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Surface IL-17A ExpressionTh17 Cells Unambiguously Identified by Phenotypical Characterization of Human

TolosaLioznov, Manuela Kolster, Christoph Hölscher and Eva Verena Brucklacher-Waldert, Karin Steinbach, Michael

http://www.jimmunol.org/content/183/9/5494doi: 10.4049/jimmunol.0901000

2009; 183:5494-5501; ;J Immunol 

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Referenceshttp://www.jimmunol.org/content/183/9/5494.full#ref-list-1

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Phenotypical Characterization of Human Th17 CellsUnambiguously Identified by Surface IL-17A Expression1

Verena Brucklacher-Waldert,*†‡ Karin Steinbach,* Michael Lioznov,§ Manuela Kolster,*Christoph Holscher,¶� and Eva Tolosa2*†

Th17 cells are involved in the defense against bacteria and fungi and play a prominent role in the pathogenesis of autoimmunediseases, but research on human Th17 cells is hindered due to the lack of a surface marker. In this study, we report that a subsetof human and mouse CD4� T cells as well as human Th17 T cell clones express IL-17A on their surface upon stimulation.Correlation of surface IL-17A expression with intracellular IL-17A production and with ROR�t mRNA expression identifiedsurface IL-17A as a specific marker for human and mouse Th17 cells. Phenotype characterization of ex vivo CD4� IL-17A� cellsshowed that the chemokines CCR6 and CCR4, costimulatory molecules, as well as CD2 and CD49d were more prominentlyexpressed on these cells than in surface IL-17A� cells, supporting the concept of Th17 cells as a potent inflammatory effectorsubtype. In addition, we generated human Th1, Th1/17 (producing both IFN-� and IL-17A), and Th17 T cell clones based on singlecell sorting of surface IL-17A�, IL-17Aint, and IL-17Ahigh CD4� T cells, respectively, and showed the plasticity of the doubleproducing clones to the cytokine milieu. The identification of surface IL-17A as a marker for Th17 cells should facilitate researchon this subset. The Journal of Immunology, 2009, 183: 5494–5501.

I L-17 is a potent inducer of inflammation by triggering therelease of cytokines and chemokines from a broad range ofstromal cells (1). Through its regulation of proinflammatory

gene expression, IL-17A has the capability to recruit neutrophils(2) and to promote antimicrobial protein synthesis (3). Until now,six structurally related isoforms for IL-17 (IL-17A, B, C, D, E, andF) have been identified and grouped into the IL-17 cytokine family(4–6). Among them, IL-17F has the highest homology with IL-17A. Both IL-17A and IL-17F are active as homodimers and canform biologically functional IL-17A/IL-17F heterodimers (7). IL-17A and IL-17F are produced by CD4� T cells, �� T cells, NKTcells, NK cells, CD8 cells, neutrophils, and eosinophils (1, 8–13).IL-17A-producing CD4� T cells, defined as Th17 cells, have beenrecently identified as a distinct Th cell lineage (14), which differ-entiates upon stimulation from naive CD4� T cells in presence ofTGF-� and IL-6 (15–17). Maintenance of Th17 cells requires inaddition IL-23 (18), and they express the lineage-specific tran-scription factor ROR�t (19). In contrast, Th1 cell differentiationrequires IL-12 and the expression of the transcription factor T-bet(20), while Th2 cells are dependent on IL-4 and the expression ofGATA-3 (21). The existence of Th1/17 cells, producing both IL-

17A and IFN-�, suggests that commitment to a specific effectortype might be more flexible than previously assumed.

Th17 cells have been linked to the pathogenesis of several inflam-matory (22, 23) and autoimmune diseases (24–26) and therefore theyare under intense investigation. Although reporter lines allow trackingof Th17 cells in mice, human Th17 cells are currently identified exvivo by intracellular IL-17 expression, which does not allow furtherfunctional analysis. Alternatively, Th17 cells can be differentiated invitro from naive CD4� T cells using combinations of cytokines, butculture conditions may change their properties.

We report in this study the unambiguous identification of Th17cells by the expression of surface IL-17A. Using surface IL-17A asa marker, we could phenotypically characterize ex vivo humanTh17 cells and generate Th17 and Th1/17 T cell clones (TCC).3

Furthermore, we show for the first time in human cells the plas-ticity of the Th1/17 cells in response to the cytokine milieu.

Materials and MethodsCells

Peripheral blood was taken from healthy donors after informed consentunder a protocol approved by the local ethics committee. CD4� cells wereisolated (�95% purity) from PBMC by negative selection using the CD4�

T Cell Isolation Kit II (Miltenyi Biotec) according to manufacturer’s in-structions. Th1 and Th17 TCC were generated from PBMC by limitingdilution (27). Clonality was assessed by TCR V�-chain staining (28). Forthe isolation of surface IL-17A� cells, CD4� T cells were stimulated with50 ng/ml PMA and 1 �g/ml ionomycin for the indicated time period,stained with anti-IL-17A Alexa647 (eBio64DEC17, eBioscience), andFACS sorted or isolated using anti-Cy5/Alexa Fluor 647 Microbeads(Miltenyi Biotec). To induce a Th17 response, C57BL/6 wild-type or IL-17R-deficient mice (29) (provided by Amgen) were immunized with 200�g MOG35–55 (NeoMPS) in CFA supplemented with 4 mg/ml Mycobac-terium tuberculosis H37RA (Difco Laboratories), followed by injection of300 ng pertussis toxin (Sigma-Aldrich) at the time point of immunization

*Institute for Neuroimmunology and Clinical Multiple Sclerosis Research, Center forMolecular Neurobiology Hamburg and †Department of Immunology, UniversityMedical Center Hamburg-Eppendorf, Hamburg, Germany; ‡Graduate School of Cel-lular and Molecular Neuroscience, University of Tubingen, Germany; §Interdiscipli-nary Clinic for Stem Cell Transplantation, University Medical Center Hamburg-Ep-pendorf, Hamburg, Germany; ¶Division of Infection Immunology, Research CenterBorstel, Borstel, Germany; and �Cluster of Excellence Inflammation at Interfaces,Borstel-Kiel-Lubeck-Plon, Germany

Received for publication March 27, 2009. Accepted for publication August 21, 2009.

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 The Institute for Neuroimmunology and Clinical Multiple Sclerosis Research andthis work are supported by the Hertie Foundation. V.B.-W. was partly supported bythe German Research Council (SFB 685) and C.H. by the Research Focus Autoim-munity at the University of Lubeck.2 Address correspondence and reprint requests to Eva Tolosa, Department of Immu-nology, University Medical Center Hamburg-Eppendorf (UKE), Martinistr. 52, 20251Hamburg, Germany. E-mail address: etolosa@uke.uni-hamburg.de

3 Abbreviations used in this paper: TCC, T cell clone; MFI, mean fluorescence intensity;ICS, intracellular cytokine staining; PD-1, programmed cell death 1; TMHMM, trans-membrane prediction using hidden Markov models; MINNOU, membrane protein iden-tification without explicit use of hydropathy profiles and alignments.

Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00

The Journal of Immunology

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and repeated 2 days later. After 12 or 25 days, CD4� T cells wereisolated from spleen and from the spinal cord and brain of the immu-nized mice as described before (30) by magnetic isolation (MiltenyiBiotec) and used for staining. All experiments involving animals wereperformed according to the German animal protection act and have beenapproved by local authorities.

