UNIT 6.25Measurement of Interleukin-17Bhanu P. Pappu1 and Chen Dong1

1M.D. Anderson Cancer Center,Houston, Texas

ABSTRACT

Upon antigenic stimulation, naive CD4+ T cells undergo proliferation and differentiateinto cytokine-producing T helper (TH) effector cells. TH1 cells secrete effector cytokineIFN-γ and regulate cell-mediated immunity, whereas TH2 cells produce IL-4, IL-5,and IL-13 cytokines, and mediate immunity against extracellular pathogens and aller-gic reactions. Recent studies have identified a novel TH subset, called TH17, THIL-17,or inflammatory TH (THi) cells, characterized by the production of a proinflammatorycytokine, IL-17, and regulating inflammatory responses. In this unit, we describe theprotocols for the differentiation of mouse IL-17-expressing T cells in vitro, detection ofIL-17-expressing T cells by intracellular cytokine staining, and measurement of IL-17 se-cretion in culture supernatants by ELISA. Generation of IL-17-expressing T cells in vitrounder defined culture conditions allows investigation of their differentiation regulation.Detection of IL-17 in cell culture and tissue samples helps in monitoring inflammatorydiseases and determining efficacy of therapeutic interventions. Curr. Protoc. Immunol.79:6.25.1-6.25.8. C© 2007 by John Wiley & Sons, Inc.

Keywords: IL-17 � helper T cells � autoimmunity � T cell differentiation

INTRODUCTION

Upon antigenic stimulation, naive CD4+ T cells undergo proliferation and differenti-ate into cytokine-producing T helper (TH) effector cells (UNIT 3.13). TH1 cells secretean important effector cytokine, IFN-γ, and regulate cell-mediated immunity. Humoralimmunity is regulated by TH2 cells, which secrete IL-4, IL-5, and IL-13 cytokines, andare critical in immunity against extracellular pathogens and in allergic reactions. Recentstudies have identified a novel TH subset, called THIL-17, TH17, or inflammatory TH

(THi) cells, characterized by the production of a proinflammatory cytokine IL-17 andregulating inflammatory responses.

IL-17, also referred as IL-17A, is the founding member of a novel cytokine family that in-cludes IL-17B, IL-17C, IL-17D, IL-17E (also called IL-25), and IL-17F. Activated CD4+T cells are the major producers of IL-17, but some CD8+ T cells, γδ T cells, and NK cellsalso produce IL-17. It has been shown that IL-17 acts on epithelial and endothelial cellsand induces various proinflammatory cytokines such as IL-6 and IL-8, as well as TNF-α,chemokines, and prostaglandins. IL-17 also promotes myeloid cell recruitment in tissuesand offers protection against certain bacteria. More importantly, elevated expression ofIL-17 was detected in patients with rheumatoid arthritis, dermatitis, multiple sclerosis,inflammatory bowel diseases, etc., thus making it a potential therapeutic candidate fortreating these inflammatory diseases.

In this unit, we describe protocols for differentiation of mouse IL-17-expressing T cellsin vitro (Basic Protocol 1), measurement of IL-17 secretion in culture supernatants byELISA (Basic Protocol 2), and detection of IL-17-expressing T cells by intracellularcytokine staining (Basic Protocol 3). Generation of IL-17-expressing T cells in vitrounder defined culture conditions allows investigation of their differentiation regulation.Detection of IL-17 in cell culture and tissue samples helps track the inflammatory diseasesand determine efficacy of therapeutic interventions.

Current Protocols in Immunology 6.25.1-6.25.8, November 2007Published online November 2007 in Wiley Interscience (www.interscience.wiley.com).DOI: 10.1002/0471142735.im0625s79Copyright C© 2007 John Wiley & Sons, Inc.

Cytokines andTheir CellularReceptors

6.25.1

Supplement 79

Measurement ofInterleukin-17

6.25.2

Supplement 79 Current Protocols in Immunology

NOTE: All solutions and equipment coming into contact with living cells must be sterile,and aseptic technique should be used accordingly.

NOTE: All cell culture incubations should be carried out in a 37◦C, 5% CO2 humidifiedincubator unless otherwise indicated.

