MethodsCulture medium

X-VIVO-15 (Cambrex Biosciences) supplemented with 10% FBS wasused for the culture of T cells and PBL.

Cytokines

All cytokines used in this study were recombinant human proteins obtainedfrom R&D Systems. For differentiation of naive T cells, the followingconcentrations were used: 65 U/ml IL-2 (stock activity, 1.3 � 104 U/�g),62 U/ml IL-12 (stock activity, 1.24 � 104 U/�g), 145 U/ml IL-4 (stockactivity, 2.9 � 104 U/�g), 32 U/ml TGF-�1 (stock activity, 3.2 � 104

U/�g), 42 U/ml IL-9 (stock activity, 8.4 � 103 U/�g), and 4500 U/mlIL-15 (stock activity, 4.5 � 105 U/�g). For PBL cultures, cytokine con-centrations are indicated.

TolerRx, Cambridge, MA 02139

Received for publication April 27, 2004. Accepted for publication November24, 2004.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 Address correspondence and reprint requests to Dr. Patricia E. Rao, TolerRx, 300Technology Square, Cambridge, MA 02139. E-mail address: dandp77@earthlink.net

2 Abbreviations used in this paper: FOXP3, forkhead/winged helix transcription fac-tor; GITR, glucocorticoid-induced TNF receptor-related gene; PKC, protein kinase C;TRITC, tetramethylrhodamine isothiocyanate; GATA3, GATA-binding protein-3;TBX21, T-box expressed in T cells.

The Journal of Immunology

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00

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Antibodies

The following Abs were obtained from BD Pharmingen: anti-CD3 (cloneUCHT1), anti-human IL-4 (clone MP4-25D2), anti-human IFN-� (cloneB27), anti-human IL-12 (clone C8.6), anti-CD25-FITC (clone M-A251),anti-CD122-PE (clone Mik-�2), anti-HLA-DR-PE (clone G46-6), anti-CD69-PE (clone FN50), and anti-CD152-biotin (clone BN13). Isotypecontrol Abs were also obtained from BD Pharmingen. Anti-CD28 (cloneANC28.1/5D10) was obtained from Ancell. Anti-CD103-biotin and anti-GITR-biotin were obtained from Beckman Coulter and R&D Systems,respectively. To detect surface TGF-�, two Abs from R&D Systems wereused: biotin-conjugated, goat anti-human TGF-� latency-associated pep-tide (catalog no. BAF 246) and biotinylated, chicken anti-human TGF-�(catalog no. BAF240). The secondary reagent streptavidin-Quantum Red(Sigma-Aldrich) was used for detection of biotinylated Abs. For immuno-fluorescence microscopy, goat anti-human FOXP3 was obtained from Ab-cam; HRP-conjugated anti-protein kinase C� (anti-PKC�) was obtainedfrom BD Transduction Laboratories; anti-��TCR was obtained from BDPharmingen; tetramethylrhodamine isothiocyanate (TRITC)-conjugatedAffinipure F(ab�)2 donkey anti-goat IgG(H�L) and TRITC-conjugated Af-finipure F(ab�)2 goat anti-HRP were obtained from Jackson Immuno-Research Laboratories. For inhibition of TGF-� or IL-10, the followingAbs from R&D Systems were used: neutralizing mouse anti-human IL-10,clone 23738; neutralizing mouse anti-human TGF-�1, clone 9016; andneutralizing anti-human TGF-�1,- 2, and -3, clone 1D11.

Preparation of cells

PBL were prepared by Ficoll density gradient centrifugation (AmershamBiosciences). PBL were washed into culture medium for direct use in as-says or were washed into sorting medium for separation of naive CD4� Tcells.

For T cell differentiation using adult peripheral naive CD4� T cells,PBL were separated using an AutoMACS cell sorter (Miltenyi Biotec).First, CD4� cells were purified by negative selection using the CD4� Mul-tisort kit (Miltenyi Biotec) according to the manufacturer’s instructions.These CD4� cells were then further sorted by positive selection usingCD45RA� microbeads (Miltenyi Biotec). The CD4�CD45RA� cells werewashed into medium for use in cell culture. The purity of sortings wasconfirmed by FACS to be between 90 and 95%.

For T cell differentiation using human cord blood naive CD4� T cells,frozen CD4�CD45RA� cells isolated using magnetic beads were obtainedfrom Allcells. Cells were thawed and washed out of DMSO into culturemedium for use in cell culture.

T cell differentiation

CD4�CD45RA� T cells from either cord blood or adult peripheral bloodwere stimulated in the presence of differentiative cytokines. Twelve-wellNunc plates were coated with 5 �g/ml anti-CD3 and 10 or 5 �g/ml anti-CD28. Briefly, wells of a tissue culture plate were coated with 0.5 ml ofPBS containing the Ab mixture and incubated overnight at 4°C or for 2 hat 37°C. Wells were then washed twice with 1 ml of PBS before platingcells at 0.5–1.0 � 106 cells/well in 2 ml of culture medium. Cytokines andneutralizing anti-cytokine Abs were added to the appropriate cultures. Allcultures contained a final IL-2 concentration of 65 U/ml. Final cultureconcentrations for each cell type were as follows: for Th1: IL-12, 62 U/ml; andanti-IL-4, 200 ng/ml; for Th2: IL-4, 145 U/ml; anti-IFN-�, 20 �g/ml; andanti-IL-12, 10 �g/ml; and for regulatory T cells: TGF-�1, 32 U/ml; IL-9,42 U/ml; IL-15, 4500 U/ml; anti-IFN-�, 20 �g/ml; and anti-IL-12, 10�g/ml. Cultures were incubated for 2–3 days at 37°C in 5% CO2. On days2–3, cells were removed from the stimulation plate and split into largerculture vessels, feeding with fresh media and cytokines. Cells were ex-panded and fed as needed for 7–10 days. In some cases a second round ofstimulation and selection was performed as described above.

