mediated pathway − pd-1 − autoimmunity via a novel non
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of March 26, 2018.This information is current as
Mediated Pathway−PD-1−Autoimmunity via a Novel Non
Differentiation and Central Nervous System 17HB7-H1 Selectively Controls T
Heinz WiendlKarin Loser, Christian Kurts, Percy Knolle, Luisa Klotz andHucke, Catharina Groß, Frank Kurth, Christoph Leder, Martin Herold, Vilmos Posevitz, Daria Chudyka, Stephanie
ol.1402746http://www.jimmunol.org/content/early/2015/09/15/jimmun
published online 16 September 2015J Immunol
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6.DCSupplementalhttp://www.jimmunol.org/content/suppl/2015/09/15/jimmunol.140274
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The Journal of Immunology
B7-H1 Selectively Controls TH17 Differentiation andCentral Nervous System Autoimmunity via a NovelNon–PD-1–Mediated Pathway
Martin Herold,*,1 Vilmos Posevitz,*,1 Daria Chudyka,* Stephanie Hucke,*
Catharina Groß,* Frank Kurth,* Christoph Leder,† Karin Loser,‡
Christian Kurts,x Percy Knolle,{ Luisa Klotz,*,2 and Heinz Wiendl*,2
It is currently acknowledged that TH17 cells are critically involved in the pathogenesis of autoimmune diseases such as multiple
sclerosis (MS). In this article, we demonstrate that signals delivered by the coinhibitory molecule B7-homologue 1 (B7-H1) via
a B7-homologue 1 mouse-IgG2aFc (B7-H1-Ig) fusion protein nearly abolish TH17, but not TH1 and TH2, differentiation via direct
interaction with the T cell. These effects were equally pronounced in the absence of programmed death-1 or B7.1 and B7.2 on the
T cell side, thus providing clear evidence that B7-H1 modulates T cell differentiation via a novel receptor. Mechanistically, B7-H1
interfered with early TCR-mediated signaling and cytokine-mediated induction of the TH17-determining transcription factors
retinoic acid-related orphan receptor g t and IFN regulator factor-4 in a programmed death-1 and B7-independent fashion. In an
animal model of MS, active myelin oligodendrocyte glycoprotein–induced experimental autoimmune encephalomyelitis, B7-H1-Ig
exhibited a significant and long-lasting effect on disease severity upon administration during the first 5 d of the priming phase,
which was accompanied by reduced TH17 responses in the periphery and within the CNS. Importantly, B7-H1-Ig was even capable
of interfering with T cell encephalitogenicity when interaction with the T cells occurred after priming using an adoptive transfer
experimental autoimmune encephalomyelitis model. In line with this, both naive human CD4+ T cells and differentiated TH17
effector cells from MS patients were highly sensitive toward B7-H1-Ig–mediated TH17 suppression. Together, we propose the
existence of a novel B7-H1–mediated immune-regulatory pathway in T cells, which selectively limits murine and human TH17 cell
responses and might be therapeutically exploited to control TH17-mediated autoimmunity. The Journal of Immunology, 2015,
195: 000–000.
During the last years, a new CD4+ Th cell lineage has beencharacterized and named TH17 subset according to theirproduction of the signature cytokine IL-17A (1, 2). TH17
cells have been implicated in the pathogenesis of T cell–mediatedautoimmunity in several animal models such as experimental
autoimmune encephalomyelitis (EAE), inflammatory bowel dis-ease, and collagen-induced arthritis (3, 4). Recently, severalstudies addressed the in vivo relevance of TH17 cells in humanCNS autoimmunity (5). In multiple sclerosis (MS), IL-17 ex-pression in CD4+ T cells within CNS lesions has been observedand more IL-17+ T cells were found in active compared with in-active lesions (6, 7). Moreover, expansion of peripheral TH17 cellsduring active MS, as well as enhanced frequencies of TH17 cells inthe cerebrospinal fluid during relapse, indicates that this pop-ulation might be involved in disease exacerbation (8, 9). Anotherstudy demonstrated increased frequencies of memory TH17 cellsin the peripheral blood of MS patients, which was further en-hanced during relapse (10). In line with this, preliminary data froman ongoing double-blind, placebo-controlled, proof-of-concepttrial with the IL-17A–neutralizing Ab secukinumab in patientswith active MS support the pathogenic role of IL-17 in MS be-cause secukinumab-treated patients exhibited a substantial re-duction in the number of new inflammatory lesions revealed bymagnetic resonance imaging (EudraCT number 2009-011626-34)(11). Based on the potent proinflammatory role of TH17 cells inautoimmunity, therapeutic targeting of this particular populationseems promising for control of TH17-mediated pathology in thecontext of CNS autoimmunity.The discovery of programmed death-1 (PD-1) and its ligand B7-
homologue 1 (B7-H1, PD-L1, CD274) provided important clueshow coinhibitory proteins maintain peripheral tolerance andcontribute to control of inflammation (12, 13). B7-H1 is consti-tutively expressed on different immune cells and can be induciblyexpressed on many parenchymal cells (14–18). Functionally, B7-H1
*Department of Neurology, University of Muenster, 48149 Muenster, Germany;†Experimental Cardiology, University of Tuebingen, 72076 Tuebingen, Germany;‡Department of Dermatology, University of Muenster, 48149 Muenster, Germany;xInstitute of Experimental Immunology, University Clinic Bonn, 53127 Bonn, Ger-many; and {Institute of Molecular Immunology, Technical University Munich, 81675Munich, Germany
1M.H. and V.P. contributed equally to this work.
2L.K. and H.W. contributed equally to this work.
Received for publication October 30, 2014. Accepted for publication August 3, 2015.
This work was supported Deutsche Forschungsgemeinschaft Grants WI 1722/7-1 andSFB 1009 TP A3 (to H.W.) and SFB 704 TP A21 and CRC 128 TP A8 (to L.K.),Interdisziplinare Zentrum f€ur Klinische Forschung Muenster Grant Wie3/011/11 (toH.W.), and Krankheitsbezogenen Kompetenznetzes Multiple Sklerose CONTROLMS
Grant 01GI0907 from the Bundesministerium f€ur Bildung und Forschung (to H.W.).
