GARP (LRRC32) is essential for the surface expressionof latent TGF-� on platelets and activated FOXP3�

regulatory T cellsDat Q. Trana,1, John Anderssona, Rui Wangb, Heather Ramseya, Derya Unutmazb, and Ethan M. Shevacha,1

aLaboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and bDepartment ofMicrobiology, New York University School of Medicine, New York, NY 10016

Edited by Dan R. Littman, New York University Medical Center, New York, NY, and approved June 2, 2009 (received for review February 21, 2009)

TGF-� family members are highly pleiotropic cytokines with di-verse regulatory functions. TGF-� is normally found in the latentform associated with latency-associated peptide (LAP). This latentcomplex can associate with latent TGF�-binding protein (LTBP) toproduce a large latent form. Latent TGF-� is also found on thesurface of activated FOXP3� regulatory T cells (Tregs), but it isunclear how it is anchored to the cell membrane. We show thatGARP or LRRC32, a leucine-rich repeat molecule of unknownfunction, is critical for tethering TGF-� to the cell surface. Wedemonstrate that platelets and activated Tregs co-express latentTGF-� and GARP on their membranes. The knockdown of GARPmRNA with siRNA prevented surface latent TGF-� expression onactivated Tregs and recombinant latent TGF-�1 is able to binddirectly with GARP. Confocal microscopy and immunoprecipitationstrongly support their interactions. The role of TGF-� on Tregsappears to have dual functions, both for Treg-mediated suppres-sion and infectious tolerance mechanism.

transforming growth factor beta � Tregs � latency-associated peptide

TGF-� family members (�1, �2, �3 isoforms) are highlypleiotropic cytokines that have critical functions in cell

differentiation, tissue morphogenesis and modulation of cellgrowth, inflammation, matrix synthesis, and apoptosis. Dysregu-lations in TGF-� function are associated with multiple patho-logical conditions including tumor cell growth, fibrosis, emphy-sema, and autoimmunity (1). All 3 TGF-� isoforms aresynthesized as homodimeric proproteins. The proproteins arecleaved in the Golgi apparatus by a furin-like convertase toproduce the dimeric propeptides called latency-associated pep-tide (LAP) that noncovalently associates with the dimeric ma-ture TGF-� to prevent its activity (2). There are multiplemechanisms of activating TGF-� from its latency by pathwaysthat include protease plasmin, matrix metalloproteases, throm-bospondin-1 (TSP1), and certain �V integrins (3). TGF-� can besecreted in a small latent form associated with LAP, or thiscomplex can further associate with latent-TGF-�-binding pro-tein (LTBP) to produce a large latent form for deposition ontothe extracellular matrix. In addition, small latent TGF-� can beexpressed on the membrane of many cell types, includingmegakaryocytes, platelets (4), immature dendritic cells (DCs)(5), and activated FOXP3� regulatory T cells (Tregs) (6–8), andhas important functions in tissue healing and immune regulation.However, it is unknown how this membrane small latent TGF-�is anchored to the cell surface.

It has been recently shown that megakaryocytes and activatedTregs expressed high levels of mRNA for a member of theleucine-rich repeat family of proteins that has been termedGARP or LRRC32 and that platelets express this molecule ontheir membrane (9, 10). The GARP or LRRC32 gene consists of662 aa and encodes an 80-kDa transmembrane protein with anextracellular region composed primarily of 20 leucine-rich re-peats (11, 12). As the Garp gene is expressed in multiple celltypes in the mouse during embryogenesis, it has been proposed

that Garp plays an important role in development, but its actualfunction is unknown (13). Since platelets and activated Tregscontain both GARP and latent TGF-� on their membranes, wehypothesized that GARP might bind and anchor latent TGF-�.Here, we show that GARP is critical for the surface expressionof latent TGF-� by binding to the complex and functioning as itscell surface receptor.

ResultsGARP or LRRC32 Is Selectively Expressed on Activated FOXP3� Tregs.Consistent with a previous publication (10), we found thatGARP mRNA is selectively expressed in fresh human Tregs andrapidly up-regulated after activation of CD4�CD25hi Tregs withanti-CD3/CD28 and IL-2 (Fig. 1A). Only very low levels ofmRNA were detected in CD4�CD25– T cells after activation for5 days. While the addition of TGF-�1 resulted in the inductionof FOXP3 mRNA in CD4�CD25– T cells (14), the level ofGARP mRNA was not dramatically increased. Cell surfaceexpression of either LAP or GARP was not significantly de-tected on freshly isolated Tregs (CD25hi), but the expression ofboth LAP and GARP was rapidly up-regulated after activation(Fig. 1B). GARP and LAP occasionally could be detected on�5% of activated CD4�CD127�CD25– T cells (Fig. S1). How-ever, when we activated the CD4�CD25int population, whichcontains mostly CD45RO�FOXP3– T cells and some FOXP3�

Tregs, the vast majority of LAP and GARP could be detected onFOXP3� Tregs. The rapid appearance and disappearance ofLAP�/GARP� FOXP3– T cells during the 36 h of activation ofCD25hi and CD25int most likely represent Tregs that havedown-regulated their FOXP3 and could be on their way to celldeath. Platelets also co-expressed LAP and GARP on theirmembranes (Fig. 1C). In contrast to previous reports (5), wewere unable to detect any significant surface expression of LAPor GARP on plasmacytoid or myeloid DCs from peripheralblood (Fig. 1D). Interestingly, the cell surface expression of bothLAP and GARP requires the Golgi apparatus, since the additionof monensin or brefeldin A during the activation culture pre-vented their surface expression (Fig. 1E).

