Journal of Immunological Methods 353 (2010) 62–70

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Journal of Immunological Methods

j ourna l homepage: www.e lsev ie r.com/ locate / j im

Research paper

Generation of monoclonal antibodies against human regulatory T cells

Christian Becker a,⁎, Heinz Hoschützky b, Wolfgang Rist c, Martin Lenter c,Franz-Joseph Schneider c, Helmut Jonuleit a

a Department of Dermatology of University Medical Center of the Johannes Gutenberg-University Mainz, Langenbeckstrasse1, 55101 Mainz, Germanyb NanoTools Antikörpertechnik GmbH, Tscheulingstrasse 21, D-79331 Teningen, Germanyc Boehringer Ingelheim Pharma GmbH & Co. KG, D-88400 Biberach, Germany

a r t i c l e i n f o

Abbreviations: Tregs, human CD4+CD25+Foxp3+ rmonoclonal antibody.⁎ Corresponding author. Tel.: +49 6131 173541; fax

E-mail addresses: christian.becker@hautklinik.klin(C. Becker), info@nanotools.de (H. Hoschützky),wolfgang.rist@boehringer-ingelheim.com (W. Rist),martin.lenter@boehringer-ingelheim.com (M. Lenter)franz-joseph.schneider@boehringer-ingelheim.com (Fjonuleit@hautklinik.klinik.uni-mainz.de (H. Jonuleit).

0022-1759/$ – see front matter © 2010 Elsevier B.V.doi:10.1016/j.jim.2010.01.002

a b s t r a c t

Article history:Received 28 August 2009Received in revised form 7 January 2010Accepted 7 January 2010Available online 22 January 2010

Natural CD4+CD25+Foxp3+ regulatory T cells (Tregs) control the activation of the immunesystem and therefore have become a major area of research in immunology. The generation ofmonoclonal antibodies against human Tregs offers the possibility to discover novel Treg-specific or Treg-associated surface markers and to identify targets for a therapeutic modulationof Tregs. Here we present amethodology optimized to efficiently induce and select mAb againsthuman Tregs by repeated immunization of mice with Tregs from a single donor and adifferential two-step flow cytometry-based hybridoma screening procedure.

© 2010 Elsevier B.V. All rights reserved.

Keywords:Regulatory T cellsSurface receptorsMonoclonal antibodies

1. Introduction

The existence of a specialized population of T cells thatcontrols immune responses remained questionable for decadesbecause of the lack of a phenotypical definition (Green andWebb, 1993). Eventually, by use of antibodies against theinterleukin-2 receptor (IL-2R)α-chain (CD25) inmice andmana subpopulation of CD4+CD25+ T cells has been identified thatcontrols the activation of the immune system (Sakaguchi et al.,1995; Jonuleit et al., 2001) and therefore has been namedregulatory T cells (Tregs). Further evaluation of the Tregphenotype with monoclonal antibodies (mAb) against knownsurface markers revealed an expression of molecules such asCD45RO, CD27 (Ruprecht et al., 2005), CTLA-4 (Takahashi et al.,2000), GITR (Shimizu et al., 2002), CD134 (Takeda et al., 2004)

egulatory T cells; mAb

:+49 6131 17474523ik.uni-mainz.de

,.-J. Schneider),

All rights reserved.

,

.

and L-selectin (CD62L) (You et al., 2004) as well as a reducedexpression of CD127 (Liu et al., 2006; Hartigan-O'Connor et al.,2007). Moreover, Tregs express the transcription factor FoxP3,which serves as a master regulator in their development andfunction (Hori et al., 2003). Although in humans none of thesemarkers is sufficient to distinguishTregs fromactivated effectorT cells (Banhamet al., 2006) they are at least helpful to reveal orincrease the purity of isolated Treg populations (Jonuleit et al.,2001; Liu et al., 2006). More recently, several studies haveshown that the human CD4+CD25+ Treg population containsseveral functionally distinct subpopulations that are distin-guishable from one another on the basis of differences inadditional cell surface determinants (Jonuleit et al., 2002;Stassen et al., 2004; Fritzsching et al., 2006; Baecher-Allan et al.,2006; Borsellino et al., 2007).