Real-time quantitative PCR assays

To isolate total RNA from differentiated cells, cells were differentiated asdescribed above, and 2–4 � 106 cells were harvested at the indicated timepoints for RNA extraction using the RNeasy kit (Qiagen, Valencia, CA)according to the manufacturer’s instructions. To measure up-regulation oftranscription factor message in activated PBL, 4 � 106 PBL were stimu-lated with plate-bound anti-CD3 (5 �g/ml) and anti-CD28 (5 �g/ml) in thepresence of 124 U/ml IL-12, 290 U/ml IL-4, or 160 U/ml TGF-�1. After48 h of culture, total RNA was extracted using the RNeasy kit accordingto the manufacturer’s instructions. For both assays, cDNA was made from5 �g of total RNA using the cDNA Archive kit (Applied Biosystems).Real-Time PCR was performed using an ABI 7600 Sequence DetectionSystem (Applied Biosystems). Primers and probes for T-box expressed in

T cells (TBX21), GATA-binding protein-3 (GATA3), and FOXP3 wereobtained as assays on demand from Applied Biosystems. For each analysis,transcription of the gene of interest was compared with transcription of thehousekeeping gene, �-actin, which was amplified in parallel.

Cytokine assays

After 7–10 days of culture, the differentiated cells were assayed for cyto-kine production. Cells were washed out of culture medium into serum-freeX-VIVO 15. Cells (0.5 ml) at 1 � 106 cells/ml were plated into 48-wellplates coated with anti-CD3 (5 �g/ml) and anti-CD28 (5 �g/ml). Cellswere incubated for 72 h, and supernatants were collected and frozen. Su-pernatant samples were sent to Pierce Endogen for multiplexed SearchlightELISA analysis of the following cytokines: IL-2, IL-4, IL-5, IL-10, IL-12,IL-13, IFN-�, TNF-�, and TGF-�. Total TGF-�, after acidification, wasmeasured in these assays.

Flow cytometry

For flow cytometric analysis, 106 cells were stained with 100 �l of appro-priately diluted Abs for 30 min on ice and washed twice with FACS buffer(PBS with 2% FBS and 0.1% sodium azide). For biotinylated Abs, cellswere additionally stained with 100 �l of a 1/10 dilution of streptavidin-Quantum Red for 30 min on ice. Cells were washed twice with FACSbuffer, resuspended in 200 �l of buffer, and analyzed on a FACScan (BDBiosciences). Intracellular staining of cells was performed after cell surfacelabeling and involved fixation and permeabilization of cells using the Fixand Perm kit (Caltag Laboratories) according to the manufacturer’sinstructions.

In vitro suppression of naive cell activation

Differentiated cells were tested for their ability to suppress the proliferationand cytokine production of naive T cells to polyclonal activation. Naiveautologous CD4� cord blood cells or adult peripheral blood cells werelabeled with the Vybrant CFSE cell tracer kit according to the manufac-turer’s instructions (Molecular Probes). The CFSE-labeled cells (1 � 106

cells/ml) were stimulated with plate-bound anti-CD3 (5 �g/ml) and anti-CD28 (5 �g/ml) in the presence or the absence of various numbers ofdifferentiated cells in a final culture volume of 200 �l. In some experimentsTGF-�-derived cells were prevented from directly contacting the CFSE-labeled cells by addition to the upper chamber of 6.5-mm Transwells with0.4-�m pore size membranes (Costar) and stimulated by the addition ofCD3/CD28 Dynabeads (Dynal Biotech), added directly to the upper well.Cultures were incubated for 4–5 days at 37°C in 5% CO2. After culture,supernatants were collected for cytokine analysis, and cells were stainedwith anti-CD25-PE and anti-CD4-biotin, followed by secondary staining ofstreptavidin-Quantum Red. Cells were resuspended in buffer and analyzedby flow cytometry using a FACScan. Equal numbers of live CFSE-labeledcells were acquired for analysis.

