doi:10.1182/blood-2012-01-407528Prepublished online April 27, 2012;2012 119: e151-e160   

 Lotta Oikari, Brigitta Stockinger, Harri Lähdesmäki and Riitta LahesmaaSoile Tuomela, Verna Salo, Subhash K. Tripathi, Zhi Chen, Kirsti Laurila, Bhawna Gupta, Tarmo Äijö, differentiationIdentification of early gene expression changes during human Th17 cell

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IMMUNOBIOLOGY

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Identification of early gene expression changes during human Th17 celldifferentiation*Soile Tuomela,1,2 *Verna Salo,1 *Subhash K. Tripathi,1,3 Zhi Chen,1 Kirsti Laurila,4,5 Bhawna Gupta,1 Tarmo Aijo,6

Lotta Oikari,1 Brigitta Stockinger,7 Harri Lahdesmaki,1,6 and Riitta Lahesmaa1

1Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, Finland; 2Turku Doctoral Programme of Biomedical Sciences, Turku,Finland; 3The National Graduate School in Informational and Structural Biology, Åbo Akademi University, Turku, Finland; 4Department of Signal Processing,Tampere University of Technology, Tampere, Finland; 5Department of Information and Service Economy, Aalto University School of Economics, Helsinki,Finland; 6Department of Information and Computer Science, Aalto University School of Science, Helsinki, Finland; and 7Division of Molecular Immunology,Medical Research Council National Institute for Medical Research, London, United Kingdom

Th17 cells play an essential role in thepathogenesis of autoimmune and inflam-matory diseases. Most of our current un-derstanding on Th17 cell differentiationrelies on studies carried out in mice,whereas the molecular mechanisms con-trolling human Th17 cell differentiationare less well defined. In this study, weidentified gene expression changes char-acterizing early stages of human Th17

cell differentiation through genome-widegene expression profiling. CD4� cells iso-lated from umbilical cord blood were usedto determine detailed kinetics of geneexpression after initiation of Th17 differen-tiation with IL1�, IL6, and TGF�. Thedifferential expression of selected candi-date genes was further validated at pro-tein level and analyzed for specificity ininitiation of Th17 compared with initiation

of other Th subsets, namely Th1, Th2, andiTreg. This first genome-wide profiling oftranscriptomics during the induction ofhuman Th17 differentiation provides astarting point for defining gene regula-tory networks and identifying new candi-dates regulating Th17 differentiation inhumans. (Blood. 2012;119(23):e151-e160)

Introduction

Naive CD4� T cells differentiate into functionally distinct lineagesin response to environmental cues and interaction with APCs. Thenature of invading pathogens determines the cytokine environmentin which the cognate Ag recognition by TCR takes place, subse-quently influencing the phenotype of differentiating CD4� Th cells.Classically, presentation of intra- or extracellular pathogens tonaive T cells leads to either a Th1 response or a Th2 response,respectively.1 Today, new functionally distinct subtypes of CD4� havebeen identified.2 Since the original identification of IL17-secretingT cells,3 further research has led to the definition of an effector Th17 celllineage.4-6 Shortly after these findings, Th17 cells were character-ized also in humans by using peripheral blood T cells and T-cellclones derived from gut tissue of patients having Crohndisease.7-9 Human Th17 cells express CCR6, CCR4, IL23R, andCD161 on their cell membrane.9,10 The characteristic cytokinesecreted by these cells is IL17A (also referred to as IL17).IL17A stimulates the secretion of wide range of proinflammatorychemokines and cytokines. As its receptor is widely expressed,many cells of the immune system as well as other cell types canrespond to it.11 In addition to IL17A, cytokines IL17F, IFN�, IL22,and IL26 have been shown to be secreted by human Th17 cells.7

Proper function of Th17 cells is needed for eradication of extracel-lular bacterial and fungal infections.11

CD4� cells isolated from peripheral or cord blood have beenused to examine Th17 polarization in human. In several studies,

differentiation of naive cells from peripheral blood has notsucceeded, or the IL17A production has been markedly lessefficient than detected by memory cells. IL17A secretion ofpolarized cord blood cells is also modest.12 Human Th17 cells havealso been shown to originate from CD161� precursor cells,10 whichrepresent a small percentage of CD4� cells present in cord blood.Recently, it has been shown that the expression of CD161 isdevelopmentally regulated, and Th17 cells can differentiate fromCD161� cells of preterm infants.13 Based on the early findings itseemed that the cytokine requirements of human and mouseTh17 cells are fundamentally different. For example, while initiallythe species specific role of TGF� in the process was activelydiscussed, it is becoming widely accepted that the human andmouse Th17 cell development is dependent on similar factors.Nevertheless, there is still an ongoing debate about the combina-tions of cytokines and cell activation needed for driving theprocess. TGF�, IL23, IL1�, and IL6 are most often reported to beeffective in in vitro differentiation of CD4� cells towardTh17 phenotype at various concentrations and in combination withother cytokines.12 In addition, prostaglandin E2 and aryl hydrocar-bon receptor ligands have been shown to modulate the process.14,15

To summarize, differentiation and function of Th17 cells is con-trolled by a complex interplay of various factors playing a role atthe distinct stages of the process and regulating different character-istics of cells secreting IL17A. To improve the understanding of

Submitted January 27, 2012; accepted April 17, 2012. Prepublished online asBlood First Edition paper, April 27, 2012; DOI 10.1182/blood-2012-01-407528.

