ARTHRITIS & RHEUMATISMVol. 64, No. 1, January 2012, pp 110–120DOI 10.1002/art.33321© 2012, American College of Rheumatology

Proinflammatory Th17 Cells Are Expanded and Inducedby Dendritic Cells in Spondylarthritis-Prone

HLA–B27–Transgenic Rats

Simon Glatigny,1 Ingrid Fert,1 Marie A. Blaton,1 Rik J. Lories,2 Luiza M. Araujo,1

Gilles Chiocchia,3 and Maxime Breban3

Objective. HLA–B27/human �2-microglobulin–transgenic (B27-transgenic) rats, a model of spondylar-thritis (SpA), develop spontaneous colitis and arthritisunder conventional conditions. CD4� T cells are pivotalin the development of inflammation in B27-transgenicrats. This study was undertaken to characterize thephenotype of CD4� T cells in this model and todetermine whether dendritic cells (DCs) induce proin-flammatory T cells.

Methods. The phenotype of CD4� T cells fromrat lymph nodes (LNs) draining the sites of inflam-mation was analyzed by flow cytometry. Immunostain-ing was used to detect interleukin-17 (IL-17)–producingcells in the rat joints. DCs from B27-transgenic orcontrol rats (transgenic for HLA–B7 or nontransgenic)were cocultured with control CD4� T cells and stimu-lated with anti–T cell receptor �/�.

Results. IL-17A– and tumor necrosis factor �(TNF�)–producing CD4� T cells were expanded inmesenteric and popliteal LNs from B27-transgenic rats.The accumulation of Th17 cells correlated with diseasedevelopment, in contrast to Th1 or Treg cells. IL-17–positive mononuclear cells were detected in the arthritic

joints of B27-transgenic rats but not in the joints ofcontrol rats. Finally, in vitro cocultures demonstratedthat Th17 cells were preferentially induced and ex-panded by DCs from B27-transgenic rats, by a processthat may involve defective engagement of costimulatorymolecules.

Conclusion. Our findings indicate that expandedCD4� T cells in B27-transgenic rats exhibit a proin-flammatory Th17 phenotype characterized by IL-17Aand TNF� production. Furthermore, this population ispreferentially induced by DCs from B27-transgenic rats.These data point toward an induction of Th17 cells as apossible pathogenic mechanism in this model of SpA.However, their pathogenic role still needs to be shown.

Spondylarthritis (SpA) is an inflammatory rheu-matic disorder characterized by axial and peripheralenthesitis and/or arthritis and frequent extraarticularmanifestations, such as uveitis, psoriasis, and inflamma-tory bowel diseases (Crohn’s disease or ulcerative coli-tis). All skeletal and extraarticular features of SpA arethought to be determined by shared genetic factors,dominated by the class I major histocompatibility com-plex (MHC) allele HLA–B27 (1). Although the strongassociation between HLA–B27 and SpA was first de-scribed more than 35 years ago, the mechanism of thisassociation has remained unexplained until now (2).

Evidence that HLA–B27 plays a direct role indetermining SpA pathogenesis came from the descrip-tion of an HLA–B27–transgenic rat model. A multi-system inflammatory disorder mimicking the moststriking aspects of SpA (rat SpA), including ankylosingspondylitis, peripheral arthritis, psoriatic lesions, andulcerative colitis, arises spontaneously in several linesof rats transgenic for HLA–B*2705 and human �2-microglobulin (h�2m), but not in the control HLA–

Supported by a DCSPA (Molecular Role of HLA-B27 inSpondylarthritis, From Animal Model to Human Disease) grant fromthe French National Agency for Research (ANR-Physiopath 2007-2011).

1Simon Glatigny, PhD, Ingrid Fert, BS, Marie A. Blaton, BSc,Luiza M. Araujo, PhD: Institut Cochin, Universite Paris Descartes,CNRS (UMR 8104), and INSERM U1016, Paris, France; 2Rik J.Lories, MD, PhD: Catholic University of Leuven, Leuven, Belgium;3Gilles Chiocchia, PhD, Maxime Breban, MD, PhD: Institut Cochin,Universite Paris Descartes, CNRS (UMR 8104), INSERM U1016,Paris, France, Hopital Ambroise Pare, AP-HP, and Universite deVersailles Saint Quentin en Yvelines, Boulogne-Billancourt, France.

Address correspondence to Maxime Breban, MD, PhD, Ser-vice de Rhumatologie, Hopital Ambroise Pare, 9 Avenue Charles deGaulle, 92104 Boulogne, France. E-mail: maxime.breban@apr.aphp.fr.

Submitted for publication July 23, 2010; accepted in revisedform August 30, 2011.

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B*0702/h�2m–transgenic line (3,4). The expression ofrat SpA is determined by several factors, including highlevels of both HLA–B27 and h�2m transgenes, a specificrat genetic background, and the presence of normalmicrobial flora (5,6). Importantly, the role of the im-mune system in this model was highlighted in severalways. First, rat SpA was induced in nontransgenic recip-ients by transferring immature hematopoietic cells, butnot mature lymph node (LN) cells, from disease-pronerats, indicating that a hematopoietic-derived cell is crit-ical for disease induction (7). Second, nude HLA–B27–transgenic rats, which lack thymically derived T cells,were protected against disease but promptly developedrat SpA upon reconstitution with CD4� T cells orthymus engraftment, whereas reconstitution with CD8�T cells was relatively inefficient (6,8). Moreover, exper-iments involving CD8� T cell depletion confirmed thatCD8� T cells were not required for the development ofrat SpA (9,10).