However, although mAb to surface markers have served asan important tool in the identification and further character-ization ofmurine andhumanTregs, no report on the generationof mAb against Tregs has been published. This is surprising,since the generation of mAb represents an unbiased discoveryapproach that offers the possibility to identify novel surfacemarkers including unusually spliced or modified versions ofknownmolecules ormolecule clusters thatmaynot bedetectedby other techniques. As an added advantage, mAb against

63C. Becker et al. / Journal of Immunological Methods 353 (2010) 62–70

surfacemolecules onTregsmaybehelpful to define targets for atherapeutic modulation of these cells (Jarvinen et al., 2003;Becker et al., 2007). However, since Tregs suppress antibodyproduction by B cells (Lim et al., 2005) and are suggested to actin a cross-species-specific manner (Tran et al., 2008) thegeneration of antibodies against Tregs may not represent amatter of course.

Here, we describe a methodology that allows to efficientlyinduce and select mAb against human Tregs consisting ofleukapheresis isolation of large numbers of human Tregs withhigh purity and yield, induction of mAb to human Tregs byrepeated immunization of mice with isolated Tregs from asingle donor and a differential two-step flow cytometry-based hybridoma screening procedure to select Treg-specificmAb. In addition, the evaluation and antigen identification isdescribed for one of the first generated antibodies.

2. Materials and methods

2.1. Mice

NMRI mice were bred and maintained under standardhousing conditions in the animal facility of the University ofMainz. Experiments were performed in accordance withrelevant laws and institutional guidelines (authorizations177-07/041-7 and G07-1-016).

2.2. Isolation of Tregs from leukapheresis products

Leukapheresis were prepared from adult healthy volunteersfollowing approval by the local ethical committee (Landesärzte-kammer Rheinlandpfalz No 837.029.05 (4687)). After prepara-tion of PBMC by density gradient centrifugation CD4+CD25high

Tregs were separated from PBMC by positive selection withmicrobeads (Miltenyi) and depletion of contaminating non Tregcells with Dynabeads (Invitrogen). In the first step, PBMC wereincubated with a limited amount of CD25 microbeads (2 µL/107

PBMC) for 20 min at 4 °C in PBS w/o Ca2+/Mg2+, 0.5% humanserum albumin and 3 mM EDTA (1×108 cells/ml) to isolate theCD25high expressing cells only. Afterwards, cells were washedtwo timeswith PBS. The CD25high cell fractionwas isolated using7–8 LS columns (Miltenyi) on midi MACS separators (Miltenyi)in parallel. The positively selected cells contain 65–80% CD4+ Tcells and 20–35% contaminating CD19+ B cells and CD8+ T cells,aswell as a fewCD14+monocytes. To deplete the contaminatingCD4 negative cells, the CD25high cell fraction was incubated withCD19 Dynabeads (2 beads/cell), CD8 Dynabeads (2 beads/cell)and CD14 Dynabeads (0,5 beads/cell) for 20 min at 4 °C on asamplemixer (5×107/ml inMEMwith 5% FCS). Afterwards, cellswere further diluted (1×107/ml) by addition of MEM with 5%FCS and incubated for additional 10 min. Depletion was carriedout according to the manufacturer's instruction by use of amagnetic particle concentrator (Dynal, Invitrogen). For highpurity of CD4+CD25high Tregs the depletion step was repeatedonce. CD4+CD25high T cell puritywas typically 95–98%. For someexperiments, Tregs were functionally activated by stimulationwith PHA-L (1 µg/ml). For later use aliquots of isolated Tregswere frozen in saline (30×106 cells/100 µL) and preserved inliquid nitrogen.

Untouched CD4+CD25− T helper cells were isolated bynegative selection from PBMC with a negative isolation kit(Miltenyi).

2.3. Immunization and monoclonal antibody production

Six to eight weeks old female NMRI mice (two to threeanimals per group) were immunized two times in 2 weeksintervals with 10×106 human Tregs in saline intraperitoneally.Mice were bled before immunization and 1 week after thesecond immunization and sera stored at−20 °C for subsequentanalysis. At day 42 mice were boosted with 10×106 Tregsintravenously. Three to 5 days after boost mice were sacrificedand the spleens removed for disaggregation and fusion.

