Surface IL-17A Expression Th17 Cells Unambiguously Identified by Phenotypical Characterization of Human

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Phenotypical Characterization of Human Th17 Cells Unambiguously Identified by Surface IL-17A Expression

This information is current as Verena Brucklacher-Waldert, Karin Steinbach, Michael of September 24, 2021. Lioznov, Manuela Kolster, Christoph Hölscher and Eva Tolosa J Immunol 2009; 183:5494-5501; ; doi: 10.4049/jimmunol.0901000

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

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

Verena Brucklacher-Waldert,*†‡ Karin Steinbach,* Michael Lioznov,§ Manuela Kolster,* Christoph Ho¨lscher,¶ʈ and Eva Tolosa2*†

Th17 cells are involved in the defense against bacteria and fungi and play a prominent role in the pathogenesis of autoimmune diseases, but research on human Th17 cells is hindered due to the lack of a surface marker. In this study, we report that a subset .of 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 identified surface IL-17A as a specific marker for human and mouse Th17 cells. Phenotype characterization of ex vivo CD4؉ IL-17A؉ cells showed that the chemokines CCR6 and CCR4, costimulatory molecules, as well as CD2 and CD49d were more prominently ؊ expressed on these cells than in surface IL-17A cells, supporting the concept of Th17 cells as a potent inflammatory effector Downloaded from subtype. In addition, we generated human Th1, Th1/17 (producing both IFN-␥ and IL-17A), and Th17 T cell clones based on single cell sorting of surface IL-17A؊, IL-17Aint, and IL-17Ahigh CD4؉ T cells, respectively, and showed the plasticity of the double producing clones to the cytokine milieu. The identification of surface IL-17A as a marker for Th17 cells should facilitate research on this subset. The Journal of Immunology, 2009, 183: 5494–5501.