Address correspondence and reprint requests to Dr. Heinz Wiendl and Dr. LuisaKlotz, Department of Neurology, University of Muenster, Albert-Schweitzer-Campus 1, A1, 48149 Muenster, Germany. E-mail addresses: [email protected] (H.W.) and [email protected] (L.K.)
The online version of this article contains supplemental material.
Abbreviations used in this article: B7-H1, B7-homologue 1; B7-H1-Ig, B7-homologue 1mouse-IgG2aFc; EAE, experimental autoimmune encephalomyelitis; IRF-4, IFN regu-lator factor-4; MFI, mean fluorescence intensity; MOG, myelin oligodendrocyte glyco-protein; MS, multiple sclerosis; PD-1, programmed death-1; rhTGF-b, recombinanthuman TGF-b; RORgt, retinoic acid-related orphan receptor g t; RR-MS, relapsing-remitting MS; Treg, regulatory T cell; WT, wild-type.
Copyright� 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00
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is crucially involved in maintenance of immunological tolerance(19, 20). PD-1 has been identified as the canonical receptor forB7-H1 and is inducibly expressed on activated lymphocytes (21).Upon PD-1 binding, TCR-mediated signaling, a prerequisite forT cell activation, is suppressed, resulting in T cell anergy andtolerance (10, 22, 23).In CNS autoimmunity, genetic deletion of either B7-H1 or PD-1
renders mice more susceptible to development of EAE (24–26).B7-H1 is upregulated in inflammatory CNS lesions and criticallydetermines local effector T cell activation, survival, and recruit-ment of regulatory T cells (Treg) into the CNS, thereby limitingCNS damage (24, 27).Based on the potent immune-regulatory role of the B7-H1/PD-1
molecular axis, therapeutic modulation of this pathway has alwaysbeen tempting. Indeed, interference with PD-1/B7-H1 interactionreinforced antitumor responses in different cancer models (28–30).Also, in humans, blockage of B7-H1/PD-1 interaction results in animproved antitumor immune response (31, 32). These studieshighlight the in vivo relevance of B7-H1–mediated immune-regulatory pathways and the potential of its targeted activationin human diseases.In this study, we investigated the role of B7-H1 during TH17
differentiation and provided evidence of a novel B7-H1–related,non–PD-1–mediated immune-inhibitory pathway in T cells thatefficiently controls TH17 lineage induction. Furthermore, we alsoprovide evidence for the in vivo relevance of this pathway usingdifferent animal models of EAE, highlighting the clinical rele-vance of this novel B7-H1–induced “anti-TH17” immune-regulatory pathway.
Materials and MethodsMice
Mice were bred under specifically pathogen-free conditions and kept in-house for experiments in individually ventilated cages under specificpathogen-free conditions. C57BL/6 mice were acquired from Harlan.BALB/c mice were kindly supplied by Prof. Karin Loser, Muenster,Germany. B7-H12/2 and PD-12/2 mice were crossed onto C57BL/6background. B7.12/2/B7.22/2 mice on C57BL/6 background werekindly provided by Profs. Percy Knolle and Christian Kurts, Bonn, Ger-many. FcRg2/2 mice (B6;129P2-Fcer1gtm1Rav/J) on C57BL/6 back-ground were kindly provided by Prof. J. Engelbert Geßner, Hanover,Germany. All mouse strains were kept under standard animal housingconditions.
Fusion protein
Themouse B7-homologue 1mouse-IgG2aFc (B7-H1-Ig) fusion protein consistsof the mouse B7-H1 protein fused to the H chain (-Ig) part of the mouse IgG2aAb. The nucleotide sequence of themouse B7-H1-Ig fusion protein is as follows:59-ATGAGGATATTTGCTGGCATATTCACAGCCTGCTGTCGCTACGG-GCGTTTACTATCACGGCTCCAAAGGACTTGTACGTGGTGGAGTA-TGGCAGCAACGTCACGATGGAGTGCAGATTCCCTGTAGAACG-GGAGCTGGACCTGCTTGCGTTAGTGGTGTACTGGGAAAAGGAAG-ATGAGCAAGTGATTCAGTTTGTGGCAGGAGAGGAGGACCTTAA-GCCTCAGCACAGCAACTTCAGGGGGAGAGCCTCGCTGCCAAA-GGACCAGCTTTTGAAGGGAAATGCTGCCCTTCAGATCACAGAC-GTCAAGCTGCAGGACGCAGGCGTTTACTGCTGCATAATCAGCT-ACGGTGGTGCGGACTACAAGCGAATCACGCTGAAAGTCAATGC-CCCATACCGCAAAATCAACCAGAGAATTTCCGTGGATCCAGCC-ACTTCTGAGCATGAACTAATATGTCAGGCCGAGGGTTATCCAGA-AGCTGAGGTAATCTGGACAAACAGTGACCACCAACCCGTGAGT-GGGAAGAGAAGTGTCACCACTTCCCGGACAGAGGGGATGCTTC-TCAATGTGACCAGCAGTCTGAGGGTCAACGCCACAGCGAATGAT-GTTTTCTACTGTACGTTTTGGAGATCACAGCCAGGGCAAAACCA-CACAGCGGAGCTGATCATCCCAGAACTGCCTGCAACACATCCT-CCACAGAACAGGACTCACATAGATCTGGAGCCCAGAGGGCCCA-CAATCAAGCCCAGTCCTCCAAGCAAAAGCCCAGCACCTAACCTC-TTGGGTGGATCATCCGTCTTCATCTTCCCTCCAAAGATCAAGG-ATGTACTCATGATCTCCCTGAGCCCCATAGTCACATGTGTGGTG-GTGGATGTGAGCGAGGATGACCCAGATGTCCAGATCAGCTGGT-
TTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCC-ATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCT-CCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAA-TGCAAGGTCAACAACAAAGACCTCCCAGCGCCCATCGAGAG-AACCATCTCAAAACCCAAAGGGTCAGTAAGAGCTCCACAGGTA-TATGTCTTGCCTCCACCAGAAGAAGAGATGACTAAGAAACAGG-TCACTCTGACCTGCATGGTCACAGACTTCATGCCTGAAGACATT-TACGTGGAGTGGACCAACAACGGGAAAACAGAGCTAAACTACA-AGAACACTGAACCAGTCCTGGACTCTGATGGTTCTTACTTCATG-TACAGCAAGCTGAGAGTGGAAAAGAAGAACTGGGTGGAAAGA-AATAGCTACTCCTGTTCAGTGGTCCACGAGGGTCTGCACAATCA-CCACACGACTAAGAGCTTCTCCCGGACTCCGGGTAAATGATCTA-GA-39. Regular characters indicate mB7-H1 protein domain, whereasunderlined characters indicate mouse IgG2a-Fc sequence.