Flow cytometry

The following anti-human Abs were used: anti-CD4 Pacific Blue (cloneMT310, DakoCytomation), anti-IFN-� PE, (clone 4S.B3), anti-IL-17AAlexa647 (clone eBio64DEC17), anti-IL-17F PE (clone SHLR17), anti-ICOS FITC (clone ISA-3), anti-CD25 PE (clone 12–1278-73), anti-CD28 PE-Cy7 (clone CD28.2), anti-CD39 PE-Cy7 (clone 25– 0399),anti-programmed cell death 1 (PD-1) FITC (clone U116), CD127 PE(clone 12–1278-73), CD45RO FITC (clone 11–0457-73) (all eBioscience),anti-CD6 FITC (clone 559491), anti-CCR6 PE (clone 11A9), anti-CTLA-4 PE(clone BNI3), anti-CD2 FITC (clone RPA-2.10), anti-CD25 PE (clone M-A251), anti-CD5 FITC (clone UCHT2), anti-CD62L PE (clone L48), anti-CD49d PE (clone 9F10), anti-CD28 PE (clone 555432), anti-CD14 PacificBlue (clone 558121), anti-CD122 PE (clone 554525), anti-CD132 PE (clone555900), anti-CD161 PE (clone HP-3G10) and isotype-matched control mAbs(all BD Biosciences), anti-IL-17R PE (clone 133617), anti-CCR4 FITC (clone205410), anti-CCR5 PE (clone 45531) (all R&D Systems), and anti-CD27Alexa750 (clone MHCD2727, Caltag). For the mouse staining protocol, weused anti-CD4 PE (GK1.5), CD11b FITC (M1/70), CD45 PE-Cy7 (30-F11),and anti-IL-17A Alexa647 (eBio17B7, all eBioscience).

For assessment of surface expression, cells were stained at different timepoints after PMA/ionomycin stimulation. For intracellular cytokine stain-ing (ICS), cells were stimulated with PMA/ionomycin in the presence ofbrefeldin A (10 �g/ml, eBioscience) for 6 h and subsequently fixed andpermeabilized with the corresponding buffers (eBioscience). Samples wereanalyzed in the LSRII flow cytometer using the DiVa software (BD Bio-sciences), and results were evaluated using the FlowJo software (TreeStar). The resulting mean fluorescence intensity (MFI) was obtained aftersubtracting the MFI of the isotype control from the MFI of the Ab ofinterest. For cell sorting we used a FACS Aria (BD Biosciences).

Expansion of surface IL-17A� cells

MACS- or FACS-sorted surface IL-17A� and surface IL-17A� cells werecultured as a bulk in X-VIVO 15 or IMDM supplemented with 5% humanserum, respectively, for 10 to 14 days. IL-17A production was assessed byICS 10 days after each restimulation and the supernatants monitored byELISA (human IL-17A ELISA kit, eBioscience).

Generation and plasticity analysis of Th1/17 cells

CD4� T cells were stimulated 6 h with PMA/ionomycin and subsequentlystained with anti-IL-17A Alexa647. Surface IL-17A�, IL-17Aint, or IL-17Ahigh CD4� T cells were sorted at one cell per well onto 96-well cultureplates containing IMDM supplemented with 5% human serum, 20 U/mlhrIL-2 and irradiated autologous PBMC (FACSAria, BD Biosciences).Cells were restimulated every second week. Cytokine production andclonality were assessed by ICS and TCR V� staining, respectively. Toassess the plasticity of the mixed clones, 2 � 104 cells were cultured inX-VIVO15 with 2.5 �g/ml PHA (Sigma-Aldrich) and irradiated feedercells in the presence of 50 ng/ml human rIL-12 or rIL-23 (both R&DSystems). After 7 days, production of IFN-� and IL-17A was assessedby ICS.

RT-PCR

RNA was isolated from magnetic bead-isolated IL-17A Alexa647� (con-taining surface IL-17A� CD4� cells) and Alexa647� (containing surfaceIL-17� CD4� cells) fractions or from Th17, Th1, and Th1/17 TCC afterovernight PMA/ionomycin stimulation. After reverse transcription, geneexpression was analyzed by real time-PCR (ABI Prism 7900, Applied Bio-systems). The following primer and probe sets were purchased from Ap-plied Biosystems: T-bet (TBX21 Hs00203436_m1), ROR�t (RORC2Hs01076112_m1), GATA-3 (Hs00231122_m1), FOXP3 (Hs01085834_m1),IL-17A (Hs99999082_m1), and IL-23R (Hs00332759_m1), IL-17R(Hs01064648_m1). Primer pairs for the other genes are as follows: TGF-�for 5�-GCCCTGGACACCAACTATTG-3�; TGF-� rev 5�-CTGGTCCAGGCTCCAAAT-3�, and IFN-� for 5�-TTCAGCTCTGCATCGTTTTG-3�;IFN-� rev 5�-CTTTCCAATTCTTCAAAATGCC-3�. Samples were run induplicates, 18S rRNA was used as endogenous control and the relativegene expression was calculated by the ��Ct method using CD4� T cellsas calibrator.

Surface biotinylation and Western blot

Purified CD4� T cells were stimulated overnight with PMA/ionomycin.Cells were then washed with PBS and surface molecules were first biotin-ylated and subsequently isolated using the cell surface protein isolation kit(Pierce) according to the manufacturer’s protocol. Collected fractions, el-uate (biotinylated proteins), and flow-through (intracellular proteins) wereseparated by SDS gel electrophoresis under reducing conditions and blot-ted onto polyvinylidine difuloride membrane. Membranes were incubatedwith polyclonal rabbit anti-IL-17A and anti-IL-17F (both PeproTech),mouse anti-GAPDH (6C5, Abcam) overnight at 4°C, followed by anti-rabbit or anti-mouse HRP-coupled secondary Ab (Sigma-Aldrich), respec-tively. Nonbiotinylated cell lysates and human rIL-17A and rIL-17F (bothPeproTech) were used as controls. Blots were developed using the ECLPlus system (Amersham Biosciences).

Prediction of transmembrane domains

Transmembrane prediction using hidden Markov models (TMHMM) (31)and membrane protein identification without explicit use of hydropathyprofiles and alignments (MINNOU) (32) were used to predict transmem-brane domains. The sequences used were AAH67505 for IL-17A andAAH70124 for IL-17F.

Statistical analysis

Two-tailed unpaired Student’s t test was use to compare surface markerexpression between surface IL-17A� and surface IL-17A� cells. Differ-ences were considered significant if p � 0.05.