L-17 is a potent inducer of inflammation by triggering the 17A and IFN-␥, suggests that commitment to a specific effector http://www.jimmunol.org/ release of cytokines and chemokines from a broad range of type might be more flexible than previously assumed. I stromal cells (1). Through its regulation of proinflammatory Th17 cells have been linked to the pathogenesis of several inflam- gene expression, IL-17A has the capability to recruit neutrophils matory (22, 23) and autoimmune diseases (24–26) and therefore they (2) and to promote antimicrobial protein synthesis (3). Until now, are under intense investigation. Although reporter lines allow tracking six structurally related isoforms for IL-17 (IL-17A, B, C, D, E, and of Th17 cells in mice, human Th17 cells are currently identified ex F) have been identified and grouped into the IL-17 cytokine family vivo by intracellular IL-17 expression, which does not allow further (4–6). Among them, IL-17F has the highest homology with IL- functional analysis. Alternatively, Th17 cells can be differentiated in 17A. Both IL-17A and IL-17F are active as homodimers and can vitro from naive CD4ϩ T cells using combinations of cytokines, but form biologically functional IL-17A/IL-17F heterodimers (7). IL- culture conditions may change their properties. by guest on September 24, 2021 17A and IL-17F are produced by CD4ϩ T cells, ␥␦ T cells, NKT We report in this study the unambiguous identification of Th17 cells, NK cells, CD8 cells, neutrophils, and eosinophils (1, 8–13). cells by the expression of surface IL-17A. Using surface IL-17A as IL-17A-producing CD4ϩ T cells, defined as Th17 cells, have been a marker, we could phenotypically characterize ex vivo human recently identified as a distinct Th cell lineage (14), which differ- Th17 cells and generate Th17 and Th1/17 T cell clones (TCC).3 entiates upon stimulation from naive CD4ϩ T cells in presence of Furthermore, we show for the first time in human cells the plas- TGF-␤ and IL-6 (15–17). Maintenance of Th17 cells requires in ticity of the Th1/17 cells in response to the cytokine milieu. addition IL-23 (18), and they express the lineage-specific transcription factor ROR␥t (19). In contrast, Th1 cell differentiation Materials and Methods requires IL-12 and the expression of the transcription factor T-bet Cells (20), while Th2 cells are dependent on IL-4 and the expression of Peripheral blood was taken from healthy donors after informed consent GATA-3 (21). The existence of Th1/17 cells, producing both IL- under a protocol approved by the local ethics committee. CD4ϩ cells were isolated (Ͼ95% purity) from PBMC by negative selection using the CD4ϩ T Cell Isolation Kit II (Miltenyi Biotec) according to manufacturer’s in- *Institute for Neuroimmunology and Clinical Multiple Sclerosis Research, Center for structions. Th1 and Th17 TCC were generated from PBMC by limiting Molecular Neurobiology Hamburg and †Department of Immunology, University ‡ dilution (27). Clonality was assessed by TCR V␤-chain staining (28). For Medical Center Hamburg-Eppendorf, Hamburg, Germany; Graduate School of Cel- ϩ ϩ lular and Molecular Neuroscience, University of Tu¨bingen, Germany; §Interdiscipli- the isolation of surface IL-17A cells, CD4 T cells were stimulated with nary Clinic for Stem Cell Transplantation, University Medical Center Hamburg-Ep- 50 ng/ml PMA and 1 ␮g/ml ionomycin for the indicated time period, pendorf, Hamburg, Germany; ¶Division of Infection Immunology, Research Center stained with anti-IL-17A Alexa647 (eBio64DEC17, eBioscience), and ʈ Borstel, Borstel, Germany; and Cluster of Excellence Inflammation at Interfaces, FACS sorted or isolated using anti-Cy5/Alexa Fluor 647 Microbeads Borstel-Kiel-Lu¨beck-Plo¨n, Germany (Miltenyi Biotec). To induce a Th17 response, C57BL/6 wild-type or IL- Received for publication March 27, 2009. Accepted for publication August 21, 2009. 17R-deficient mice (29) (provided by Amgen) were immunized with 200 ␮g MOG (NeoMPS) in CFA supplemented with 4 mg/ml Mycobac- The costs of publication of this article were defrayed in part by the payment of page 35–55 charges. This article must therefore be hereby marked advertisement in accordance terium tuberculosis H37RA (Difco Laboratories), followed by injection of with 18 U.S.C. Section 1734 solely to indicate this fact. 300 ng pertussis toxin (Sigma-Aldrich) at the time point of immunization 1 The Institute for Neuroimmunology and Clinical Multiple Sclerosis Research and this work are supported by the Hertie Foundation. V.B.-W. was partly supported by 3 Abbreviations used in this paper: TCC, T cell clone; MFI, mean fluorescence intensity; the German Research Council (SFB 685) and C.H. by the Research Focus Autoim- ICS, intracellular cytokine staining; PD-1, programmed cell death 1; TMHMM, trans- munity at the University of Lu¨beck. membrane prediction using hidden Markov models; MINNOU, membrane protein iden- 2 Address correspondence and reprint requests to Eva Tolosa, Department of Immu- tification without explicit use of hydropathy profiles and alignments. nology, University Medical Center Hamburg-Eppendorf (UKE), Martinistr. 52, 20251 Hamburg, Germany. E-mail address: [email protected] Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901000 The Journal of Immunology 5495 and repeated 2 days later. After 12 or 25 days, CD4ϩ T cells were Surface biotinylation and Western blot isolated from spleen and from the spinal cord and brain of the immu- ϩ nized mice as described before (30) by magnetic isolation (Miltenyi Purified CD4 T cells were stimulated overnight with PMA/ionomycin. Biotec) and used for staining. All experiments involving animals were Cells were then washed with PBS and surface molecules were first biotin- performed according to the German animal protection act and have been ylated and subsequently isolated using the cell surface protein isolation kit approved by local authorities. (Pierce) according to the manufacturer’s protocol. Collected fractions, el- uate (biotinylated proteins), and flow-through (intracellular proteins) were Flow cytometry separated by SDS gel electrophoresis under reducing conditions and blot- ted onto polyvinylidine difuloride membrane. Membranes were incubated The following anti-human Abs were used: anti-CD4 Pacific Blue (clone with polyclonal rabbit anti-IL-17A and anti-IL-17F (both PeproTech), MT310, DakoCytomation), anti-IFN-␥ PE, (clone 4S.B3), anti-IL-17A mouse anti-GAPDH (6C5, Abcam) overnight at 4°C, followed by anti- Alexa647 (clone eBio64DEC17), anti-IL-17F PE (clone SHLR17), anti- rabbit or anti-mouse HRP-coupled secondary Ab (Sigma-Aldrich), respec- ICOS FITC (clone ISA-3), anti-CD25 PE (clone 12–1278-73), anti- tively. Nonbiotinylated cell lysates and human rIL-17A and rIL-17F (both CD28 PE-Cy7 (clone CD28.2), anti-CD39 PE-Cy7 (clone 25–0399), PeproTech) were used as controls. Blots were developed using the ECL anti-programmed cell death 1 (PD-1) FITC (clone U116), CD127 PE Plus system (Amersham Biosciences). (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- Prediction of transmembrane domains A251), anti-CD5 FITC (clone UCHT2), anti-CD62L PE (clone L48), anti- Transmembrane prediction using hidden Markov models (TMHMM) (31) CD49d PE (clone 9F10), anti-CD28 PE (clone 555432), anti-CD14 Pacific and membrane protein identification without explicit use of hydropathy Blue (clone 558121), anti-CD122 PE (clone 554525), anti-CD132 PE (clone profiles and alignments (MINNOU) (32) were used to predict transmem- 555900), anti-CD161 PE (clone HP-3G10) and isotype-matched control mAbs brane domains. The sequences used were AAH67505 for IL-17A and