Subcloning was performed using mammalian expression vector pcDNA3.1(Invitrogen), and the protein was expressed in suspension HEK 293‐6E cellculture (GenScript). Cells were grown in serum-free Freestyle 293 ex-pression medium (Invitrogen, Carlsbad, CA). Transfection was carried outapplying mixed DNA and polyethylenimine (Polysciences, Eppelheim,Germany) to the cells. The supernatant was collected on day 6 and usedfor purification. Filtered supernatant was loaded onto a 5 ml HiTrapProtein A HP column (GE Healthcare, Uppsala, Sweden) at 1.0 ml/min.The fractions were collected and neutralized with 1M Tris-HCl, pH 9.0.
Murine TH cell differentiation
Differentiation was performed as described previously (33). In brief, mu-rine CD4+ T cells were isolated (spleen and lymph node) using magneticbeads (Miltenyi; negative selection protocol). Purified CD4+ cells werecultured in IMDM (10% FCS, 1% penicillin/streptavidin, 50 mM 2-ME,1% L-glutamine) and stimulated with 5 mg/ml plate-bound anti-CD3 (145-2C11) and 1 mg/ml soluble anti-CD28 (37.51; BD). For TH17 differenti-ation, cells were cultured in the presence of recombinant human TGF-b(rhTGF-b) (5 ng/ml; R&D Systems), murine IL-6 (20 ng/ml; eBioscience),anti–IFN-g mAb (10 mg/ml, XMG1.2; eBioscience), and anti–IL-4 mAb(10 mg/ml, 11B11; eBioscience). For TH1 differentiation, cells were cul-tured for up to 7 d with 10 ng/ml IL-12 and anti–IL-4 mAb (10 mg/ml).Plate-bound B7-H1-Ig and mouse IgG2a isotype control (G155-178; BD)were coated overnight at 4˚C in PBS. The IgG2a-Fc control fragment(BIOMOL) was preincubated with the T cells for 7 min at 37˚C beforeseeding them to the plates. B7-H1-Ig protein was typically used at 10 mg/mlunless otherwise indicated. Control IgG2a isotype and IgG2a-Fc controlswere used at 10 mg/ml concentration. The PD-1–specific blocking Ab (J43;eBioscience) and the according isotype control were used at 20 mg/ml. Theanti–B7-H1 mAb (10F.9G2; Biolegend) and control rat IgG2b, k isotypewere applied at 20 mg/ml and incubated on plates for 30 min at 37˚Cbefore seeding the cells. For determination of proliferation, cell prolifera-tion dye eFluor 670 (eBioscience) was applied and cell survival was de-termined using Hoechst (Molecular Probes).
Murine Treg induction
Purified mouse CD4+ T cells were stimulated with 5 mg/ml plate-boundanti-CD3 and 1 mg/ml soluble anti-CD28 Abs in the presence of 100 U/mlrIL-2 and rhTGF-b (2 ng/ml) for 72 h. Intracellular Foxp3 staining wasperformed using intranuclear staining kit (eBioscience) and Foxp3-allophycocyanin Ab (FJK-16s; eBioscience) and corresponding isotypecontrol (eBioscience).
Murine TH2 differentiation
Purified CD4+ T cells from BALB/c mice were stimulated with 5 mg/mlplate-bound anti-CD3 and 1 mg/ml soluble anti-CD28 Abs in the presenceof IL-4 (10 ng/ml) and anti–IFN-g mAb (10 mg/ml) for up to 72 h.Intranuclear staining was performed using the intranuclear staining kit(eBioscience) and GATA3-allophycocyanin Ab (TWAJ; eBioscience) andthe corresponding isotype control (eBioscience).
Flow cytometry
Cells were restimulated with 5 ng/ml PMA/ionomycin (Cayman Chemical)and 200 ng/ml ionomycin (Cayman Chemical) for 4 h in the presence ofGolgiPlug (BD Pharmingen). Subsequently, surface staining was performedat 4˚C for 30 min using the following Abs: CD4 (GK1.5; Biolegend),CD11b (M1/70; Biolegend), CD25 (PC61.5; eBioscience), CD45 (30-F11;Biolegend), PD-1 (29F.1A12; Biolegend), and CD69 (H1.2F3; eBio-science). Cells were fixed and permeabilized using Cytofix/Cytoperm PlusFixation/Permeabilization Kit (BD) and stained with IL-17A (eBio17B7;eBioscience) and IFN-g (XMG1.2; BD Pharmingen) or correspondingisotype controls. For analysis of IFN regulator factor-4 (IRF-4) and
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retinoic acid-related orphan receptor g t (RORgt) expression, purifiedCD4+ T cells were subjected to TH17 differentiation for indicated timepoints as described earlier and stained for CD4+ and IRF-4 (3E4; eBio-science) or RORgt (AFKJS-9; eBioscience) or corresponding isotype Abusing the intranuclear staining kit. Cells subjected to TH1 differentiationwere stained for the transcription factors Tbet (4B10; eBioscience) usingthe intranuclear staining kit. Stained cells were assayed on a FACSGalliosflow cytometer using Kaluza software (Beckman Coulter) and FlowJosoftware (Tree Star).