BASICPROTOCOL 1

IN VITRO DIFFERENTIATION OF MURINE IL-17-PRODUCING CELLS

Differentiation of TH1 cells is promoted by IL-12 and inhibited by the TH2 signaturecytokine IL-4. Similarly, TH2 lineage development is promoted by IL-4 and inhibitedby the TH1 signature cytokine IFN-γ. Interestingly, both TH1 and TH2 cytokines havenegative effects on the development of IL-17-producing effector T cells. Hence, it iscritical to block IFN-γ and IL-4 action in T cell culture for optimal differentiation ofIL-17-expressing T cells. In addition, several recent studies have shown that TGF-β andIL-6 are the critical cytokines driving the development of IL-17-expressing T cells whileinhibiting TH1 and TH2 differentiation. TNF-α and IL-1β have been shown to furtherenhance this differentiation. Another important cytokine, IL-23, has been shown to bedispensable for the in vitro generation of IL-17-expressing T cells, but required in vivo,possibly for the maintenance and expansion of these cells.

Materials

Mice on desirable genetic background as a source of naive CD4+ T cells (C57BL/6or OT-II TCR transgenic mice on C57BL/6 genetic background are routinelyused)

Phosphate-buffered saline (PBS; APPENDIX 2A)0.1 mM sodium carbonate buffer, pH 9.5Anti-CD3ε (2C11, BD Pharmingen) and anti-CD28 (37.51, BD Pharmingen)Complete RPMI-1640 medium containing 10% FBS (APPENDIX 2A)Anti-IFN-γ antibody, clone XMG1.1 (BD Pharmingen)Anti-IL-4 antibody, clone 11B11 (BD Pharmingen)Murine TGF-β and IL-6 (PeproTech)Murine recombinant IL-23 (R&D systems)6- or 24-well tissue culture plates

Additional reagents and equipment for harvesting lymphoid organs of mice(UNIT 1.9) preparing single-cell suspensions from mouse lymphoid organs(UNIT 3.1), depletion of red blood cells from cell suspensions (UNIT 7.2),purification of T cells (UNIT 3.5A), counting of viable cells by trypan blueexclusion (APPENDIX 3B), preparation of APC by pulsing BMDC with cognatepeptide (Lanzavecchia and Sallusto, 2001) or irradiation of T cell–depletedsplenocytes (UNIT 3.12), and stimulation of naive CD4+ T cells using APC orantibody-coated plates (UNIT 3.13)

Prepare naive CD4+ T cells1. Harvest lymph nodes and spleen from the mice (UNIT 1.9) and prepare a single-cell

suspension (UNIT 3.1). Remove red blood cells by treatment with ammonium chloride–potassium (ACK) lysis buffer (UNIT 7.2) and purify the naive CD4+ T cells (UNIT 3.5A).Count the live cells by trypan blue exclusion (APPENDIX 3B) and adjust concentrationto 4 × 106 cells/ml in complete RPMI-1640 medium containing 10% FBS.

Purification of naive CD4+ T cells can be achieved by flow cytometric sorting (Chapter5) of CD4+CD62LhiCD44loCD25– population or by isolation of CD4+ T cells from TcRtransgenic mice on Rag–/– background.

Prepare APC or antibody-coated plates for stimulation of naive CD4+ T cells2a. For stimulation of polyclonal naive CD4+ T cells using APC: Pulse syngenic bone

marrow–derived dendritic cells (BMDC) with cognate peptide (usually in the range

Cytokines andTheir CellularReceptors

6.25.3

Current Protocols in Immunology Supplement 79

of 1 to 10 µg/ml) for 2 hr at 37◦C and use them as APC (Lanzavecchia and Sallusto,2001). Alternatively, irradiate T cell–depleted splenocytes with 3000 rad and usethem as APC (UNIT 3.12).

Even though BMDC provide the most potent activation stimuli for the naive T cells, splenicAPC are easier to prepare and work well in promoting IL-17-expressing T cells. Theconcentration of cognate peptide must be titrated to determine the optimal concentrationfor inducing naive T cell differentiation (Lanzavecchia and Sallusto, 2001).

2b. For stimulation of polyclonal naive CD4+ T cells using antibody-coated plates: Coattissue culture plates by incubating overnight at 4◦C with PBS or 0.1 mM sodiumcarbonate buffer (pH 9.5) containing 10 µg/ml anti-CD3ε and 2 µg/ml anti-CD28.Use 500 µl per well for 6-well plates or 250 µl per well for 24-well plates Washplates thoroughly with PBS.