GARP Associates with Latent TGF-� Complex and Is Critical for ItsSurface Expression. Since the kinetics of induction and pattern ofexpression of LAP and GARP on activated Tregs appeared tobe similar, we used siRNA technology to knockdown TGF-�1 orGARP mRNA to assess if the expression of these molecules is

Author contributions: D.Q.T. designed research; D.Q.T., J.A., and H.R. performed research;R.W. and D.U. contributed new reagents/analytic tools; D.Q.T. and E.M.S. analyzed data;and D.Q.T. and E.M.S. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

1To whom correspondence may be addressed. E-mail: dtran@niaid.nih.gov oreshevach@niaid.nih.gov.

This article contains supporting information online at www.pnas.org/cgi/content/full/0901944106/DCSupplemental.

www.pnas.org�cgi�doi�10.1073�pnas.0901944106 PNAS � August 11, 2009 � vol. 106 � no. 32 � 13445–13450

IMM

UN

OLO

GY

Dow

nloa

ded

by g

uest

on

Mar

ch 1

5, 2

020

related. Freshly isolated Tregs transfected with TGF-�1 siRNAand activated for 48 h expressed GARP, but not LAP on theirsurface, while Tregs transfected with GARP siRNA failed toexpress either molecule. Expression of GARP or LAP was notaffected when Tregs were transfected with a control nonspecificsiRNA (Fig. 2A). This result suggested that GARP was requiredfor the expression of latent TGF-�1 on the cell surface. To testwhether latent TGF-�1 can associate with GARP, we incubatedthe 2 activated Treg populations (TGF-�1 siRNA or GARPsiRNA) with recombinant human (rh) TGF-�1, LAP, LAP plusTGF-�1, or latent TGF-�1. LAP was only detected on theTGF-�1 siRNA transfected GARP�LAP– Tregs when they wereincubated with either LAP mixed with TGF-�1 or latent TGF-�1, but not mature TGF-�1 or LAP alone, indicating that theTGF-�1/LAP complex was needed for interaction with GARP(Fig. 2B). We next tested whether the interaction of TGF-�2 orTGF-�3 with LAP of TGF-�1 results in binding to GARP.Interestingly, the mixture of TGF-�2 and LAP resulted inbinding to GARP, while the mixture of TGF-�3 and LAP failedto bind to GARP (Fig. 2C). Although different isoforms ofTGF-� naturally associate with their own distinct LAPs, it hasbeen reported that LAP of TGF-�1 can bind and inactivateTGF-�2 and TGF-�3 with apparent Kd values of 1.9 and 0.4 nM,respectively (15). However, our result suggests that eitherTGF-�3 does not interact with LAP of TGF-�1 or their inter-actions produce a conformation that does not bind to GARP.Moreover, while TGF-�2 could interact with LAP to bind toGARP, it appears to be less efficient than with TGF-�1 based onthe lower mean fluorescence intensity of LAP detection. Theinteraction of various molecules including �V integrins andTSP1 with the RGD sequence in LAP has been implicated inreleasing and activating TGF-� (4, 16, 17). Using Jurkat cellsexpressing surface GARP, we tested whether TSP1, RGD, orRGDS peptides can block the binding of latent TGF-�1 toGARP. Preincubation of rhTSP1 or RGD/RGDS peptides did

not block the binding of latent TGF-�1 to GARP (Fig. 2D). Thisresult suggested that it is unlikely that the binding of GARP tolatent TGF-�1 occurs via the RGD site on LAP or that latentTGF-�1 binds via the RGD site to another cell surface moleculethat then interacts with GARP. To further support the associ-ation of GARP with LAP, we performed immunoprecipitationand colocalization confocal imaging studies. When LAP wasimmunoprecipitated from the surface of 48 h activated Tregs,GARP could be detected by immunoblot (Fig. 2E). Likewise,confocal imaging of surface-stained LAP and GARP on 48 hactivated Tregs strongly demonstrated their colocalization (Fig.3). Finally, to determine whether GARP and latent TGF-�1 candirectly bind to each other, we performed a flow cytometricprotein-protein interaction assay. Dynabeads were conjugated toeither anti-LAP or anti-GARP mAbs and then incubated with amixture of latent TGF-�1 and GARP-Fc fusion proteins, LAPand GARP-Fc, or TGF-�1 and GARP-Fc followed by flowcytometric detection of the anti-LAP Dynabead complex withfluorochrome conjugated anti-hFc or anti-GARP Dynabeadcomplex with anti-LAP Abs. Beads conjugated with GARPbound latent TGF-�1, but not LAP and beads conjugated withlatent TGF-�1, but not LAP, bound GARP (Fig. 2F). This resultindicates that GARP can directly bind to latent TGF-�1.