Spleen cells were fused at a 1:1 ratio with the mouseP3X63.Ag8.653.1 (P3) hybridoma fusion partner using stan-dard techniques (Kohler and Milstein, 1975). Hybridomaswere selected in complete DMEM-10% FCS with 1× HATsupplement for 10–14 days. Supernatants were screened byFACS. Candidate clones were isolated by limiting dilution inDMEM/10% FCS/0.5×HAT/0.5×HT. Single clones werescreened by FACS as described above. Positive clones wereexpanded in DMEM/10% FCS/HT, subsequently adapted toDMEM/10% FCS and cryopreserved.

Selected candidate antibodies were purified from serum-free cell culture supernatants by ultrafiltration followed by(NH4)2SO4 precipitation and gel filtration on Superdex 200.

2.4. Screening of sera from immunized mice

Human Tregs or CD4+CD25− T helper cells were incubatedwith serially diluted immune and pre-immune serum fromindividual animals, washed and counterstained with a FITC-labeled F(ab′)2 fragment specific to mouse immunoglobulins(MP Biomedicals). To exclude reactivity of the secondaryantibody against antibodies used in the T cell isolation process,Tregs used in this assaywere isolatedwithmicrobeads coupledto a humanized anti-CD25 antibody (Miltenyi, #130-090-445)and untouched CD4+CD25− T cellswere generated bynegativeselection.

2.5. Screening of hybridoma supernatants

Hybridoma supernatants (12 plates per fusion) werescreened 10–14 days after fusion and cloning, working back-wards fromplates 9–12 (3–5 clones perwell) to plates 5–8 (2–3clones per well) and plates 1–4 (1 clone per well). Screeningconsisted of a two-step flow cytometry-based hybridomascreening procedure using PBMC. In a primary screening stepindividual hybridoma supernatants were analyzed for theircapacity to stain less than10%of theCD4+Tcells in PBMC. PBMCwere incubated with 100 µL of undiluted supernatants for30 min, washed two times with PBS and counterstained for30 min with a FITC-labeled goat anti-mouse (GAM) F(ab′)2fragment to mouse immunoglobulins (MP Biomedicals). After-wards, cells were stained with rat anti-CD4 mAb (Serotec),washed and counterstainedwith anti-rat-IgG-PE (Dianova). Thefollowing mAb were used as control antibodies: IgG1 (clone B-21, Diaclone), IgG2a (clone B-22, Diaclone), IgG2b (clone BE4,Diaclone), IgM (clone B-N6, Diaclone) and anti-CD3mAb (cloneOKT-3). PBMC stained with supernatants or controls were

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analyzed by flow cytometry (FACSCalibur; BD Biosciences)gating on lymphocytes and collecting at least 2×104 events foreach supernatant. Supernatants that stained aminimumof 2–3%but less than 10% of CD4+ cells were additionally analyzed fortheir capacity to stain Foxp3+ T cells. Therefore, PBMCwere firststained with supernatant and goat anti-mouse-IgG-FITC asdescribed above, fixed/permealized and stained with anti-Foxp3-PE (clone PCH101, eBioscience) according to manufac-turer's instructions. Hybridomas obtained by immunization ofmice with activated Tregs were first screened on freshlyprepared PBMC and subsequently re-screened on PBMC pre-activatedwith PHA-L (1 µg/ml) for 24 h. The collected datawereevaluated using the CellQuest® software.