ResultsA CD4� T cell subset and Th17 TCC express IL-17A on thecell surface upon stimulation

Although trying to identify human IL-17A-producing CD4� Tcells using a cytokine-capture/detection system, we observed thatthe anti-IL-17A detection Ab was binding to �1% of CD4� Tcells even in the absence of the capture anti-IL-17 Ab, indicatingthat IL-17A was expressed on the membrane of some activatedCD4� T cells. To verify this finding, we stimulated purified CD4�

T cells with PMA/ionomycin and stained the surface for IL-17A.Flow cytometry analysis revealed that IL-17A was displayed at thesurface of a small population of CD4� T cells (0.89% � 0.10, n 11) (Fig. 1A), in all donors tested. Unstimulated cells and cellstreated with brefeldin A were completely devoid of surface IL-17A(data not shown), suggesting that active transport from the endo-plasmic reticulum to the cell surface is required for membraneexpression of IL-17A. After PMA/ionomycin stimulation, the per-centage of cells expressing IL-17A on the surface was maximal at6 h and declined steadily to minimum levels after 48 h of stimu-lation (Fig. 1B), and always correlated with the frequency of IL-17A-producing cells detected by ICS. Surface IL-17A expressionwas also detected on CD4� T cells isolated from the spleen and theCNS of immunized mice 6 h after PMA/ionomycin stimulation(Fig. 1C), but again not in unstimulated cells or in the presence ofbrefeldin A. In the CNS, the proportion of CD4� T cells withintracellular IL-17A was 24.09%, and the frequency of surfaceIL-17A-positive cells was 24.07%, indicating that surface IL-17Afaithfully reflects the presence of IL-17A-producing cells in dif-ferent compartments. Moreover, biotinylation of cell surface pro-teins confirmed the presence of IL-17A on the cell membrane ofPMA/ionomycin-stimulated human CD4� T cells (Fig. 1D).

To assess whether all IL-17A producing CD4� T cells alsoexpress IL-17A on their cell surface, we analyzed surface IL-17A expression on Th1 and Th17 TCC that we had previouslygenerated (V. Brucklacher-Waldert, K. Sturner, M. Kolster, J.Wolthausen, and E. Tolosa; submitted for publication). Wefound that all Th17 TCC displayed high amounts of IL-17A atthe surface, while none of the Th1 TCC did (Fig. 1E). Th1/17

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clones, producing simultaneously IFN-� and IL-17A also ex-pressed surface IL-17A at the cell surface, although the inten-sity of the staining was lower than for bona fide Th17 TCC (datanot shown).

Surface IL-17A-expressing CD4� T cells are Th17 cells

We performed the following experiments to demonstrate that sur-face IL-17A positive CD4� cells are indeed Th17 cells. First, weseparated CD4� T cells on the basis of their surface IL-17A ex-pression using magnetic beads (Fig. 2, A and B), or FACS sorting(Fig. 2C) and analyzed the positive and negative fractions for theexpression of T cell lineage specific transcription factors. SurfaceIL-17A� CD4� T cells expressed ROR�t mRNA, while this tran-scription factor was barely detectable in surface IL-17� CD4� Tcells. T-bet, by contrast, was expressed at similar levels by Th1and Th17 TCC (Fig. 2B), as already described (26). Second, after10 days expansion of the purified subsets, we analyzed the surfaceIL-17A� sorted subset by ICS and still found 35.5% of Th17 cells(Fig. 2C). As expected, 1% of the expanded surface IL-17A�

population contained Th17 cells. Finally, we measured IL-17A inthe supernatants of surface IL-17A� and surface IL-17� popula-tions during PMA/ionomycin expansion. IL-17A could be detectedin the supernatant of the surface IL-17A� fractions for nearly 2 wkafter restimulation, whereas surface IL-17� cells production wasnegligible (Fig. 2D). In summary, the expression of ROR�t and thestable production of IL-17A by expanded surface IL-17A� cellsidentified surface IL-17A� cells as Th17 cells.

Phenotypic characterization of surface IL-17A� CD4� T cells

The expression levels of surface molecules in ex vivo Th17 cellscould be easily analyzed using surface IL-17A as a marker. Westimulated CD4� T cells with PMA/ionomycin and analyzed ex-pression of surface markers 8 h after stimulation, when surfaceIL-17A expression is maximal, and again after 48 h, when cells arefully activated (Fig. 3, supplemental Fig. 1).4 Flow cytometricanalysis showed that surface IL-17A� cells are exclusivelyCD45RO� memory cells, whereas surface IL-17A� cells were inaverage 69.07% � 9.82 CD45RO� memory cells, 30.13% � 9.95CD45RO�CD27� naive T cells and 0.8% � 0.28 CD45RO�

CD27� effector cells (n 4), representing altogether not onlyother polarized cells, but also the naive compartment (Fig. 3A).

Next, we analyzed molecules involved in the recruitment intoinflammatory tissues. We found that surface IL-17A� CD4� Tcells expressed significantly more CCR4, as reported for Th17cells (26, 33, 34), while CCR5 expression was comparable in bothpopulations (Fig. 3B). As expected, CCR6 was expressed signifi-cantly higher on surface IL-17A� cells than on surface IL-17A�

4 The online version of this article contains supplementary material.

FIGURE 1. IL-17A is displayed on the cell surface of a subset of humanand mouse CD4� T cells and in Th17 TCC. A, Purified human CD4� Tcells were stimulated with PMA/ionomycin and stained 6 h later withanti-IL-17A without cell permeabilization (left) or after permeabilization(right). Brefeldin A was added 1 h after stimulation where indicated. Thenumbers in the plots indicate the percentage of CD4� T cells expressingsurface IL-17A (left) and of intracellular IL-17A (right). Data are repre-sentative of eleven experiments. B, Time course of surface IL-17A expres-sion. Purified CD4� T cells were stimulated with PMA/ionomycin for theindicated time points and stained for surface expression of IL-17A. Thegraph shows the mean percentage � SEM of surface IL-17A� cells at eachtime point (n 3). C, Purified mouse CD4� T cells isolated from spleen(top) and from the CNS (bottom) 25 days after MOG35–55 immunizationwere stimulated with PMA/ionomycin and stained and analyzed as in A.Data are representative of three experiments. D, Detection of IL-17A in theintracellular and isolated membrane fraction of membrane-biotinylated andnonbiotinylated PMA/ionomycin-stimulated human CD4� T cell lysate.Human rIL-17A was used as positive control and GAPDH as a loadingcontrol for intracellular proteins. E, Resting Th1 (top) and Th17 (bottom)TCC were stimulated overnight with PMA/ionomycin and stained for sur-face IL-17A (left) or after permeabilization (right). Brefeldin A was addedfor intracellular cytokine detection. Data are representative of eight clones.

FIGURE 2. Surface IL-17A-expressing CD4� T cells are Th17 cells. A,Isolated CD4� T cells were stimulated overnight with PMA/ionomycin,surface stained with anti-IL-17A Alexa647, and subsequently purified us-ing magnetic beads coupled to anti-Alexa647 Abs. The dot plots show thesurface staining for IL-17A in the original fraction before MACS (left),in the negative fraction (middle), and in the positively selected fraction(right). The numbers in the plots indicate the percentage of surface IL-17A� cells in comparison to the isotype control. B, ROR�t and T-betmRNA expression analysis of the surface IL-17A� and surface IL-17A�

fractions immediately after purification (n 3). Data are shown as mean �SEM. C, PMA/ionomycin-stimulated CD4� T cells (left) were sorted ac-cording to their surface IL-17A expression and cultured as bulk in theabsence of polarizing cytokines. After 10 days, surface IL-17A� cells (up-per panel) and surface IL-17A� cells (lower panel) were stained for in-tracellular cytokines. D, Detection of IL-17A in the supernatant of MACS-isolated surface IL-17A� (black line) and surface IL-17A� (gray line) atthe indicated time points after magnetic isolation.