Quantitative real-time RT-PCR
Isolation of mRNA from T cells was performed with RNeasy Mini Kit(Qiagen) according to the manufacturer’s protocol. A total of 1000 ngmRNA was transcribed into cDNA by reverse transcription using Super-script III (Invitrogen) according to the manufacturer’s protocol. Expressionof mRNA was quantified in triplicates by real-time PCR using customgene-specific probes for IL-17F, IL-21, and IL-23R (Invitrogen) accordingto the manufacturer’s recommendations using TaqMan Gene ExpressionMaster Mix (Applied Biosystems). Amplification of cDNAwas carried outon a StepOne Plus real-time PCR cycler (Applied Biosystems). Dataquantification was performed by the DD cycle threshold method andnormalized to 18S expression.
Adoptive transfer EAE
C57BL/6 or PD-1 knockout donor mice were immunized s.c. with 50 mgmyelin oligodendrocyte glycoprotein (MOG) 35–55 (MOG35–55) in CFA(BD Difco). After 10 d, mice were euthanized and splenocytes wererestimulated in vitro with MOG35–55 (20 mg/ml; Biotrend) and IL-12(20 ng/ml) for 3 d in the presence of plate-bound B7-H1-Ig (10 mg/ml) orIgG2a isotype (10 mg/ml) control Ab. After 48 h, cells were againrestimulated with MOG35–55 and IL-12 (20 ng/ml) for the last 24 h. Subse-quently, in vitro restimulated splenocytes (8 3 106/per mouse) were injected(i.p.) into healthy C57BL/6 recipient mice, and mice were subsequentlytreated with pertussis toxin (17.5 ml/mouse; Sigma) at days 0 and 2 aftertransfer. Clinical EAE score was monitored daily. Disease severity was de-termined using a scale from 0 to 8: 0, no paralysis; 1, limp tail; 2, ataxia orunilateral hind-limb paresis; 3, severe unilateral or weak bilateral hind-limbparesis; 4, severe bilateral hind-limb paresis; 5, complete bilateral hind-limbplegia; 6, complete bilateral hind-limb plegia and partial forelimb paresis; 7,severe tetraparesis/plegia; and 8, moribund/dead animals.
Active MOG EAE
EAE was induced in C57BL/6 mice or in PD-1 knockout mice by im-munization with 50 mg MOG35–55 as described previously (33). From theday of immunization mice received daily i.p. injections of either B7-H1-Ig(60 mg/mouse) or corresponding IgG2a isotype control (60 mg/mouse) for5 consecutive days. Clinical EAE score was monitored daily.
To determine numbers of cytokine-producing cells in the CNS and spleenof EAE-diseased mice, we sacrificed mice on day 15 of EAE. Formononuclear cell isolation from the CNS, mice were euthanized withcarbon dioxide, perfused with PBS solution and heparin (Ratiopharm), andbrains and spinal cords were removed carefully. Tissue was mechanicallydissected and digested with 1 mg/ml collagenase A (Sigma) in PBS for 30min at 37˚C. The tissue was homogenized, washed, and cells were isolatedfrom the interface of 30–50% Percoll after centrifugation for 30 min at2500 rpm and used for further analysis by flow cytometry.
Single-cell suspensions of splenocytes were stained for flow cytometry asdescribed or seeded and restimulated in the presence of 5 mg/ml plate-bound anti-CD3 for up to 72 h. Cytokines in cell culture supernatants weredetected using ELISA Ready-SET-Go! (eBioscience) according to themanufacturer’s instructions.
Human TH17 differentiation
PBMCs were obtained from peripheral blood of healthy volunteers or frompatients with clinically definite relapsing-remitting MS (RR-MS) accordingto the McDonald criteria (34) who were clinically stable at the time ofblood withdrawal, in accordance with the local Ethics Committee. HumanCD4+ T cells were isolated with the RosetteSep Kit (STEMCELL) andstimulated with 1.5 mg/ml plate-bound anti-CD3 Ab (clone OKT3), 1 mg/mlsoluble anti-CD28 Ab (clone 28.2), 2.5 ng/ml human TGF-b (R&Dsystems), and 12.5 ng/ml human IL-21 (cell systems) for up to 7 d inserum-free X-VIVO 15 medium (BioWhittaker) (35). Plate-bound B7-H1-Ig or IgG2a isotype control were coated overnight at 4˚C in PBS. B7-H1-Igproteins were typically used (10 mg/ml) unless otherwise indicated. Hu-man IL-17A protein levels in cell culture supernatants were determined byELISA according to the manufacturer’s instructions (R&D Systems).
Restimulation of human effector T cells
Human PBMCs from RR-MS patients were directly ex vivo stimulated with1.5 mg/ml plate-bound anti-CD3 Ab (clone OKT3) plus 1 mg/ml solubleanti-CD28 Ab (clone 28.2) in the presence of brefeldin A for 4 h. After-ward, surface staining for CD3 and CD4 and subsequent intracellularstaining for IL-17A (eBioscience) and IFN-g (Biolegend) was performedas described earlier. Coated B7-H1-Ig protein was used as described.Successful TCR stimulation–induced cytokine response was defined as atleast 4-fold increase in cytokine-expressing CD4+ T cells; these patients(13/17 patients) were included in the analysis.
ELISA assays
Cell culture supernatants were analyzed using ELISA Ready-SET-Go!(eBioscience) for levels of IFN-g, IL-2, IL-4, IL-10, IL-22 (mouse), orIL-17A (mouse or human) and were performed according to the manu-facturer’s instructions.
Analysis of STAT-3, Smad2, and Smad3 signaling
Mouse CD4+ T cells were preincubated with 10 mg/ml B7-H1-Ig for 10min at 4˚C in RPMI 1640 medium. When indicated, cells were stimulatedwith different concentrations of rhTGF-b (20 ng/ml) or with recombinantmouse IL-6 (20 ng/ml) for 30 min at 37˚C. After stimulation, cells wereimmediately lysed [lysis buffer: 1% Nonidet P-40, 1% laurylmaltoside (N-dodecyl-D-maltoside), 50 mM Tris (pH 7.5), 165 mM NaCl, 10 mM EDTA,10 mM NaF, 1 mM phenyl-methylsulfonylfluoride, and 1 mM Na3VO4].Postnuclear lysates were subjected to SDS-PAGE and proteins wereelectrotransferred onto nitrocellulose membranes. Membranes were probedwith anti–p-Smad2 (Ser645/647, cl.138D4; Cell Signaling), anti–p-STAT3(Tyr705; Cell Signaling), and anti–p-Smad3 (Ser423/425; Millipore) Abs.Equal protein loading was controlled by probing the stripped membraneswith anti-mouse b-actin Ab (cl. AC-15; Sigma).