Immunofluorescence microscopy

Differentiated cells were counted and set at 1 � 106 cells/ml. One hundredmicroliters of cell suspension was loaded into the funnel of a cytospin, andthe cells were spun onto microscope slides. Slides were air-dried overnight,fixed in acetone for 5 min, then air-dried for 1 h. Slides were blocked with10% FBS in PBS for 15 min in a humid chamber, then stained with anti-FOXP3, HRP-conjugated anti-PKC�, or appropriate isotype-matched neg-ative control Ab for 2 h in a humid chamber. Slides were washed for 10min in PBS with 0.2% gelatin. For detection of FOXP3 Ab, the slides werethen stained with TRITC-conjugated donkey anti-goat IgG (1/200) dilutedin donkey serum for 30 min in a humid chamber in the dark. For detectionof PKC�, slides were stained with TRITC-conjugated goat anti-HRP (1/200 in blocking buffer). Slides stained for PKC� were also stained withFITC-conjugated anti-��TCR (BD Pharmingen). Slides were againwashed for 10 min in PBS with 0.2% gelatin. A single drop of Vectashieldwas placed over the cells, and a coverslip was applied. The slides were thenread under the immunofluorescence microscope.

Inhibition of PKC�

To analyze the effects of PKC� inhibitors on the proliferative response ofthe differentiated cells, 96-well, flat-bottom plates (Nunc) were coated with50 �l of anti-CD3 (5 �g/ml) and anti-CD28 (10 �g/ml) diluted in PBS,incubated overnight, then washed with PBS. Cells were plated at 2 � 105

cells/well in a final volume of 200 �l in the presence or the absence of PKCinhibitors. Rottlerin, the novel PKC inhibitor, was purchased from Calbiochemand used at final concentrations of 1, 5, and 50 �M. The antennapedia-PKC�

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inhibitor peptide (NH2-RQIKIWFQNRRMKWKKMDQNMFRNFSFMNP-COOH) was purchased from New England Peptide and used at final concen-trations of 2, 20, and 40 �M. After 3 days of culture, cells were pulsed with0.5 �Ci/well [3H]thymidine and harvested 16 h later.

ResultsAddition of TGF-� to stimulated PBL up-regulates FOXP3message

Human PBL were stimulated by CD3 and CD28 bound to thetissue culture plate for 48 or 72 h. Quantitative PCR performed onmRNA from these samples indicated that with no additional se-lective pressures, transcription factors associated with the differ-entiation of Th1 (TBX21), Th2 (GATA3), and regulatory T cells(FOXP3) were readily detected, although GATA3 was relativelymore prevalent than the other two transcription factors. When cul-tures were established with stimulating Abs and the addition ofexogenous IL-4, IL-12, or TGF-�, increases in one transcriptionfactor message relative to the others was observed (Fig. 1a). Ad-dition of IL-4 resulted in a relative increase in GATA3 (�240% ofthe expression levels in PBL stimulated under control conditions),IL-12 resulted in a 235% relative increase in TBX21, and additionof TGF-� resulted in a 190% relative increase in FOXP3. Additionof other cytokines associated with T cell differentiation, such asIFN-� or IL-13, or cytokines previously reported to augment invitro differentiation of regulatory cells or survival of differentiatedcells (IL-7, IL-1�, or IL-9) did not result in a similar relative in-crease in the transcription factors associated with T cell differen-tiation (Fig. 1b). Only addition of IL-10 produced a skewing oftranscription factor usage in the population. Addition of IL-10 re-sulted in relative suppression of TBX21 and FOXP3 messagesbelow the level normally seen in stimulated PBL, but levels ofGATA3 remained at the control levels.

Optimal growth and differentiation of naive human lymphocyteswith TGF-�

Because activation of human PBL in the presence of TGF-� ap-peared to induce increased levels of FOXP3 message, similar tothe induction of TBX21 and GATA3 induced by IL-12 and IL-4,respectively, naive human lymphocytes were stimulated in thepresence of TGF-� to determine whether regulatory T cells couldbe differentiated under these conditions. Human CD4�CD45RA�

(naive) cord blood or PBL, isolated by magnetic bead separationsand 90–99 or �93% pure, respectively, were stimulated by plate-bound CD3 and CD28 in the presence of IL-12 and anti-IL-4 (Th1conditions); IL-4, anti-IL-12, and anti-IFN-� (Th2 conditions); orTGF-� (�IL-9), anti-IL-4, anti-IFN-�, and anti-IL-12. IL-2 wasadded to all cultures.

Lymphocytes differentiated in the presence of TGF-� dividedmore slowly than cells differentiated under Th1 or Th2 conditions.In developing the culture conditions for TGF-�-derived cells, weinvestigated the effects of various additional cytokines on the cells.IL-15 was absolutely required for expansion of these cells. Asshown in Fig. 2, addition of IL-15 to Th1 or Th2 cell culturesimproved cell expansion marginally in the final days of cultureafter stimulation. However, TGF-�-derived cells failed to expandbeyond 2- to 3-fold from their initial numbers without addition ofIL-15, and viable cell numbers dropped steadily after day 3 (datanot shown). Addition of IL-15 in the absence of IL-2 supportedexpansion of these cells, but significantly less well than when IL-2was added ( p � 0.03, by Student’s t test). The combination of IL-9and IL-15 provided an additional, although statistically nonsignif-icant, boost to cell growth, but the best expansion of TGF-� cellswas observed when IL-15 and IL-2 or IL-15, IL-9, and IL-2 werecombined. Under these conditions, TGF-�-derived cells expandedto 20-fold their starting numbers.