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cells (Fig. 3B) (26, 33, 34). CCR6 expression was highest after 8 hof stimulation and was later down-regulated. Although nearly allof the IL-17A� cells were CCR6�, only 20% of the IL-17A�

population expressed this chemokine receptor. To assess whethersorting of CD4� T cells on the basis of CCR6 expression wouldgenerate exclusively Th17 cells, we sorted CCR6�CD4� andCCR6�CD4� T cells and analyzed both subsets immediately forintracellular production of IFN-� and IL-17A. We found that IL-17A-producing cells were exclusively present in the CCR6� sub-set, whereas IFN-� producing CD4� T cells could be detected inboth CCR6� and CCR6� populations (Fig. 3G), indicating thatCCR6 is not an exclusive marker for Th17 cells. We next analyzed

the differential expression of molecules involved in adhesion toendothelial tissue. CD49d was expressed at much higher levels onsurface IL-17A� cells early after activation, whereas CD6, theligand for activated leukocyte cell adhesion molecule-1, expres-sion was comparable in both subsets (Fig. 3C).

Proliferation and survival of T cells depend on the combinationof signals provided by costimulatory molecules. Although CD28 isconstitutively present in most T cells, CTLA-4 (CD152) is up-regulated relatively late after activation. We found that expressionof CD28 was significantly higher in surface IL-17A� cells com-pared with surface IL-17A� cells both 8 and 48 h after activation(Fig. 3D). Remarkably, the T cell proliferation inhibitor CTLA-4was expressed by nearly 70% of the surface IL-17A� cells alreadyat the early activation time point (supplemental Fig. 1), suggestingconstitutive CTLA-4 expression in Th17 cells. In contrast,CTLA-4 expression in surface IL-17A� cells was expressed atlower levels, and stayed low after 48 h of stimulation. Two furthermembers of the CD28 family, ICOS and PD-1, were expressed atlow levels after 8 h and showed after 48 h stimulation a higherexpression on surface IL-17A� cells (Fig. 3D). The receptors forthe T cell growth factors IL-2 and IL-7 were also compared be-tween surface IL-17A� and surface IL-17A� cells (Fig. 3E).CD25 (IL-2R�-chain) was barely detectable in both cell subsets8 h after stimulation, but was up-regulated after 48 h, and at ahigher extent in the surface IL-17A� compartment. CD122 (IL-2R�-chain) was also up-regulated later after activation, and morenoticeably in the surface IL-17A� cells, whereas CD132 (IL-2R�-chain) was up-regulated in the same cells already 8 h after stim-ulation. CD127, the IL-7R�-chain, was constitutively expressed inall T cells except regulatory cells, and down-regulated upon acti-vation. The levels of CD127 were slightly higher also in Th17 cells(Fig. 3E). Finally, we investigated T cell activation markers. Theearly activation markers CD5 and CD69 were expressed at com-parable levels in both populations, while the T cell-APC contactpromoting CD2 was expressed at significantly higher levels onsurface IL-17A� cells than on surface IL-17A� cells. CD62L (L-selectin), which is down-regulated after activation, was expressedat similarly low levels in both (Fig. 3F). It has been recently pub-lished that Th17 cells express CD161, a C-lectin present on NKcells (35). We checked for coexpression of CD161 and surfaceIL-17A, however, because CD161 is down-regulated upon activa-tion and surface IL-17A only appears after stimulation, it was notpossible to prove directly that both molecules are coexpressed. Wethen sorted CD161 negative and positive cells, stimulated themwith PMA/ionomycin, and proceeded with IL-17A staining. Sur-face IL-17A� cells are mostly contained in the CD161� subset(3.39% in contrast to 0.19% in the CD161- subset), similar to Th17cells defined by cytoplasmic expression of IL-17A. In contrast,Th1 cells are similarly distributed in the CD161� and CD161�

compartments (16.5 and 13%, respectively) (Fig. 3H). Altogether,our results indicate that surface IL-17A� cells show a higher levelof basal activation, and also express higher levels of costimulatoryand adhesion molecules than cells expressing no IL-17A. In addi-tion, we show that surface IL-17A� are contained in the CCR6�

and CD161� fractions of CD4� T cells.

Th1/17 TCC generated on the basis of surface IL-17Aexpression show plasticity to the environment

As mentioned above, we have observed that bona fide Th17 TCCexpress high amounts of IL-17A on their surface whereas Th1/17TCC, producing simultaneously IL-17 and IFN-�, express less sur-face IL-17A and Th1 clones are devoid of surface IL-17A. To testwhether we could generate TCC with different effector phenotypesfrom cells expressing different levels of surface IL-17A, we sorted

FIGURE 3. Phenotypic characterization of surface IL-17A cells. Puri-fied CD4� T cells were stimulated with PMA/ionomycin and stained 8 and48 h later with anti-IL-17A-Alexa647 together with other surface markers,as indicated in each graph. A, Percentage of naive (CD45RO� CD27�),effector (CD45RO�CD27�) and memory cells (CD45RO�CD27�/�) cellsin the surface IL-17A� (left) and surface IL-17A� (right) subsets eighthours after PMA/ionomycin. Data are representative of four experiments.Expression of chemokine receptors CCR4, CCR5, and CCR6 (B); adhesionmolecules CD49d and CD6 (C); costimulatory molecules CD28, CTLA-4,ICOS, PD-1 (D); IL-2R and IL-7R subunits (E); and activation markers (F)in surface IL-17A� cells (f) and surface IL-17A� cells (u). Data aredisplayed as the average MFI or percentage of positive cells as indicated(n 4). G, sorted CD4�CCR6� and CD4�CCR6� T cells were indepen-dently stimulated with PMA/ionomycin and stained intracellularly forIFN-� and IL-17A. H, sorted CD4�CD161� and CD4�CD161� cells wereindependently stimulated with PMA/ionomycin and stained for surface IL-17A (left) and intracellularly for IFN-� and IL-17A (right).

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surface IL-17A�, surface IL-17Aint, and surface IL-17A� cellsonto 96-well plates at one cell per well (Fig. 4A). After restimu-lation, the cytokine profile and clonality of these cells lines wereanalyzed. Intracellular cytokine staining showed that surface IL-17A� cells gave rise exclusively to Th1 cell lines (n 13), whilesurface IL-17Ahigh resulted in 13 Th17 cell lines and three T celllines producing both IL-17A and IFN-�. In contrast, cells express-