*S.T., V.S., and S.K.T. contributed equally to this study.

This article contains a data supplement.

The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is herebymarked ‘‘advertisement’’ in accordance with 18 USC section 1734.

© 2012 by The American Society of Hematology

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Th17 differentiation as a whole, it would be important to movefrom a factor- or a gene-centric view to a holistic one revealingpathways and gene regulatory pathways controlling the process.

The aim of the present study was to identify new candidategenes to be tested for their role in regulating Th17 cell polarization.Th17 cells have been extensively studied by using mouse differen-tiation and disease models. As there is a clear link between manyautoimmune diseases and IL17A similarly in humans, it is crucialto identify the factors controlling differentiation and function ofTh17 subset. Interestingly, the dysregulation of Th17 responses isassociated with certain forms of asthma, which is classicallythought to be mediated by Th2 cells. Th17 cells have also beenconnected to regulation of immune reactions against cancer cells.16

In this study, by using CD4� T cells isolated from human umbilicalcord blood, we report, for the first time, genome-wide profiling ofgene expression throughout human Th17 cell priming. We demon-strate that gene expression at the very early stage of human Th17differentiation is highly dynamic. We further validated the differen-tial expression of the selected genes at the protein level andanalyzed their expression in Th1, Th2, and inducible regulatoryT cell (iTreg) Th subsets. The results indicate that KDSR, ATP1B1,CXCR5, and IL2RB are selectively regulated in Th17 conditionduring the early priming toward Th17 phenotype. On the otherhand, CD52, VDR, and CTSL1 were highly regulated also inresponse to initiation of some of the other Th programs. Insummary, our study not only provides an overview of genes andpathways regulated in response to induction of Th17 differentiationin humans, but also provides several candidates for modulation ofTh17 responses, which will be of interest for further studies.

Methods

Human CD4� T-cell isolation and culture

CD4� T cells were purified from umbilical cord blood of healthy neonates(Turku University Central Hospital, Turku, Finland). Mononuclear cellswere isolated (Ficoll-Paque PLUS; GE Healthcare) after which CD4� cellswere collected (Dynal CD4 Positive Isolation Kit; Invitrogen). Cells wereactivated with plate-bound anti-CD3 (750 ng/24-well culture plate well;Immunotech) and soluble anti-CD28 (1 �g/mL; Immunotech) in a densityof 0.5 � 106 cells/mL of X-vivo 20 serum-free medium (Lonza) supple-mented with 2mM L-glutamine (Sigma-Aldrich), and 50 U/mL penicillinand 50 �g/mL streptomycin (Sigma-Aldrich) and cultured at 37°C in 5%CO2. Th17 polarization was initiated with IL6 (20 ng/mL; Roche), IL1�(10 ng/mL) and TGF� (10 ng/mL) in the presence of neutralizing anti-IFN�(1 �g/mL) and anti-IL4 (1 �g/mL). Control Th0 cell were cultured in amedium containing only the neutralizing Abs. Th1, Th2, and iTregdifferentiation was initiated with IL12 (2.5 ng/mL), IL4 (10 ng/mL), andTGF� (10 ng/mL), respectively. All cytokines and neutralizing Abs werefrom R&D Systems unless otherwise stated. Comparisons between thedifferent polarization conditions were always done with the same pool ofaliquoted cells. The usage of blood of unknown donors was approved by theFinnish Ethics Committee.

Transcriptional profiling

Samples were collected at 0-, 0.5-, 1-, 2-, 4-, 6-, 12-, 24-, 48-, and 72-hourtime points of culture. Total RNA from 3 cultures was DNase treated(RNase-Free Dnase Set; QIAGEN) during the isolation (RNeasy Kit;QIAGEN), processed, and hybridized on Illumina Sentrix HumanHT-12Expression BeadChip Version 3.

The microarray data were analyzed using a Bioconductor packagebeadarray. The time-series microarray data were filtered by choosing toanalyze only the probes with detection P values � .05 at least in 1 time pointat 1 cell type. In addition, only the probes having an SD � 0.15 over all the

samples were used in the analysis. The data were normalized using quantilenormalization. For each time point, differentially expressed genes wereidentified using the Bioconductor limma package with moderated t-statisticwith false discovery rate (FDR) � 0.1. Annotations of genes were gatheredusing Ingenuity Pathways Analysis (Ingenuity Systems, www.ingenuity.com). Clusters were identified using k-means clustering.