Based on the results described above, we postu-lated that rat SpA could arise as a consequence of aninteraction between antigen-presenting cells (APCs) ex-pressing high levels of HLA–B27 and CD4� T cells, andcarried out experiments to examine the consequences ofsuch interaction. Interestingly, these experiments re-vealed several aberrant functions in APCs from HLA–B27/h�2m–transgenic rat lines. Most notably, maturesplenic and LN dendritic cells (DCs) exhibited a de-creased capacity to stimulate an allogeneic or a synge-neic T cell response, which paralleled disease suscepti-bility in a broad variety of lines, and was not aconsequence of the inflammatory disease (11–13). Thisaltered function could be linked to a defective engage-ment of costimulatory molecules, such as CD86, andalso to a decreased capacity to form an antigen-independent immunologic synapse with CD4� T cells(12,14). Other aberrant functions in HLA–B27–transgenic rat DCs included altered cytoskeletal dynam-ics, decreased class II MHC expression, and enhancedapoptotic susceptibility, all potentially contributing totheir decreased stimulatory function (15).

DCs are professional APCs that are capable ofactivating naive CD4� T cells to differentiate and toproliferate. However, besides promoting T cell re-sponses to antigen, evidence indicates that DCs play arole in establishing tolerance toward self antigens and inthe maintenance of peripheral tolerance (16). Accord-ingly, spontaneous inflammation in this model couldresult from a breakdown of peripheral tolerance.

Upon stimulation by DCs, naive CD4� T cellsare known to differentiate into distinct lineages of

effector cells. While interleukin-12 (IL-12) drives thedevelopment of Th1 cells, producing predominantlyinterferon-� (IFN�) and IL-2 and eliciting cell-mediatedimmunity against intracellular pathogens (17), Th2 cellsdifferentiate in response to IL-4; produce IL-4, IL-5, andIL-13; and are involved in the humoral response againstparasites and allergy (17,18). More recently, Th17 cells,a subset of T cells producing IL-17A, IL-17F, and IL-22,have been identified (19–21) and shown to be critical forthe induction of several autoimmune disease models,such as collagen-induced arthritis and experimental auto-immune encephalomyelitis (22,23). Th17 cells differen-tiate in mice in response to transforming growth factor �(TGF�) and IL-6 or IL-21 (24) and differentiate inhumans in response to a mixture of cytokines, includingIL-1�, IL-6, tumor necrosis factor � (TNF�), IL-23, andTGF� (25,26). Finally, a CD4� Treg cell populationexpressing the FoxP3 transcription factor has also beendescribed (27). This Treg cell population appears toinhibit the proliferation of all of the former effector Thcells (28).

In the present study, we examined the phenotypeof CD4� T cells in a disease-prone HLA–B27–transgenic rat line and the putative influence of DCsfrom these rats on the differentiation of CD4� T cells.First, we observed that CD4� T cells with a proinflam-matory Th17 phenotype accumulate in B27-transgenicrats, in parallel with the development of rat SpA.Furthermore, we showed that Th17 cells were preferen-tially induced and expanded by DCs from B27-transgenic rats, by a contact-dependent mechanism thatmay involve a previously described defective engage-ment of costimulatory molecules (12,14).

MATERIALS AND METHODS

Rats. The HLA–B27–transgenic rat line 33-3 and theHLA–B7–transgenic rat line 120-4 were originally produced atthe University of Texas Southwestern Medical Center (Dallas,TX). Disease-prone rats of the 33-3 line, bearing 55 copies ofHLA–B*2705 and 29 copies of h�2m, and disease-free ho-mozygous rats of the 120-4 line, bearing 52 copies of HLA–B*0702 and 31 copies of h�2m, both on a Fischer background,were bred and maintained under conventional conditions(12,14). Nontransgenic littermates from the 33-3 rat line wereused as controls. Age- and sex-matched rats (1–12 months ofage) were used in each experiment. Study procedures wereapproved by the institutional animal care committee.

Cell culture medium, monoclonal antibodies (mAb),and other reagents. Cell cultures were performed in RPMI1640 medium with Glutamax I (Life Technologies) supple-mented with 10% fetal calf serum, streptomycin (100 �g/ml),2% sodium pyruvate, 0.05 mM 2-mercaptoethanol, and 5 mMHEPES (complete medium), unless otherwise stated. The

INDUCTION OF Th17 CELLS IN HLA–B27–TRANSGENIC RATS 111

following mouse IgG1 or IgG2a anti-rat antigen mAb wereobtained from European Collection of Cell Cultures or pur-chased from BD PharMingen: R73 (T cell receptor �/�[TCR�/�]), OX35 (CD4), OX8 (CD8� chain), 3.2.3 (naturalkiller cell receptor protein 1A), OX62 (�E2 integrin presenton CD103� DCs), OX33 (CD45 epitope specific to B cellsand a subset of DCs), OX12 (Ig� chain), OX42 (C3bR[macrophages]), OX39 (CD25), 24F (CD86), JJ316 (CD28),and FJK-16s (FoxP3). Fluorescein isothiocyanate (FITC)–conjugated goat anti-mouse (GAM) IgG2a was fromCaltag, and Cy5-conjugated GAM IgG1 was from JacksonImmunoResearch. Allophycocyanin-conjugated anti-rat CD4,Cy7-conjugated anti-rat TNF�, phycoerythrin-conjugatedanti-mouse IL-17A, and FITC-conjugated anti-rat IFN�were from BD PharMingen. Rabbit anti-human IL-17A (sc-7927), goat anti-rat IL-23 p19 (sc-21083), and goat anti-human TGF�1 (sc-31608) were from Santa Cruz Biotech-nology. Peroxidase-conjugated goat anti-rabbit IgG was fromJackson ImmunoResearch. Recombinant rat IFN� was fromImmunoTools. Phorbol 12-myristate 13-acetate (PMA), 5,6-carboxyfluorescein diacetate N-succinimidyl ester (CFSE),ionomycin, and brefeldin A were from Sigma.