Culture the CD4+ T cells3. Prepare a mixture of the CD4+ T cells with antigen-pulsed APCs (ratio of APC to

T cells should be empirically determined; usually a 1:20 ratio of DC to T cells ora 1:1 ratio of irradiated spleen cells to T cells should be used; also see UNIT 3.13) oradd the CD4+ T cells to the antibody-coated wells; in either case, prepare the cellsuspension RPMI-10 containing the following (final) concentrations of cytokinesand blocking antibodies:

10 µg/ml anti-IFN-γ antibody10 µg/ml anti-IL-4 antibody5 ng/ml TGF-β20 ng/ml IL-620 ng/ml IL-23.

The above cocktail is the most optimal for inducing IL-17+ T cells. Depending on exper-imental purposes, one may differentiate T cells without blocking antibodies or IL-23. Donot exceed 2 × 106 CD4+ T cells per ml. Culture volume should be adjusted to 2 ml in6-well plates and as low as 500 µl in 24-well plates. If the medium turns yellow by day 3or 4, supplement with fresh RPMI medium (one-fourth of original culture volume).

4. On day 4 to 7, harvest the cells and wash with fresh RPMI-10 medium before furtherfunctional analysis.

Unless otherwise indicated, cells are usually washed with 4 to 5 volumes of fresh RPMImedium or PBS in a 15-ml conical centrifuge tube, centrifuging 5 min at 1500 rpm in atabletop centrifuge at room temperature.

BASICPROTOCOL 2

QUANTITATION OF HUMAN AND MURINE IL-17 USING ELISA

This protocol is based on the sandwich ELISA technique in which the cytokine issandwiched between a capture antibody bound to the plastic surface and a detectionantibody that activates an enzymatic reaction. The concentration of IL-17 in the testsamples is determined using a standard curve plotted based on the known concentrationof IL-17 standards.

Materials

Capture antibody: anti–mouse IL-17, clone TC11-18H10 (BD Pharmingen) oranti–human IL-17, clone eBio64CAP17 (eBioscience)

0.1 mM sodium carbonate buffer, pH 9.5Phosphate-buffered saline (PBS; APPENDIX 2A)PBS (APPENDIX 2A) containing 5% FBS or BSAWashing solution: PBS (APPENDIX 2A) containing 0.05% (v/v) Tween 20Human or murine recombinant IL-17 (Peprotech)Assay diluent: 1% (v/v) FBS in PBS

Measurement ofInterleukin-17

6.25.4

Supplement 79 Current Protocols in Immunology

Samples to be assayed for IL-17Detection antibody: biotinylated anti–mouse IL-17, clone TC11-8H4.1 (BD

Pharmingen) or biotinylated anti–human IL-17, clone eBio64CAP17(eBioscience)

Avidin-conjugated horseradish peroxidase (avidin-HRP; Vector Laboratories)ELISA substrate: SigmaFAST OPD (o-phenylenediamine dihydrochloride; Sigma)ELISA stop solution: 2 M sulfuric acid

96-well flat-bottom MaxiSorp plates (Nunc)Microtiter plate sealers (R&D systems)ELISA plate reader with 450 and 570 nm filters

NOTE: It is recommended that the samples be run in triplicate and standards in duplicate.

Quantitation of IL-171. Coat Maxisorp 96-well plates by adding 100 µl per well of 1 µg/ml anti-IL-17

capture antibody diluted in 0.1 mM sodium carbonate buffer, pH 9.5, sealing theplate tightly with a microtiter plate sealer, and incubating at 4◦C overnight.

Plates can also be coated at 37◦C for 2 hr instead of overnight. Plates must be sealedtightly to avoid evaporation.

2. Wash the plate twice with washing solution. Block by adding 250 µl/well PBScontaining 5% FBS or BSA and incubating at 37◦C for 1 hr.

Proper blocking is necessary to avoid high background readings.

3. Wash the plate twice with washing solution. Prepare serial 2× dilutions of IL-17standard in assay diluent, starting from 10 ng/ml, in successive wells in duplicate.Add samples to be assayed to the designated wells without further dilution, or dilutedin assay diluent, if necessary. Incubate at 4◦C overnight or 3 hr at 37◦C.

Culture supernatants and serum samples that contain high levels of IL-17 can be dilutedin assay diluent. Culture supernatants should be centrifuged to remove cells and debris.Samples can be used immediately or frozen at −20◦C for future use.

4. Wash the plate six times with washing solution. Add 100 µl of 0.5 µg/ml detectionantibody, diluted in assay diluent, to each well and incubate at 37◦C for 1 hr.