Quantitative RT-PCR

RNA was isolated (RNeasy Mini Kit; QIAGEN) and treated in-column withDNase (RNase-Free Dnase Set; QIAGEN) for 15 minutes. The removal ofgenomic DNA was ascertained by treating the samples with DNaseI (Invitrogen) before cDNA synthesis either with SuperScript II ReverseTranscriptase (Invitrogen) or with Transcriptor First Strand cDNA Synthe-sis Kit (Roche). Quantitative RT-PCR (qPCR) was performed usingUniversal ProbeLibrary probes (Roche Applied Science) or with FAM(reporter), TAMRA (quencher) double-labeled probes in a 10-�L reactionvolume. Reaction mix used was Absolute QPCR ROX Mix (ThermoScientific) and amplification was monitored with Applied Biosystems7900HT Fast Real-Time PCR System (15 minutes enzyme activation and40 cycles of 15 seconds 95°C, 1 minute 60°C). The cycle threshold (Ct)values of the transcripts studied were normalized against the signal acquiredwith EF1�.17 The primers and probes are listed in supplemental Table 1 (seethe Supplemental Materials link at the top of the article).

Western blotting

Samples were lysed in Triton-X sample buffer (50mM Tris-HCl, pH7.5; 150mM NaCl; 0.5% Triton-X-100; 5% glycerol; 1% SDS), containingproteinase (Roche) and phosphate inhibitors (Roche) and sonicated (Biorup-tor UCD-200; Diagenode). Sonicated samples were centrifuged at maxi-mum speed for 20 minutes at 4°C and supernatants were collected. Sampleswere quantified (DC Protein Assay; Bio-Rad) and boiled with 6� loadingdye (330mM Tris-HCl, pH 6.8; 330mM SDS; 6% �-ME; 170�M bromophe-nol blue; 30% glycerol). Samples were loaded on 10% or 12% SDS-PAGEgels, transferred to nitrocellulose membranes, and probed with Abs listed insupplemental Table 2.

Flow cytometry

CCR6 detection was done at day 3. Cells were washed with PBS andstaining was done in 0.5% FBS/0.1% atzide/PBS for 15 minutes at 4°C.Cells were fixed with 1% paraformaldehyde, and an LSRII flow cytometer(BD Biosciences) was used in data acquisition. Living cells were gated foranalysis based on forward and side scattering. CD52, IL2RB, and CXCR5detections were done as CCR6 detection except the Ab incubation lasted30 minutes. Cells were fixed with 4% paraformaldehyde before analysis ofITM2A, and in addition permeabilized with methanol before analysis ofLMNA. Thirty-minute incubation with primary Ab was followed with15-minute incubations with secondary Ab if needed. The stainings werecontrolled with isotype Ab or by staining the cells only with the secondaryAb. The Abs used are listed in supplemental Table 2.

IL17A secretion

Differentiation of CD4� T cells toward the Th17 phenotype was character-ized by detection of IL17A production in cell-culture supernatant using theMillipore Human Cytokine/Chemokine, 96-Well Plate Assay on day3. IL17A secretion was normalized with the number of living cells detectedbased on forward and side scattering in flow cytometric analysis (LSRIIflow cytometer; BD Biosciences).

Ethical aspects

The usage of blood of unknown donors was approved by the EthicsCommittee of the Hospital District of Southwest Finland.

Accession numbers

Microarray data can be found at the NCBI Gene Expression Omnibus withaccession number GSE35103.

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Results

Genome-wide transcriptional regulation at the early stage ofhuman Th17 differentiation

Because the essential role of Th17 cells in the pathogenesis ofinflammation, autoimmunity, and cancer has been well documentedby several studies in recent years,16 it has been of great interest todefine the molecular mechanisms involved in the fate decision ofnaive CD4� T cells to acquire the highly inflammed Th17 pheno-type. To address this question, we performed a genome-wide geneexpression profiling of in vitro–cultured human CD4� cells duringthe initiation of Th17 polarization. First, we evaluated the polariza-tion conditions by analyzing IL17A secretion (Figure 1A), IL17F,RORC, and CCR6 expression (Figure 1B-C). The up-regulation ofthese marker genes showed that the cell population was success-fully biased toward Th17 phenotype. We were interested in theinitiation of Th17 differentiation and hence focused our study onthe early changes in transcriptomics during human Th17 polariza-tion. CD4� T cells isolated from umbilical cord blood wereactivated with anti-CD3 and anti-CD28 in the presence of acytokine cocktail containing IL1�, IL6, and TGF� as well asneutralizing Abs against IL4 and IFN�. The samples for microarrayanalysis were collected at 9 time points during the culture startingfrom 0.5 hours to 72 hours (Figure 1D).