To analyze the eligibility of newly identified Treg mAb foridentification of T cells with regulatory activity, negativelyisolated CD4+ T cells were incubated with 5 µg/ml of thepurified candidate antibody for 30 min,washed two timeswithPBS and counterstained for 30 min with a PE-labeled goat anti-mouse-Ig (GAM) F(ab′)2 fragment (MP Biomedicals) and FITC-labeled rat anti-CD4. For isolation of labeled CD4+ T cells, anti-

Fig. 1. Large scale preparation of human Tregs from leukapheresis. A) Overviewmagnetically isolated from PBMC using limited amounts of CD25 microbeads for powith the indicated amounts of Dynabeads. Recovery rates refer to initial PBMC numbTregs were stained with anti-CD4, -CD45RO, -CD27, -CD127, as well as anti-Foxp3 anafter positive selection with CD25 microbeads, a PE-labeled anti-CD25 antibody wmicrobeads to anti-CD25-PE=3:1). C) Suppressive activity of isolated CD4+CD25+

irradiated PBMC (50 Gy, 3×105/well) and 0.5 µg/ml anti-CD3mAb in absence (gray bon amicrotiter plate (Costar). Cultures of Tregs with PBMC (white bar) served to conon day 3 for a 16-h pulse.

PE microbeads (2 µL/107 cells) were used. The positive andnegative cell fraction were re-analyzed by flow cytometry andsubjected to a suppression assay (Section 2.8).

2.6. Antibody isotyping and purification

To prevent multiple antibody specificities in single wells,selected hybridoma candidates were re-cloned at a range offive to one cells per well on 96 well plates and supernatantsfrom wells were re-analyzed as described. Antibody isotypeswere determined using the mouse IsoStrip™ monoclonalantibody isotyping kit (Santa Cruz Biotechnology, Inc., CA).

2.7. Antigen identification

Purified antibody candidates were evaluated on a numberof human T lymphoma cell lines. Antigens recognized bymonoclonal antibodies were identified by immunoprecipita-tion and peptide sequencing using either isolated Tregs or ahuman T lymphoma cell line expressing the antigen.

of the isolation technique for human Tregs from leukapheresis. Tregs weresitive selection and subsequently depletion of contaminating non Treg cellsers. B) Example of the phenotypic characterization of isolated Tregs. Isolatedd analyzed by flow cytometry. Because CD25 molecules are difficult to assessas added to the CD25 microbeads during the separation procedure (ratio ofT cells. CD4+CD25− T helper cells (1×105/well) were cultured together withar) or presence of different Treg numbers (black bars, ratios starting with 1:1)trol Treg anergy. To determine proliferation 37 kBq [3H]thymidine was added

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The eluate of the pull-down was separated by SDS-PAGEand visualized by staining with coomassie. In-gel digestionusing modified porcine trypsin (Promega, Madison, USA) wasperformed as described (Shevchenko et al., 1997). Extractedpeptides were dried using a vacuum concentrator. NanoLC-MS/MS analysis was performed with an UltiMate nanoHPLCsystem (LC Packings, Amsterdam, Netherlands) connected toa QSTAR XL quadrupole TOF hybrid mass spectrometer (ABISciex, Toronto, Canada). The dried peptide sample wasdissolved in 10 µL of 2% ACN, 0.1% TFA, applied to aprecolumn (PepMap C18, 0.3×5 mm; Dionex) and separatedusing an analytical column (PepMap C18, 0.075×150 mm;Dionex) at a flow rate of 250 nL/min. The mobile phaseswere: A=2% ACN, 0.1% formic acid and B=80% ACN, 0.1%formic acid. The gradient for separation was 5–50% B in30 min, 50–100% B in 2 min. A survey scan from m/z 380 to1300 was performed for 1 s with subsequent two MS/MSscans for 2 s each. Precursor ions were dynamically excludedfor 120 s. Under script control (Analyst, ABI Sciex) theacquired product ion spectra were submitted to the MASCOTdatabase search engine (Matrix Science, London, UK) againstthe Swiss-Prot database with the following search para-meters: maximum of one missed trypsin cleavage, cysteinecarbamidomethylation, methionine oxidation, and a maxi-mum 0.15 Da error tolerance in both, theMS andMS/MS data.All hits were manually verified using accepted rules forpeptide fragmentation (Biemann, 1990).