5. Wash the plate six times with washing solution. Add 100 µl of avidin-HRP (diluted1:2000 in assay diluent) and incubate at 37◦C for 30 to 45 min.

6. Prepare the OPD-substrate solution by dissolving buffer and substrate pellets in20 ml of distilled water. Wash the plate six times with washing solution, then add100 µl of the substrate solution. Incubate the plates in dark at room temperature andperiodically check for color development.

7. Stop the reaction by adding 50 µl of ELISA stop solution (2 M sulfuric acid). Readthe optical density within 30 min at 450 nm with 570-nm background correction.

If the plate reader program does not include automatic 570-nm wavelength correction,manually subtract the 570-nm values from 450-nm values. Plot the standard curve anddetermine the actual value of IL-17 by taking the dilution factor into account. Accuratevalues are usually obtained in the range of 50 pg/ml to 1 ng/ml.

BASICPROTOCOL 3

INTRACELLULAR CYTOKINE STAINING ANALYSIS OF MOUSEIL-17-PRODUCING CELLS

This protocol allows the detection of IL-17-producing cells obtained from both in vitroand in vivo stimulation conditions. Intracellular staining for IL-17 in conjunction withIFN-γ, IL-4, or other markers will allow the simultaneous analysis of different T cellsubsets.

Cytokines andTheir CellularReceptors

6.25.5

Current Protocols in Immunology Supplement 79

Materials

In vitro–differentiated mouse IL-17-producing cells (Basic Protocol 1)Complete RPMI-1640 medium containing 10% FBS (APPENDIX 2A)Phorbol myristate acetate (PMA; Sigma)Ionomycin (Sigma)Staining buffer: PBS (APPENDIX 2A) containing 1% (v/v) FBS and 0.05% (w/v)

sodium azideFixation/Permeabilization kit containing:

GolgiPlug (BD Biosciences)Cytofix/Cytoperm solution10× Perm/Wash solution

PerCP/Cy5.5-conjugated anti-CD4 antibody, clone GK1.5 (eBioscience)Anti–mouse CD16/CD32 antibody, clone 2.4G2 (also referred to as Fc-block; BD

Pharmingen)APC-conjugated anti–mouse IFN-γ antibody, clone XMG1.2 (BD Pharmingen)PE-conjugated anti–mouse IL-17 antibody, clone TC11-18H10 (BD Pharmingen)

Additional reagents and equipment for flow cytometry (Chapter 5)

1. Wash T cells with complete RPMI-10 after in vitro differentiation or direct isolationfrom mouse tissues. Restimulate in complete RPMI-10 containing PMA (50 ng/mlfinal concentration) and ionomycin (500 ng/ml final concentration) in the presenceof GolgiPlug (1:1000 dilution) for 5 to 6 hr at 37◦C.

It is important that cells not be left in GolgiPlug (which containing protein transportinhibitor brefeldin A) for more than 12 hr. Comparable staining of IL-17 has also beenachieved with GolgiStop(BD Pharmingen), a protein transport inhibitor containingmonensin A.

Typically, up to 2 × 106 cells can be stained in 100 µl of staining buffer.

2. Wash cells with 1 ml of staining buffer, then resuspend in 100 µl of staining buffercontaining a 1:100 dilution of Fc-block and incubate 10 min at 4◦C. Next, incubatewith anti-CD4 antibody diluted in staining buffer at 4◦C for 30 min, then wash twicewith 1 ml of staining buffer and fix the cells by resuspending them in 250 µl ofCytofix/Cytoperm solution.

Concentration of the antibody should be determined empirically by the investigator foreach application. Manufacturer’s recommended concentrations are a good starting pointfor determining the optimal concentration.

Alternatively, 4% paraformaldehyde solution can be used to fix the cells and a 1% solutionof saponin in BSA can be for permeabilization.

3. Dilute the 10× Perm/Wash solution to 1× in distilled water and wash the fixed cellstwice, each time in 500 µl of 1× Perm/Wash solution according to the manufacturer’sinstructions. Resuspend the fixed cells in 50 µl of 1× Perm/Wash solution. Add 50 µlof fluorochrome-conjugated antibody against IL-17 and/or IFN-γ and incubate at 4◦Cfor 30 min.

Optimal concentration of staining antibodies against IL-17 and IFN-γ must be determinedempirically. Dilutions of 1:100 or 1:200 are usually recommended as starting dilutions.Each investigator should adjust the optimal antibody concentration depending of the celltype and batch of the antibody.