Regulation of GARP Is Independent of FOXP3. Since GARP and LAPexpression was observed in FOXP3� Tregs, we next evaluatedwhether the de novo induction of FOXP3 in CD4�FOXP3– Tcells was sufficient to induce GARP and LAP expression on thecell surface. Naïve CD45RA� and memory CD45RO� T cellsactivated with anti-CD3/CD28 in the presence of TGF-�1 can beinduced to express FOXP3 (Fig. 4A) although such cells lackregulatory function (18). In contrast to Tregs, activation of naïveor memory T cells in the presence or absence of TGF-�1 failedto express significant levels of surface LAP or GARP on primarystimulation (Fig. S1 A) or following multiple rounds of restimu-

Fig. 1. GARP and LAP are selectively expressed on activated FOXP3� Tregs and platelets. (A) Level of FOXP3 and GARP mRNA on fresh (0 h) and activatedCD4�CD25– T cells and Tregs (CD25hi) at 12, 24, 120 h, or 120 h with TGF-�1. Tregs were rested until day 14 (0 h) and restimulated for 18 h. (B) Flow cytometricanalysis of surface LAP, GARP and intracellular FOXP3 on fresh (0 h) and activated Tregs (CD25hi) and CD4�CD25int T cells. (C) Surface staining of LAP and GARPon platelets based on FSC/SSC and CD61 expression. (D) LAP and GARP surface staining of plasmacytoid and myeloid DCs from PBMCs by gating on CD303�Lin-1–

and CD1c�, respectively. (E) Surface LAP and GARP expression on Tregs after 12 h activation in the absence (none) or presence of monensin or brefeldin A forthe last 8 h. Data are representative of 3 independent experiments. Numbers indicate percentage in each quadrant for B–E.

13446 � www.pnas.org�cgi�doi�10.1073�pnas.0901944106 Tran et al.

Dow

nloa

ded

by g

uest

on

Mar

ch 1

5, 2

020

lation without TGF-�1 (Fig. 4A and Fig. S1B). It has been shownthat high level and prolonged expression of FOXP3 followinglentiviral-mediated transfection can result in the complete ac-quisition of the Treg phenotype in CD4�FOXP3– T cells (19).Although the TGF�-induced FOXP3� cells expressed similar

level of FOXP3 as the Tregs upon restimulation, they fail toexpress surface latent TGF-� or GARP (Fig. S1B) and continueto lack Treg phenotype. Therefore, it does not appear thatexpression of FOXP3 in non-Tregs is sufficient to drive thesurface expression of GARP and LAP.

To evaluate whether FOXP3 was essential for the expressionof GARP and LAP on Tregs, we first knocked down the level ofFOXP3 on CD25hi cells with siRNA for 5 days and determinedthe induction of GARP and LAP after 48 h of restimulation.Although FOXP3 expression was completely suppressed withthe siRNA compared to the nonspecific control, the expressionof GARP and LAP was not affected (Fig. 4B). This resultindicates that the rapid expression of GARP and LAP was notcontrolled by an immediate downstream effect of FOXP3.However, it remains possible that FOXP3 might have an indirecteffect on GARP expression after a more prolonged period ofsuppression of FOXP3 expression. Since virtually all T cellsexpress TGF-�1 mRNA and are capable of secreting TGF-�1,we evaluated whether forced expression of GARP inCD4�FOXP3� T cells was sufficient to permit surface expres-sion of latent TGF-�. Indeed, transduction of GARP intoCD4�FOXP3� T cells allows for the surface expression of LAP,but not an increase in the percentage of FOXP3� cells (Fig. 4C).Therefore it appears that GARP associates with latent TGF-�intracellularly and transports it to the cell surface via the Golgiapparatus. Furthermore, this result demonstrates that the failureto detect latent TGF-� on activated non-Tregs was due to thelack of GARP expression in these cells.

Dual Role of TGF-� on Tregs for Mediating Suppression and InfectiousTolerance. As the kinetics for induction of cell surface expressionof GARP and LAP closely resembled the kinetics for activation

Fig. 2. Surface expression of latent TGF-�1 requires GARP association. (A) Tregs were transfected with nonspecific (siNS), TGF-�1 (siTGF�1), or GARP (siGARP) siRNAand rested in IL-2 culture medium for 24 h before stimulation for 48 h to assess surface expression of GARP and LAP. (B) Tregs transfected with TGF-�1 or GARP siRNAwere activated for 48 h then incubated for 30 min at 37 °C with 5 �g/mL mature rhTGF-�1, LAP, LAP � TGF-�1, or latent TGF-�1 and then surface stained for LAP. (C)GARP transfected Jurkat cells were incubated for 30 min at 37 °C without (NONE) or with 5 �g/mL LAP, TGF-�1, TGF-�2, or TGF-�3 alone or mixed with LAP and thenstained for surface LAP. Jurkat cells transfected with the RFP vector were used as a control. (D) GARP-transfected Jurkat cells were incubated with 5 �g/mL latent TGF-�1(LTGF�1) or preincubated for 20 min at 37 °C with a 10-fold excess of TSP1, RGD, or RGDS peptides before latent TGF-�1 incubation and then stained for surface LAP.(E) Tregs were expanded in vitro for 14 days and then restimulated for 48 h before immunoprecipitation with anti-LAP (left lane) or isotype (right lane) followed byimmunoblot with anti-GARP. (F) Dynabeads conjugated to �GARP (Left) or �LAP (Right) were incubated with a mixture of GARP-Fc � TGF-�1 (shaded histogram),GARP-Fc � LAP (dashed histogram), or GARP-Fc � latent TGF-�1 (solid histogram) followed by flow cytometric analysis with PE-labeled anti-hLAP (Left) or anti-hFc(Right). Data are representative of 3 independent experiments for A, B, C, D, and F and 2 for E. Numbers indicate percentage in each quadrant for A–D.