2.8. Suppression assays

CD4+CD25− T helper cells were plated at 1×105/welltogether with 3×105/well irradiated (50 Gy) PBMC in thepresence of 0.5 µg/ml anti-CD3 mAb. Tregs were added to thecultures at different ratios starting with 1:1. To determineproliferation 37 kBq [3H]thymidine was added on day 3 of theculture for a 16-h pulse. All assays exhibited less than 10%SEM and were repeated at least three times using cellsisolated from different donors.

Fig. 2. Immunization schedules and analyses of sera from immunized mice. A) Immsecond immunization were assayed for the presence of antibodies to human Tregs anmicrobeads coupled to a humanized anti-CD25 antibody were incubated with serialFITC-labeled F(ab′)2 fragment specific to mouse immunoglobulins. A representativ

2.9. Statistical analysis

Functional experiments were performed at least threetimes. Differences in proliferation between samples wereanalyzed by using the Student's t-test. A P-value of≤0.05 wasconsidered significant.

3. Results and discussion

3.1. Large scale preparation of human regulatory T cells from asingle donor for immunization

The generation of mAb against human Treg surfacemolecules in their native form first and foremost requiresaccess to substantial numbers of isolated Tregs for repeatedimmunization. However, because Tregs are a rare populationin human blood, only limited numbers can be obtained fromwhole blood samples or buffy coats. Unfavorably, immuniza-tion of mice with cells from different donors broadens thespectrum of foreign determinants which may hamper theinduction and identification of mAb against Tregs. In order tolimit the number of determinants in immunization we usedTregs from a single donor throughout all immunizations. Wetherefore performed leukapheresis on healthy volunteers andisolated Tregs and CD4+CD25− T helper cells using acombination of isolation and depletion steps as illustratedin Fig. 1A. This procedure resulted in isolation of up to 108

CD4+CD25highFoxp3+CD127low human Tregs with 96–98%purity (Fig. 1B and C). Upon isolation and phenotypicalcharacterization aliquots were either immediately preservedin liquid nitrogen for later use or activated for 24 h bystimulation with PHA-L beforehand.

3.2. Immunization schedules and analysis of sera fromimmunized mice

Since no standard methods for the induction andscreening of Treg-reactive mAb were available, we had to

unization schedules. B) Sera taken before immunization and 1 week after thed CD4+CD25− T cells. Untouched CD4+CD25− T cells and Tregs isolated withly diluted immune and pre-immune sera, washed and counterstained with ae result from a mouse immunized with resting Tregs is shown.

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explore different immunization strategies and establish ascreening method.

Initially we aimed at obtaining mAb to native surfacemolecules on resting Tregs by injecting intact unstimulatedTregs into mice without adjuvant (immunization I). Based onthe common finding that human Tregs use a cell contact-dependent suppressor mechanism that requires their activa-tion (Jonuleit et al., 2001; Dieckmann et al., 2001; Taams et al.,2001; Baecher-Allan et al., 2001) andmight involve an alteredexpression of surface molecules, we also performed animmunization with activated Tregs (immunization II). Alter-natively, mice were immunized with a 5:1 mix of activatedTregs and resting CD4+CD25− T cells to include moleculesthat are possibly induced during interaction of Tregs with

Fig. 3. Selection of antibody candidates. A) Flow chart of the screening procedure. Nselected in primary screening. Screening was performed on PBMC stimulated with PHB) Examples of staining patterns that led to rejection or selection in the screening pthe percentage of CD4+ T cells and Foxp3+ T cells stained with individual superna

CD4+CD25− T helper cells (immunization III) (Fig. 2A). Aftertwo rounds of immunization and a subsequent boost, spleencells from immunized animals were fused with hybridomacells.

Immunization efficacy and Treg reactivity were evaluatedby analyzing sera from animals before immunization and1 week after the second immunization. We observed prom-inent reactivity of sera from Treg-immunized mice withhuman Tregs compared to CD4+CD25− T helper cells(Fig. 2B). This difference was apparent even at a 104 foldserum dilution, indicating that the serum from Treg-immu-nized mice contained antibodies to surface antigens onhuman Tregs not expressed by CD4+CD25− T helper cells.No antibodies to Tregs or CD4+CD25− T helper cells were

ote, secondary screening was restricted to hybridoma supernatants that wereA-L (1 µg/ml) for 24 h (activated PBMC) or left unstimulated (resting PBMC).rocedure. Examples belong to the screening on resting PBMC, dot blots showtants.