The overall gene expression changes revealed 2 waves oftranscriptional changes in response to the Th17 stimulation usedcompared with TCR activation alone; the first one took placewithin the first 4 hours of culture, and the second one started after6 hours (Figure 2). The selected time points captured the earliestchanges in the overall transcriptome, because there were only veryfew genes differentially expressed at the earliest 0.5-hour timepoint after which the number of changes increased (supplemental

Table 3). After 1 hour of stimulation, 28 probes representing25 genes were already differentially expressed in the cells culturedunder the Th17 polarizing condition compared with the cellscultured in the Th0 condition (Figure 1D). The first peak of changesof gene expression was observed after 2 hours of culture,256 probes representing 228 genes were differentially expressedduring the maximal primary transcriptional response after theinitiation of polarization. Among the differentially expressedgenes, most genes were up-regulated, suggesting many signalingpathways to be turned on in response to the stimulation by theTh17 polarization inducing cytokine cocktail. From 6 hours to72 hours, the number of differentially expressed genes increased,resulting in up to 799 differentially expressed probes at the 72-hourtime point (Figure 2). Within them, 416 probes were up-regulated

Figure 1. In vitro differentiation of cord blood CD4� cells for Th17 gene expression profiling. (A) IL17A secretion after 72 hours of culture. The detection was donedirectly from the culture supernatant with fluorescent beads. The values are normalized with the number of living cells determined based on cell size and granularity detectedwith flow cytometer. The data shown are the average of 6 cultures with the SEM and statistical significance was determined with the Student t test. (B) The expression of IL17Fand RORC in the cells cultured toward Th17 phenotype or left as control (Th0). The data are presented as fold change over the expression level in Th0 control cells at 24 hours.The data show the average expressions and the SEM. Statistical significance of the results has been determined with the Student t test. (C) Representative CCR6 expressionafter 72 hours of culturing. (D) Schematic presentation of the microarray profiling showing the culturing conditions used and the sampling time points.

Figure 2. The genes regulated during the early stages of human Th17differentiation. Three biologic replicates of time-series data of Th17 polarized orcontrol cord blood CD4� cells were hybridized to Illumina Sentrix Human HT-12Expression Version 3 BeadChips. The differential expression analysis was performedfor the probes having a detection P � .05 at least in 1 time point at 1 cell type andSD � 0.15 over all the samples. The differentially expressed genes between Th17and Th0 conditions were identified with the false discovery rate (� 0.1). Thedifferentially expressed probes are classified according to the appearance of thedifference.

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and 383 were down-regulated, suggesting that Th17 polarization isregulated both by actively up-regulating the signaling pathwaysdriving the differentiation and by shutting down the interferinggene expression.

Dynamic changes of gene expression during the early stage ofhuman Th17 cell differentiation

Interestingly and importantly, a considerable number of the differ-entially expressed genes were enzymes, kinases, and transcriptionregulators. Statistical enrichment of cytokines (Table 1) andlocalization of the differentially expressed genes preferentially onthe cell surface or on extracellular space (Table 2) indicate that,even at the early stages during the polarization process, cells start tofocus on activating the pathways needed for signaling with theneighboring cells. Nevertheless, acquisition of fully competentphenotype continues actively beyond the first 3 days of polariza-tion as evidenced by the appearance of new differentially expressedgenes at all time points studied (Figure 2).

CD4� cells vigorously regulate the gene expression patternduring the early polarization toward Th17 differentiation. This isclearly demonstrated by following the gene expression throughoutthe selected timeframe; for example, most of the genes differen-tially expressed at the 2-hour time point are significantly differen-tially regulated only at that time point. Similarly, there are manydifferentially expressed genes only regulated at the 72-hour timepoint (supplemental Table 3). The expression pattern of the geneswith the highest signal log-ratio between Th17 and Th0 culturingcondition at 2 and 72 hours are visualized in Figure 3A and B,respectively.Among the differentially expressed genes, there are severalgenes known to be preferentially expressed in Th17 cells, such as CCR6,IL17F, RORC, RORA, IL9, IL23R, BATF, and VDR9,18-21 which

validates the cultures and the analysis done in this study. Of note,the differential expression of IL17A is not detectable with the probeon the microarray used while its expression was clearly up-regulated in response to Th17-inducing cytokines in our sampleswhen measured with other methods. Most importantly, our microar-ray study also revealed novel genes in this context.

Next, we clustered the genes identified to be differentiallyexpressed between our Th17 and Th0 culturing conditions, basedon their gene expression difference to the freshly isolated unacti-vated CD4� T cells (Figure 4, supplemental Table 4). The cluster-ing reveals 3 main expression patterns: up-regulation, down-regulation, and somewhat steady expression throughout the analyzedtimeframe after exposure to the Th17-polarizing cytokines. Inaddition to these predominant patterns, the level of expressioncategorizes the genes into different clusters. Compared with thedifferential expression between Th17 and Th0 control cells (Figure3, supplemental Table 3), the regulation in Th17 cells comparedwith the freshly isolated CD4� cells is more straightforward. Onaverage, there is only some fluctuation in expression during thevery first time points and after this, the gene expression follows theselected direction. This indicates that the differential expression inTh17 polarized cells compared with Th0 cells is not only becauseof either active up- or down-regulation of the genes, but alsocounterregulation to the stimulus caused by activation withoutpolarizing cytokines.