(all BD Biosciences), anti-IL-17R PE (clone 133617), anti-CCR4 FITC (clone AAH70124 for IL-17F. Downloaded from 205410), anti-CCR5 PE (clone 45531) (all R&D Systems), and anti-CD27 Alexa750 (clone MHCD2727, Caltag). For the mouse staining protocol, we used anti-CD4 PE (GK1.5), CD11b FITC (M1/70), CD45 PE-Cy7 (30-F11), Statistical analysis and anti-IL-17A Alexa647 (eBio17B7, all eBioscience). Two-tailed unpaired Student’s t test was use to compare surface marker For assessment of surface expression, cells were stained at different time ϩ Ϫ expression between surface IL-17A and surface IL-17A cells. Differ- points after PMA/ionomycin stimulation. For intracellular cytokine stain- ences were considered significant if p Յ 0.05. ing (ICS), cells were stimulated with PMA/ionomycin in the presence of brefeldin A (10 ␮g/ml, eBioscience) for 6 h and subsequently fixed and http://www.jimmunol.org/ permeabilized with the corresponding buffers (eBioscience). Samples were Results analyzed in the LSRII flow cytometer using the DiVa software (BD Bio- ϩ sciences), and results were evaluated using the FlowJo software (Tree A CD4 T cell subset and Th17 TCC express IL-17A on the Star). The resulting mean fluorescence intensity (MFI) was obtained after cell surface upon stimulation subtracting the MFI of the isotype control from the MFI of the Ab of ϩ interest. For cell sorting we used a FACS Aria (BD Biosciences). Although trying to identify human IL-17A-producing CD4 T cells using a cytokine-capture/detection system, we observed that Expansion of surface IL-17Aϩ cells the anti-IL-17A detection Ab was binding to ϳ1% of CD4ϩ T MACS- or FACS-sorted surface IL-17Aϩ and surface IL-17AϪ cells were cells even in the absence of the capture anti-IL-17 Ab, indicating cultured as a bulk in X-VIVO 15 or IMDM supplemented with 5% human that IL-17A was expressed on the membrane of some activated by guest on September 24, 2021 serum, respectively, for 10 to 14 days. IL-17A production was assessed by CD4ϩ T cells. To verify this finding, we stimulated purified CD4ϩ ICS 10 days after each restimulation and the supernatants monitored by T cells with PMA/ionomycin and stained the surface for IL-17A. ELISA (human IL-17A ELISA kit, eBioscience). Flow cytometry analysis revealed that IL-17A was displayed at the ϩ Ϯ ϭ Generation and plasticity analysis of Th1/17 cells surface of a small population of CD4 T cells (0.89% 0.10, n ϩ 11) (Fig. 1A), in all donors tested. Unstimulated cells and cells CD4 T cells were stimulated 6 h with PMA/ionomycin and subsequently treated with brefeldin A were completely devoid of surface IL-17A stained with anti-IL-17A Alexa647. Surface IL-17AϪ, IL-17Aint,orIL- 17Ahigh CD4ϩ T cells were sorted at one cell per well onto 96-well culture (data not shown), suggesting that active transport from the endo- plates containing IMDM supplemented with 5% human serum, 20 U/ml plasmic reticulum to the cell surface is required for membrane hrIL-2 and irradiated autologous PBMC (FACSAria, BD Biosciences). expression of IL-17A. After PMA/ionomycin stimulation, the per- Cells were restimulated every second week. Cytokine production and centage of cells expressing IL-17A on the surface was maximal at ␤ clonality were assessed by ICS and TCR V staining, respectively. To 6 h and declined steadily to minimum levels after 48 h of stimu- assess the plasticity of the mixed clones, 2 ϫ 104 cells were cultured in X-VIVO15 with 2.5 ␮g/ml PHA (Sigma-Aldrich) and irradiated feeder lation (Fig. 1B), and always correlated with the frequency of IL- cells in the presence of 50 ng/ml human rIL-12 or rIL-23 (both R&D 17A-producing cells detected by ICS. Surface IL-17A expression Systems). After 7 days, production of IFN-␥ and IL-17A was assessed was also detected on CD4ϩ T cells isolated from the spleen and the by ICS. CNS of immunized mice 6 h after PMA/ionomycin stimulation RT-PCR (Fig. 1C), but again not in unstimulated cells or in the presence of brefeldin A. In the CNS, the proportion of CD4ϩ T cells with ϩ RNA was isolated from magnetic bead-isolated IL-17A Alexa647 (con- intracellular IL-17A was 24.09%, and the frequency of surface taining surface IL-17Aϩ CD4ϩ cells) and Alexa647Ϫ (containing surface IL-17Ϫ CD4ϩ cells) fractions or from Th17, Th1, and Th1/17 TCC after IL-17A-positive cells was 24.07%, indicating that surface IL-17A overnight PMA/ionomycin stimulation. After reverse transcription, gene faithfully reflects the presence of IL-17A-producing cells in dif- expression was analyzed by real time-PCR (ABI Prism 7900, Applied Bio- ferent compartments. Moreover, biotinylation of cell surface pro- systems). The following primer and probe sets were purchased from Ap- teins confirmed the presence of IL-17A on the cell membrane of ␥ plied Biosystems: T-bet (TBX21 Hs00203436_m1), ROR t (RORC2 PMA/ionomycin-stimulated human CD4ϩ T cells (Fig. 1D). Hs01076112_m1), GATA-3 (Hs00231122_m1), FOXP3 (Hs01085834_m1), ϩ IL-17A (Hs99999082_m1), and IL-23R (Hs00332759_m1), IL-17R To assess whether all IL-17A producing CD4 T cells also (Hs01064648_m1). Primer pairs for the other genes are as follows: TGF-␤ express IL-17A on their cell surface, we analyzed surface IL- for 5Ј-GCCCTGGACACCAACTATTG-3Ј; TGF-␤ rev 5Ј-CTGGTCCAG 17A expression on Th1 and Th17 TCC that we had previously GCTCCAAAT-3Ј, and IFN-␥ for 5Ј-TTCAGCTCTGCATCGTTTTG-3Ј; generated (V. Brucklacher-Waldert, K. Stu¨rner, M. Kolster, J. IFN-␥ rev 5Ј-CTTTCCAATTCTTCAAAATGCC-3Ј. Samples were run in duplicates, 18S rRNA was used as endogenous control and the relative Wolthausen, and E. Tolosa; submitted for publication). We gene expression was calculated by the ⌬⌬Ct method using CD4ϩ T cells found that all Th17 TCC displayed high amounts of IL-17A at as calibrator. the surface, while none of the Th1 TCC did (Fig. 1E). Th1/17 5496 Th17 CELLS IDENTIFIED BY SURFACE IL-17A