FIGURE 1. Enhancement of tran-scription factor usage by PBL stimu-lated in the presence of exogenous cy-tokines. PBL were stimulated byincubation with CD3 and CD28 Absin the absence or the presence of cy-tokines for 72 h, and total RNA wasprepared. Quantitative PCR was per-formed on all samples, and the num-ber of copies of message for FOXP3,GATA3, or TBX21 present in sam-ples derived from cells stimulatedwith cytokines was expressed relativeto the number of copies found in PBLstimulated in the absence of cyto-kines, which was arbitrarily set at100. Comparison of transcription fac-tor message induction in the presenceof IL-4, IL-12, and TGF-� (a) isshown. The effects of addition ofother cytokines on induction of mes-sages for the three transcription fac-tors are also shown in representativeexperiments (b).

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Cytokines produced by TGF-�-derived cells

Cells were stimulated and allowed to expand and differentiate un-der these conditions for 7–12 days. Once they returned to a restingstate, cells were restimulated on plate-bound CD3 and CD28 inserum-free medium to assess cytokine production. Supernatantscollected at 72 h were analyzed for cytokine production. As ex-pected, cells differentiated under Th1 conditions (Fig. 3) producedIFN-�, and those differentiated under Th2 conditions produced pri-marily IL-13 and IL-10, with lesser and variable amounts of IL-5and TGF-�. Cells differentiated in the presence of TGF-� supple-mented with IL-9 and IL-15 produced primarily TGF-�, withlesser and variable amounts of IFN-�, IL-13, and IL-10. All celltypes produced TNF-�; notably, IL-2 was produced at some level

by all cell types. Greater variability in production of the less abun-dant cytokines was seen when cultures were derived from adultnaive lymphocytes rather than cord blood naive lymphocytes (datanot shown). Restimulation of differentiated cells with plate-boundCD3 alone or plate-bound CD3 and CD28 in the presence of ex-ogenous IL-2 and in the presence or the absence of the differen-tiative cytokine/Ab conditions described above was also tested. Asecond round of stimulation in the differentiative cytokines en-hanced cell type-specific (e.g., IFN-� for Th1 cells) cytokine pro-duction for each of the cell types. Stimulation of all cell types wassuccessful using either CD3 or CD3� CD28 and in the presence orthe absence of exogenous IL-2 (data not shown). Restimulation ofTGF-�-derived cells by CD3 and CD28 without addition of thedifferentiative cytokine and Ab mixture produced cells that whenstimulated still produced the cytokine profile associated with reg-ulatory T cells (data not shown).

FIGURE 2. Effects of addition of exogenous cytokines on the expansionof Th1, Th2, or TGF-�-derived cells in culture. Cultures were initiated with1 � 106 naive human CD4� cells (CD4�CD45RA�) sorted from cordblood and were stimulated by plate-bound CD3 and CD28 Abs in thepresence of Th1- or Th2-polarizing conditions or TGF-�. All cultures re-ceived exogenous IL-2. The effect of addition of exogenous IL-15 to Th1(a) or Th2 (b) cells on expansion of the cells in culture is shown. The effectof the addition of IL-15 and IL-9, in the presence or the absence of IL-2,on the growth of TGF-�-cultured cells is shown in c.

FIGURE 3. Cytokines produced by Th1, Th2, and TGF-�-derived cells.Th1, Th2, and TGF-�-derived cells were prepared from cord blood asdescribed in Materials and Methods. After expansion of the cells underdifferentiating conditions, rested cells were restimulated by plate-boundCD3 and CD28 in X-Vivo serum-free medium, and supernatants were an-alyzed after 72 h by multiplexed ELISA to identify and measure cytokinesproduced by Th1 (a), Th2 (b), or TGF-�-derived (c) cells.

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Phenotype of differentiated cells

After one round of stimulation and differentiation, Th1, Th2, orTGF-�-differentiated cells were stained with a variety of Abs rec-ognizing surface molecules associated with lymphocyte activationand/or regulatory T cells (days 7–12). The results of this pheno-typing are shown in Fig. 4. At the peak of activation, all cell typesexpressed high levels of CD25, CD69, and CD122 (data notshown). Resting, differentiated Th1 and Th2 cells down-regulatedthe expression of CD25, CD122, and CD69; however, the expres-sion of these markers, although diminished from their expressionat the peak of stimulation, remained well above background levelson the cells differentiated in the presence of TGF-�. As shown inFig. 4, addition of IL-15 to cultures of TGF-�-derived cells re-duced the expression of CD122. Intracellular expression ofCTLA-4 was detected in all cell types, although TGF-�-derivedcells expressed somewhat higher levels than Th1 or Th2 cells. Nodifferences were observed in extracellular expression of CTLA-4among the differentiated cell types. Staining for surface expressionof TGF-� was consistently negative whether Abs detecting TGF-�or the TGF-� latency-associated peptide were used. HLA class IIAg expression remained elevated on subsets of rested Th1, Th2,and TGF-�-derived cells; GITR remained elevated on rested Th1and TGF-�-derived cells, although GITR was strongly expressedby all three cell types at the peak of activation. CD103 was ex-pressed on essentially all TGF-�-derived cells as well as on asubset of Th2 cells and a very small subset of Th1 cells. Additionalculture of TGF-�-derived cells in the absence of TGF-� resulted inthe expression of CD103 on the same percentage of cells as that

seen in control cultures with TGF-�, but with reduced fluorescenceintensity.