Validation of selected differentially expressed genes

We selected some of the genes with the highest and long-lastinggene expression difference between Th17 and Th0 conditions to bestudied further at protein level (supplemental Figure 1). Previouslyknown function of the genes was also used as a criterion in

Table 1. Functional classification of the differentially expressed genes

0.5 h 1 h 2 h 4 h 6 h 12 h 24 h 48 h 72 h

Cytokine 0 0 1 1 0 3 9* 12* 18*

Enzyme 1 4 43 20 2 10 34 52 120

G-protein coupled receptor 0 0 3 4 1 4 7 12* 11

Growth factor 0 0 0 1 0 0 0 0 1

Ion channel 0 0 1 0 0 1 2 4 5

Kinase 0 0 17 9 2 12 16 14 34

Ligand-dependent nuclear receptor 0 0 1 0 1 2 2 3 4

MicroRNA 0 0 0 0 0 0 0 0 0

Other 1 13 107 62 13 57 110 159 370

Peptidase 0 0 6 2 3 6 10 13 19

Phosphatase 0 0 4 0 0 0 5 5 15

Transcription regulator 1 5 27 15 6 7 11 24 52

Translation regulator 0 0 0 1 0 0 0 2 4

Transmembrane receptor 0 0 3 6 3 3 7 7 10

Transporter 0 3 12 7 0 7 9 18 29

The number of unannotated probes: 25.*Enrichment of the functional class (false discovery rate � 0.05).

Table 2. Location of the differentially expressed genes

0.5 h 1 h 2 h 4 h 6 h 12 h 24 h 48 h 72 h

Extracellular space 0 1 5 3 2 11* 20* 30* 51*

Plasma membrane 0 3 31 24* 6 31* 49* 72* 95*

Cytoplasm 1 8 74 38 11 34 70 100 242

Nucleus 1 10 56 29 7 16 41 51 148

Unknown 1 3 59 34 5 20 42 72 156

The number of unannotated probes: 25.*Enrichment of the cellular compartment (false discovery rate � 0.05).

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selecting the targets for validation. Western blotting results showedthat BASP1, CTSL1, RUNX1, BATF, KDSR, NOTCH1, VDR, andATP1B1 were highly expressed in Th17 cells compared with their

expression in Th0 cells at various time points during the first 3 daysof polarization (Figure 5A). CXCR5, IL2RB, and LMNA wereup-regulated in CD4� T cells cultured under Th17-polarizing

Figure 3. Dynamic regulation of the genes in cord blood CD4� cells during Th17 differentiation. (A) Heatmap presentation of the expression kinetics of the differentiallyexpressed genes between Th17 and Th0 control cells at 2 hours. The genes with signal log ratio � 1 or � �1 are presented. (B) Heatmap presentation of the expressionkinetics of the differentially expressed genes at 72 hours. Signal log ratio � 1.5 or � �1.5 was used as cutoff. Gene up-regulation in Th17 culturing condition is shown with red,and down-regulation with blue as indicated with the scales below the heatmaps.

Figure 4. Gene expression profiles of the identified Th17 differentiation–associated genes. The differentially regulated genes between Th17 polarized and Th0 controlcord blood CD4� cells were clustered based on their expression profile during Th17 polarization over the analyzed timeframe. For each gene, the probe found to bedifferentially expressed for the first time was used in the analysis. The number of genes belonging to each cluster is marked to the figure. Clustering was done with the k-meansmethod. Gene expression patterns were drawn by using cluster center values.

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conditions compared with Th0 cells in our microarray study, andthis difference is reflected to protein level (Figure 5B). On the otherhand, the flow cytometric detection of CD52 and ITM2A at 48 and72 hours showed down-regulation of these 2 proteins in CD4�

T cells cultured under a Th17-polarizing condition compared withcells cultured under a Th0 condition (Figure 5B), which is alsoconsistent with the patterns seen in the microarray data. Themicroarray data from this study showed the up-regulation ofCOL6A3 and MIAT genes, and down-regulation of the BTBD11gene in Th17-polarizing cells. These findings were confirmedwith RT-PCR detections at 24-, 48-, and 72-hour time points(Figure 5C).

Selective expression of genes during Th-subset differentiation

To further characterize the selectivity of the differentially ex-pressed genes in the Th17-culturing condition, cord blood CD4�

T cells were cultured under Th0, Th1, Th2, iTreg, or Th17conditions. Cells were harvested at 72 hours and the expression ofselected proteins was analyzed with Western blotting or flowcytometry. As shown in Figure 6A, the expression of TBX21,GATA3, and FOXP3 the key transcription factors for Th1, Th2, oriTreg differentiation were detected preferentially expressed in thecorresponding subset of helper T cells. IL17A was also selectivelyexpressed in the cells polarized to Th17 direction (Figure 6B).