Statistical analysis
All data are expressed as mean 6 SEM. Statistical analysis was performedusing Student t test. A p value , 0.05 was considered significant and p ,0.01 was considered highly significant.
ResultsTo elucidate the role of B7-H1 during TH17 differentiation, weused an APC-free TH17 differentiation system to exclude con-founding effects by B7-H1 on APCs. We generated a B7-H1-Igfusion protein by fusing the mouse B7-H1 molecule to the mouseIgG2a-Fc domain. Purified murine CD4+ T cells were cultured inthe presence of plate-bound B7-H1-Ig or corresponding isotypecontrol under TH17-inducing conditions. Interestingly, in thepresence of B7-H1-Ig protein, TH17 differentiation (assessed byproduction of IL-17A after 72 h) was almost abolished (Fig. 1A,1B). Control experiments revealed that the suppressive effect ofB7-H1-Ig on TH17 differentiation was abolished upon coincuba-tion with a blocking Ab against B7-H1, thus indicating that theseeffects are indeed mediated by the B7-H1 part of the protein(Fig. 1C). Furthermore, both plate-bound mouse IgG2a isotypecontrol Ab and preincubation of cells with control IgG2a-Fcprotein did not affect TH17 differentiation, thus ruling out thatparts other than the B7-H1 domain are responsible for the strikinginhibitory effect (Fig. 1A, 1B). In addition, the suppressive effectof the B7-H1-Ig protein was preserved in Fc receptor knockoutT cells, thus excluding confounding effects mediated by the Fcpart of our fusion protein (data not shown). IL-17A productionwas suppressed by B7-H1-Ig in a dose-dependent manner(Fig. 1D). Notably, in vitro TH17 differentiation experiments in thepresence of B7-H1-Ig were performed independently in two dif-ferent laboratories and yielded consistent results; data analysiswas performed in a blinded fashion.In addition, B7-H1-Ig affects TH17 differentiation per se as B7-
H1-Ig treatment clearly interfered with cytokine-induced RORgtprotein expression (Fig. 1E), whereas both T cell proliferation andsurvival were not altered (Fig. 1F). Expression of early activation
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+IgG2a Isotype
+B7-H1-Ig
34%
1.4%
+IgG2a-Fc
+B7-H1-Ig
1.7%
33%
+IgG2a Isotype+IgG2a-Fc
A w/o
+IgG2a-Fc
35%
36%
IL-1
7AIL
-17A
0
5
10
15
20
25
30
35 **
RO
Rγt
+ c
ells
(%
)
W/O
+ B7-
H1-Ig
RORγt
2%
+B7-H1-Ig
37%
W/O
E
proli. index % divided01234
Th17+B7-H1-Ig
20406080
100
05
1015202530354045
+ IgG
2a
+ B7-H
1-Ig
αCD3/α
CD28
surv
ival
(%)
Th17-differentiated
F
C
αCD3/α
CD28
+ IgG
2a +
IgG2b
+ IgG
2a +
αB7-
H1
+ B7-
H1-Ig
+ αB7-
H1
+ B7-
H1-Ig
+ IgG
2b
Th17-differentiated
50100150200250300350400450 ++
n.s.
0
IL-1
7A p
g/m
l
αCD3/α
CD28
+ B7-
H1-Ig
+ IgG
2a Is
otype
+ IgG
2a-F
c
+ IgG
2a Is
otype
+ IgG
2a-F
c+ B
7-H1 I
g
+ IGg2
a-Fc
0
10
20
30
40
Th17-differentiated
***
W/O
B
IL-1
7A+
cells
(%)
W/O
0
5
10
15
20
25
30
35
B7-H1-Ig
°°
0.5 μg/m
l
10 μg/m
l
4 μg/m
l
2 μg/m
l
1 μg/m
l
°° °°
D
IL-1
7A+
cells
(%)
H
Th2
+B7-H
1-Ig
0
5
10
15
20
25
30
35
CD
4+ G
ATA
3+ c
ells
(%)
αCD3/α
CD28 Th1
+ B7-H
1-Ig
0
10
20
30
40
50
CD
4+ T
bet+
cel
ls (%
)
αCD3/α
CD28
I
Th1
+B7-H
1-Ig
Th1
+B7-H
1-Ig
010
30
50
70
90
110
48h
72h
IFN
γ n
g/m
l
Th2
+B7-H
1-Ig
Th2
+B7-H
1-Ig
0
2000
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FIGURE 1. B7-H1-Ig inhibits murine in vitro CD4+ TH17 differentiation. (A–D) Purified mouse CD4+ T cells were subjected to in vitro TH17 differ-
entiation in the presence or absence of plate-bound B7-H1-Ig protein (10 mg/ml), mouse IgG2a isotype (10 mg/ml), or mouse IgG2a-Fc protein (10 mg/ml)
(or combined) for 72 h and subsequently stained for IL-17A expression or supernatant was analyzed for IL-17A secretion (mean 6 SEM; *p = 0.0157,
**p , 0.0029, ssp , 0.0072). B7-H1 neutralizing Ab (20 mg/ml) or isotype control was incubated on the coated plate for 30 min before seeding of cells
(mean 6 SEM; **p = 0.0075). Representative data from at least three independent experiments are shown. (E) Representative RORgt intracellular staining
profiles of B7-H1-Ig–treated (right panel) versus nontreated (left panel) CD4+ T cells 16 h after initiation of TH17 differentiation. Bar graph shows
representative data obtained from one of three independent experiments (mean 6 SEM; **p = 0.0041). (F) Division index and percentage of divided cells
from three independent experiments (left) and percentage of survival from two independent experiments (right) measured 72 h after induction of TH17
differentiation. (G) Percentage of CD69+ and PD-1+ cells from WT (left) or PD-12/2 (right) mice 72 h after TH17 polarization. Graphs summarize unified
results of nine experiments using WT cells and three experiments using PD-12/2 cells (mean6 SEM; ***p = 0.0002). (H and I) Purified CD4+ T cells were
subjected to in vitro TH1 and TH2 differentiation for up to 72 h in the presence or absence of plate-coated B7-H1-Ig protein and (Figure legend continues)