FIGURE 2. Surface IL-17A-expressing CD4ϩ T cells are Th17 cells. A, Isolated CD4ϩ T cells were stimulated overnight with PMA/ionomycin, surface stained with anti-IL-17A Alexa647, and subsequently purified us- Downloaded from ing magnetic beads coupled to anti-Alexa647 Abs. The dot plots show the surface 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- ϩ FIGURE 1. IL-17A is displayed on the cell surface of a subset of human 17A cells in comparison to the isotype control. B, ROR␥t and T-bet ϩ ϩ mRNA expression analysis of the surface IL-17Aϩ and surface IL-17AϪ and mouse CD4 T cells and in Th17 TCC. A, Purified human CD4 T http://www.jimmunol.org/ cells were stimulated with PMA/ionomycin and stained 6 h later with fractions immediately after purification (n ϭ 3). Data are shown as mean Ϯ ϩ anti-IL-17A without cell permeabilization (left) or after permeabilization SEM. C, PMA/ionomycin-stimulated CD4 T cells (left) were sorted ac- (right). Brefeldin A was added 1 h after stimulation where indicated. The cording to their surface IL-17A expression and cultured as bulk in the Ϫ numbers in the plots indicate the percentage of CD4ϩ T cells expressing absence of polarizing cytokines. After 10 days, surface IL-17A cells (up- ϩ surface IL-17A (left) and of intracellular IL-17A (right). Data are repre- per panel) and surface IL-17A cells (lower panel) were stained for in- sentative of eleven experiments. B, Time course of surface IL-17A expres- tracellular cytokines. D, Detection of IL-17A in the supernatant of MACS- ϩ Ϫ sion. Purified CD4ϩ T cells were stimulated with PMA/ionomycin for the isolated surface IL-17A (black line) and surface IL-17A (gray line)at indicated time points and stained for surface expression of IL-17A. The the indicated time points after magnetic isolation. graph shows the mean percentage Ϯ SEM of surface IL-17Aϩ cells at each time point (n ϭ 3). C, Purified mouse CD4ϩ T cells isolated from spleen by guest on September 24, 2021 population contained Th17 cells. Finally, we measured IL-17A in (top) and from the CNS (bottom) 25 days after MOG35–55 immunization ϩ Ϫ were stimulated with PMA/ionomycin and stained and analyzed as in A. the supernatants of surface IL-17A and surface IL-17 popula- Data are representative of three experiments. D, Detection of IL-17A in the tions during PMA/ionomycin expansion. IL-17A could be detected ϩ intracellular and isolated membrane fraction of membrane-biotinylated and in the supernatant of the surface IL-17A fractions for nearly 2 wk nonbiotinylated PMA/ionomycin-stimulated human CD4ϩ T cell lysate. after restimulation, whereas surface IL-17Ϫ cells production was Human rIL-17A was used as positive control and GAPDH as a loading negligible (Fig. 2D). In summary, the expression of ROR␥t and the control for intracellular proteins. E, Resting Th1 (top) and Th17 (bottom) stable production of IL-17A by expanded surface IL-17Aϩ cells TCC were stimulated overnight with PMA/ionomycin and stained for sur- identified surface IL-17Aϩ cells as Th17 cells. face IL-17A (left) or after permeabilization (right). Brefeldin A was added for intracellular cytokine detection. Data are representative of eight clones. Phenotypic characterization of surface IL-17Aϩ CD4ϩ T cells The expression levels of surface molecules in ex vivo Th17 cells clones, producing simultaneously IFN-␥ and IL-17A also ex- could be easily analyzed using surface IL-17A as a marker. We ϩ pressed surface IL-17A at the cell surface, although the inten- stimulated CD4 T cells with PMA/ionomycin and analyzed ex- sity of the staining was lower than for bona fide Th17 TCC (data pression of surface markers 8 h after stimulation, when surface not shown). IL-17A expression is maximal, and again after 48 h, when cells are fully activated (Fig. 3, supplemental Fig. 1).4 Flow cytometric ϩ Surface IL-17A-expressing CD4 T cells are Th17 cells analysis showed that surface IL-17Aϩ cells are exclusively CD45ROϩ memory cells, whereas surface IL-17AϪ cells were in We performed the following experiments to demonstrate that sur- ϩ ϩ average 69.07% Ϯ 9.82 CD45RO memory cells, 30.13% Ϯ 9.95 face IL-17A positive CD4 cells are indeed Th17 cells. First, we Ϫ ϩ Ϫ ϩ Ϯ separated CD4 T cells on the basis of their surface IL-17A ex- CD45RO CD27 naive T cells and 0.8% 0.28 CD45RO Ϫ ϭ pression using magnetic beads (Fig. 2, A and B), or FACS sorting CD27 effector cells (n 4), representing altogether not only (Fig. 2C) and analyzed the positive and negative fractions for the other polarized cells, but also the naive compartment (Fig. 3A). Next, we analyzed molecules involved in the recruitment into expression of T cell lineage specific transcription factors. Surface ϩ ϩ IL-17Aϩ CD4ϩ T cells expressed ROR␥t mRNA, while this tran- inflammatory tissues. We found that surface IL-17A CD4 T scription factor was barely detectable in surface IL-17Ϫ CD4ϩ T cells expressed significantly more CCR4, as reported for Th17 cells. T-bet, by contrast, was expressed at similar levels by Th1 cells (26, 33, 34), while CCR5 expression was comparable in both populations (Fig. 3B). As expected, CCR6 was expressed signifi- and Th17 TCC (Fig. 2B), as already described (26). Second, after ϩ Ϫ 10 days expansion of the purified subsets, we analyzed the surface cantly higher on surface IL-17A cells than on surface IL-17A IL-17Aϩ sorted subset by ICS and still found 35.5% of Th17 cells Ϫ (Fig. 2C). As expected, Ͻ1% of the expanded surface IL-17A 4 The online version of this article contains supplementary material. The Journal of Immunology 5497