Figure 5. Validation of the selected genes. (A) Representative Western blot detection of RUNX1, CTSL1, BATF, BASP1, KDSR, NOTCH1, VDR, and ATP1B1 at 0, 4, 12, 24, 48, and72 hour time points after culturing of cord blood CD4� cells in Th17-polarizing medium or Th0 control condition. Histone H2B was analyzed to confirm equal loading. (B) Flow cytometricdetection of CD52, IL2RB, CXCR5, LMNA, and ITM2A after culturing for 48 and 72 hours. Analysis of the stained cells was done with an LSRII flow cytometer (BD Biosciences).(C) RT-PCR detection of MIAT, BTBD11, and COL6A3 at 24-, 48-, and 72-hour time points. EF1� was used as an endogenous normalization control. Fold changes were calculated bycomparing the normalized expression values to the corresponding expression in Th0 control samples at the 24-hour time point. SEM, statistical significance were determined with Studentt test, and the average fold change is shown in the figure.

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Based on the kinetic profiling study, the expression of KDSR,CTSL1, ATP1B1, BASP1, NOTCH1, VDR, RUNX1, IL2RB, andCXCR5 was increased after Th17 polarization compared with thecells cultured under a Th0 condition. In contrast, the expression ofCD52 was reduced in the cells cultured under a Th17 conditioncompared with those cultured in a Th0 condition. Consistent withthe findings from the microarray study as well as the validation(Figure 5A-B), ATP1B1, KDSR, IL2RB, and CXCR5 were ex-pressed at a higher level in the cells cultured under a Th17condition compared with Th0 cells. In addition, the expression ofthese proteins was higher after Th17 polarization than after Th1,Th2, or iTreg induction (Figure 6C-D). However, although theexpression of VDR was higher in cells under Th17-polarizingconditions than in Th0 cells, which was consistent with the resultsfrom the microarray study, it was also highly expressed in the cellscultured toward the iTreg (Figure 6C) phenotype. Again, thedetection of CTSL1 confirmed the result from the gene profilingstudy, but the highest expression of CTSL1 was detected in thecells cultured under the iTreg condition (Figure 6C). The down-

regulation of the expression of CD52 by a Th17-culturing conditionwas observed in the gene expression profiling study. However, theexpression of CD52 was found to be lowest in Th2 cells (Figure6D). Clear cell-type specificity could not be determined forBASP1, RUNX1, or NOTCH1 (data not shown). In summary,further analysis revealed that some of the identified genes regulatedduring Th17 polarization are selectively expressed during thedifferentiation of this particular lineage. However, some of thegenes were not specific for Th17 cells as the differential expressionwas also observed in other Th subset–polarizing conditions.

Discussion

Th17 cells play essential roles in the pathogenesis of both autoim-mune and allergic inflammatory diseases. There is also accumula-tive evidence linking Th17 cells to cancer biology. In the past fewyears, studies focusing on this subset of Th cells have thus drawngreat attention in the field of immunology. However, compared

Figure 6. Expression of the validated proteins throughout Th-cell subsets. CD4� T cells isolated from umbilical cord blood were activated with plate-bound anti-CD3 andsoluble anti-CD28. The only activated cells cultured with neutralizing anti-IL4 and anti-IFN� were used as Th0 control. Cells were stimulated with IL12 (2.5 ng/mL) for initiationof Th1, IL4 (10 ng/mL) for Th2, TGF� (10 ng/mL) for iTreg, and IL1� (10 ng/mL) � IL6 (20 ng/mL) � TGF� (10 ng/mL) � anti-IL4 (1 �g/mL) � anti-IFN� (1 �g/mL) for Th17differentiation. Cells were harvested after 72 hours of culture. (A) Polarization toward different Th-cell subtypes was examined by analyzing the expression of key transcriptionfactors of each subtype; TBX21 for Th1, GATA3 for Th2, and FOXP3 for iTreg cells with Western blotting. (B) Cytokine secretion was used to validate the selective expression ofIL17A in the cells polarized toward the Th17 phenotype. The representative result from 2 experiments is shown. (C) The expression of CTSL1, ATP1B1, KDSR, and VDR wasanalyzed with Western blotting. (D) CD52, IL2RB, and CXCR5 were detected with flow cytometry. GAPDH detection was used as a loading control in Western blotting analysis.

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with the comprehensive studies carried out in mouse models, thestudies on molecular mechanisms and signaling pathways involvedin regulating human Th17 differentiation from naive CD4� T-cellprogenitors have not been as thorough. The strength of our study isthe usage of CD4� T cells isolated from umbilical cord blood ofnewborn babies. As the majority of these cells are naive T cells,cord blood is the best material available for large profiling studiesdone with human primary cells without prior expansion or cloning.Of note, there is a possibility, albeit remote, that non-naive Th17precursors present in small numbers in cord blood could influencethe results. To capture and characterize the upstream molecularmechanisms of Th17 fate decision, we followed the detailed kineticchanges of gene expression starting from 0.5 hours to 72 hoursafter the cells were exposed to TGF�, IL6, and IL1�. We identifiedthe immediate target genes responding to Th17-polarizing stimula-tion peaking at 2 hours. More and more genes were regulated after24 hours, indicating increasing deviation of the Th17 polarizedlineage from the control cells and accumulation of primary andsecondary target genes of the polarizing cytokines. As such, thepresented dataset is the first comprehensive, genome-wide transcrip-tomics study of the first steps of human Th17 differentiation.