Staining TGF-�-differentiated cells for FOXP3 expression iden-tified FOXP3 protein in the nuclear region of TGF-�-differentiatedcells, but not in Th1- or Th2-differentiated cells (Fig. 4c). Mor-phologically, cells differentiated in the presence of TGF-� weresmaller than either Th1 or Th2 cells, as seen microscopically (Fig.4b) or by their forward and side scatter profile on a flow cytometer(data not shown).

Transcription factor usage by differentiated cells

After one round of stimulation/differentiation as described above,total RNA was isolated from resting populations of Th1, Th2, orTGF-�-derived cells. Relative usage of the TBX21, GATA3, andFOXP3 transcription factors was determined by quantitative PCR.As shown in Fig. 5, the differentiated Th1 and Th2 cells prefer-entially expressed TBX21 and GATA3 message, respectively.TGF-�-derived cells preferentially expressed FOXP3 message.The level of FOXP3 relative to 10,000 copies of �-actin detectedusing these techniques from human CD4�CD25� cells freshly iso-lated from PBL was 1597 � 293.

Inhibition of naive cell stimulation by addition of TGF-�-differentiated cells

Cells derived in the presence of TGF-� resembled regulatory Tcells both phenotypically and in their cytokine profile. We nextexamined the cells for regulatory function in vitro. Th1, Th2, orTGF-�-differentiated cells were added in increasing proportions to

FIGURE 4. Phenotype of Th1, Th2,and TGF-�-derived cells. Th1, Th2, orTGF-�-derived cells, produced as de-scribed in Materials and Methods, werestained for expression of the surfacemolecules CD25, CD122, HLA-DR,CD103, GITR, TGF-� (sTGF-�), andCTLA-4 (sCTLA-4) and for intracellu-lar expression of CTLA-4 by flow cy-tometry on day 7 after primary stimu-lation and differentiation. Staining bythe isotype control is indicated by thesingle line. Staining by the specific Abis indicated by the filled shape. Thedashed line in the histogram of TGF-�-derived cells stained for CD122 indi-cates the staining of cells grown in theabsence of IL-15, whereas the filledshape indicates staining of cells grownin IL-15. Staining with a polyclonalgoat anti-human TGF-� Ab is shown(a). Differentiated Th1, Th2, and TGF-�-derived cells were stained withWright’s stain and examined micro-scopically (�200; b) or were spun ontomicroscope slides, dried, fixed, andstained as described for the expressionof FOXP3 using a goat-anti-humanFOXP3 Ab. Detection of this Ab wasmade using TRITC-conjugated donkeyanti-goat IgG and fluorescent micros-copy at �400 magnification (c).

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autologous, CFSE-labeled, cord blood CD4�CD45RA� lympho-cytes, or autologous CD4�CD45RA� PBL stimulated with plate-bound CD3 or CD3�CD28. The effects of the addition of thesecells were examined on both cytokine production and proliferationof CFSE-labeled cells.

Addition of differentiated Th1 cells to CFSE-labeled cells atratios of Th1 cells to naive cells between 1:2 (50%) to 1:10 (10%)resulted in a general increase in the level of production of mostcytokines measured (Fig. 6). In three independent experiments,addition of Th1 cells produced mean increases in levels of IL-10,TNF-�, and IFN-� up to five times the levels produced by stim-ulated CFSE-labeled cells alone. Addition of Th2 cells at the sameratios strongly suppressed the production of IL-2 and IFN-�, butincreased the mean production of Th2 cytokines IL-4 and IL-10 togreater than five times the levels produced by CFSE-labeled cellsalone. Addition of TGF-�-derived cells at the same ratios resultedin suppression of all cytokines except TGF-�. Only TGF-�showed a mean increase in expression of 1.6-fold above control

levels; all other cytokines were suppressed. Experiments per-formed using the addition of differentiated cell types to alter cy-tokine production by allogeneic CD4�CD45RA� PBL yieldedsimilar results, although the TGF-�-derived cells suppressed cy-tokine production by stimulated allogeneic cells by only 50% (datanot shown).

Labeling naive cells with CFSE before activation and culturingthem with unlabeled, differentiated cells permitted monitoring thedivision of the naive cells specifically, even in the presence ofactivating differentiated cells. The results of the addition of differ-entiated cells on the division of naive cells are shown in Fig. 7. Inthree independent experiments, addition of Th1 cells, even at aratio of 1:2, resulted in normal proliferation of the naive cells(97 � 10% of control proliferation). Addition of Th2 cells alsoresulted in no suppression of proliferation (79 � 21% of controlproliferation) at a ratio of 1:2. Addition of TGF-�-derived cells,however, resulted in significant suppression of PBL proliferation ata ratio of 1:2 to 47 � 33% of control levels (range, 27–85%; p �0.01, by Student’s t test). TGF-�-derived cells that were restimu-lated in a second round in the absence of the differentiative cyto-kine and Ab mixture retained the ability to suppress proliferation

FIGURE 5. Relative levels of message for TBX21, GATA3, or FOXP3in differentiated T cells. Total RNA was extracted from differentiated Th1,Th2, or TGF-�-derived cells, and the relative number of copies of messagefor the transcription factors TBX21, GATA3, and FOXP3 was determinedby real-time quantitative PCR for each cell type. Copy numbers were nor-malized such that the most prevalent message in a cell type was assigneda value of 100. A representative result of five experiments is shown.