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detectable before immunization (Fig. 2B). Similar resultswere obtained from all immunization approaches demon-strating that all three protocols were efficient to induce Treg-reactive mAb.

3.3. Identification of Treg-reactive antibodies in hybridomasupernatants

More than 6800 hybridomas were generated from immu-nized mice and characterized in a two-step flow cytometryscreening using freshly isolated PBMC (Fig. 3). 6–12% of thehybridoma supernatants (SN) reactedwith all human lympho-cytes, with a majority of pan lymphocyte antibodies found inhybridomas from mice immunized with resting Tregs (12%)

Fig. 4. Immunization schedules and characterization of hybridoma supernatants. A)in different immunization schedules. B) Staining profiles of selected hybridoma sup

(Fig. 4A). Notably, all immunizations consistently resulted insimilar numbers of candidates after primary and secondaryscreening (Fig. 4A). In the primary screening around 5% of allhybridomas fulfilled the first selection criterion by staining lessthan 10% of the CD4+ T lymphocytes. Among these primarycandidates 8–15%were selected in the secondary screening forstaining more than 10% of the Foxp3+ T cells in PBMC. Thelowest number of final candidates was obtained from miceimmunized with a mix of activated Tregs and CD4+CD25− Thelper cells.

In general, all immunizations generated antibodies againsthuman Tregs. Notably, the frequency of hybridoma super-natants reacting with all human lymphocytes did not seem tointerfere with the generation and identification of antibodies

Number and percentages of hybridoma supernatants generated and selectedernatants in resting and activated PBMCs.

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against human Tregs, suggesting that the use of a single humandonor helped to limit the number of foreign determinants inimmunization. Fig. 4B shows the staining pattern of severalhybridoma supernatants as obtained during screening onresting PBMC. All candidate antibodies recognize a fraction ofthe CD4+ T cells and different numbers of Foxp3+ T cells.

Together these results demonstrate that murine antibodiesto human Tregs can be obtained by immunization of mice withhuman Tregs. In contrast to the recent observation that humanTregs suppressmouse T cell activation in vitro (Tranet al., 2008)our results indicate that in vivo human Tregs do not stronglyinterferewithmouse B cell activation and antibody production.This is corroborated by the fact that all antibody candidateswere of the mouse IgG1 or IgG2a isotype.

3.4. Antibody purification and antigen identification

As shown in Figs. 3 and 4 several hybridomas secretedantibodies that reacted with CD4+Foxp3+ T cells in PBMC.The corresponding hybridomas were re-cloned and re-screened following the outlined screening procedure. Con-firmed hybridomas were subjected to large scale productionand purification and their isotype determined.

Fig. 5. Functional analyses of CD4+ T cells stained by the antibody 9H6E10. A) Staininanalysis of CD4+ cells stained with the mAb 9H6E10 and magnetically sorted intantibody, followed by a PE-labeled anti-mouse mAb and counterstained with anti-CDwere magnetically sorted into negative and positive cells using anti-PE microbeads.to the cultures at different numbers starting with 1×105/well and CD4+CD25− T cel0,5 µg/ml anti-CD3 mAb. Numbers of sorted cells and CD4+CD25− T cells are givenfor a 16-h pulse.

As illustrated in Fig. 4B for three of the newly identifiedantibody candidates, staining of resting and activated PBMCallowed to reveal whether the recognized surface moleculeswere up-regulated on activated cells (Fig. 5A). Whereas inresting PBMC some candidates seemed to preferentially stainCD4+Foxp3+ cells, in activated PBMC cells outside of the CD4and Foxp3 compartment were additionally stained, suggest-ing that Treg-associated markers were recognized. Althoughthe screening criteria were set up to identify monoclonalantibodies against cell surface markers on CD4+Foxp3+