Fig. 3. Colocalization of GARP and latent TGF-�1 on the surface of activatedTregs. Tregs activated for 48 h from 3 different donors (D1, D2, D3) weresurface-stained with anti-GARP and anti-LAP or isotype controls. The cellswere imaged with a Leica SP2-AOBS confocal microscope.

Tran et al. PNAS � August 11, 2009 � vol. 106 � no. 32 � 13447

IMM

UN

OLO

GY

Dow

nloa

ded

by g

uest

on

Mar

ch 1

5, 2

020

of Treg suppressor function (20), we next evaluated the role ofthese molecules in Treg-mediated suppression. Tregs that lackedthe surface expression of LAP following treatment with TGF-�1

siRNA or the expression of GARP and LAP following treatmentwith GARP siRNA were significantly less suppressive than Tregstreated with the control siRNA in an in vitro suppression assay(Fig. 5A and B). Although the role of cell surface or secretedTGF-�1 as a major effector molecule of Treg suppression in vitrohas remained controversial (6, 21), these results indicate thatTGF-� contributes moderately to Treg-mediated suppression invitro, but TGF-� does not appear to be the dominant mechanismsince suppression was not completely abrogated. We have shownpreviously that activated mouse Tregs can induce Foxp3 expres-sion in CD4�FOXP3� T cells during a 4-day co-culture (7).Similarly, co-culture of activated human Tregs treated withnonspecific, but not TGF-�1 or GARP siRNA, induced FOXP3in CD4�FOXP3� T cells (Fig. 5C). We have not tested whetherthese induced FOXP3� T cells have regulatory functions, as weare unable to separate them from the large population ofFOXP3� T cells in the culture.

DiscussionOur results clearly demonstrate that GARP or LRRC32 func-tions as a carrier and cell surface receptor for latent TGF-�1.Using siRNA technology, we have shown in vitro thatGARP�LAP� cells can bind latent TGF-�1, but we have notbeen able to determine whether in vivo the binding of latentTGF-�1 to GARP occurs exclusively intracellularly or whetherGARP can also bind secreted latent TGF-�1 or latent TGF-�associated with LTBP. Although the GARP/LAP complex isexpressed by platelets, within the immune system GARP/LAPexpression is mostly observed on activated functional FOXP3�

Tregs (9). We did not observe significant surface expression ofGARP or LAP in CD19�/CD20� B cells, CD14� monocytes,CD8� T cells, natural killer (NK) cells, NK T cells, and imma-ture/mature monocyte-derived DCs. However, it is possible thatunder certain conditions and with activation, these cells mightexpress GARP. Thus far, the predominant function of GARP/LAP on human Tregs remains elusive. Our functional studies

Fig. 4. GARP is required for the surface expression of latent TGF-�1. (A)FACS-sorted Tregs (CD25hi), naïve (CD45RA�), and memory (CD45RO�) T cellswere activated for 5 days in the absence or presence of TGF-�1 then rested for 7days and restimulated for 48 h without TGF-�1 before analysis of FOXP3 withsurface GARP and LAP expressions. (B) Tregs were transfected with nonspecific(siNS) or FOXP3 (siFOXP3) siRNA and cultured for 5 days before activation for 48 hand analysis of FOXP3 with surface GARP and LAP expressions. (C) Naïve T cellswere transduced with control (vector) or GARP encoded RFP-expressing lentiviralvectors and expanded for 7 days with anti-CD3/CD28 Dynabeads before restimu-lationfor48htoevaluateforsurfaceGARPandLAP(Left)andintracellularFOXP3expressions among RFP� and RFP� cells (Right). Data are representative of 3independent experiments. Numbers indicate percentage in each quadrant.

Fig. 5. Dual functions of TGF-� in Treg-mediated suppression and infectious tolerance mechanism. (A) Tregs were transfected with siNS, siGARP, or siTGF�1and preactivated for 24 h before assessing their suppression of the proliferation of CD4�CD25– T cells stimulated with HLA-DR� APCs and soluble anti-CD3 for3 days and pulsed with 3H-TdR. The top panel is the FOXP3 expression, and the middle panel is the surface GARP and LAP expression of the 3 Treg populationsat the end of the 3-day suppression assay. *, P � 0.05 between the siNS and siGARP or siTGF�1 Treg suppression. (B) Similarly, CFSE-labeled CD4�CD25� responderswere activated with HLA-DR� APCs and soluble anti-CD3 for 3 days alone (top row) or in the presence of Tregs transfected with siNS, siGARP, or siTGF�1 at aratio of 2:1, 4:1, and 8:1 responder:Treg (bottom rows). (C) CFSE-labeled CD4�CD25�CD127�CD45RA� T cells were stimulated with anti-CD3/CD28 Dynabeadsfor 5 days alone (top panel) or with 24-h preactivated Tregs transfected with siNS, siGARP, or siTGF�1 and analyzed for FOXP3 induction in the CFSE-labeled cells.Data are representative of 3 independent experiments. Numbers indicate percentage in each quadrant.