Cell preparation. Splenic DCs were obtained by twodifferent methods, as previously described (12,14). The firstmethod was adapted from that described by Knight et al (29).Briefly, a single-cell suspension prepared from spleen wascultured overnight in RPMI Medium 1640 (Dutch Modifica-tion) (Life Technologies). Recovered nonadherent cells weresubjected to a 14.5% (weight/volume) metrizamide gradient.Low-density cells were collected at the interface and consistedmostly of DCs.

The second method was derived from the techniquedescribed by Josien et al (30), as follows. A single-cell suspen-sion was prepared from spleen, minced, and digested in 2mg/ml collagenase D (Roche Diagnostics). Low-density cellswere then collected after centrifugation on a 14.5% Nycodenzgradient (Nycomed). Cells were then incubated with OX62mAb–coated microbeads (Miltenyi Biotec) and positively se-lected on MS-type selection columns (Miltenyi Biotec). Thispopulation of OX62� DCs was used after overnight incuba-tion in complete medium containing 4% culture supernatantfrom murine hybridoma transfected with rat granulocyte–macrophage colony-stimulating factor.

T cells from HLA–B7–transgenic or nontransgeniccontrol rats were isolated from LN single-cell suspensions bymagnetic-activated cell sorting. Negative selection was per-formed, using combinations of OX33, OX42, 3.2.3, OX8, andOX39 to purify naive CD4�CD25� T cells. The purity of theselected populations was routinely in the range of 95–98% asdetermined by fluorescence-activated cell sorting (FACS)analysis.

Cell cultures. Splenic DC populations were coculturedwith CD4�CD25� T cells labeled with 5 �M CFSE withanti-TCR�/� (R73) mAb for 4 days in 24-well flat-bottomedplates in 500 �l complete medium. The DC:T cell ratio was 1:1.Proliferation of T cells was assessed by FACS, by evaluatingthe dilution of CFSE in TCR�/�� cells.

In experiments using a Transwell apparatus (CorningCostar), CD4�CD25� T cells (5 � 105/well) were stimulatedwith DCs (5 � 105/well) in lower compartments of 24-wellculture dishes, in a volume of 250 �l, in the presence of a

Transwell insert (6.5-mm diameter, 0.4-�m pore size) contain-ing either DCs and T cells (2 � 105 of each cell type per insert)or DCs alone, in 250 �l of medium. After 4 days of culture,cells from the lower compartments were separately transferredto 96-well plates before intracellular staining. In blockingexperiments, anti-CD86 and anti-CD28 mAb were used at5-�g/ml saturating concentrations.

Determination of cytokine levels. The cytokines IL-4,IL-10, IL-17A, TNF�, and IFN� were measured in rat sera andculture supernatants by specific enzyme-linked immunosor-bent assays, according to the recommendations of the manu-facturer (eBioscience). Results are expressed as the mean �SEM.

Immunohistochemical analysis. Paraffin-embeddedsections of paws were dewaxed with Histo-Clear (twice for 5minutes each time) and methanol (twice for 5 minutes eachtime) and subsequently added to distilled water. Sections werequenched with 3% H2O2 in Milli-Q water (twice for 5 minuteseach time) to block endogenous peroxidase activity. Afterwashing 3 times in phosphate buffered saline (PBS)–0.1%Tween, sections were incubated with goat serum diluted 1:5 (inPBS–0.1% Tween) for 30 minutes at room temperature toblock nonspecific binding. Sections were subsequently incu-bated overnight at 4°C with 5 �g/ml rabbit anti-human IL-17Aantibody or 5 �g/ml normal rabbit IgG as a negative control. Inadditional experiments, the primary antibody was first incu-bated for 2 hours with 250 ng murine recombinant IL-17A(eBioscience). After washing 3 times, a second blocking stepwith goat serum diluted 1:5 was applied. Sections were subse-quently incubated with peroxidase-conjugated goat anti-rabbitIgG diluted 1:300 in PBS–0.1% Tween for 30 minutes at roomtemperature. After washing, enzyme activity was detected withdiaminobenzidine chromogen, and sections were counter-stained with hematoxylin.