the differential expression of molecules involved in adhesion to endothelial tissue. CD49d was expressed at much higher levels on surface IL-17Aϩ cells early after activation, whereas CD6, the ligand for activated leukocyte cell adhesion molecule-1, expression was comparable in both subsets (Fig. 3C). Proliferation and survival of T cells depend on the combination of signals provided by costimulatory molecules. Although CD28 is constitutively present in most T cells, CTLA-4 (CD152) is up- regulated relatively late after activation. We found that expression of CD28 was signiﬁcantly higher in surface IL-17Aϩ cells compared with surface IL-17AϪ cells both 8 and 48 h after activation (Fig. 3D). Remarkably, the T cell proliferation inhibitor CTLA-4 was expressed by nearly 70% of the surface IL-17Aϩ cells already at the early activation time point (supplemental Fig. 1), suggesting constitutive CTLA-4 expression in Th17 cells. In contrast, CTLA-4 expression in surface IL-17AϪ cells was expressed at lower levels, and stayed low after 48 h of stimulation. Two further members of the CD28 family, ICOS and PD-1, were expressed at

low levels after 8 h and showed after 48 h stimulation a higher Downloaded from expression on surface IL-17Aϩ cells (Fig. 3D). The receptors for the T cell growth factors IL-2 and IL-7 were also compared between surface IL-17AϪ and surface IL-17Aϩ cells (Fig. 3E). CD25 (IL-2R␣-chain) was barely detectable in both cell subsets 8 h after stimulation, but was up-regulated after 48 h, and at a ϩ

higher extent in the surface IL-17A compartment. CD122 (IL- http://www.jimmunol.org/ 2R␤-chain) was also up-regulated later after activation, and more noticeably in the surface IL-17Aϩ cells, whereas CD132 (IL-2R␥- chain) was up-regulated in the same cells already 8 h after stimulation. CD127, the IL-7R␣-chain, was constitutively expressed in all T cells except regulatory cells, and down-regulated upon activation. The levels of CD127 were slightly higher also in Th17 cells (Fig. 3E). Finally, we investigated T cell activation markers. The early activation markers CD5 and CD69 were expressed at comparable levels in both populations, while the T cell-APC contact by guest on September 24, 2021 promoting CD2 was expressed at significantly higher levels on FIGURE 3. Phenotypic characterization of surface IL-17A cells. Puri- surface IL-17Aϩ cells than on surface IL-17AϪ cells. CD62L (L- fied CD4ϩ T cells were stimulated with PMA/ionomycin and stained 8 and selectin), which is down-regulated after activation, was expressed 48 h later with anti-IL-17A-Alexa647 together with other surface markers, as indicated in each graph. A, Percentage of naive (CD45ROϪ CD27ϩ), at similarly low levels in both (Fig. 3F). It has been recently pub- effector (CD45ROϪCD27Ϫ) and memory cells (CD45ROϩCD27ϩ/Ϫ) cells lished that Th17 cells express CD161, a C-lectin present on NK in the surface IL-17AϪ (left) and surface IL-17Aϩ (right) subsets eight cells (35). We checked for coexpression of CD161 and surface hours after PMA/ionomycin. Data are representative of four experiments. IL-17A, however, because CD161 is down-regulated upon activa- Expression of chemokine receptors CCR4, CCR5, and CCR6 (B); adhesion tion and surface IL-17A only appears after stimulation, it was not molecules CD49d and CD6 (C); costimulatory molecules CD28, CTLA-4, possible to prove directly that both molecules are coexpressed. We ICOS, PD-1 (D); IL-2R and IL-7R subunits (E); and activation markers (F) then sorted CD161 negative and positive cells, stimulated them ϩ f Ϫ u in surface IL-17A cells ( ) and surface IL-17A cells ( ). Data are with PMA/ionomycin, and proceeded with IL-17A staining. Sur- displayed as the average MFI or percentage of positive cells as indicated face IL-17Aϩ cells are mostly contained in the CD161ϩ subset (n ϭ 4). G, sorted CD4ϩCCR6ϩ and CD4ϩCCR6Ϫ T cells were indepen- (3.39% in contrast to 0.19% in the CD161- subset), similar to Th17 dently stimulated with PMA/ionomycin and stained intracellularly for ␥ ϩ ϩ ϩ Ϫ cells defined by cytoplasmic expression of IL-17A. In contrast, IFN- and IL-17A. H, sorted CD4 CD161 and CD4 CD161 cells were Ϫ ϩ independently stimulated with PMA/ionomycin and stained for surface IL- Th1 cells are similarly distributed in the CD161 and CD161 17A (left) and intracellularly for IFN-␥ and IL-17A (right). compartments (16.5 and 13%, respectively) (Fig. 3H). Altogether, our results indicate that surface IL-17Aϩ cells show a higher level of basal activation, and also express higher levels of costimulatory cells (Fig. 3B) (26, 33, 34). CCR6 expression was highest after 8 h and adhesion molecules than cells expressing no IL-17A. In addi- of stimulation and was later down-regulated. Although nearly all tion, we show that surface IL-17Aϩ are contained in the CCR6ϩ of the IL-17Aϩ cells were CCR6ϩ, only Ͻ20% of the IL-17AϪ and CD161ϩ fractions of CD4ϩ T cells. population expressed this chemokine receptor. To assess whether sorting of CD4ϩ T cells on the basis of CCR6 expression would Th1/17 TCC generated on the basis of surface IL-17A generate exclusively Th17 cells, we sorted CCR6ϩCD4ϩ and expression show plasticity to the environment CCR6ϪCD4ϩ T cells and analyzed both subsets immediately for As mentioned above, we have observed that bona fide Th17 TCC intracellular production of IFN-␥ and IL-17A. We found that IL- express high amounts of IL-17A on their surface whereas Th1/17 17A-producing cells were exclusively present in the CCR6ϩ sub- TCC, producing simultaneously IL-17 and IFN-␥, express less sur- set, whereas IFN-␥ producing CD4ϩ T cells could be detected in face IL-17A and Th1 clones are devoid of surface IL-17A. To test both CCR6ϩ and CCR6Ϫ populations (Fig. 3G), indicating that whether we could generate TCC with different effector phenotypes CCR6 is not an exclusive marker for Th17 cells. We next analyzed from cells expressing different levels of surface IL-17A, we sorted 5498 Th17 CELLS IDENTIFIED BY SURFACE IL-17A