13448 � www.pnas.org�cgi�doi�10.1073�pnas.0901944106 Tran et al.

Dow

nloa

ded

by g

uest

on

Mar

ch 1

5, 2

020

indicate that the GARP/LAP complex does contribute to Treg-mediated suppression in vitro but whether this result also holdstrue in vivo is unknown and remains controversial. We haverecently proposed (7) that a major role of latent TGF-� on thesurface of murine Tregs is to convert responder T cells intoFOXP3� Tregs through a mechanism of infectious tolerancewhen both populations are activated in concert via their TCRs.This TGF�-mediated infectious tolerance mechanism appears toalso be involved in human Tregs. The regulation of expression ofGARP at the mRNA level (10) in the mouse and the regulationof the expression of cell surface LAP are similar to that seen onhuman Tregs. GARP expression at the protein level in the mousehas not been evaluated due to the lack of anti-murine GARPantibodies. As GARP and LAP are only expressed on TCR-activated FOXP3� Tregs, 1 major function of this complex is totarget delivery of TGF-�1 to sites of ongoing immune responseswhere Tregs can be activated by recognition of their cognateantigens on APCs. Following release of active TGF-�1, it mightact locally on FOXP3– T cells to convert them to FOXP3� Tregs(7), to generate Th17 effectors if an inflammatory milieu ispresent (22), to act directly on the DCs to modulate theirfunctions, or to signal in an autocrine manner to maintain Tregfunctions. A major question that remains to be addressed is themechanism by which active TGF-�1 is generated from the latentGARP/LAP complex either by cell-associated molecules such asthe �V integrins or by soluble factors.

Mutations in both GARP and LAP have been reported andresult in complex clinical conditions. A base substitution fromarginine to tryptophan in the coding region of the GARP genehas been identified in a large Samaritan kindred with Ushersyndrome type 1, an autosomal recessive disease characterizedby profound congenital sensorineural deafness, vestibular dys-function, and progressive visual loss (23). It is unclear whetherthis mutation would result in a defective GARP protein thatwould fail to associate with LAP or potentially lead to enhancedrelease of active TGF-�. Mutations in the TGF-�1 signal peptideor the LAP coding region result in enhanced TGF-� activity asseen in Camurati-Engelmann disease, a rare, autosomal domi-nant condition characterized by sclerosing bone dysplasia andneurological deficiencies (24). Detailed studies of lymphocytefunction, platelet function, or studies of tissue healing, remod-eling, and fibrosis have not been performed in these patients.Mutations or deletions in GARP would only affect membranebound TGF-�, and not the secreted pool, and therefore offer anopportunity to understand the unique contribution of membraneTGF-� to the regulation of immunity and inflammation. Arecent study using antisense morpholino oligonucleotide toknockdown GARP in zebrafish demonstrated that GARP mightbe important in thrombus initiation on platelets (25). GARP alsoappears to be associated with infertility since up-regulation ofGARP transcripts were observed in infertile human endome-trium compared with fertile controls (26). Lastly, GARP mRNAis highly amplified in different tumors (27–34), but surfaceexpression of GARP and its association with TGF-� in tumorshas not been studied. Tumor cells may use GARP to expressTGF-� or to capture TGF-� from their surroundings resulting inlocal suppression of anti-tumor immune responses or the induc-tion of Tregs. Further studies of the regulation of GARPexpression may lead to the development of drugs that canenhance or suppress the expression of GARP and membraneTGF-� and might be useful to treat autoimmunity or cancers andfibrotic diseases, respectively. Therefore, our discovery of thecritical role of GARP in controlling surface expression of latentTGF-� will provide insights into another importance pathway ofTGF-� regulation of morphogenesis, immune homeostasis, in-f lammation, and tissue remodeling.

Materials and MethodsCell Purification. Peripheral blood was obtained from healthy adult donorsthrough the Department of Transfusion Medicine at the National Institutes ofHealth and approved by the NIAID institutional review board. The study wasconducted in accordance with the Declaration of Helsinki. PBMCs were pre-pared over Ficoll-Paque Plus gradients (GE Healthcare). The CD4� cells wereenriched over the AutoMACS with CD4 microbead (Miltenyi). The cells wereFACS-sorted with the FACSVantage DiVa or FACSAria for CD4�CD127�CD25�,CD4�CD25int (intermediate), and CD4�CD127–CD25hi (high). In some experiments,the cells were FACS-sorted for CD4�CD127�CD25–CD45RA� or CD45RO�.Human APCs for in vitro suppression assay were obtained by depleting T cellsfrom PBMCs with CD3 microbead (Miltenyi) using the AutoMACS followed bypositive selection with HLA-DR microbead.

Flow Cytometric Analysis. FOXP3 expression was detected with anti-FOXP3mAbs after fixing and permeabilizing the cells with a Fixation/Permeabiliza-tion kit (eBioscience). LAP and GARP expression was surface stained withanti-LAP and anti-GARP mAbs before fixation/permeabilizing for FOXP3 de-tection. For GARP, a secondary detection with fluorochrome conjugatedanti-mouse IgG2b was needed.