Real-time polymerase chain reaction (PCR). TotalRNA was isolated from LN CD4� T cell pellets using anRNeasy Mini kit (Qiagen). Genomic DNA was removed usingthe RNase-Free DNase set for DNA digestion during RNApurification (Qiagen). First-strand complementary DNA syn-thesis was performed using a Sensiscript RT kit with randomhexamer primers (Qiagen). Levels of messenger RNA(mRNA) for the tested genes were quantified by real-timePCR (ABI 7900HT Sequence Detection System; AppliedBiosystems) using SYBR Green Master Mix (Applied Biosys-tems). Thermocycling included incubation at 95°C for 10minutes, followed by a 2-step PCR program of 95°C for 15seconds and 55°C for 60 seconds and extension at 72°C for 10seconds, for a total of 45 cycles. The total amount of mRNAwas normalized across samples according to endogenous Hprt1mRNA. The primer sequences (forward and reverse, respec-tively) were as follows: for Hprt1, 5�-TTTGTGTCATCAGC-GAAAGTG-3� and 5�-ATGGCCACAGGACTAGAAC-3�;for IL-21, 5�-AAGGCCAGATCACCTTCTGA-3� and 5�-GC-CCCTTTACATCTTGTGGA-3�; for IL-22, 5�-ACCAAGCT-CAGCAGTCACCT-3� and 5�-AGTTCCCCGATCGCTTTA-AT-3�; for Tbx21, 5�-AGCCCACTGGATGCGACAGGA-3�and 5�-GCGGCTGGTACTTATGGA-3�; for IFN�, 5�-GGC-CATCAGCAACAACATAAG-3� and 5�-TGGGTTGTTCA-CCTCGAACTT-3�; and for Rorc, 5�-GGATGAGATTGCCC-TCTACAC-3� and 5�-GGAGGCCTTGTCGATGAGTC-3�.

112 GLATIGNY ET AL

Cytofluorometry. For FACS analysis of surface anti-gen, cells were incubated with saturating concentrations of theappropriate primary mAb (mouse IgG1 or IgG2a isotype) for30 minutes, washed, and then incubated with secondary Cy5-conjugated GAM IgG1 or FITC-conjugated IgG2a, as needed,for 30 minutes. Cytokine-producing cells were determined bysurface staining using allophycocyanin-conjugated anti-ratCD4 and intracellular staining using fluorescent anticytokinemAb. Briefly, cells were stimulated with PMA and ionomycin(both at 0.5 �g/ml) in the presence of brefeldin A (10 �g/ml)for 4 hours. Cells were fixed in 2% paraformaldehyde, perme-abilized with 0.1% saponin, and stained with fluorescent mAb.Then, the cells were analyzed using an FC500 cytometer(Beckman Coulter) and CXP analysis software (BeckmanCoulter).

Statistical analysis. Statistical comparisons betweengroups were performed using Student’s t-tests. For analysis ofreal-time reverse transcriptase–PCR experiments, the ratios ofthe gene transcript levels in the B27-transgenic rats to those inthe nontransgenic control rats were compared to the theoret-

ical value of 1.0 (i.e., no difference) by 1-sample t-test. P valuesless than 0.05 were considered significant.

RESULTS

Increased CD4� T cell numbers in HLA–B27–transgenic rat LNs. To investigate the role of CD4� Tcells in rat SpA, we enumerated CD4� T cells inmesenteric LNs (MLNs) and popliteal LNs from B27-transgenic rats and control rats (either nontransgenic ortransgenic for HLA–B7). There was a notable increasein the number of CD4� T cells obtained from the MLNsof HLA–B27–transgenic rats compared to their non-transgenic littermates; this difference was statisticallysignificant at any time point (Figure 1A). Similar statis-tically significant differences were also observed in pop-liteal LNs, but to a lesser extent (data are available from

Figure 1. Increased numbers of CD4� and CD4�CD25� T cells in the lymph nodes (LNs) of HLA–B27–transgenic (B27) rats. Cell suspensionswere prepared from the mesenteric LNs (MLNs) and peripheral LNs (PLNs) of age-matched control rats (nontransgenic [NTG] rats orB7-transgenic rats) and B27-transgenic rats. Cells were labeled with anti-CD4, anti-CD25, and anti-FoxP3 monoclonal antibodies to discriminatebetween activated T cells and Treg cells by fluorescence-activated cell sorting. A, Absolute numbers of CD4� T cells in the MLNs of nontransgenicrats (shaded bars) and B27-transgenic rats (solid bars) at the indicated ages. Bars show the mean � SEM (n � 5–7 rats per group). There wasa significant difference between the nontransgenic group and the B27-transgenic group at each time point. B, Absolute numbers of CD4�CD25�T cells in the peripheral LNs and MLNs of 6-month-old nontransgenic and B7-transgenic control rats (shaded bars) and B27-transgenic rats (solidbars). Bars show the mean � SEM (n � 6 rats per group). C, Dot-blots showing the expression of CD25 and FoxP3 in CD4�-gated T cells fromthe MLNs of 6-month-old nontransgenic and B27-transgenic rats. D, Ratios of FoxP3� cells (activated cells) to FoxP3� cells (Treg cells) amongCD4�CD25�-gated cells in the peripheral LNs and MLNs of 6-month-old nontransgenic and B7-transgenic control rats (shaded bars) andB27-transgenic rats (solid bars). Bars show the mean � SEM (n � 6 rats per group). � � P � 0.05; �� � P � 0.005.

INDUCTION OF Th17 CELLS IN HLA–B27–TRANSGENIC RATS 113

the author upon request). There was no difference in thenumber of CD4� T cells between HLA–B7–transgenicrats and nontransgenic rats, confirming the specificity oftheir expansion to disease-prone B27-transgenic rats andnot to any HLA–B–transgenic rat line (data not shown).