4. Wash cells twice with 1 ml of 1× Perm/Wash solution and resuspend in 150 µl ofstaining buffer. Proceed to flow cytometric analysis (Chapter 5).

Measurement ofInterleukin-17

6.25.6

Supplement 79 Current Protocols in Immunology

COMMENTARY

Background InformationIL-17 is the founding of member of the

six-member cytokine family that includes cy-tokines IL-17A to F. Among these mem-bers, IL-17A and F are closely related andcoexpressed in T cells. IL-17A acts on abroad range of cells including epithelialcells, endothelial cells, osteoblasts, fibrob-lasts, and monocytes/macrophages (Fossiezet al., 1996). IL-17 induces secretion of sev-eral proinflammatory cytokines, chemokines(CXCL1 and 8), colony-stimulating factors(GM-CSF, G-CSF), IL-6, IL-8, IL-1β, andmatrix metalloproteinases (Kolls and Linden,2004; Happel et al., 2005). IL-17 signalingactivates NF-κB and MAP kinase pathways;this activation is dependent on adaptor proteinsTRAF6 and Act1 (Schwandner et al., 2000;Chang et al., 2006). IL-17 has been shown tomediate tissue recruitment of neutrophils andmacrophages and to promote granulopoiesis.It is also important in host protection againstcertain extracellular bacteria. Moreover, ele-vated levels of IL-17 expression have been as-sociated with several autoimmune and inflam-matory diseases (Tartour et al., 1999; Iwakuraand Ishigame, 2006; Langowski et al., 2006;Weaver et al., 2006).

For a long time, TH1 and TH2 cells havebeen considered to represent two mutually ex-clusive developmental programs undertakenby naive CD4+ T cells upon antigenic stim-ulation (Mosmann and Coffman, 1989; Dongand Flavell, 2000; Dong, 2006). Several re-cent studies have identified another subset ofTH cells that produce a signature cytokine,IL-17 (Dong, 2006). First, costimulatory re-ceptor ICOS (Dong and Nurieva, 2003)and cytokine IL-23 (Murphy et al., 2003;Langrish et al., 2005) were found to selec-tively impact IL-17 expression in T cellsin autoimmune-disease models. Subsequently,Harrington et al. (2005) and Park et al. (2005)provided conclusive evidence to show that de-velopment of IL-17-expressing T cells fol-lowed a pathway that is distinct from TH1 andTH2 lineage development. Presence of the TH1cytokine IFN-γ and the TH2 cytokine IL-4inhibits IL-17 expression (Harrington et al.,2005; Park et al., 2005). Furthermore, T cellsdeficient in STAT1 and STAT6, mediators ofIFN-γ and IL-4 signaling, respectively,are more amenable to IL-17 production(Harrington et al., 2005). Several recentstudies have shown that TGF-β and IL-6play essential roles in the differentiation ofIL-17-expressing effector T cells (Bettelli

et al., 2006; Mangan et al., 2006; Veldhoenet al., 2006a), in which IL-23 plays a syner-gistic role (Yang et al., 2007). Moreover, re-cent results suggest that IL-2 signaling throughSTAT5 suppresses the development of TH17cells (Laurence et al., 2007). Additionally,proinflammatory cytokines TNF-α and IL-1β

have been reported to enhance this differ-entiation process, even though they are notabsolutely required for the TH17 differenti-ation (Sutton et al., 2006; Veldhoen et al.,2006a).

Several recent studies have provided criti-cal insights into the differentiation of mouseTH17 cells from naive CD4+ T cells (Dong,2006; Weaver et al., 2007). Unlike the murinesystem, the details regarding the developmentof IL-17-producing human CD4+ cells are rel-atively unclear and slowly emerging. A recentpaper demonstrated that naive human CD4+

can be differentiated into TH17 cells in thepresence of TGF-β, IL-6, IL-1β, and TNF-α,in the presence of blocking antibodies forIFN-γ and IL-4. Additionally, the human or-tholog of murine RORγt, a critical transcrip-tion factor driving TH17 differentiation inmice, is up-regulated in human TH17 cells(Acosta-Rodriguez et al., 2007). However, fur-ther studies are needed to define the preciserequirements for the development of humanTH17-lineage cells.