Comparing the selectively expressed genes in Th17 cells fromour study with the published studies done with mouse cellsindicates that a large amount of genes are similarly regulated inboth species, for example, IL23R, RORA, BATF, RUNX1, andVDR.9,18-21 This indicates that the studies characterizing the func-tion of these genes done with mouse models are likely to berelevant and applicable in medical research. However, findingsshould be validated in both systems and extrapolation directly fromone organism to another should be carefully considered.22 This isclearly a challenge because of genetic variation and restrictedavailability of human cells compared with cells from inbred modelorganisms. An additional level of intricacy arises from complexinteractions of cytokines promoting Th17 cell development. Theknown cytokines promoting Th17 cell differentiation provide allspecific features to a differentiating cell. Single-cell analysis havealso shown that cells expressing IL17A are heterogeneous for theircytokine expression profile and cytokines associated with Th17 cellscan be secreted without IL17A. An additional level of complexitycomes from the fact that Th-cell differentiation is known to beflexible leading to plasticity of the acquired phenotype.2 Although alot of effort has been put on identification of an optimal cytokinecocktail for in vitro differentiation of human Th17 cells, theconditions needed for robust IL17A secretion remain elusive.12

Despite challenges, identification of genes regulated during humanTh17 priming is an important step needed to understand, and to beable to modulate, this convoluted process in humans.

In this study, profiling of human Th17 differentiation revealed� 1000 probes differentially expressed in response to the Th17culture condition compared with the Th0 control condition. Inaddition, we took a step forward in identifying new candidate genesputatively important for Th17 differentiation by validating thedifferential expression of the selected genes at the protein level. Weshow that the expression of transcription regulators BASP1,NOTCH1, RUNX1, and VDR is increased after TGF�, IL6, andIL1� stimulation in cord blood CD4� cells. The function of BASP1in T cells is not known, but there are indications that it might beinvolved in modulation of the transcriptional program during T-cellapoptosis.23 In contrast to BASP1, the role of NOTCH1, RUNX1,and VDR in T cells has been intensively studied. Recently, it hasbeen reported that NOTCH1, along with HIF-1, controls humanTh17 cell survival and apoptosis.24 It has also been shown that

inhibition of Notch signaling directly, and indirectly via regulationof RORC, turns down the production of IL17A.25 RUNX1 interactswith FOXP3 and RORC, and is needed for Treg and Th17 cellfunction, respectively.26,27 Calcitriol-activated VDR constrainsIL17A transcription by inhibiting NFAT, recruiting HDAC, andsequestrating RUNX1.28 We showed that BTBD11 is down-regulated during Th17 polarization. BTBD11 is a retinoic acid–inducible gene in the neuroblastoma cell line.29 As retinoic acidinhibits TGF�- and IL6-driven Th17 cell differentiation andpromotes induction of regulatory T cells,30 BTBD11 may be anovel transcriptional regulator determining the selection betweenTh17 and iTreg lineages.

In addition, we validated the up-regulation of membraneproteins ATP1B1, IL2RB, and CXCR5, and down-regulation ofITM2A and CD52 in Th17 cells compared with control cells. TheATP1B1 subunit regulates the assembly of the Na�/K�-ATPaseprotein pump, and eventually the number of functional proteincomplexes on the plasma membrane. Interestingly, apart from itsclassic function, ATP1B1 has been shown to interact with the E2Atranscription factor regulating its nuclear localization.31 In epithe-lial cells, Na�/K�-ATPase subunits have been shown to play a rolein regulation of tight junctions, cell polarity, actin dynamics, cellmovement, and cell signaling.32 It remains to be shown whethersimilar atypical functions can also be found in T cells. IL2RB isinstead one of the 3 chains forming a receptor for IL2. It is also apart of IL15 receptor complex. Interestingly, IL15 has been shownto increase IL17A production by the T-cell lines derived fromsynovial fluid of rheumatoid arthritis patients.33 CXCR5 is one ofthe markers of the newly identified lineage of follicular helperT cells providing help for B cells and adaptive immunity.34

CXCR5� cells in human blood contain heterogeneously differentTh-cell subsets capable of producing Th1, Th2, and Th17 markercytokines. In the systemic autoimmune disease juvenile dermato-myositis, CXCR5� Th cells were biased toward Th2 and Th17phenotypes and this correlated with disease activity.35 It has beenshown that activated CD4� T cells can express CXCR5,36 butimportantly our data show that during in vitro polarization towardthe Th17 phenotype, cord blood Th cells are skewed to up-regulateCXCR5. ITM2A is a membrane protein suggested to regulatechondrocyte differentiation.37 In T cells, ITM2A expression isinduced by activation and it down-regulates CD8 expression duringpositive selection.38 Alemtuzumab, also known as Campath-1, anAb specifically recognizing CD52, has been extensively studiedand used in cancer therapy. Recently, this humanized Ab has alsobeen examined for treatment of multiple sclerosis patients.39 Thereare indications that CD52 is involved in costimulation of T cellsand induces the differentiation of Treg cells.40 However, precisefunction of this glycoprotein, mainly expressed on mature T andB lymphocytes, remains obscure.