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markers such as CD69 was not suppressed by B7-H1-Ig, furthersupporting the notion that B7-H1 binding does not impair generalT cell activation (Fig. 1G). In contrast to the effect of B7-H1-Ig onTH17 differentiation, IL-12–induced TH1 and IL-4–induced TH2differentiation as assessed by expression of the transcription fac-tors Tbet and GATA3, as well as production of IFN-g and IL-10,was completely unaffected (Fig. 1H, 1I). Further time-courseanalysis for up to 168 h did not reveal any suppressive effectsof B7-H1-Ig on TH1 differentiation (Supplemental Fig. 1A).We next investigated whether B7-H1-Ig exerted its suppressive
effect on TH17 differentiation via B7-H1-PD-1 interaction, be-cause PD-1 is the canonical receptor for B7-H1 on the T cell sideand our B7-H1-Ig protein has been shown to bind to PD-1 in anin vitro binding assay (Supplemental Fig. 2A). Surprisingly,a neutralizing Ab against PD-1 did not affect the B7-H1-Ig–mediated suppression of TH17 differentiation (Fig. 2A). Moreover,the inhibitory effect of B7-H1-Ig remained fully preserved whenTH17 differentiation experiments were performed using PD-1-KOT cells (Fig. 2B, middle column), also shown on mRNA levels ofTH17-related genes such as IL-17F, IL-21, and IL-23R (Fig. 2C).These results demonstrate that the observed B7-H1-Ig effect is notmediated via PD-1 expressed on T cells.Recently, B7.1 (CD80) has been identified as an alternative B7-
H1 binding receptor (36). However, we observed that B7-H1equally suppressed TH17 differentiation of B7.1/B7.2-deficientCD4+ T cells compared with wild-type (WT) T cells (Fig. 2B).Importantly, upon combined ablation of signaling via PD-1 andB7.1/B7.2 by using a neutralizing PD-1 Ab in B7.1/B7.2-deficientCD4+ T cells, the inhibitory effect of B7-H1-Ig is fully preserved(Fig. 2D, 2E). Taken together, we provide evidence that B7-H1–mediated suppression of TH17 differentiation in vitro is neithermediated via PD-1 nor via B7.1 nor B7.2, which strongly suggeststhe presence of a novel yet unknown receptor for B7-H1 onT cells. Notably, B7-H1-Ig reciprocally facilitated TGF-b–induced differentiation of Treg (Fig. 2F).Based on these findings, we investigated potential molecular
mechanisms by which B7-H1-Ig influences TH17 differentiation.The transcription factor IRF-4 is not only crucial for TH17 dif-ferentiation but also tightly regulates TH17 versus Treg lineagedecision (37). We therefore addressed the impact of B7-H1-Ig onIRF-4. Indeed, we observed that early during TH17 differentiationB7-H1-Ig interfered with IRF-4 expression, as the proportion ofIRF-4–expressing cells is significantly reduced (Fig. 3A–C). Im-portantly, also on a per-cell basis, IRF-4 expression reflected bymean fluorescence intensity (MFI) levels within the population ofIRF-4+ cells was significantly reduced upon B7-H1-Ig treatment(Fig. 3B). The reduction in IRF-4 expression by B7-H1-Ig wasequally pronounced in WT T cells and in PD12/2 or B7.12/2/B7.22/2 T cells (Fig. 3B, 3C). This clearly reveals that B7-H1-Iginterferes with cytokine-mediated induction of IRF-4 during TH17differentiation on a single-cell level, and this effect is independentfrom expression of PD12/2 or B7.12/2/B7.22/2 on T cells. In-terestingly, it has been shown that IRF-4 not only induces RORgtexpression, but also controls Foxp3 expression under TH17-inducing conditions, and reduction in IRF-4 levels results in aug-mented Foxp3 levels in TH17 cells (37). Along this line, we alsoobserved enhanced Foxp3 expression in T cells subjected to TH17differentiation in the presence of B7-H1-Ig (Fig. 3D). Anotherfeature of IRF4-mediated effects during TH17 differentiation isdiscordant regulation of IL-17A and IL-22 production (38). We
therefore compared the effect of B7-H1-Ig on IL-22 and IL-17Asecretion by TH17 cells (Fig. 3E). Strikingly, whereas B7-H1-Igreduced IL-17A levels by .90% compared with isotype controltreatment, IL-22 levels were reduced only slightly by ∼38%.Together, this strongly supports the notion that B7-H1 influencesTH17 differentiation via reducing IRF-4 levels. Notably, cytokinesignaling was not significantly affected by B7-H1-Ig during T celldifferentiation, because neither STAT-3 signaling nor smad 2/3signaling was altered by B7-H1-Ig (Fig. 3F, 3G).We next addressed the in vivo relevance of B7-H1-Ig–mediated
interference with TH17 cell responses during CNS autoimmunityby using two different EAE approaches. In active MOG EAE,early treatment of immunized WT mice with B7-H1-Ig for 5 dresulted in a long-lasting significant disease amelioration (Fig. 4A)compared with mIgG2a isotype control-treated mice.In a second approach, we aimed at investigating whether B7-H1-
Ig has the capacity to reduce the encephalitogenic potential ofalready primed MOG-specific WT T cells. To this end, werestimulated spleen and lymph node cells from immunized WTmice in the presence of B7-H1-Ig or corresponding isotype controlAb and adoptively transferred these cells into WT recipients in anadoptive transfer EAE approach. Also in this setting, B7-H1-Igtreatment of WT T cells during in vitro MOG recall signifi-cantly alleviated EAE symptoms (Fig. 4B). In accordance withour in vitro data, the alleviating effect of B7-H1-Ig during MOG-specific recall was equally pronounced when using T cells fromPD-1KO mice (Fig. 4C), indicating that also in vivo, PD-1 is dis-pensable for B7-H1-Ig–mediated effects during CNS autoimmunity.We next addressed the impact of B7-H1-Ig on Th cell responses