ing intermediate levels of IL-17A generated 15 Th1/17 double- producing cell lines and three Th1 cell lines. We assessed the TCR V␤ from eight selected Th1/17 cell lines and, after confirming their clonality, used them for further studies. As shown in Fig. 4A, some of these Th1/17 TCC contained individual cells producing either IFN-␥ or IL-17A or both cytokines simultaneously (TCC 051 and TCC 049), while some clones gave rise to cells producing only IFN-␥ and cells producing both cytokines (TCC 045) and finally some TCC had individual cells producing only IL-17 and cells producing both cytokines (TCC068). To determine whether the heterogeneous cytokine profile of Th1/17 clones cells was mir- rored at transcriptional level, we performed RT-PCR analysis of the relevant transcription factors and cytokines (Fig. 4B). As expected, bona fide Th17 clones expressed ROR␥t and IL-17A mRNA, while Th1 TCC were negative for both of them and expressed instead IFN-␥. T-bet was equally expressed in Th1 and Th17 TCC, as we had already seen in ex vivo Th17 cells. The receptor for IL-23, a cytokine involved in the maintenance of Th17 cells, was undetectable in Th1 TCC, but clearly present in Th17 Downloaded from TCC. Remarkably, Th1/17 clones expressed the highest levels of ROR␥t, IL-17A and IL-23R, but also of IFN-␥, T-bet, and FOXP3. The Th2 cell specific transcription factor GATA-3 was equally expressed at low levels in all TCC. By following up the sorted TCC over several rounds of restimulation, we observed that, in the absence of cytokines, the phenotype of Th1 and Th17 TCC re- http://www.jimmunol.org/ mained stable, while Th1/17 clones changed their cytokine profile toward less IL-17A and more IFN-␥ production, suggesting that they are in a transitional phase rather than being a stable phenotype (Fig. 4C). We addressed their capacity to respond to environment by culturing these cells in the presence IL-23 or IL-12, cytokines that promote or stabilize the development of Th17 and Th1 cells, respectively. As shown in Fig. 4D, IL-23 moderately increased the

percentage of cells producing IL-17A, whereas the Th1 cell-pro- by guest on September 24, 2021 moting IL-12 markedly decreased the expression of IL-17A and increased that of IFN-␥. IFN-␥ production by Th1 TCC was not drastically affected by either the addition of IL-23 or IL-12, while addition of IL-12 to the Th17 TCC resulted in the decrease of cells producing solely IL-17A and a corresponding increase in the percentage of double producers. Thus, Th1/17 and Th17 clones re- FIGURE 4. Generation and plasticity of Th1/17 double producer T cell spond to changes in their cytokine environment. clones. A, CD4ϩ T cells were sorted in surface IL-17AϪ (1), surface IL- int high 17A (2), and surface IL-17A (3) cells according to the gates shown in ϩ the left panel to generate TCC. After one round of restimulation, cells were Surface IL-17A on CD4 T cells is not presented by IL-17R stained for intracellular IFN-␥ and IL-17A. The figure shows overlays of Cytokines can be displayed on the cell surface either bound to a 13 TCC originating from surface IL-17AϪ cells (1), 15 TCC obtained from int high receptor or directly anchored at the cell membrane by means of a surface IL-17A cells (2), and 16 TCC generated from surface IL-17A transmembrane domain. First, we looked for expression of the IL- (3) CD4ϩ T cells. The lower panels show four individual examples of 17R that could present IL-17A on the surface of CD4ϩ T cells. Th1/17 double producer clones obtained from surface IL-17Aint sorted cells. B, mRNA analysis of indicated genes of Th1 (n ϭ 3, u), Th17 (n ϭ Flow cytometry analysis revealed that all monocytes expressed 3, f), and Th1/17 (n ϭ 4, Ⅺ) TCC generated in A. Data are shown as IL-17R, while only a minority of unstimulated (Fig. 5A) and PMA/ ϩ mean Ϯ SEM. C, Cytokine profile of two double producer Th1/17 clones ionomycin-stimulated (data not shown) human CD4 T cells ex- (TCC121 and TCC062) after consecutive restimulations in the absence of pressed IL-17R on their membrane. When stimulated cells were polarizing cytokines. D, Cytokine profiles of two double producers Th1/17 double stained for surface IL-17A and IL-17R, we found no co- TCC (TCC157 and TCC099), one Th1 (TCC011), and one Th17 (TCC323) expression of the two molecules (Fig. 5B). In addition, blocking of after three restimulations in the presence of IL-12 or IL-23. the IL-17R by anti-IL-17R Abs did not prevent surface IL-17A expression (data not shown). PCR analysis of TCC showed similarly low expression of IL-17R in both Th17 and Th1 cells (Fig. surface IL-17AϪ, surface IL-17Aint, and surface IL-17Aϩ cells 5C). Moreover, surface IL-17A expression could also be detected ϩ onto 96-well plates at one cell per well (Fig. 4A). After restimu- in CD4 T cells of the IL-17R-deficient mice (Fig. 5D). These lation, the cytokine profile and clonality of these cells lines were results suggest IL-17A is not bound to the IL-17R. Next, we pre- analyzed. Intracellular cytokine staining showed that surface IL- dicted potential transmembrane domains using the TMHMM and 17AϪ cells gave rise exclusively to Th1 cell lines (n ϭ 13), while MINNOU programs and revealed that IL-17A generally lacks a surface IL-17Ahigh resulted in 13 Th17 cell lines and three T cell transmembrane domain and appears to be a soluble protein (Fig. lines producing both IL-17A and IFN-␥. In contrast, cells express- 5E). Given that IL-17A is functional in vivo as a heterodimer of The Journal of Immunology 5499