Enzymes CTSL1 and KDSR were found to be up-regulated inTh17 cells. CTSL1 is a ubiquitously expressed protease, which hasbeen linked to the regulation of immune responses at the level ofMHC complex maturation and Ag presentation influencing differ-entiation of CD4� cells and autoimmune reactions.41 Recent stud-ies have shown that CTSL1 also has T cell–specific functions;cathepsin L inhibition prevents the cytotoxic CD8� T-cell responsein autoimmune diabetes NOD mice.42 We found 3-ketodihydrosph-ingosine reductase, KDSR, to be up-regulated already after an hourof culture in the Th17-polarizing condition. KDSR regulatessphingolipid biosynthesis by reducing 3-ketodihydrosphingosine todihydrosphingosine.43 Sphingolipids are expressed on the plasma

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membranes ubiquitously, and are involved in structural organiza-tion of lipid rafts, regulation of apoptosis, differentiation, andproliferation. The role of the T-cell sphingolipid metabolism on theTh17 differentiation or function has not been explored. Interest-ingly though, altered sphingolipid metabolism is associated withpathogenesis of multiple sclerosis,44 and externally added sphingo-lipids augment Th17 differentiation.45

We also validated the Th17 cell–associated up-regulation oflong noncoding RNA MIAT, mRNA of structural protein COL6A3encoding -3 chain of the type VI collagen, and LMNA which isone of the nuclear lamina proteins. Previous studies have indicatedthat MIAT plays a role in the development of the nervous system,46

retinal cells,47 and regulation of pluripotency,48 but so far, there hasnot been implication on its function in the immune system cells.COL6A3 is an important organizer of the extracellular matrixproteins. Mutations in the type VI collagen genes are associatedwith Bethlem myopathy and Ullrich congenital muscular dystro-phy. In humans, COL6A3 expression is increased with body massindex and it contributes to adipose tissue inflammation.49 Muta-tions in LMNA lead to severe laminopathies. Lmna�/� mice displayreduced thymus and spleen size, but so far no cell-intrinsic immunedefect related to this gene has been found.50

The present study was carried out by using primary CD4�

T cells isolated from cord blood. The dataset describing thedetailed kinetic changes at a very early stage of human Th17differentiation provides a valuable resource for characterization ofa variety of molecular mechanisms and signal networks involved inpolarization of human Th17 cells. Although further functionalanalysis is needed to show whether the candidate genes identifiedin this study play a specific role in the Th17 differentiation processand function, the study provides a valuable starting point foridentifying new pharmaceutical targets possibly regulating theprocess. This knowledge is essential for tackling the Th17 cell–mediated human diseases.

Acknowledgments

The authors thank all voluntary blood donors and the personnel ofthe Department of Obstetrics and Gynaecology, Maternity Ward,Turku University Hospital (Hospital District of Southwest Finland)for the cord blood collection. Sumedha Gattani and Omid Rasoolare acknowledged for their technical help. Microarray hybridiza-tions were done at Finnish Microarray and Sequencing Center,Turku, Finland.

This work was supported by the Academy of Finland (Center ofExcellence in Molecular Systems Immunology and PhysiologyResearch, 2012-2017, Decision no. 250114, and grants 207490SYSBIO, 116639, 115939, 140019), the European CommissionSeventh Framework grants (EC-FP7-SYBILLA-201106, EC-FP7-NANOMMUNE-214281 and EC-FP7-DIABIMMUNE-202063),the JDRF, the Sigrid Juselius Foundation, a Turku UniversityHospital grant, and the Turku University Foundation.

Authorship

Contribution: S.T., Z.C., B.S., H.L., and R.L. designed research;V.S. and B.G. set up Th17 culturing; S.T., V.S., S.K.T., B.G., andL.O. performed experiments; S.T., V.S., S.K.T., Z.C., K.L., T.A.,H.L., and R.L. analyzed and interpreted data; B.S. providedexpertise and guidance; and S.T., Z.C., and R.L. wrote themanuscript.

Conflict-of-interest disclosure: The authors declare no compet-ing financial interests.

Correspondence: Riitta Lahesmaa, Turku Centre for Biotechnol-ogy, University of Turku and Åbo Akademi University, PO Box123, FIN-20521 Turku, Finland; e-mail: riitta.lahesmaa@btk.fi.

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