Antibodies and Reagents. CD4, CD25, CD45RA, CD45RO, anti-mouse IgG1,anti-mouse IgG2b, goat (Fab�)2 anti-hFc, and carboxyfluorescein succinimidylester (CFSE) were from Invitrogen. Linage-1 mixture, CD127, streptavidin,monensin, and brefeldin A were from BD. CD1c and CD303 were from Milte-nyi. Anti-LAP unconjugated and PE-conjugated IgG1 mAbs (clone 27232),biotinylated goat anti-hLAP, recombinant human (rh) LAP, latent TGF-�1, andthrombospondin-1 were from R&D Systems. Unconjugated anti-GARP IgG2bmAbs (clone Plato-1) and rhGARP (amino acid 20–627) fused to the Fc portionof human IgG1 were from Alexis Biochemicals. rhTGF-�1, -�2, -�3, and rhIL-2were from Peprotech. RGD and RGDS peptides were from Sigma-Aldrich.Anti-CD3 (UCHT1), -CD28, and -FOXP3 (clone 236A/E7) mAbs were fromeBioscience. FACSCalibur was used for data acquisition and the data wereanalyzed with FlowJo software (Tree Star).

Cell Culture. For cell stimulation, 24-well culture plates (Corning) were coatedwith anti-CD3/CD28 at 5 �g/mL each for 2–4 h at 37 °C. The cells were culturedin complete RPMI 1640 supplemented with 2% heat-inactivated autologoushuman serum and 100 U/mL rhIL-2 (Peprotech). For induction of FOXP3, naïveCD4�CD127�CD25–CD45RA� and memory CD45RO� T cells were stimulatedfor 5 d with plate-bound anti-CD3/CD28 (5 �g/mL) � 5 ng/mL rhTGF-�1(Peprotech). For expansion, Tregs were stimulated with anti-CD3/CD28 TregDynabead (Invitrogen) and 100 U/mL IL-2. For in vitro suppression assay,50,000 FACS-sorted allogeneic CD4�CD25– T cells were unlabeled or labeledwith 2 �M CFSE and stimulated with 25,000 nonirradiated autologous CD3-depleted HLA-DR� APCs and 0.25 �g/mL OKT3 (Ortho Biotech) alone or withvarying numbers of preactivated (24 h) Tregs. The cells were cultured for 3 daysin 96-well flat bottom plates (Corning) and pulsed with 3H-TdR (1 �Ci/well) forthe last 6–8 h or FACS analysis for CFSE labeled experiment. For the infectioustolerance experiments, 50,000 CFSE-labeled CD4�CD25–CD127�CD45RA� Tcells were stimulated with anti-CD3/CD28 Dynabeads at 2:1 cell-to-bead ratiofor 5 days alone (top panel) or with 25,000 preactivated (24 h) Tregs trans-fected with siNS, siGARP, or siTGF�1 and analyzed for FOXP3 induction in theCFSE-labeled cells. The cells were placed in 96-well flat bottom plates andcultured in X-VIVO 15 (Lonza) serum-free medium with 100 U/mL IL-2. For boththe suppression and infectious tolerance experiments, the Tregs were firsttransfected by electroporation then rested for 24 h in 100 U/mL IL-2 beforepreactivation with plate-bound anti-CD3/CD28 for 24 h before testing theirfunction.

Quantitative Real-Time PCR Analysis. Total RNA was extracted from cells withan RNeasy Plus Kit (Qiagen). RT-PCR was performed with �1 �g of isolatedRNA for cDNA synthesis using SuperScript II RNase H- Reverse Transcriptase(Invitrogen). Real-time PCR was performed in triplicate according to theTaqman Universal 2� master mix and run on the ABI/PRISM 7900 SequenceDetector System (Applied Biosystems). The amount of FOXP3 and GARP mRNAexpression was normalized to the 18S rRNA and calculated according to thecomparative Ct method as described by Applied Biosystems. The TagManGene Expression Assays from FOXP3 (Hs01085834_m1) and GARP(Hs00194136_m1) were from Applied Biosystems.

siRNA Experiments. GARP, TGF-�1, FOXP3, and nonsilencing control siRNAswere from Invitrogen (Stealth Select RNAi). To transfect the Tregs, 200 pmolssiRNA were mixed with 100 �L human T cell Nucleofector solution (Amaxa

Tran et al. PNAS � August 11, 2009 � vol. 106 � no. 32 � 13449

IMM

UN

OLO

GY

Dow

nloa

ded

by g

uest

on

Mar

ch 1

5, 2

020

Biosystems), and 5 � 106 cells were resuspended in this mixture. The cellsuspension was immediately electroporated by the Nucleofector II instrument(Amaxa Biosystems) and placed in 37 °C prewarmed 100 IU/mL IL-2 culturemedium. For the GARP and TGF-�1 knockdown experiments, the transfectedTregs were rested in 100 U/mL IL-2 for 24 h before stimulation with plate-bound anti-CD3/CD28. For the FOXP3 knockdown experiments, the trans-fected Tregs were rested for 5 d before stimulation with anti-CD3/CD28.