Higher numbers of activated CD4� T cells thanTreg cells in HLA–B27–transgenic rats. Similar to thewhole CD4� T cell population, the subset of CD4�CD25� T cells was significantly expanded in the MLNsand peripheral LNs of HLA–B27–transgenic rats (Fig-ure 1B). Both activated CD4� T cells and Treg cellsmay express CD25, but they can be distinguished onthe basis of FoxP3 expression, which is restricted tothe latter population. Both CD25�FoxP3� (Treg) andCD25�FoxP3� (activated) CD4� T cells were increasedin the LNs of B27-transgenic rats (Figure 1C). However,this increase was more pronounced among activated cellsthan Treg cells (Figures 1C and D).

Preferential increase in Th17 cell number inparallel with disease development in the B27-transgenicrat. To investigate the phenotype of expanded CD4� Tcells, we performed intracellular staining and monitoredthe proportions of MLN CD4� T cells expressing IFN�,IL-17A, and TNF�, alone or in combination, in rats ages1 month (prior to rat SpA development) up to 1 year(Figure 2A). Th1 cells producing IFN� alone predomi-nated in control rats, and the number of these cellsincreased with age in both B27-transgenic rats andcontrols, without any differences between the 2 groups(Figure 2A). The proportion of Th17 cells producingIL-17A remained low (�1%) across all ages in controls(Figure 2A). In contrast, in the MLNs of B27-transgenicrats, the proportion of IL-17A�CD4� cells, which wasas low as that in controls at 1 month, rose steadilythereafter, reaching 20% at 1 year. This pattern wasobserved for the major population of IL-17A�IFN��cells as well as for a minor subset of IL-17A�IFN��cells (Figure 2A).

In addition, we quantified CD4� T cells pro-ducing TNF�, a cytokine that can be produced by eitherTh1 or Th17 cells. The proportion of TNF��CD4� Tcells was significantly increased in the MLNs of B27-transgenic rats (Figure 2A). Interestingly, this TNF��CD4� T cell population contained a majority of theIL-17A� cells in B27-transgenic rat LNs, and a minorsubset of the TNF��IL-17A� cells were also IFN��(data are available from the author upon request).A similar pattern, consisting of an increased proportionof IL-17A� and TNF�� CD4� T cells, but a normalproportion of IFN��IL-17A� CD4� T cells, was ob-served in the popliteal LNs of B27-transgenic rats (Fig-

ure 2B). Notably, this increase appeared later in thepopliteal LNs than in the MLNs (i.e., at 6 months versus3 months), parallel to the development of arthritis,which follows that of colitis in this model.

Figure 2. CD4� T cells are biased toward a proinflammatory Th17phenotype in the B27-transgenic rat. In vitro–stimulated MLN (A) andpopliteal LN (B) cells from age-matched nontransgenic rats, B7-transgenic rats, and B27-transgenic rats at the indicated ages (n � 5rats per group) were first surface-labeled with anti-CD4 and thenstained intracellularly with fluorescent anti–interferon-� (anti-IFN�),anti–tumor necrosis factor � (anti-TNF�), and anti–interleukin-17A(anti–IL-17A) and analyzed by fluorescence-activated cell sorting.Results are the percentages of CD4�-gated cells and represent theevolution of Th1 cells (IFN��IL-17�), Th17 cells (IFN��IL-17�),Th1/17 cells (IFN��IL-17�), and TNF�-producing CD4 T cells overtime. Symbols represent individual rats; horizontal lines show themean � SEM. �� � P � 0.005; ��� � P � 0.001, B27-transgenic ratsversus pooled controls (B7-transgenic and nontransgenic rats). SeeFigure 1 for other definitions.

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The preferential increase in the number of Th17cells was further evaluated by transcription analysis ofMLN CD4� T cells. Using real-time PCR, we quantified

the expression of IL-21, IL-22, and IFN� mRNA. IL-21and IL-22 are typically produced by Th17 cells, andIFN� is a Th1-specific cytokine. We also quantified theexpression of retinoic acid receptor–related orphan nu-clear receptor (coded by Rorc) and T-bet (coded byTbx21) as typical Th17 and Th1 transcription factors,respectively. The 5 transcripts were detected in allCD4� T cell samples purified from the MLNs of 5nontransgenic and 8 diseased B27-transgenic rats ages6–9 months. The transcription levels of IL-21, IL-22, andRorc, the combination of which is characteristic of aTh17 phenotype, were significantly increased in theB27-transgenic rats relative to the nontransgenic con-trols, with mean ratios of 1.8 (95% confidence interval[95% CI] 1.2–2.4) (P � 0.02), 32 (95% CI 14.5–49.5)(P � 0.004), and 8.8 (95% CI 3.2–14.3) (P � 0.013),respectively. In contrast, the expression levels of IFN�and Tbx21, which are representative of a Th1 phenotype,did not differ between B27-transgenic and nontrans-genic rats (mean ratio 1.0 [95% CI 0.6–1.5] [P � 0.92]and 1.4 [95% CI 0.5–2.3] [P � 0.4], respectively) (dataare available from the author upon request).

Consistent with the results described above, IL-17A was detected at significantly higher levels in the seraof 6-month-old B27-transgenic rats (mean � SEM32.2 � 4.7 pg/ml; n � 6), than in B7-transgenic controls(2.85 � 1.6 pg/ml; n � 6) (P � 0.005) and nontransgeniccontrols (0 � 0 pg/ml; n � 6) (P � 0.001). These levelswere higher than the levels of IFN� and TNF� in theB27-transgenic rats, which were not significantly differ-ent from those observed in the B7-transgenic and non-transgenic controls (data are available from the authorupon request). Neither IL-4 nor IL-10 was detected inany of the sera (data not shown).