FIGURE 6. Effect of the addition of Th1, Th2, or TGF-�-derived cellson the cytokines produced by CD3�CD28-stimulated naive cells. Differ-entiated Th1 (a), Th2 (b), or TGF-�-derived (c) cells were added to cul-tures of PBL stimulated with CD3�CD28 at the percentages indicated (per-centages represent the number of differentiated cells as a percentage of thetotal number of naive cells in the culture). Supernatants were removed fromcultures after 5 days and assayed for cytokines. Cytokines produced byactivated naive cells with no differentiated cells added were taken to rep-resent 100%. In the experiment shown, production of IL-13, TNF-�, andIFN-� exceeded 10-fold the control levels in cultures to which Th1 cellswere added. The data shown represent one of three experiments performedusing either cells differentiated from cord blood or adult peripheral bloodnaive cells.

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of naive cells (data not shown). In some experiments blockinganti-IL-10 or anti-TGF-� Abs were included in cultures to whichTGF-�-derived cells had been added to determine whether block-ade of either of these cytokines would affect the inhibitory abilityof the TGF-�-derived cells. Anti-IL-10 had no effect on the abilityof TGF-�-derived cells to inhibit naive cell proliferation. Anti-TGF-� resulted in a slight reduction in the inhibition seen withaddition of TGF-�-derived cells, but it also enhanced the prolif-eration of naive cells to which no TGF-�-derived cells were added.Blockade of IL-10 or TGF-� did not inhibit the suppression pro-duced by addition of TGF-�-derived cells.

To determine whether the TGF-�-derived cells mediated theirsuppressive effects through cell-cell contact, TGF-�-derived cellswere added to CFSE-labeled naive cells at a 1:2 ratio (50%) eitherin the same well or separated from the naive cells in a 0.4-�m poresize Transwell. Under these conditions, naive cells were sup-pressed by only �50% compared with direct addition of the sup-pressive cells (Fig. 7g). No additional blockade of the remainingsuppressive activity of TGF-�-derived cells was obtained by ad-dition of anti-IL-10 or anti-TGF-�.

Cells in these experiments were also stained for CD25 expres-sion. Addition of TGF-�-derived cells at a ratio of 1:2 suppressedCD25 up-regulation by activated naive cells from 79% of cellsexpressing CD25 with a mean channel fluorescence of 161 to only34% of cells expressing CD25 with a mean channel fluorescenceof 87.

TGF-�-derived cells are less dependent on PKC�

Recently, using knockout mice, Berg-Brown et al. (32) demon-strated that the PKC� pathway of NF-�B activation is importantprimarily for the development of effector Th cell responses. Basedon these findings, we examined the expression of PKC� by Th1,Th2, and TGF-�-derived cells using anti-PKC� Ab and immuno-histochemistry. As shown in Fig. 8, PKC� was readily detectablein the cytoplasm of Th1 and Th2 cells. In contrast, PKC� expres-sion in TGF-�-derived cells was barely detectable.

We therefore examined the role of PKC� in the proliferation ofTh1, Th2, and TGF-�-derived cells using the inhibitor Rottlerin.As shown in Fig. 8, Rottlerin, within ranges established to specif-ically inhibit PKC� functions in T cells (33, 34), inhibited theproliferation of Th1 and Th2 cells, but had minimal effects onTGF-�-derived cell proliferation driven by CD3 and CD28. Athigh concentrations, the proliferation of all cell types was inhib-ited, consistent with the known ability of Rottlerin to inhibit manycellular enzymes at high concentration (35). To further demon-strate that these effects were due to specific inhibition of PKC�, apeptide that specifically blocks the interaction of PKC� and itsscaffolding proteins was synthesized, incorporating an antennape-dia helical domain to permit cell entry (36), and its effect on cellproliferation was examined. This more specific inhibitor of PKC�also inhibited the proliferation of Th1 and Th2 cells to 40–50% ofcontrol levels, but spared TGF-�-derived cells at concentrationsbetween 12 and 50 �M (Fig. 8b).

DiscussionThese data demonstrate that human naive peripheral CD4� cells,stimulated through their TCR in the presence of TGF-� with ex-ogenous IL-2 and IL-15, can expand and differentiate into cellsexpressing a surface phenotype consistent with thymus-derivedregulatory cells. The TGF-�-derived cells up-regulate message forFOXP3, and the translated protein is present in the nucleus of thecells. Like thymus-derived CD4�CD25� regulatory cells, TGF-�-derived cells are capable of suppressing proliferation and cytokineproduction of normal, naive cells in vitro. Recently, Chen et al.(31) demonstrated that activation of murine CD4�CD25�cellsthrough the TCR by Ag or anti-CD3 Ab stimulation in the pres-ence of TGF-� and exogenous IL-2 resulted in differentiation ofCD4�CD25� cells expressing FOXP3 and capable of suppressingimmune responses in vitro and in vivo.