ing intermediate levels of IL-17A generated 15 Th1/17 double-producing cell lines and three Th1 cell lines. We assessed the TCRV� from eight selected Th1/17 cell lines and, after confirming theirclonality, used them for further studies. As shown in Fig. 4A, someof these Th1/17 TCC contained individual cells producing eitherIFN-� or IL-17A or both cytokines simultaneously (TCC 051 andTCC 049), while some clones gave rise to cells producing onlyIFN-� and cells producing both cytokines (TCC 045) and finallysome TCC had individual cells producing only IL-17 and cellsproducing both cytokines (TCC068). To determine whether theheterogeneous cytokine profile of Th1/17 clones cells was mir-rored at transcriptional level, we performed RT-PCR analysis ofthe relevant transcription factors and cytokines (Fig. 4B). As ex-pected, bona fide Th17 clones expressed ROR�t and IL-17AmRNA, while Th1 TCC were negative for both of them and ex-pressed instead IFN-�. T-bet was equally expressed in Th1 andTh17 TCC, as we had already seen in ex vivo Th17 cells. Thereceptor for IL-23, a cytokine involved in the maintenance of Th17cells, was undetectable in Th1 TCC, but clearly present in Th17TCC. Remarkably, Th1/17 clones expressed the highest levels ofROR�t, IL-17A and IL-23R, but also of IFN-�, T-bet, and FOXP3.The Th2 cell specific transcription factor GATA-3 was equallyexpressed at low levels in all TCC. By following up the sortedTCC over several rounds of restimulation, we observed that, in theabsence of cytokines, the phenotype of Th1 and Th17 TCC re-mained stable, while Th1/17 clones changed their cytokine profiletoward less IL-17A and more IFN-� production, suggesting thatthey are in a transitional phase rather than being a stable phenotype(Fig. 4C). We addressed their capacity to respond to environmentby culturing these cells in the presence IL-23 or IL-12, cytokinesthat promote or stabilize the development of Th17 and Th1 cells,respectively. As shown in Fig. 4D, IL-23 moderately increased thepercentage of cells producing IL-17A, whereas the Th1 cell-pro-moting IL-12 markedly decreased the expression of IL-17A andincreased that of IFN-�. IFN-� production by Th1 TCC was notdrastically affected by either the addition of IL-23 or IL-12, whileaddition of IL-12 to the Th17 TCC resulted in the decrease of cellsproducing solely IL-17A and a corresponding increase in the per-centage of double producers. Thus, Th1/17 and Th17 clones re-spond to changes in their cytokine environment.

Surface IL-17A on CD4� T cells is not presented by IL-17R

Cytokines can be displayed on the cell surface either bound to areceptor or directly anchored at the cell membrane by means of atransmembrane domain. First, we looked for expression of the IL-17R that could present IL-17A on the surface of CD4� T cells.Flow cytometry analysis revealed that all monocytes expressedIL-17R, while only a minority of unstimulated (Fig. 5A) and PMA/ionomycin-stimulated (data not shown) human CD4� T cells ex-pressed IL-17R on their membrane. When stimulated cells weredouble stained for surface IL-17A and IL-17R, we found no co-expression of the two molecules (Fig. 5B). In addition, blocking ofthe IL-17R by anti-IL-17R Abs did not prevent surface IL-17Aexpression (data not shown). PCR analysis of TCC showed simi-larly low expression of IL-17R in both Th17 and Th1 cells (Fig.5C). Moreover, surface IL-17A expression could also be detectedin CD4� T cells of the IL-17R-deficient mice (Fig. 5D). Theseresults suggest IL-17A is not bound to the IL-17R. Next, we pre-dicted potential transmembrane domains using the TMHMM andMINNOU programs and revealed that IL-17A generally lacks atransmembrane domain and appears to be a soluble protein (Fig.5E). Given that IL-17A is functional in vivo as a heterodimer of

FIGURE 4. Generation and plasticity of Th1/17 double producer T cellclones. A, CD4� T cells were sorted in surface IL-17A� (1), surface IL-17Aint (2), and surface IL-17Ahigh (3) cells according to the gates shown inthe left panel to generate TCC. After one round of restimulation, cells werestained for intracellular IFN-� and IL-17A. The figure shows overlays of13 TCC originating from surface IL-17A� cells (1), 15 TCC obtained fromsurface IL-17Aint cells (2), and 16 TCC generated from surface IL-17Ahigh

(3) CD4� T cells. The lower panels show four individual examples ofTh1/17 double producer clones obtained from surface IL-17Aint sortedcells. B, mRNA analysis of indicated genes of Th1 (n 3, u), Th17 (n 3, f), and Th1/17 (n 4, �) TCC generated in A. Data are shown asmean � SEM. C, Cytokine profile of two double producer Th1/17 clones(TCC121 and TCC062) after consecutive restimulations in the absence ofpolarizing cytokines. D, Cytokine profiles of two double producers Th1/17TCC (TCC157 and TCC099), one Th1 (TCC011), and one Th17 (TCC323)after three restimulations in the presence of IL-12 or IL-23.

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IL-17A and IL-17F (7, 36), we tested the presence of a transmem-brane domain in the IL-17F sequence. Interestingly, IL-17F con-tains a sequence compatible with a classical transmembrane do-main, as predicted by TMHMM and MINNOU (Fig. 5E). Analysisof PMA/ionomycin-stimulated Th17 TCC showed that Th17 cellsexpress IL-17F in addition to IL-17A (Fig. 5, F and G). Moredefinitively, we found IL-17F expressed on the surface of a subsetof CD4� T cells (Fig. 5H). Simultaneous surface staining of IL-17A and IL-17F did show very dim staining of both moleculeswhen compared with the individual stainings, and coexpressionwas not detectable (data not shown), possibly because of stericalhindrance of anti-IL-17A and anti-IL-17F Abs due to a spatialassociation of both cytokines. To overcome this problem, wesorted CD4� T cells into surface IL-17A� and IL-17F� cells.After 10 days to allow cells to rest, both sorted populations werestimulated again with PMA/ionomycin and subsequently stainedwith the two Abs (single staining for the surface, double stainingfor the cytoplasm, Fig. 5H). Sorted IL-17A� cells were clearlypositive for both IL-17A, and at a lesser extent, for IL-17F, whileIL-17A� cells were negative for both IL-17 molecules, both in thesurface and in the cytoplasm. The reciprocal experiment showedsimilar results, although the levels of expression of the IL-17A andIL-17F molecules were lower. Intracellular staining of both sortedIL-17A� and IL-17F� populations showed that the majority ofcells coexpress intracellularly both cytokines. Remarkably, insome cells, only IL-17A could be detected intracellularly, possiblybecause anti-IL-17A and anti-IL-17F Abs compete on alreadydimerized IL-17A/IL-17F complexes. In sorted IL-17� cells nei-ther IL-17A nor IL-17F were detected on the surface or intracel-lularly. Even though we cannot discard that IL-17A is bound to ayet unknown IL-17R or is in association with other members of theIL-17 family, our data support the hypothesis that IL-17A is tran-siently displayed at the cell surface forming a heterodimer withmembrane-bound IL-17F.