Finally, IL-17A–positive mononuclear cells weredetected in the hyperplastic lining layer and in synovialinfiltrates of the arthritic paws of 7-month-old HLA–B27–transgenic rats (Figures 3A, B, and D). In contrast,the paucicellular synovium of the nontransgenic controls(Figures 3C and D) yielded negative signals, as did thatof the nonarthritic HLA–B7–transgenic controls (Fig-ures 3C and D).

DCs from HLA–B27–transgenic rats favorTh17 cell induction. We next addressed whether DCsin B27-transgenic rats, which display aberrant function(14), contributed to the notable increase in the numberof Th17 cells. To test this possibility, we coculturedsplenic DCs with CFSE-labeled syngeneic CD4�CD25� T cells from control rats (either B7-transgenicor nontransgenic) and stimulated the coculture withanti-TCR�/�. In these assays, we used 2 different splenic

Figure 3. Presence of interleukin-17 (IL-17)–positive mononuclearcells in the arthritic joints of HLA–B27–transgenic rats. Immunohis-tochemical analysis for IL-17A was performed in paws from 7-month-old rats. A and B, IL-17–positive cells in the synovium of an arthriticfemale HLA–B27–transgenic rat. Negative controls showing preincu-bation of the antibody with recombinant IL-17 or control IgG substi-tution of the primary antibody are also shown. C, Near absence ofIL-17–positive mononuclear cells in the paucicellular normal synoviumfrom an HLA–B7–transgenic (HLAB7tg) rat and a nontransgenic(wild-type) control rat. Original magnification � 400. D, IL-17–positive cells in nontransgenic, HLA–B7–transgenic, and HLA–B27–transgenic rats. Six randomly chosen zones of the synovium from 2 ratsin each group were scored in a blinded manner. Bars show the mean �SEM. P � 0.0006, HLA–B27–transgenic rats versus nontransgenicrats; P � 0.0005, HLA–B27–transgenic rats versus HLA–B7–transgenic rats.

INDUCTION OF Th17 CELLS IN HLA–B27–TRANSGENIC RATS 115

DC populations: CD103� DCs or DCs isolated by themethod described by Knight et al (29) (Figure 4). Bothtypes of DCs showed the expansion and/or differentia-tion of IL-17A–producing T cells, which were detectedamong the CD4� T cells that divided the most (Figure4A). This effect was 3- to 15-fold greater with B27-transgenic rat DCs than with control DCs (Figures 4Aand B). In this coculture assay, both control and B27-transgenic rat DCs also induced the production of IFN�and TNF� by control T cells (Figure 4C). Most interest-ingly, the proportion of T cells producing those cyto-kines was considerably greater after coculture with B27-transgenic rat DCs (Figure 4C). Furthermore, as shownin the MLNs of B27-transgenic rats, the majority ofIL-17A–producing T cells induced by B27-transgenic rat

DCs also produced TNF� (Figure 4D). Thus, B27-transgenic rat DCs directly participate in the inductionof proinflammatory cytokine–producing T cells.

Th17 induction is contact dependent. B27-transgenic rat DCs may favor the expansion of Th17 cellsby an increased secretion of stimulatory factors or adecreased capacity to produce inhibitory factors. Toinvestigate whether soluble factors such as cytokineswere involved in this process, we used a Transwell assay(Figure 5). The proportion of Th17 cells induced uponcontact with control DCs in the lower well was notmodified by the presence of B27-transgenic rat DCs inthe upper well, whether alone or in coculture withcontrol T cells (0.1% of proliferative Th17 cells, with orwithout B27-transgenic rat DCs in the upper compart-

Figure 4. Splenic dendritic cells (DCs) from HLA–B27–transgenic (B27) rats induce a biased expansion of Th17 cells. Mature nontransgenic (NTG)or B27-transgenic rat CD103� DCs or DCs isolated by a method adapted from Knight et al (29) (Knight DCs) were cocultured with5,6-carboxyfluorescein diacetate N-succinimidyl ester (CFSE)–labeled lymph node CD4�CD25� T cells from nontransgenic or B7-transgeniccontrol rats with anti–T cell receptor �/� (anti-TCR�/�) (R73 monoclonal antibody; 1 �g/ml) stimulation. After 4 days, cells were surface-labeledwith anti-TCR�/�, then stained intracellularly with anti–IL-17A and analyzed by fluorescence-activated cell sorting (FACS). Results are shown forTCR�/��-gated cells. A, Dot-blots showing results with Knight DCs and with CD103� DCs. Results are representative of 10 independentexperiments using T cells from nontransgenic or B7-transgenic control rats. B, IL-17A�CSFEintermediate/low T cells resulting from coculture withKnight DCs and CD103� DCs from nontransgenic and B27-transgenic rats. Bars show the mean � SEM (n � 10 experiments with Knight DCs and7 experiments with CD103� DCs). ��� � P � 0.001. C, Dot-blots showing results after DCs isolated by the method adapted from Knight et al werecocultured as described above with T cells from nontransgenic control rats. After 4 days, cells were surface-labeled with anti-TCR�/� and thenstained intracellularly with anti-IFN�, anti–IL-17A, and anti-TNF� and analyzed by FACS. Results are representative of 3 independent experiments.D, Dot-blots showing the expression of TNF� and IL-17A in TCR�/��-gated cells (same experiment as described in C). See Figure 2 for otherdefinitions.