Even though IL-23 is critical for in vivoinduction and effector function of IL-17-expressing T cells, it is not essential in theinduction of IL-17-expressing cells in vitro.It has been suggested that IL-23 may playcritical role in the maintenance or expansionof differentiated TH17 cells (Langrish et al.,2005; Veldhoen et al., 2006b). Despite thesereports regarding cytokine requirements in thedevelopment of IL-17-producing T cells, thetranscriptional program that controls this dif-ferentiation remains to be investigated. Ivanovet al. (2006) have shown that nuclear orphanreceptor RORγt is restrictedly expressed inIL-17-producing T cells and is critical for theirdifferentiation; mice deficient in RORγt areresistant to autoimmune-disease development(Ivanov et al., 2006).

In addition to IL-17, IL-17F, and IL-22have been shown to be expressed in IL-17-expressing T cells, at least those generatedin vitro (Laan et al., 2001; Langrish et al.,2005; Liang et al., 2006). There are a lim-ited number of commercial reagents availablefor measuring these two cytokines, althougha mouse IL-22 ELISA kit was recently made

Cytokines andTheir CellularReceptors

6.25.7

Current Protocols in Immunology Supplement 79

available by Antigenix America (http://www.antigenix.com).

Critical ParametersThere are several factors that influence the

differentiation of IL-17-expressing T cells.Since TH1 and TH2 cytokines inhibit TH17cell differentiation, the blocking of IFN-γ,IL-4, and IL-12 effects is critical to promotingoptimal differentiation of IL-17-expressingT cells. Use of flow cytometrically sorted naiveCD4+ T cells (CD25–veCD62LhighCD44low) ispreferred over total CD4+ T cells, since the lat-ter population may contain some memory cellsthat already express IL-17. Since the presenceof TGF-β and IL-6 is critical for the develop-ment of the IL-17-expressing cell lineage, onemust ensure the biological activity of thesecytokines. Repeated freeze-thawing of the cy-tokines is not recommended, as it results in theloss of their activity. Additionally, the batch offetal bovine serum (FBS) should be carefullytested, because the quality of the serum hasa significant effect on naive T cell activationand differentiation. One should not use toomany T cells (>2 × 106/ml), since it is diffi-cult to block the effects of IFN-γ and IL-4. Ifblocking IFN-γ-producing cells becomes dif-ficult and percentage of IL-17-producing cellsis low, adding the blocking antibody to IL-12and IFN-γ on each day during the first 3 daysof culture should be considered, in order topromote optimal differentiation of TH17 cells.Once generated, IL-17-expressing T cells canbe maintained in low-dose IL-23 (20 ng/ml)for up to 2 weeks.

During the intracellular cytokine staininganalysis, costaining for IFNγ or IL-4 andIL-17 is highly recommended, since this pro-vides information regarding polarized differ-entiation with respect to competing TH1/TH2differentiation programs. Intracellular cyto-kine staining should be used in combinationwith ELISA-based detection of IL-17 in theculture supernatants. The quality and repro-ducibility of results for intracellular cytokinestaining is highly dependent on the quality ofreagents. Even though home-made reagentscan be used instead of the commercially avail-able kit, the quality of these reagents must beconfirmed using positive controls before theyare used in critical experiments.

Care must be taken in interpreting theELISA results and calculating the protein con-centrations. If the OD value of culture super-natants used for ELISA is beyond the standardcurve, the samples should be rediluted and re-

analyzed for more accurate determination ofIL-17 concentration. Repeated freeze-thawingof samples should be avoided, and samplesshould be stored frozen at −20◦C. More infor-mation and general troubleshooting informa-tion regarding ELISA is found in UNIT 2.1.

Anticipated ResultsTypical in vitro culture to generate TH17

cells results in a cell population containing20% to 40% IL-17-producing cells and <2%IFN-γ-producing cells. However, these resultsmay vary greatly depending on the geneticbackground of the mice from which the CD4+

T cells were purified. Even though IL-17-producing cells can be detected by day 4 afteractivation, the most optimal time to analyze isbetween day 5 and 7.

The amount of IL-17 detected in the cul-ture supernatants is highly variable and depen-dent on the cell density and culture conditions.Based on the protocol described in this unit,the amount of IL-17 detected has varied be-tween 1 and 10 ng/ml, and the lower limit ofdetection is in the range of 25 to 50 pg/ml.