DiscussionWe report in this study that both IL-17A and IL-17F are displayedon the cell surface of a subset of activated CD4� T cells. Theexpression of surface IL-17A correlates with intracellular IL-17Aexpression in human and murine CD4� T cells in different com-partments, however, there is always some degree of discrepancybetween the percentage of positive cells in the surface and theintracellular staining. Indeed, the MFI of the surface staining isalways lower than intracellular, probably indicating that there areless IL-17A molecules present at the cell surface than inside thecell. In the first case, IL-17A is only transiently expressed on itsway to secretion, while the amount of intracellular IL-17A is ar-tificially enhanced by brefeldin A, which blocks the transport tothe cell surface. The lower MFI results in less resolution betweenpositive and negative cells, and thus in differences in the percent-ages. IL-10 and IFN-� have also been described on the surface ofactivated T cells in the absence of a specific receptor (37, 38).However, in both cases a sensitivity-enhancing staining methodwas required for detection, probably due to a very low expression,while surface IL-17A can be detected by conventional flow cy-tometry using commercially available fluorochrome-labeled anti-IL-17A Abs. Our data show that the surface IL-17A� cells arebona fide Th17 cells, and thus surface IL-17A can be used as aspecific surface marker for this Th subset. Moreover, we have usedsurface IL-17A for the isolation of ex vivo Th17 cells by magnetic-and fluorescence-activated cell sorting. A method for ex vivo iso-lation of human Th17 cells using a bispecific Ab against CD45 andIL-17A for capturing IL-17-secreting cells has been recently re-ported (39). However, this method requires previous preparation of

FIGURE 5. IL-17A is not bound to the specific receptor IL-17R at thecell surface. A, Surface staining of PBMC of IL-17R and CD14� cells(upper panel) or CD4� T cells (lower panel). B, Surface staining ofIL-17A and IL-17R in human purified CD4� T cells. C, mRNA analysisfor IL-17R of two Th1 (u) and two Th17 (f) TCC. As calibratormRNA of LPS-stimulated monocyte-derived dendritic cells was used.Data are shown as mean � SEM. D, Purified mouse CD4� T cellsisolated from the spleen of IL-17R-deficient mice 12 days afterMOG35–55 immunization were stimulated with PMA/ionomycin andstained as in Fig. 1A. E, Alignment with ClustaW2 and transmembranedomain prediction of human IL-17A (AAH67505) and IL-17F(AAH70124) using the MINNOU server. Black frame indicates the pu-tative transmembrane domain in IL-17F. �, Identical nucleotides; :,Conserved substitutions. F, mRNA analysis for ROR�t, IL-17A andIL-17F of two Th1 (u) and two Th17 (f) TCC. As calibrator mRNA ofPMA/ionomycin-stimulated CD4� T cells was used. Data are shown asmean � SEM. G, Western blot analysis for IL-17A (upper panel) andIL-17F (lower panel) of overnight stimulated PMA/ionomycin CD4� Tcells. Human recombinant cytokines were used as positive controls. H,CD4� T cells were sorted on the basis of surface IL-17A and IL-17Fexpression, allowed to rest for 10 days and then stimulated with PMA/ionomycin for 6 h and stained for surface IL-17A and IL-17F indepen-dently (left and middle, respectively) or together (right) for intracellulardetection. The histograms show the levels of staining of the indicated surfacecytokine (f) in comparison to the isotype control (u) staining. The dot plotshows the intracellular staining for both cytokines for each population.

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reagents, needs long handling times, and has the risk of unspecificbystander detection.

Using surface IL-17A expression as a marker for Th17 cells, wehave analyzed the ex vivo phenotype of this subset in human pe-ripheral blood in comparison to IL-17A-negative cells. The higherexpression and up-regulation of CD2, costimulatory molecules,activation markers, and of CD49d on surface IL-17A� indicatesthat Th17 cells are better equipped for recruitment into inflamedtissues, activation, and proliferation. Higher expression of CCR6,CD49d, CD28, CTLA-4, CD25, and CD2 was also observed in Th17TCC when compared with Th1 TCC generated from peripheral bloodand cerebrospinal fluid of a patient with multiple sclerosis (V.Brucklacher-Waldert, K. Sturner, M. Kolster, J. Wolthausen, and E.Tolosa; submitted for publication), supporting the view of Th17 cellsas a potent effector T cells subset.

Our analysis confirmed the higher expression of CCR6 and thecoexpression of CD161 by Th17 cells already described by others(26, 33–35). CCR6 and CD161, alone and in combination, arecurrently used as markers for the enrichment of Th17 cells, how-ever, both CD161� and CCR6� and even the double-positive cellscan give rise to Th1 cells in addition to IL-17A-producing cells. Incontrast, cells selected on the basis of high surface IL-17A gen-erated only Th17 or Th1/17 clones, but never Th1 cells. Veryinterestingly, the levels of surface expression of IL-17A seem toindicate the commitment to the Th phenotype: surface IL-17A�

cells gave rise exclusively to Th1 clones; surface IL-17Ahigh cellsproduced mostly Th17 TCC and a few Th1/17 TCC. In contrast,sorting on the basis of surface IL-17Aint cells produced mostlyTCC expressing both cytokines. When we analyzed the TCC fortranscription factor expression, we found that ROR�t was highlyexpressed in Th17 TCC and nearly absent from the Th1 TCC.T-bet, by contrast, was found at similar levels in Th1 and Th17clones, indicating that T-bet is not exclusive of Th1 cells, in agree-ment with data showing that T-bet can directly regulate transcrip-tion of the IL-23R, and therefore influence the fate of Th17 cells(40). Interestingly, Th1/17 double producer TCC expressed higherlevels of ROR�t, IL17A, and IL23R, and also T-bet and IFN-�mRNA than bona fide Th17 or Th1 cells, respectively. FoxP3 wasagain highest in this population, whereas GATA-3 showed similarlow levels in all subsets. The high expression of Th1- and Th17-specific transcription factors suggested the capability of Th1/17cells to differentiate into one of these T cell lineages. We charac-terized these clones assuming that they would show some plastic-ity in response to the provided cytokine milieu, and indeed wefound that the presence of IL-12 drove cells to more IFN-� pro-duction, as it has already been described (26), whereas IL-23would preserve and increase IL-17A expression. These resultsdemonstrate a certain degree of plasticity of human Th1/17 andalso Th17 clones in response to the cytokine milieu.

The mechanism of cytokine surface expression is not clear, be-cause IL-17A does not harbor a classical transmembrane domain.The presence of IL-17A at the cell surface was only detectable afew hours after stimulation, and was blocked in the presence ofbrefeldin A, suggesting that the cytokine is actively transported tothe cell membrane, where it stays transiently. Our data excludedthe presentation of IL-17A on the surface by the known specificreceptors, and the specificity of surface IL-17A expression by IL-17A-producing cells makes it unlikely that IL-17A binds nonspe-cifically to sugar moieties or other molecules on the surface of thecells. IL-17F has a transmembrane domain, is expressed by acti-vated CD4� T cells (our own data and Ref. 41), and it is presentat the cell surface of cells previously sorted for high IL-17A ex-pression. Thus, we suggest that IL-17A is displayed at the cellmembrane forming a complex together with IL-17F, as it has been

described for lymphotoxin �, which does not contain a transmem-brane domain and it appears at the cell surface anchored via lym-photoxin � (42). A heterodimeric form of IL-17 comprising IL-17A and IL-17F and its biological function has already beendescribed (7). The biological activity of this IL-17A surface formis still unknown, but it may have distinct functions, for instance itmight serve as a ligand for IL-17R expressed on APCs to prolongcytokine signaling in T/APC clusters (43), or on endothelial cells(44). This may provide a cell contact-dependent signal for trans-migration of surface IL-17A positive cells through vascular endo-thelial cells expressing IL-17 receptor (44).

Our data show that surface IL-17A constitutes a unique markerfor the unambiguous identification and isolation of viable Th17cell. By analyzing phenotypically surface IL-17A� CD4� T cells,we show that human ex vivo Th17 cells express high levels ofcostimulatory and adhesion molecules as well as activation mark-ers, indicating the potential of Th17 cells as potent effector T cells.In addition, we could consistently generate Th1/17 T cell clonesderived from intermediate surface IL-17A-expressing cells, andthese clones adapted their phenotype to the cytokine milieu, pro-viding evidence for the plasticity of human Th1/17 cells.