Immunoprecipitation. Day 14 expanded Tregs (20 � 106) were preactivated for48 h with anti-CD3/CD28, then incubated with anti-LAP or IgG (JacksonImmunoresearch) for 1 h at 4 °C and then washed to remove unbound Abs.The cells were detergent solubilized in 1% Brij 96 lysis buffer containingprotease inhibitors. LAP complexes were precipitated with anti-mouse IgGDynabeads. Immunoprecipitates were resolved on 10%–14% acrylamide gels(Invitrogen) under reducing conditions and transferred to nitrocellulose mem-branes (Amersham). Blots were incubated with the anti-GARP and thenhorseradish peroxidase-conjugated anti-IgG2b. Reactivity was revealed byenhanced chemiluminescence. For flow cytometric protein-protein interac-tion experiment, 4.5 �m (1 � 106) anti-mouse IgG Dynabeads (Invitrogen)were incubated in PBS with either 20 �g/mL anti-LAP or anti-GARP mAbs atroom temperature (RT) for 20 min and placed on magnet to wash away thesupernatant. The anti-LAP or anti-GARP conjugated Dynabeads were incu-bated with a mixture of 20 �g/mL GARP-Fc � 20 �g/mL LAP, GARP-Fc � latentTGF-�1 or GARP-Fc � TGF-�1 recombinant proteins for 20 min at RT in PBS andthen placed on magnet to wash away the supernatant. Finally the anti-LAPDynabead samples were stained with goat anti-hFc PE and the anti-GARPDynabead samples were stained with biotinylated goat anti-hLAP followed bystreptavidin PE and then analyzed by flow cytometry.

Confocal Microscopy. Tregs were activated for 48 h then surface-stained withanti-GARP IgG2b and anti-LAP IgG1 mAbs followed by anti-mouse IgG2b AF568 and IgG1 AF 647. For background controls, isotype staining and anti-GARPIgG2b with anti-IgG1 AF 647 or anti-LAP IgG1 with anti-IgG2b AF 568 wereused. Tregs were then fixed with 4% paraformaldehyde in PBS for 30 min at4 °C and cytospun onto slides, permeabilized with 0.05% Triton X-100 for 5min at RT. After 3 PBS washes, the nuclei were stained with 40 ng/mL Hoechst33342 (Invitrogen) for 5 min. Slides were rinsed and mounted with a coverslipusing Fluoromount-G (Southern Biotechnology). Images were collected on aSP2-AOBS confocal microscope (Leica Microsystems) by the NIAID BiologicalImaging Facility.

GARP Transduction. CD4� T cells were purified using magnetic Dynabeads.CD45RO�CD25� population was sorted via FACSAria (BD). The naive cells wereactivated by anti-CD3/CD28-coated Dynabeads and transduced with controlHIV-derived HDV expressing red fluorescent protein (RFP) lentiviral vectors(Clontech Laboratories) or encoding GARP gene as previously described (10)and expanded for 7 days in 200 U/mL IL-2 culture medium. The cells were thenrestimulated for 48 h with plate-bound anti-CD3/CD28 and analyzed forsurface expression of GARP and LAP. The Jurkat cells were transduced with thesame vectors and maintained in regular RPMI media with 10% FCS.

ACKNOWLEDGMENTS. We thank Carol Henry, Tom Moyer, and Calvin Eigstiin the National Institute of Allergy and Infectious Diseases Flow CytometrySection for sorting our cells; Cynthia Matthews in the Department of Trans-fusion Medicine for the leukapheresis; and Lily Koo in the National Institute ofAllergy and Infectious Diseases Biological Imaging Facility for performing theconfocal microscopy. This work was supported by the National Institute ofAllergy and Infectious Diseases Intramural Research Program and NationalInstitutes of Health Grant R01 AI065303 (to D.U.).

1. Wan YY, Flavell RA (2008) TGF-beta and regulatory T cell in immunity and autoimmu-nity. J Clin Immunol 28:647–659.

2. Gleizes PE, et al. (1997) TGF-beta latency: Biological significance and mechanisms ofactivation. Stem Cells 15:190–197.

3. Lawrence DA (2001) Latent-TGF-beta: An overview. Mol Cell Biochem 219:163–170.4. Grainger DJ, Wakefield L, Bethell HW, Farndale RW, Metcalfe JC (1995) Release and

activation of platelet latent TGF-beta in blood clots during dissolution with plasmin.Nat Med 1:932–937.

5. Gandhi R, Anderson DE, Weiner HL (2007) Cutting edge: Immature human dendriticcells express latency-associated peptide and inhibit T cell activation in a TGF-beta-dependent manner. J Immunol 178:4017–4021.

6. Nakamura K, et al. (2004) TGF-beta 1 plays an important role in the mechanism ofCD4�CD25� regulatory T cell activity in both humans and mice. J Immunol 172:834–842.

7. Andersson J, et al. (2008) CD4� FoxP3� regulatory T cells confer infectious tolerancein a TGF-beta-dependent manner. J Exp Med 205(9):1975–1981.

8. Tran DQ, et al. (2009) Selective expression of latency-associated peptide (LAP) and IL-1receptor type I/II (CD121a/CD121b) on activated human FOXP3� regulatory T cellsallows for their purification from expansion cultures. Blood 113:5125–5133.

9. Macaulay IC, et al. (2007) Comparative gene expression profiling of in vitro differen-tiated megakaryocytes and erythroblasts identifies novel activatory and inhibitoryplatelet membrane proteins. Blood 109:3260–3269.

10. Wang R, Wan Q, Kozhaya L, Fujii H, Unutmaz D (2008) Identification of a regulatory Tcell specific cell surface molecule that mediates suppressive signals and induces Foxp3expression. PLoS ONE 3:e2705.