In vitro differentiation of T cells with regulatory function hasbeen reported using numerous methods. Coculture of T cells withdendritic cells or other APCs in different states of maturity has

FIGURE 7. Effect of the addition of Th1, Th2, or TGF-�-derived cells on proliferation of naive cells stimulated by CD3. Naive cells from either cordblood or adult peripheral blood were labeled with CFSE and stimulated with plate-bound CD3 and CD28 Abs. At initiation of the cultures, Th1, Th2, orTGF-�-derived cells were added at the indicated percentages of total naive cell numbers. Dot plots of CFSE staining intensity vs CD25 expression areshown for unstimulated naive cells (a), stimulated naive cells with no differentiated cells added (b), naive cells with addition of Th1 cells (50%; c), naivecells with addition of Th2 cells (50%; d), and naive cells with addition of TGF-�-derived cells (50%; e) after 4 days of culture. A graphical representationof the results of this experiment is shown in f. The requirement for cell-cell contact was examined by combining CFSE-labeled responding cells withTGF-�-derived cells at a 2:1 ratio in the same well or with the TGF-�-derived cells separated from the responders by a 0.4-�m pore size membrane (g).Division of responder cells was determined after 4 days. These results are representative of seven experiments.

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resulted in the differentiation of T cells with suppressive function(37–39). Addition of exogenous IL-10 and TGF-� was demon-strated to induce murine CD4�CD25� T cells stimulated in anMLR to produce cells capable of inhibiting new primary MLR

reactions and inhibiting graft-vs-host disease in vivo (40). Othersystems used culture conditions based on purified T cells and cy-tokines to derive cells with apparent regulatory function. TGF-�has been demonstrated to induce expansion of regulatory popula-tions resembling thymic-derived CD4�CD25� cells or Th3 cellswhen either CD4�CD45RA� cells or CD4�CD25�human PBL,respectively, were stimulated with Ag in the presence of the cy-tokine (29, 41).

Other cytokines have also been used to differentiate cells in vitrowith some regulatory properties. The first regulatory T cell popu-lations to be derived from peripheral T cells in vitro were the TR1

cells. TR1 cells were derived initially from mouse or human CD4�

T cells stimulated through their Ag receptors in the presence ofexogenous IL-10 (27). Interestingly, Bacchetta et al. (42) demon-strated, that like the TGF-�-derived cells described in this study,TR1 cells required IL-15 for efficient expansion. These cells weredemonstrated in vitro and in vivo to possess clear regulatory T cellfunctions, but examination of their transcription factor usage in-dicated that GATA3 was the primary T cell differentiation-relatedtranscription factor in these cells. Our finding that human PBLstimulated in the presence of exogenous IL-10 results in relativeenhancement of GATA3 message usage is consistent with the po-tential to derive human TR1 cells.

Stimulation of murine CD4�CD62high cells with Ag in the pres-ence of APCs, vitamin D3, and dexamethasone resulted in T cellssimilar to TR1 cells, producing high levels of IL-10 (43). In thehuman system, TR1 cells have been differentiated in vitro fromCD4� cord blood cells or naive, CD4�CD45RO�, peripheralblood T cells by stimulation through the TCR in the presence ofexogenous IL-10 and IFN-� (44). Addition of TGF-� to thesecells, with or without IL-10, did not result in the outgrowth of cellswith typical TR1 cytokine profiles, and TGF-� appeared to gener-ally inhibit IL-10, IL-4, IFN-�, and IL-2 production. Only lowlevels of IFN-� were produced by TGF-�-treated cultures. Theseresults and the conclusion that TGF-� treatment did not enhancedifferentiation of TR1 cells are consistent with our findings

Phenotypically, we have demonstrated that humanCD4�CD45RA� cells activated in vitro in the presence of TGF-�express cell surface molecules consistent with those expressed byboth in vitro-derived and thymus-derived regulatory T cells. Hu-man and mouse CD4�CD25� thymic-derived regulatory T cellsare consistently reported to express CD4, CD25, CD69, CD103,CD122, CTLA-4, HLA-DR, and GITR (9, 22, 25, 45–49). Be-cause these molecules are all activation Ags on lymphocytes, dur-ing the in vitro differentiation of Th1, Th2, and TGF-�-derivedcells, all these molecules appeared on all cell types at some pointduring activation. However, as shown, when the differentiatedcells returned to a resting state, TGF-�-derived cells retained ex-pression of these cell surface proteins of this phenotype, similar tothe phenotype of natural thymus-derived CD4�CD25� cells. Reg-ulatory T cells from both mice and humans are described as gen-erally producing primarily IL-10 and/or TGF-� when stimulated,with variable reports of low levels of IL-4, IL-5, and IFN-�. Spe-cifically, TR1 cells appear to produce primarily IL-10 (27, 42, 50),whereas Th3 cells and CD4�CD25� cells produce more TGF-�(51, 52). Like TR1 cells, the TGF-�-derived cells described in thisstudy require exogenous IL-15 (42) for efficient proliferation.However, based on cytokine production, the cells described in thisstudy are more similar to Th3 or CD4�CD25� cells than to TR1

cells. Most in vitro assays of the suppressive abilities of regulatoryT cells have demonstrated that addition of regulatory cells to re-sponding cells in ratios ranging from 1:10 to 1:1 produces sup-pression of mitosis by the responders. The TGF-�-derived cells