Time ConsiderationsGeneration and analysis of IL-17-

expressing T cells usually take about 7 days.The intracellular staining protocol usuallytakes 8 to 12 hr. However, if desired, surface-stained cells fixed in paraformaldehydesolution can be stored at 4◦C for a fewdays before performing permeabilization andintracellular staining. The ELISA procedurecan be completed in 1 to 2 days, or it canbe adjusted, since coating and incubating thesamples can be done at 4◦C overnight.

Literature CitedAcosta-Rodriguez, E.V., Rivino, L., Geginat, J.,

Jarrossay, D., Gattorno, M., Lanzavecchia, A.,Sallusto, F., and Napolitani, G. 2007. Surfacephenotype and antigenic specificity of human in-terleukin 17–producing T helper memory cells.Nat. Immunol. 8:639-646.

Bettelli, E., Carrier, Y., Gao, W., Korn, T., Strom,T.B., Oukka, M., Weiner, H.L., and Kuchroo,V.K. 2006. Reciprocal developmental pathwaysfor the generation of pathogenic effector TH17and regulatory T cells. Nature 441:235-238.

Chang, S.H., Park, H., and Dong, C. 2006. Act1adaptor protein is an immediate and essentialsignaling component of interleukin-17 receptor.J. Biol. Chem. 281:35603-35607.

Dong, C. 2006. Diversification of T-helper-celllineages: Finding the family root of IL-17-producing cells. Nat. Rev. Immunol. 6:329-333.

Dong, C. and Flavell, R.A. 2000. Cell fate decision:T-helper 1 and 2 subsets in immune responses.Arthritis Res. 2:179-188.

Measurement ofInterleukin-17

6.25.8

Supplement 79 Current Protocols in Immunology

Dong, C. and Nurieva, R.I. 2003. Regulation ofimmune and autoimmune responses by ICOS.J. Autoimmun. 21:255-260.

Fossiez, F., Djossou, O., Chomarat, P., Flores-Romo, L., Ait-Yahia, S., Maat, C., Pin, J.J.,Garrone, P., Garcia, E., Saeland, S., Blanchard,D., Gaillard, C., Das Mahapatra, B., Rouvier, E.,Golstein, P., Banchereau, J., and Lebecque, S.1996. T cell interleukin-17 induces stromal cellsto produce proinflammatory and hematopoieticcytokines. J. Exp. Med. 183:2593-2603.

Happel, K.I., Dubin, P.J., Zheng, M., Ghilardi,N., Lockhart, C., Quinton, L.J., Odden, A.R.,Shellito, J.E., Bagby, G.J., Nelson, S., and Kolls,J.K. 2005. Divergent roles of IL-23 and IL-12in host defense against Klebsiella pneumoniae.J. Exp. Med. 202:761-769.

Harrington, L.E., Hatton, R.D., Mangan, P.R.,Turner, H., Murphy, T.L., Murphy, K.M., andWeaver, C.T. 2005. Interleukin 17-producingCD4+ effector T cells develop via a lineagedistinct from the T helper type 1 and 2 lineages.Nat. Immunol. 6:1123-1132.

Ivanov, I.I., McKenzie, B.S., Zhou, L., Tadokoro,C.E., Lepelley, A., Lafaille, J.J., Cua, D.J., andLittman, D.R. 2006. The orphan nuclear recep-tor RORγt directs the differentiation programof proinflammatory IL-17+ T helper cells. Cell126:1121-1133.

Iwakura, Y. and Ishigame, H. 2006. The IL-23/IL-17 axis in inflammation. J. Clin. Invest.116:1218-1222.

Kolls, J.K. and Linden, A. 2004. Interleukin-17family members and inflammation. Immunity21:467-476.

Laan, M., Lotvall, J., Chung, K.F., and Linden,A. 2001. IL-17-induced cytokine release in hu-man bronchial epithelial cells in vitro: Role ofmitogen-activated protein (MAP) kinases. Br. J.Pharmacol. 133:200-206.

Langowski, J.L., Zhang, X., Wu, L., Mattson, J.D.,Chen, T., Smith, K., Basham, B., McClanahan,T., Kastelein, R.A., and Oft, M. 2006. IL-23promotes tumour incidence and growth. Nature442:461-465.

Langrish, C.L., Chen, Y., Blumenschein, W.M.,Mattson, J., Basham, B., Sedgwick, J.D., Mc-Clanahan, T., Kastelein, R.A., and Cua, D.J.2005. IL-23 drives a pathogenic T cell popu-lation that induces autoimmune inflammation.J. Exp. Med. 201:233-240.