11. Ollendorff V, Szepetowski P, Mattei MG, Gaudray P, Birnbaum D (1992) New gene inthe homologous human 11q13–q14 and mouse 7F chromosomal regions. MammGenome 2:195–200.

12. Ollendorff V, Noguchi T, deLapeyriere O, Birnbaum D (1994) The GARP gene encodesa new member of the family of leucine-rich repeat-containing proteins. Cell GrowthDiffer 5:213–219.

13. Roubin R, et al. (1996) Structure and developmental expression of mouse Garp, a geneencoding a new leucine-rich repeat-containing protein. Int J Dev Biol 40:545–555.

14. Chen W, et al. (2003) Conversion of peripheral CD4�CD25- naive T cells to CD4�CD25�

regulatory T cells by TGF-beta induction of transcription factor Foxp3. J Exp Med198:1875–1886.

15. Miller DM, et al. (1992) Characterization of the binding of transforming growthfactor-beta 1, -beta 2, and -beta 3 to recombinant beta 1-latency-associated peptide.Mol Endocrinol 6:694–702.

16. Murphy-Ullrich JE, Poczatek M (2000) Activation of latent TGF-beta by throm-bospondin-1: Mechanisms and physiology. Cytokine Growth Factor Rev 11:59–69.

17. Yang Z, et al. (2007) Absence of integrin-mediated TGFbeta1 activation in vivo reca-pitulates the phenotype of TGFbeta1-null mice. J Cell Biol 176:787–793.

18. Tran DQ, Ramsey H, Shevach EM (2007) Induction of FOXP3 expression in naive humanCD4�FOXP3- T cells by T-cell receptor stimulation is transforming growth factor-betadependent but does not confer a regulatory phenotype. Blood 110:2983–2990.

19. Allan SE, Song-Zhao GX, Abraham T, McMurchy AN, Levings MK (2008) Induciblereprogramming of human T cells into Treg cells by a conditionally active form ofFOXP3. Eur J Immunol 38:3282–3289.

20. Thornton AM, Piccirillo CA, Shevach EM (2004) Activation requirements for the induc-tion of CD4�CD25� T cell suppressor function. Eur J Immunol 34:366–376.

21. Piccirillo CA, et al. (2002) CD4(�)CD25(�) regulatory T cells can mediate suppressorfunction in the absence of transforming growth factor beta1 production and respon-siveness. J Exp Med 196:237–246.

22. Xu L, Kitani A, Fuss I, Strober W (2007) Cutting edge: Regulatory T cells induceCD4�CD25-Foxp3- T cells or are self-induced to become Th17 cells in the absence ofexogenous TGF-beta. J Immunol 178:6725–6729.

23. Bonne-Tamir B, et al. (1997) Usher syndrome in the Samaritans: Strengths and limita-tions of using inbred isolated populations to identify genes causing recessive disorders.Am J Phys Anthropol 104:193–200.

24. Janssens K, et al. (2000) Mutations in the gene encoding the latency-associated peptideof TGF-beta 1 cause Camurati-Engelmann disease. Nat Genet 26:273–275.

25. O’Connor MN, et al. (2009) Functional genomics in zebrafish permits rapid character-ization of novel platelet membrane proteins. Blood 113:4754–4762.

26. Feroze-Zaidi F, et al. (2007) Role and regulation of the serum- and glucocorticoid-regulated kinase 1 in fertile and infertile human endometrium. Endocrinology148:5020–5029.

27. Liu CJ, Lin SC, Chen YJ, Chang KM, Chang KW (2006) Array-comparative genomichybridization to detect genomewide changes in microdissected primary and meta-static oral squamous cell carcinomas. Mol Carcinog 45:721–731.

28. Martinez-Cardus A, et al. (2009) Pharmacogenomic approach for the identification ofnovel determinants of acquired resistance to oxaliplatin in colorectal cancer. Mol CancerTher 8:194–202.

29. Lassmann S, et al. (2007) Array CGH identifies distinct DNA copy number profiles ofoncogenes and tumor suppressor genes in chromosomal- and microsatellite-unstablesporadic colorectal carcinomas. J Mol Med 85:293–304.

30. Bekri S, et al. (1997) Detailed map of a region commonly amplified at 11q13–�q14 inhuman breast carcinoma. Cytogenet Cell Genet 79:125–131.

31. Rodriguez C, et al. (2004) Amplification of the BRCA2 pathway gene EMSY in sporadicbreast cancer is related to negative outcome. Clin Cancer Res 10:5785–5791.

32. Maire G, et al. (2003) 11q13 alterations in two cases of hibernoma: Large heterozygousdeletions and rearrangement breakpoints near GARP in 11q13.5. Genes ChromosomesCancer 37:389–395.

33. Schraml P, et al. (2003) Combined array comparative genomic hybridization and tissuemicroarray analysis suggest PAK1 at 11q13.5-q14 as a critical oncogene target inovarian carcinoma. Am J Pathol 163:985–992.

34. Edwards J, Krishna NS, Witton CJ, Bartlett JM (2003) Gene amplifications associatedwith the development of hormone-resistant prostate cancer. Clin Cancer Res 9:5271–5281.

13450 � www.pnas.org�cgi�doi�10.1073�pnas.0901944106 Tran et al.

Dow

nloa

ded

by g

uest

on

Mar

ch 1

5, 2

020