FIGURE 8. Less PKC� is present in the cytoplasm of TGF-�-derivedcells, and inhibition of PKC� has less effect on proliferation of TGF-�-derived cells than Th1 or Th2 cells. a, Th1, Th2, and TGF-�-derived cellswere differentiated as described. Differentiated cells were centrifuged ontomicroscope slides, fixed, and stained with anti-��TCR-FITC and anti-PKC�-HRP. Bound anti-PKC� Ab was visualized by staining with goat-anti-HRP-biotin, followed by streptavidin-TRITC. Cells were examinedusing fluorescent microscopy at �400 magnification. b, Differentiated Th1,Th2, and TGF-�-derived cells were restimulated with plate-bound CD3and CD28 in the presence of medium containing 1, 5, 10, or 50 �M Rot-tlerin in DMSO or medium with DMSO (upper panel) or in 0–50 �Mantennapedia-PKC�-blocking peptide (ANT-PKC�). After 3 days, cell pro-liferation was quantified by uptake of [3H]thymidine and scintillationcounting. Proliferation, as a percentage of the DMSO control, is shown inthe upper panel, and cpm minus background cpm (no CD3�CD28) � 1 SDof triplicate determinations is shown in the lower panel of these represen-tative experiments (one of four each).

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appear to function similarly. Therefore, in all respects these cellsappear to represent regulatory T cells.

The mechanisms by which regulatory cells suppress T cell pro-liferation varies by the type of regulatory cell studied. Thymic-derived CD4�CD25� cells appear to require cell-cell contact tosuppress proliferation (53, 54), whereas TR1 cells appear to useIL-10 (28, 55, 56). Inhibition of cell-cell contact reduces suppres-sion by the TGF-�-derived cells described in this report by �50%,whereas addition of anti-TGF-� or anti-IL-10 has no effect. Theseresults appear to be more consistent with the mechanism of actiondescribed for Th3 cells than that for TR1 cells. They also suggestthat some other soluble factor produced by the cells may accountfor some of the suppressive activity. Because the cells used inthese experiments are lines and not clones, it is not possible todetermine whether these results are produced by a single type ofregulatory cells using both cell-cell contact and secretion of anunidentified suppressive factor or whether differentiation of cells inthe presence of TGF-� results in the development of more than onetype of regulatory cell.

The ability of TGF-� to alter the cytokine production and tran-scription factor usage of differentiated T cells has been described.TGF-� is known to inhibit both IFN-� production by inhibitingexpression of TBX21 (57) and the production of Th2 cytokinesand GATA3 by Th2 cells (58, 59). TGF-� is produced by a varietyof cell types in the body and is often produced in association withhealing. The observations in this paper suggest that naive lympho-cytes, drawn to a site of previous injury, might be activated duringthe healing process in the presence of sufficient quantities ofTGF-� to result in the differentiation of a population of peripher-ally derived regulatory T cells. These cells could then play animportant physiological role in containing the natural, protective,immune responses and promoting normal healing, augmenting amore homeostatic role possibly belonging to the thymus-derivedregulatory T cells.

PKC� has been shown to play an important role in the prolif-eration of peripheral T cells. Peripheral T cells, but not thymo-cytes, from PKC� knockout mice proliferate poorly in vitro whenstimulated with CD3, CD3�CD28, TPA plus ionomycin, and al-loantigens in MLR; in vivo responses to keyhole limpet hemocy-anin were also impaired (60). Various data strongly suggest thateffector T cells (Th1 and Th2) use the caspase-recruitment domain-containing 11 (CARD11)/PKC� pathway to activate NF-�B to turnon the production of inflammatory cytokines (61, 62). The expres-sion of NF-�B and components of this transcription factor hasbeen shown to increase during allograft rejection. Treatment ofanimals with CD154 Ab resulting in permanent graft acceptanceand tolerance inhibited the expression of NF-�B and related pro-teins (63). Other inhibitors of the NF-�B pathway, such as deox-yspergualin (64), have been used successfully in vivo to inducelong-term allograft survival and tolerance (65). Recently, datashowing that PKC� knockout mice readily developed tolerant oranergic T cell responses have more closely linked the inhibition orabsence of PKC�, resulting in decreased activation of NF-�B, tothe development of tolerance (32). Our data suggest that humanTh1 and Th2 cells may also more heavily use the PKC� activationpathway for proliferation than do TGF-�-derived regulatory cells.Taken together, these data suggest that regulatory T cells may notbe as dependent on the PKC� pathway for activation of NF-�B toexpand and respond to stimuli. Additional examinations of thebiochemical pathways used by effector and regulatory T cells maypermit the identification of targets for new therapeutic agents thatuniquely target effector T cells and spare regulatory T cells, en-hancing the development of tolerance.

AcknowledgmentsWe thank Dr. Herman Waldmann for his comments on this manuscript andgeneral scientific guidance. We also thank Shinji Oyama for his excellenttechnical assistance.

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