T cells, they could not ensure a regulatory function of therecognized cells. Several studies have reported that Foxp3expression, as measured by flow cytometry, is transiently up-regulated by activated human non-regulatory CD4+ T cells(Mantel et al., 2006; Gavin et al., 2006). Moreover, a numberof other cell surface markers on Tregs such as GITR and CTLA-4 are similarly up-regulated during T cell activation and arethus expressed on effector T cells. It was therefore necessaryto analyze the eligibility of newly identified Treg antibodiesfor identification of CD4+CD25+Foxp3+ T cell with regula-tory activity in resting or activated PBMC. To reveal thefunction of CD4+ T cells stained by newly generated Tregantibodies negative and positive selected CD4+ T cells were

g profile of the antibody 9H6E10 in resting and activated PBMC. B) Functionalo negative and positive cells. Briefly, PBMC were stained with the 9H6E104-FITC. After negative isolation of CD4 T cells using negative isolation kit, cellsDot blots of separated populations are shown. Sorted populations were addedls (1×105/well) and irradiated (50 Gy) PBMC (3×105/well) in the presence ofas ratios. To determine proliferation 37 kBq [3H]thymidine was added on day

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magnetically sorted from stained CD4+ T cells, coculturedwith CD4+CD25− T cells and proliferation examined(Fig. 5B). Only antibodies that were found to stain CD4+ Tcells with suppressive activity were finally subjected toepitope identification.

In order to identify an additional source for large cellnumbers needed in immune precipitation, we stained severalhuman T lymphoma cell lines with selected antibody candi-dates against Treg-associated surface molecules. As shown inFig. 6 for the candidate antibody 9H6E10 individual Tlymphoma cell lines expressed Treg-associated surface mole-cules and were therefore subsequently used to simplify andaccelerate the identification of the respective molecules(Fig. 6A). For antigen identification immunoprecipitationexperiments and peptide sequencing were carried out with

Fig. 6. Antigen identification of the antibody 9H6E10. A) Binding of the antibody 9H6Karpas299 and Jurkat cells were incubated with mAb 9H6E10 conjugated to CNBr-Seby SDS-PAGE under reducing conditions and stained with Coomassie. Molecular maasterisk was subjected to in gel digestion. C) Search result from ProteinPilot after m(asterisk) revealing three peptides (dark background) within the protein sequence

lysates of Tregs (Treg-specific molecules) or, as depicted, ofhuman T lymphoma cell lines (Treg-associated molecules).

Among the targets of selected antibody candidates againsthuman Tregs several prominent Treg cell surface moleculessuch as CD25 and MHC class II were identified (Fig. 6B and C).However, other candidates showed expression profilesdifferent from most known Treg markers suggesting thatthey recognize novel or previously unnoticed Treg surfacemarkers. These antibodies are under thorough investigation.

4. Concluding remarks

Together our data demonstrate that the outlined strate-gy is successful for the generation of mAb to moleculeson mature suppressive CD4+CD25+Foxp3+ T cells. Since

E10 to different human T cell lymphoma cell lines. B) Detergent extracts frompharose. Bound material was eluted with SDS-PAGE sample buffer, separatedsses (in kDa) are shown on the left. The 55 kDa protein band marked by anass spectrometric analysis of the tryptic peptides of the 55 kDa protein bandof human IL-2 receptor alpha subunit (CD25).

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antibodies are able to recognize a wide variety of antigensincluding lipids and sugars, the approach followed here maycomplement gene-expression studies in search of new cellsurface markers for human Tregs, providing new tools tostudy the biology of Tregs.

Role of funding source

Boehringer Ingelheim Pharma GmbH and Co. KG wasinvolved in study design; data collection, analysis and inter-pretation; the writing of the report; and in the decision tosubmit the paper for publication.

Acknowledgements

The authors thank L. Paragnik, and P. Hoelter for their experttechnical assistance. This work was supported by the DeutscheForschungsgemeinschaft, grant A2 TR52 (to H. Jonuleit and A.Tüttenberg) and grant BE 3685/1-1 (to C. Becker). H.J. receivedresearch support from Boehringer Ingelheim Pharma GmbHand Co. KG. W.R., M. L. and F.J.S. are employees of BoehringerIngelheim Pharma GmbH and Co. KG.

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