during CNS autoimmunity. Analysis of restimulated splenic im-mune cells from MOG-immunized WT mice receiving B7-H1-Igfrom days 1–5 after immunization revealed a selective reductionin IL-17A but not IFN-g production as assessed by flow cytometryat day 15 after immunization (Fig. 4D, upper panel). Moreover,polyclonal recall of splenic cells from these mice demonstrateda significant reduction of IL-17A secretion, whereas IFN-g, IL-2,IL-4, and IL-10 secretion were not altered (Fig. 4D, lower panel).Moreover, CNS analysis of WT EAE mice that received B7-H1-Igdemonstrated a reduction in the number of CNS-infiltrating CD4+
T cells, the percentage of CD4+ T cells producing IL-17A uponrestimulation, as well as the total number of IL-17A–producingCD4+ T cells within the CNS, whereas both total cell numbers andthe percentage of IFN-g–producing CD4+ T cells were not altered(Fig. 4E).Furthermore, we observed a reduction in the expression of
several TH17-related but not TH1-related proinflammatory medi-ators within the CNS of B7-H1-Ig–treated EAE mice (Fig. 4F).Together, our in vivo data allow three important conclusions: 1)
mB7-H1-Ig is capable of controlling T cell–mediated CNS auto-immunity, 2) B7-H1-Ig limits autoreactive T cell responses evenafter in vivo priming as demonstrated by the adoptive transferEAE experiments, and 3) expression of PD-1 on T cells is dis-pensable for the disease-alleviating effect of B7-H1 during EAE.Finally, we investigated whether human CD4+ T cells are also
responsive to B7-H1-Ig–mediated control of TH17 differentiation,given the high homology of murine and human B7H1 and the factthat B7-H1-Ig also binds to human PD-1 (Supplemental Fig. 2B).Indeed, human CD4+ T cells were also responsive toward B7-H1-Ig–mediated suppression during TH17 but not TH1 differentiation(Fig. 5A, 5B, and not shown). Notably, the in vitro suppressive
subsequently analyzed for Tbet and GATA3 expression by flow cytometry; moreover, IFN-g and IL-10 secretion were measured in the according
supernatants. Graph shows one representative experiment of four independent experiments and depicts mean 6 SEM.
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effect on CD4+ T cells derived from healthy donors was stable overseveral days (Fig. 5C). Furthermore, pretreatment of CD4+ T cellswith an anti–PD-1 blocking Ab did not alter the suppressive capacity
of mB7-H1-Ig on human TH17 differentiation, thus indicating thatthis effect is, in analogy to the murine system, not mediated via PD-1(Fig. 5B). Importantly, CD4+ T cells from patients suffering from
FIGURE 2. B7-H1 interferes with TH17 lineage development via a novel B7-H1 receptor. (A) Purified mouse CD4+ T cells were subjected to TH17
differentiation in the presence or absence of plate-bound B7-H1-Ig protein (10 mg/ml) and a neutralizing Ab against mouse PD-1 (10 mg/ml) for 72 h and
subsequently stained for IL-17A expression and analyzed by flow cytometry. Representative dot plots of two independent experiments are shown. (B)
Purified mouse CD4+ T cells from WT, PD-12/2, and B7.12/2/B7.22/2 mice were subjected to TH17 differentiation in the presence or absence of plate-
bound B7-H1-Ig protein (10 mg/ml) as described before. Representative FACS profiles of one of three independent experiments are shown. (C) Purified
mouse PD-12/2 CD4+ T cells were subjected to in vitro TH17 differentiation in the presence or absence of plate-bound B7-H1-Ig protein (10 mg/ml). After
48 h cells were harvested, mRNA was isolated and transcribed to cDNA, and the relative expression of IL-17F, IL-21, and IL-23R was determined in
relation to expression in undifferentiated CD4+ T cells by quantitative real-time PCR. Bar graphs show pooled results from three independent experiments
(mean 6 SEM; *p = 0.0351, **p , 0.0062). (D and E) Purified mouse B7.12/2/B7.22/2 CD4+ T cells were subjected to TH17 differentiation in the
presence or absence of plate-bound B7-H1-Ig protein (10 mg/ml) and a neutralizing Ab against mouse PD-1 (10 mg/ml) for 72 h and subsequently stained
for IL-17A expression and analyzed by flow cytometry. Dot plots and graph show representative data of four separate experiments (mean 6 SEM; WT,
***p = 0.0004, B7.1/B7.22/2, **p = 0.0049). (F) Purified mouse CD4+ T cells from WT or PD12/2 mice were subjected to in vitro inducible Treg (iTreg)
differentiation with 0.5 ng/ml TGF-b in the presence or absence of plate-bound B7-H1-Ig protein (10 mg/ml). After 72 h, surface (CD4 and CD25) along
with intracellular Foxp3 staining was performed. Representative dot plot profiles show percentage of CD25+Foxp3+ out of CD4+ T cells. Bar graph shows
one representative experiment out of three independent experiments and depicts mean 6 SEM (WT, **p = 0.0013, PD-12/2, *p = 0.0236).