Lanzavecchia, A. and Sallusto, F. 2001. Regula-tion of T cell immunity by dendritic cells. Cell106:263-266.

Laurence, A., Tato, C.M., Davidson, T.S., Kanno,Y., Chen, Z., Yao, Z., Blank, R.B., Meylan, F.,Siegel, R., Hennighausen, L., Shevach, E.M.,and O’Shea, J.J. 2007. Interleukin-2 signalingvia STAT5 constrains T helper 17 cell genera-tion. Immunity 26:371-381.

Liang, S.C., Tan, X.Y., Luxenberg, D.P., Karim,R., Dunussi-Joannopoulos, K., Collins, M., andFouser, L.A. 2006. Interleukin (IL)-22 and IL-17are coexpressed by Th17 cells and cooperatively

enhance expression of antimicrobial peptides. J.Exp. Med. 203:2271-2279.

Mangan, P.R., Harrington, L.E., O’Quinn, D.B.,Helms, W.S., Bullard, D.C., Elson, C.O., Hatton,R.D., Wahl, S.M., Schoeb, T.R., and Weaver,C.T. 2006. Transforming growth factor-betainduces development of the T(H)17 lineage.Nature 441:231-234.

Mosmann, T.R. and Coffman, R.L. 1989. TH1 andTH2 cells: Different patterns of lymphokine se-cretion lead to different functional properties.Annu. Rev. Immunol. 7:145-173.

Murphy, C.A., Langrish, C.L., Chen, Y.,Blumenschein, W., McClanahan, T., Kastelein,R.A., Sedgwick, J.D., and Cua, D.J. 2003. Diver-gent pro- and antiinflammatory roles for IL-23and IL-12 in joint autoimmune inflammation.J. Exp. Med. 198:1951-1957.

Park, H., Li, Z., Yang, X.O., Chang, S.H., Nurieva,R., Wang, Y.H., Wang, Y., Hood, L., Zhu, Z.,Tian, Q., and Dong, C. 2005. A distinct lineageof CD4 T cells regulates tissue inflammation byproducing interleukin 17. Nat. Immunol. 6:1133-1141.

Schwandner, R., Yamaguchi, K., and Cao, Z. 2000.Requirement of tumor necrosis factor receptor-associated factor (TRAF)6 in interleukin 17 sig-nal transduction. J. Exp. Med. 191:1233-1240.

Sutton, C., Brereton, C., Keogh, B., Mills, K.H., andLavelle, E.C. 2006. A crucial role for interleukin(IL)-1 in the induction of IL-17-producingT cells that mediate autoimmune en-cephalomyelitis. J. Exp. Med. 203:1685-1691.

Tartour, E., Fossiez, F., Joyeux, I., Galinha, A.,Gey, A., Claret, E., Sastre-Garau, X., Couturier,J., Mosseri, V., Vives, V., Banchereau, J.,Fridman, W.H., Wijdenes, J., Lebecque, S.,and Sautes-Fridman, C. 1999. Interleukin 17, aT-cell-derived cytokine, promotes tumorigenic-ity of human cervical tumors in nude mice.Cancer Res. 59:3698-3704.

Veldhoen, M., Hocking, R.J., Atkins, C.J.,Locksley, R.M., and Stockinger, B. 2006a. TGF-beta in the context of an inflammatory cy-tokine milieu supports de novo differentiation ofIL-17-producing T cells. Immunity 24:179-189.

Veldhoen, M., Hocking, R.J., Flavell, R.A., andStockinger, B. 2006b. Signals mediated bytransforming growth factor-beta initiate autoim-mune encephalomyelitis, but chronic inflamma-tion is needed to sustain disease. Nat. Immunol.7:1151-1156.

Weaver, C.T., Harrington, L.E., Mangan, P.R.,Gavrieli, M., and Murphy, K.M. 2006. Th17:An effector CD4 T cell lineage with regulatoryT cell ties. Immunity 24:677-688.

Weaver, C.T., Hatton, R.D., Mangan, P.R., andHarrington, L.E. 2007. IL-17 family cytokinesand the expanding diversity of effector T Celllineages. Annu. Rev. Immunol. 25:821-852.

Yang, X.O., Panopoulos, A.D., Nurieva, R., Chang,S.H., Wang, D., Watowich, S.S., and Dong, C.2007. STAT3 regulates cytokine-mediated gen-eration of inflammatory helper T cells. J. Biol.Chem. 282:9358-9363.