116 GLATIGNY ET AL

ment) (Figure 5). Nor did the presence of nontransgenicDCs in the upper well, alone or in coculture with controlT cells, influence the induction of Th17 cells uponcontact with B27-transgenic rat DCs in the lower well(0.4–0.5% of proliferative Th17 cells, with or withoutnontransgenic DCs in the upper compartment) (Figure5). Moreover, neither the addition of anti–IL-23 oranti-TGF� neutralizing antibodies to the coculture toblock major soluble factors known to stabilize or toinduce Th17 phenotype, nor the addition of IFN�, whichmay oppose Th17 cell differentiation, influenced theobserved pattern (results not shown).

Correlation of Th17 cell induction with impairedengagement of costimulatory molecules. We have previ-ously shown that DCs from B27-transgenic rats have animpaired capacity to form a mature antigen-independent immunologic synapse with CD4� T cells,which involves a defective engagement of costimulatory

molecules such as CD86 (12). Thus, in the present studywe examined whether such abnormal function could beinvolved in the expansion of Th17 cells. We performedcocultures in the presence of either a neutralizing anti-CD86 mAb or an anti-CD28 “superagonist,” mAb JJ316,which stimulates T cell proliferation independently ofDC–T cell contact (31) (Figure 6). As expected, T cellproliferation resulting from the coculture of nontrans-genic DCs with T cells from control rats (either B7-transgenic or nontransgenic rats) was strongly inhibitedby the addition of anti-CD86 but not by the addition ofJJ316. Most interestingly, the proportion of Th17 cellswas increased in the presence of both mAb, irrespectiveof the level of T cell proliferation (P � 0.0018). Incontrast, the proliferation of nontransgenic T cells sup-ported by B27-transgenic rat DCs was much weaker thanthat supported by nontransgenic DCs and further de-creased in the presence of anti-CD86, which was consis-tent with the findings of our previous study (14). Never-theless, strong T cell proliferation was induced in thepresence of B27-transgenic rat DCs when JJ316 wasadded (Figure 6). Finally, the increased proportion ofTh17 cells detected in the coculture with B27-transgenicrat DCs remained unchanged in the presence of eithermAb (Figure 6). Thus, the expansion of Th17 cells duringthe coculture appeared to be independent of the level of

Figure 6. Th17 cell induction by rat DCs correlates with CD86/CD28blockade. Mature nontransgenic or B27-transgenic rat DCs isolated bythe method adapted from Knight et al (29) were cocultured withCFSE-labeled lymph node CD4�CD25� T cells from nontransgeniccontrol rats with anti-TCR�/� (R73 monoclonal antibody; 1 �g/ml)stimulation in the presence of anti-CD86 neutralizing monoclonalantibody, anti-CD28 superagonist JJ316, or isotype control. After 4days of culture, cells were harvested, labeled, and analyzed by FACS asdescribed in the Figure 4 legend. Dot-blots are shown for TCR�/��-gated cells. Results are representative of 3 independent experiments.IL-17A � interleukin-17A (see Figure 4 for other definitions).

Figure 5. Th17 cell induction by B27-transgenic rat DCs is contactdependent. Mature nontransgenic or B27-transgenic rat DCs isolatedby a method adapted from Knight et al (29) were cocultured withCFSE-labeled lymph node CD4�CD25� T cells from a nontransgeniccontrol rat with anti-TCR�/� (R73 monoclonal antibody; 1 �g/ml)stimulation, in the lower compartment of a Transwell apparatus. Theupper compartment contained DCs alone or DCs and T cells, asindicated below each panel. After 4 days, cells in the lower compart-ment were harvested, labeled, and analyzed by FACS as described inthe Figure 4 legend. Dot-blots are shown for TCR�/��-gated cells.Data are representative of 3 independent experiments. IL-17A �interleukin-17A (see Figure 4 for other definitions).

INDUCTION OF Th17 CELLS IN HLA–B27–TRANSGENIC RATS 117

proliferation induced but correlated notably with theimpairment of CD86–CD28 engagement (Figure 6).

DISCUSSION

A critical role for T cells in the B27-transgenic ratmodel of SpA was previously established (7). Further-more, several lines of evidence strongly support a directrole for CD4� T cells, whereas CD8� T cells appear tobe dispensable (8–10). However, the phenotype ofCD4� T cells that might drive the disease process hasnot been previously thoroughly ascertained. In the pres-ent study, we observed an expansion of CD4� T cells inthe LNs draining the sites of inflammation in B27-transgenic rats. Interestingly, the frequency of CD4� Tcells has also been shown to be increased in the peri-pheral blood of SpA patients (32).

Among CD4� T cells, the expansion was moststriking in the CD4�CD25� subset, which may corre-spond to activated or Treg cells. We showed a preferen-tial expansion of FoxP3� activated cells compared toFoxP3� Treg cells. We had previously shown that DCsfrom B27-transgenic rats have a decreased capacity toactivate naive CD4� T cells, by a mechanism implyingdefective costimulatory function (12,14). We had pro-posed that such abnormal function could predominantlyimpair the capacity of B27-transgenic rat APCs toactivate Treg cells. Our present results corroborate thishypothesis by showing an unbalanced control of CD4�T cell activation.