IL-17A and IL-17F (7, 36), we tested the presence of a transmembrane domain in the IL-17F sequence. Interestingly, IL-17F contains a sequence compatible with a classical transmembrane domain, as predicted by TMHMM and MINNOU (Fig. 5E). Analysis of PMA/ionomycin-stimulated Th17 TCC showed that Th17 cells express IL-17F in addition to IL-17A (Fig. 5, F and G). More deﬁnitively, we found IL-17F expressed on the surface of a subset of CD4ϩ T cells (Fig. 5H). Simultaneous surface staining of IL- 17A and IL-17F did show very dim staining of both molecules when compared with the individual stainings, and coexpression was not detectable (data not shown), possibly because of sterical hindrance of anti-IL-17A and anti-IL-17F Abs due to a spatial association of both cytokines. To overcome this problem, we sorted CD4ϩ T cells into surface IL-17Aϩ and IL-17Fϩ cells. After 10 days to allow cells to rest, both sorted populations were stimulated again with PMA/ionomycin and subsequently stained with the two Abs (single staining for the surface, double staining for the cytoplasm, Fig. 5H). Sorted IL-17Aϩ cells were clearly

positive for both IL-17A, and at a lesser extent, for IL-17F, while Downloaded from IL-17AϪ cells were negative for both IL-17 molecules, both in the surface and in the cytoplasm. The reciprocal experiment showed similar results, although the levels of expression of the IL-17A and IL-17F molecules were lower. Intracellular staining of both sorted IL-17Aϩ and IL-17Fϩ populations showed that the majority of

cells coexpress intracellularly both cytokines. Remarkably, in http://www.jimmunol.org/ some cells, only IL-17A could be detected intracellularly, possibly because anti-IL-17A and anti-IL-17F Abs compete on already dimerized IL-17A/IL-17F complexes. In sorted IL-17Ϫ cells nei- ther IL-17A nor IL-17F were detected on the surface or intracellularly. Even though we cannot discard that IL-17A is bound to a yet unknown IL-17R or is in association with other members of the IL-17 family, our data support the hypothesis that IL-17A is transiently displayed at the cell surface forming a heterodimer with membrane-bound IL-17F. by guest on September 24, 2021

Discussion We report in this study that both IL-17A and IL-17F are displayed on the cell surface of a subset of activated CD4ϩ T cells. The expression of surface IL-17A correlates with intracellular IL-17A expression in human and murine CD4ϩ T cells in different com- FIGURE 5. IL-17A is not bound to the specific receptor IL-17R at the ϩ partments, however, there is always some degree of discrepancy cell surface. A, Surface staining of PBMC of IL-17R and CD14 cells between the percentage of positive cells in the surface and the (upper panel)orCD4ϩ T cells (lower panel). B, Surface staining of ϩ intracellular staining. Indeed, the MFI of the surface staining is IL-17A and IL-17R in human purified CD4 T cells. C, mRNA analysis for IL-17R of two Th1 (u) and two Th17 (f) TCC. As calibrator always lower than intracellular, probably indicating that there are mRNA of LPS-stimulated monocyte-derived dendritic cells was used. less IL-17A molecules present at the cell surface than inside the Data are shown as mean Ϯ SEM. D, Purified mouse CD4ϩ T cells cell. In the first case, IL-17A is only transiently expressed on its isolated from the spleen of IL-17R-deficient mice 12 days after way to secretion, while the amount of intracellular IL-17A is ar-