AcknowledgmentsWe thank all blood donors for their cooperation, Roland Martin for thesupport given to this project, Christoph Ogrodowczyk for help with thebiotinylation experiments, and F. Koch-Nolte, F. Haag, and C. Stoeckle forsuggestions and critical reading of the manuscript.

DisclosuresThe authors have no financial conflict of interest.

References1. Fossiez, F., O. Djossou, P. Chomarat, L. Flores-Romo, S. Ait-Yahia, C. Maat,

J. J. Pin, P. Garrone, E. Garcia, S. Saeland, et al. 1996. T cell interleukin-17induces stromal cells to produce proinflammatory and hematopoietic cytokines.J. Exp. Med. 183: 2593–2603.

2. Laan, M., Z. H. Cui, H. Hoshino, J. Lotvall, M. Sjostrand, D. C. Gruenert,B. E. Skoogh, and A. Linden. 1999. Neutrophil recruitment by human IL-17 viaC-X-C chemokine release in the airways. J. Immunol. 162: 2347–2352.

3. Peric, M., S. Koglin, S. M. Kim, S. Morizane, R. Besch, J. C. Prinz, T. Ruzicka,R. L. Gallo, and J. Schauber. 2008. IL-17A enhances vitamin D3-induced ex-pression of cathelicidin antimicrobial peptide in human keratinocytes. J. Immu-nol. 181: 8504–8512.

4. Li, H., J. Chen, A. Huang, J. Stinson, S. Heldens, J. Foster, P. Dowd,A. L. Gurney, and W. I. Wood. 2000. Cloning and characterization of IL-17B andIL-17C, two new members of the IL-17 cytokine family. Proc. Natl. Acad. Sci.USA 97: 773–778.

5. Lee, J., W. H. Ho, M. Maruoka, R. T. Corpuz, D. T. Baldwin, J. S. Foster,A. D. Goddard, D. G. Yansura, R. L. Vandlen, W. I. Wood, and A. L. Gurney.2001. IL-17E, a novel proinflammatory ligand for the IL-17 receptor homologIL-17Rh1. J. Biol. Chem. 276: 1660–1664.

6. Shi, Y., S. J. Ullrich, J. Zhang, K. Connolly, K. J. Grzegorzewski, M. C. Barber,W. Wang, K. Wathen, V. Hodge, C. L. Fisher, et al. 2000. A novel cytokinereceptor-ligand pair: identification, molecular characterization, and in vivo im-munomodulatory activity. J. Biol. Chem. 275: 19167–19176.

7. Wright, J. F., Y. Guo, A. Quazi, D. P. Luxenberg, F. Bennett, J. F. Ross, Y. Qiu,M. J. Whitters, K. N. Tomkinson, K. Dunussi-Joannopoulos, et al. 2007. Iden-tification of an interleukin 17F/17A heterodimer in activated human CD4� Tcells. J. Biol. Chem. 282: 13447–13455.

8. Lockhart, E., A. M. Green, and J. L. Flynn. 2006. IL-17 production is dominatedby �� T cells rather than CD4 T cells during Mycobacterium tuberculosis infec-tion. J. Immunol. 177: 4662–4669.

9. Rachitskaya, A. V., A. M. Hansen, R. Horai, Z. Li, R. Villasmil, D. Luger,R. B. Nussenblatt, and R. R. Caspi. 2008. Cutting edge: NKT cells constitutivelyexpress IL-23 receptor and ROR�t and rapidly produce IL-17 upon receptorligation in an IL-6-independent fashion. J. Immunol. 180: 5167–5171.

10. Cupedo, T., N. K. Crellin, N. Papazian, E. J. Rombouts, K. Weijer, J. L. Grogan,W. E. Fibbe, J. J. Cornelissen, and H. Spits. 2009. Human fetal lymphoid tissue-inducer cells are interleukin 17-producing precursors to RORC� CD127� naturalkiller-like cells. Nat. Immunol. 10: 66–74.

11. Liu, S. J., J. P. Tsai, C. R. Shen, Y. P. Sher, C. L. Hsieh, Y. C. Yeh, A. H. Chou,S. R. Chang, K. N. Hsiao, F. W. Yu, and H. W. Chen. 2007. Induction of adistinct CD8 Tnc17 subset by transforming growth factor-� and interleukin-6.J. Leukocyte Biol. 82: 354–360.

5500 Th17 CELLS IDENTIFIED BY SURFACE IL-17A

by guest on February 1, 2019http://w

ww

.jimm

unol.org/D

ownloaded from

12. Ferretti, S., O. Bonneau, G. R. Dubois, C. E. Jones, and A. Trifilieff. 2003. IL-17,produced by lymphocytes and neutrophils, is necessary for lipopolysaccharide-induced airway neutrophilia: IL-15 as a possible trigger. J. Immunol. 170:2106–2112.

13. Molet, S., Q. Hamid, F. Davoine, E. Nutku, R. Taha, N. Page, R. Olivenstein,J. Elias, and J. Chakir. 2001. IL-17 is increased in asthmatic airways and induceshuman bronchial fibroblasts to produce cytokines. J. Allergy Clin. Immunol. 108:430–438.

14. Harrington, L. E., R. D. Hatton, P. R. Mangan, H. Turner, T. L. Murphy,K. M. Murphy, and C. T. Weaver. 2005. Interleukin 17-producing CD4� effectorT cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat.Immunol. 6: 1123–1132.

15. Mangan, P. R., L. E. Harrington, D. B. O’Quinn, W. S. Helms, D. C. Bullard,C. O. Elson, R. D. Hatton, S. M. Wahl, T. R. Schoeb, and C. T. Weaver. 2006.Transforming growth factor-� induces development of the TH17 lineage. Nature441: 231–234.

16. Veldhoen, M., R. J. Hocking, C. J. Atkins, R. M. Locksley, and B. Stockinger.2006. TGF� in the context of an inflammatory cytokine milieu supports de novodifferentiation of IL-17-producing T cells. Immunity 24: 179–189.

17. Yang, J., E. A. James, L. Huston, N. A. Danke, A. W. Liu, and W. W. Kwok.2006. Multiplex mapping of CD4 T cell epitopes using class II tetramers. Clin.Immunol. 120: 21–32.

18. Wilson, N. J., K. Boniface, J. R. Chan, B. S. McKenzie, W. M. Blumenschein,J. D. Mattson, B. Basham, K. Smith, T. Chen, F. Morel, et al. 2007. Development,cytokine profile and function of human interleukin 17-producing helper T cells.Nat. Immunol. 8: 950–957.

19. Ivanov, I. I., B. S. McKenzie, L. Zhou, C. E. Tadokoro, A. Lepelley, J. J. Lafaille,D. J. Cua, and D. R. Littman. 2006. The orphan nuclear receptor ROR�t directsthe differentiation program of proinflammatory IL-17� T helper cells. Cell 126:1121–1133.

20. Szabo, S. J., S. T. Kim, G. L. Costa, X. Zhang, C. G. Fathman, andL. H. Glimcher. 2000. A novel transcription factor, T-bet, directs Th1 lineagecommitment. Cell 100: 655–669.

21. Zheng, W., and R. A. Flavell. 1997. The transcription factor GATA-3 is neces-sary and sufficient for Th2 cytokine gene expression in CD4 T cells. Cell 89:587–596.