We next characterized the cytokines produced byCD4� T cells in the LNs draining the sites of inflam-mation. Notably, we observed a preferential expansionof TNF�- and IL-17A–producing cells, which paralleleddisease development, likely corresponding to Th17 cells(20,26,33). Such an interpretation is strongly supportedby the increased transcription of Rorc, IL-21, and IL-22(which are involved in the Th17 lineage) in the LNCD4� T cells from B27-transgenic rats (34,35), and bythe detection of IL-17A–positive mononuclear cells inarthritic joints from B27-transgenic rats (although wecannot exclude the possibility that at least some of theseIL-17–producing cells were not T cells). Accordingly,up-regulation of IL-17A in CD4� T cells from thecolonic lamina propria was recently shown in the sameB27-transgenic rats (36). We also detected increasedlevels of IL-17A in the serum of the B27-transgenic rats,similar to findings reported in human SpA (37). All ofthese findings support a direct role of Th17 cells in thismodel, although this remains to be formally demon-strated by experiments such as those involving cell

transfer. (Of note, it has been impossible until now topurify live Th17 cells in the rat.)

Former studies of T cell cytokines produced inthe B27-transgenic rat model of SpA demonstrated anearly increase in IFN� and IL-2 levels in the inflamma-tory colonic mucosa, suggesting a Th1-mediated disor-der (5). Interestingly, in the present study the proportionof Th1 cells producing IFN� alone and the expressionlevels of IFN� and Tbx21 transcripts in LN CD4� T cellsfrom B27-transgenic rats were comparable to that incontrols, suggesting that Th1 cells may not be the mostcritical T cells mediating the disease process. Similarobservations have been reported in the context of hu-man SpA (38). Nevertheless, we also detected a minorpopulation of CD4� T cells that produced IFN�, TNF�,and IL-17A together, which was specific to the B27-transgenic rats and appeared late during the diseaseprocess. A similar phenotype of polyfunctional T cellsproducing both Th1 and Th17 cytokines has previouslybeen described as selectively enriched in the peripheralblood of SpA patients (38,39).

The role of Th17 cells has recently emerged aspivotal in several models of autoimmune and inflamma-tory diseases (40). Our results suggest that Th17 cellscould contribute to both intestinal and joint inflamma-tion in the B27-transgenic rat model of SpA. Such aninterpretation is consistent with recent findings in hu-man SpA. Genetics studies have identified several poly-morphisms in the IL23R gene, which codes for a recep-tor involved in Th17 cell differentiation, as beingassociated with susceptibility to ankylosing spondylitis(41). Furthermore, the expansion of cells producing bothTNF� and IL-17A is consistent with the striking efficacyof anti-TNF� agents, both in the B27-transgenic ratmodel and in human SpA (42,43). In contrast, anti-ratIL-17 treatment failed to prevent disease in B27-transgenic rats (Glatigny S, et al: unpublished observa-tions). Nevertheless, this result does not rule out a rolefor Th17 cells in rat SpA pathogenesis, since we ob-served an increased number of IL-17–producing T cellsin the LNs of the anti–IL-17–treated rats. It is knownthat IL-17 negatively regulates its own production byway of negative feedback (44). Therefore, blocking IL-17in the B27-transgenic rat led to an increase in theproportion of Th17 cells, which could still exert apathogenic effect via the production of other mediators.

We next showed that interaction between DCsfrom B27-transgenic rats and CD4� T cells from con-trol rats contributes to the expansion of Th17 cells,despite their defective capacity to support a T cell–proliferative response (11–13). Furthermore, those IL-

118 GLATIGNY ET AL

17A–producing cells were detected among the T cellsthat divided the most, suggesting that the B27-transgenicrat DCs stimulated potentially autoreactive Th17 cells,similar to what has been described with DCs isolatedfrom human psoriatic lesions (45).

Regarding the mechanism responsible for thebiased Th17 cell induction by B27-transgenic rat DCs,Transwell experiments indicated that it was not ex-plained by a difference in soluble factor production,such as an excess of IL-23 or TGF�, or a lack of IFN�(results not shown). Alternatively, blocking the inter-action between CD86 and CD28 during DC–T cellcoculture resulted in an increased induction of Th17cells in the nontransgenic DC condition. This effect wasnot correlated with the level of T cell proliferation,which was inhibited by the anti-CD86 mAb, but wasconversely enhanced by the anti-CD28 superagonistmAb JJ316. Taken together, these results are consistentwith a critical role played by the defective costimulatorycapacity of B27-transgenic rat DCs in the biased induc-tion of Th17 cells and could link aberrant characteristicsof B27-transgenic rat DCs to their putative pathogenicrole in this model.

In conclusion, these results provide a mechanismthat could explain how defective function in HLA–B27–transgenic rat DCs would contribute to disease develop-ment by skewing CD4� T cell differentiation toward aproinflammatory over a regulatory phenotype. Whethersimilar mechanisms also apply to SpA in humans re-mains to be demonstrated. However, it is worth notingthat a defective stimulatory capacity of autologous andheterologous CD4� T cells has been observed in humanDCs from HLA–B27–positive ankylosing spondylitis pa-tients (46).

AUTHOR CONTRIBUTIONS

All authors were involved in drafting the article or revising itcritically for important intellectual content, and all authors approvedthe final version to be published. Dr. Breban had full access to all ofthe data in the study and takes responsibility for the integrity of thedata and the accuracy of the data analysis.Study conception and design. Glatigny, Fert, Lories, Chiocchia, Bre-ban.Acquisition of data. Glatigny, Fert, Blaton, Lories, Araujo.Analysis and interpretation of data. Glatigny, Fert, Blaton, Lories,Araujo, Chiocchia, Breban.

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