MOG35–55 immunization were stimulated with PMA/ionomycin and tificially enhanced by brefeldin A, which blocks the transport to stained as in Fig. 1A. E, Alignment with ClustaW2 and transmembrane the cell surface. The lower MFI results in less resolution between domain prediction of human IL-17A (AAH67505) and IL-17F positive and negative cells, and thus in differences in the percent- (AAH70124) using the MINNOU server. Black frame indicates the pu- ages. IL-10 and IFN-␥ have also been described on the surface of ء tative transmembrane domain in IL-17F. , Identical nucleotides; :, activated T cells in the absence of a specific receptor (37, 38). Conserved substitutions. F, mRNA analysis for ROR␥t, IL-17A and However, in both cases a sensitivity-enhancing staining method IL-17F of two Th1 (u) and two Th17 (f) TCC. As calibrator mRNA of PMA/ionomycin-stimulated CD4ϩ T cells was used. Data are shown as was required for detection, probably due to a very low expression, mean Ϯ SEM. G, Western blot analysis for IL-17A (upper panel) and while surface IL-17A can be detected by conventional flow cy- IL-17F (lower panel) of overnight stimulated PMA/ionomycin CD4ϩ T tometry using commercially available fluorochrome-labeled anti- ϩ cells. Human recombinant cytokines were used as positive controls. H, IL-17A Abs. Our data show that the surface IL-17A cells are CD4ϩ T cells were sorted on the basis of surface IL-17A and IL-17F bona fide Th17 cells, and thus surface IL-17A can be used as a expression, allowed to rest for 10 days and then stimulated with PMA/ specific surface marker for this Th subset. Moreover, we have used ionomycin for 6 h and stained for surface IL-17A and IL-17F indepen- surface IL-17A for the isolation of ex vivo Th17 cells by magnetic- dently (left and middle, respectively) or together (right) for intracellular and fluorescence-activated cell sorting. A method for ex vivo iso- detection. The histograms show the levels of staining of the indicated surface lation of human Th17 cells using a bispecific Ab against CD45 and cytokine (f) in comparison to the isotype control (u) staining. The dot plot IL-17A for capturing IL-17-secreting cells has been recently re- shows the intracellular staining for both cytokines for each population. ported (39). However, this method requires previous preparation of 5500 Th17 CELLS IDENTIFIED BY SURFACE IL-17A reagents, needs long handling times, and has the risk of unspecific described for lymphotoxin ␣, which does not contain a transmem- bystander detection. brane domain and it appears at the cell surface anchored via lym- Using surface IL-17A expression as a marker for Th17 cells, we photoxin ␤ (42). A heterodimeric form of IL-17 comprising IL- have analyzed the ex vivo phenotype of this subset in human pe- 17A and IL-17F and its biological function has already been ripheral blood in comparison to IL-17A-negative cells. The higher described (7). The biological activity of this IL-17A surface form expression and up-regulation of CD2, costimulatory molecules, is still unknown, but it may have distinct functions, for instance it activation markers, and of CD49d on surface IL-17Aϩ indicates might serve as a ligand for IL-17R expressed on APCs to prolong that Th17 cells are better equipped for recruitment into inflamed cytokine signaling in T/APC clusters (43), or on endothelial cells tissues, activation, and proliferation. Higher expression of CCR6, (44). This may provide a cell contact-dependent signal for trans- CD49d, CD28, CTLA-4, CD25, and CD2 was also observed in Th17 migration of surface IL-17A positive cells through vascular endo- TCC when compared with Th1 TCC generated from peripheral blood thelial cells expressing IL-17 receptor (44). and cerebrospinal fluid of a patient with multiple sclerosis (V. Our data show that surface IL-17A constitutes a unique marker Brucklacher-Waldert, K. Stu¨rner, M. Kolster, J. Wolthausen, and E. for the unambiguous identification and isolation of viable Th17 Tolosa; submitted for publication), supporting the view of Th17 cells cell. By analyzing phenotypically surface IL-17Aϩ CD4ϩ T cells, as a potent effector T cells subset. we show that human ex vivo Th17 cells express high levels of Our analysis confirmed the higher expression of CCR6 and the costimulatory and adhesion molecules as well as activation mark- coexpression of CD161 by Th17 cells already described by others ers, indicating the potential of Th17 cells as potent effector T cells. (26, 33–35). CCR6 and CD161, alone and in combination, are In addition, we could consistently generate Th1/17 T cell clones currently used as markers for the enrichment of Th17 cells, how- derived from intermediate surface IL-17A-expressing cells, and Downloaded from ever, both CD161ϩ and CCR6ϩ and even the double-positive cells these clones adapted their phenotype to the cytokine milieu, pro- can give rise to Th1 cells in addition to IL-17A-producing cells. In viding evidence for the plasticity of human Th1/17 cells. contrast, cells selected on the basis of high surface IL-17A generated only Th17 or Th1/17 clones, but never Th1 cells. Very Acknowledgments interestingly, the levels of surface expression of IL-17A seem to Ϫ We thank all blood donors for their cooperation, Roland Martin for the indicate the commitment to the Th phenotype: surface IL-17A support given to this project, Christoph Ogrodowczyk for help with the http://www.jimmunol.org/ high cells gave rise exclusively to Th1 clones; surface IL-17A cells biotinylation experiments, and F. Koch-Nolte, F. Haag, and C. Stoeckle for produced mostly Th17 TCC and a few Th1/17 TCC. In contrast, suggestions and critical reading of the manuscript. sorting on the basis of surface IL-17Aint cells produced mostly TCC expressing both cytokines. When we analyzed the TCC for Disclosures transcription factor expression, we found that ROR␥t was highly The authors have no financial conflict of interest. expressed in Th17 TCC and nearly absent from the Th1 TCC. 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