22. Infante-Duarte, C., H. F. Horton, M. C. Byrne, and T. Kamradt. 2000. Microbiallipopeptides induce the production of IL-17 in Th cells. J. Immunol. 165:6107–6115.

23. Rudner, X. L., K. I. Happel, E. A. Young, and J. E. Shellito. 2007. Interleukin-23(IL-23)-IL-17 cytokine axis in murine Pneumocystis carinii infection. Infect. Im-mun. 75: 3055–3061.

24. Kebir, H., K. Kreymborg, I. Ifergan, A. Dodelet-Devillers, R. Cayrol,M. Bernard, F. Giuliani, N. Arbour, B. Becher, and A. Prat. 2007. Human TH17lymphocytes promote blood-brain barrier disruption and central nervous systeminflammation. Nat. Med. 13: 1173–1175.

25. Tzartos, J. S., M. A. Friese, M. J. Craner, J. Palace, J. Newcombe, M. M. Esiri,and L. Fugger. 2008. Interleukin-17 production in central nervous system-infil-trating T cells and glial cells is associated with active disease in multiple scle-rosis. Am. J. Pathol. 172: 146–155.

26. Annunziato, F., L. Cosmi, V. Santarlasci, L. Maggi, F. Liotta, B. Mazzinghi,E. Parente, L. Fili, S. Ferri, F. Frosali, et al. 2007. Phenotypic and functionalfeatures of human Th17 cells. J. Exp. Med. 204: 1849–1861.

27. Sospedra, M., P. A. Muraro, I. Stefanova, Y. Zhao, K. Chung, Y. Li,M. Giulianotti, R. Simon, R. Mariuzza, C. Pinilla, and R. Martin. 2006. Redun-dancy in antigen-presenting function of the HLA-DR and -DQ molecules in themultiple sclerosis-associated HLA-DR2 haplotype. J. Immunol. 176: 1951–1961.

28. Muraro, P. A., M. Jacobsen, A. Necker, J. W. Nagle, R. Gaber, N. Sommer,W. H. Oertel, R. Martin, and B. Hemmer. 2000. Rapid identification of local T

cell expansion in inflammatory organ diseases by flow cytometric T cell receptorV� analysis. J. Immunol. Methods 246: 131–143.

29. Ye, P., F. H. Rodriguez, S. Kanaly, K. L. Stocking, J. Schurr,P. Schwarzenberger, P. Oliver, W. Huang, P. Zhang, J. Zhang, et al. 2001. Re-quirement of interleukin 17 receptor signaling for lung CXC chemokine andgranulocyte colony-stimulating factor expression, neutrophil recruitment, andhost defense. J. Exp. Med. 194: 519–527.

30. Gelderblom, M., F. Leypoldt, K. Steinbach, D. Behrens, C. U. Choe, D. A. Siler,T. V. Arumugam, E. Orthey, C. Gerloff, E. Tolosa, and T. Magnus. 2009. Tem-poral and spatial dynamics of cerebral immune cell accumulation in stroke.Stroke 40: 1849–1857.

31. Sonnhammer, E. L., G. von Heijne, and A. Krogh. 1998. A hidden Markov modelfor predicting transmembrane helices in protein sequences. Proceedings of theInternational Conference on Intelligent Systems for Molecular Biology; ISMB 6:175–182.

32. Cao, B., A. Porollo, R. Adamczak, M. Jarrell, and J. Meller. 2006. Enhancedrecognition of protein transmembrane domains with prediction-based structuralprofiles. Bioinformatics 22: 303–309.

33. Acosta-Rodriguez, E. V., L. Rivino, J. Geginat, D. Jarrossay, M. Gattorno,A. Lanzavecchia, F. Sallusto, and G. Napolitani. 2007. Surface phenotype andantigenic specificity of human interleukin 17-producing T helper memory cells.Nat. Immunol. 8: 639–646.

34. Singh, S. P., H. H. Zhang, J. F. Foley, M. N. Hedrick, and J. M. Farber. 2008.Human T cells that are able to produce IL-17 express the chemokine receptorCCR6. J. Immunol. 180: 214–221.

35. Cosmi, L., R. De Palma, V. Santarlasci, L. Maggi, M. Capone, F. Frosali,G. Rodolico, V. Querci, G. Abbate, R. Angeli, et al. 2008. Human interleukin17-producing cells originate from a CD161�CD4� T cell precursor. J. Exp. Med.205: 1903–1916.

36. Liang, S. C., A. J. Long, F. Bennett, M. J. Whitters, R. Karim, M. Collins,S. J. Goldman, K. Dunussi-Joannopoulos, C. M. Williams, J. F. Wright, andL. A. Fouser. 2007. An IL-17F/A heterodimer protein is produced by mouse Th17cells and induces airway neutrophil recruitment. J. Immunol. 179: 7791–7799.

37. Scheffold, A., M. Assenmacher, L. Reiners-Schramm, R. Lauster, andA. Radbruch. 2000. High-sensitivity immunofluorescence for detection of thepro- and anti-inflammatory cytokines � interferon and interleukin-10 on the sur-face of cytokine-secreting cells. Nat. Med. 6: 107–110.

38. Assenmacher, M., A. Scheffold, J. Schmitz, J. A. Segura Checa, S. Miltenyi, andA. Radbruch. 1996. Specific expression of surface interferon-� on interferon-�producing T cells from mouse and man. Eur. J. Immunol. 26: 263–267.

39. Streeck, H., K. W. Cohen, J. S. Jolin, M. A. Brockman, A. Meier, K. A. Power,M. T. Waring, G. Alter, and M. Altfeld. 2008. Rapid ex vivo isolation and long-term culture of human Th17 cells. J. Immunol. Methods 333: 115–125.

40. Gocke, A. R., P. D. Cravens, L. H. Ben, R. Z. Hussain, S. C. Northrop,M. K. Racke, and A. E. Lovett-Racke. 2007. T-bet regulates the fate of Th1 andTh17 lymphocytes in autoimmunity. J. Immunol. 178: 1341–1348.

41. Hymowitz, S. G., E. H. Filvaroff, J. P. Yin, J. Lee, L. Cai, P. Risser, M. Maruoka,W. Mao, J. Foster, R. F. Kelley, et al. 2001. IL-17s adopt a cystine knot fold:structure and activity of a novel cytokine, IL-17F, and implications for receptorbinding. EMBO J. 20: 5332–5341.

42. Browning, J. L., A. Ngam-ek, P. Lawton, J. DeMarinis, R. Tizard, E. P. Chow,C. Hession, B. O’Brine-Greco, S. F. Foley, and C. F. Ware. 1993. Lymphotoxin�, a novel member of the TNF family that forms a heteromeric complex withlymphotoxin on the cell surface. Cell 72: 847–856.

43. Yao, Z., S. L. Painter, W. C. Fanslow, D. Ulrich, B. M. Macduff, M. K. Spriggs,and R. J. Armitage. 1995. Human IL-17: a novel cytokine derived from T cells.J. Immunol. 155: 5483–5486.

44. Moseley, T. A., D. R. Haudenschild, L. Rose, and A. H. Reddi. 2003. Interleu-kin-17 family and IL-17 receptors. Cytokine Growth Factor Rev. 14: 155–174.

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