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FIGURE 3. B7-H1-Ig interferes with IRF-4 induction during TH17 differentiation. (A–C) Early IRF-4 expression in murine CD4+ T cells subjected to
TH17 differentiation in the presence of plate-bound B7-H1-Ig or corresponding isotype control as described before. (A) Dot plots show percentages of
IRF4+ CD4+ cells at 7 h after cytokine-induced TH17 differentiation; dot plot on the right depicts isotype staining control for IRF-4. One representative
experiment of three is shown. Bar graphs (B) show corresponding IRF-4 MFI values from one representative of three experiments. White bars represent WT,
gray bars represent PD-12/2, and black bars indicate B7.12/2/B7.22/2 CD4+ T cells (***p = 0.0001). (C) Time course of IRF-4 expression in CD4+ T cells
upon TH17 induction; bar graph depicts percentages of CD4+IRF4+ cells at different time points (mean 6 SEM values), significance for untreated versus
treated groups: ˚˚˚p, 0.0013, +++p, 0.0004, **p = 0.0018, ***p, 0.0007. (D) Murine CD4+ T cells were subjected to TH17 differentiation in the presence
of plate-bound B7-H1-Ig or corresponding isotype control as described and analyzed for Foxp3 expression by flow cytometry. Representative dot plots from
one out of three experiments are shown depicting the percentage of Foxp3-expressing CD4+ T cells. Bar graph shows corresponding average Foxp3 MFI
values (*p = 0.0102). (E) Purified mouse CD4+ T cells from WT mice were subjected to TH17 differentiation as described in the presence of plate-bound
B7-H1-Ig (10 mg/ml) protein versus IgG2a isotype control (10 mg/ml). After 72 h, supernatants were collected and IL-22 and IL-17A levels were analyzed
by ELISA. Graph shows pooled data of IL-22 and IL-17A levels from three independent experiments (mean 6 SEM; **p = 0.0058). (F and G) Purified
CD4+ T cells from WT, PD-12/2, and B7.12/2/B7.22/2 mice were stimulated with recombinant mouse IL-6 (20 ng/ml) (F) or with rhTGF-b (20 ng/ml) (G)
in the presence or absence of B7-H1-Ig (10 mg/ml) protein for 30 min at 37˚C. After stimulation, cells were lysed immediately, and postnuclear lysates were
transferred onto nitrocellulose membrane and probed with anti–p-STAT3 (Tyr705), anti–p-Smad2 (Ser465/467) and anti–p-Smad3 (Ser425/427) Abs, respec-
tively. Equal protein loading was controlled by probing the membranes with anti-mouse b-actin. Asterisks indicate unspecific signal caused by the Ig
domain of B7-H1-Ig fusion protein that is recognized by the anti-mouse HRP secondary Ab.
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FIGURE 3. Continued
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FIGURE 4. B7-H1-Ig alleviates active and adoptive transfer EAE. (A) Active MOG35–55 EAE was induced in WT C57BL6. Immunized mice received
daily i.p. injections of either B7-H1-Ig (60 mg/mouse) or corresponding mIgG2a isotype Ab (60 mg/mouse) for 5 consecutive days. Animals were
monitored daily and scored for clinical EAE symptoms. Graphs show mean clinical scores (seven mice/group) 6 SEM. (B and C) For the adoptive transfer
EAE, splenocytes were isolated from MOG35–55–immunized WT (B) and PD-12/2 (C) mice at day 10 after immunization. Cells were in vitro restimulated
with MOG35–55 (10 mM) for 72 h in the presence of plate-bound 10 mg/ml B7-H1-Ig or 10 mg/ml IgG2a isotype control and adoptively transferred into
healthy C57/BL6 recipient mice (n = 8 mice/group). Mice were monitored daily for clinical EAE signs. Mean clinical scores at the individual time points
are shown with SEM values (*p = 0.05, **p = 0.01, ***p = 0.001). (D) Polyclonal restimulated for 4 h at 37˚C in the presence of PMA/ionomycin and
GolgiPlug for subsequent flow-cytometric quantification of CD4+ IFN-g+ and CD4+ IL-17A+ T cells (**p = 0.0056) from the spleens of active EAE-
diseased mice (left) and ELISA-based quantification of IFN-g, IL-17A (**p = 0.0015), IL-2, IL-4, and IL-10 secretion of restimulated splenocytes derived
from active EAE mice (right). Graphs are shown with SEM (day 15: n = 9 IgG2a, n = 10 B7-H1-Ig). (E) Flow-cytometric quantification of CD4+, CD4+ IFN-g,
and CD4+ IL-17A+ T cells from the CNS of active EAE-diseased WT mice (day 15: n = 9 IgG2a, n = 10 B7-H1-Ig). Scatterplots depict cell numbers 6 SEM
(*p = 0.0179, **p = 0.0021), graphs show mean percentage6 SEM (**p = 0.0059). (F) Expression of inflammatory mediators in the CNS of EAE-diseased WT
with or without B7-H1-Ig treatment was determined by quantitative real-time reverse transcriptase PCR on day 15 (n = 6/group). Data were normalized to
endogenous 18s and are displayed as rate of expression compared with healthy WT CNS tissue. Data are expressed as mean 6 SEM.
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RR-MS (stable disease, untreated; Supplemental Table 1, firstcohort) were equally responsive toward B7-H1-Ig–mediatedsuppression of TH17 differentiation (Fig. 5A, filled bars), althoughthese cells showed an increased propensity toward TH17 differen-tiation compared with healthy individuals (Fig. 5A, open bars).
To address whether B7-H1-Ig was also capable of suppressingIL-17A production by already primed effector T cells from RR-MSpatients (Supplemental Table 1, second cohort), we restimulatedPBMCs with anti-CD3 and anti-CD28 for 4 h in the presence orabsence of B7-H1-Ig and subsequently assessed cytokine pro-
FIGURE 5. B7-H1-Ig suppresses TH17 differentiation of human CD4+ T cells from healthy control subjects and MS patients. (A) Human CD4+ T cells from healthy
donors (HD) and RR-MS patients were subjected to TH17 differentiation in the presence of plate-bound B7-H1-Ig (10 mg/ml) protein or corresponding IgG2a isotype
control. As control, CD4+ cells were stimulated without addition of polarizing cytokines. Supernatants were collected on day 7 and IL-17A ELISAwas performed. Graph
shows pooled results obtained from nine healthy control subjects (***p = 0.0009) and nine MS patients (+++p = 0.0008, **p = 0.0021). Error bars indicate SEM values. (B)
Human purified CD4+ T cells from healthy donors (n = 3) were either preincubated with 10 mg/ml human anti–PD-1 neutralizing Ab for 7 min or left untreated. Sub-
sequently, cells were subjected to TH17 differentiation in the presence or absence of plate-bound B7-H1-Ig (10 mg/ml) or corresponding IgG2a isotype control. Supernatants
were collected on day 5, and IL-17A secretion was determined by ELISA. Graphs are shown as mean 6 SEM (**p = 0.0076, ++p = 0.0081). (C) Purified human CD4+
T cells from healthy donors (n = 3) were subjected to TH17 differentiation in the presence of plate-bound B7-H1-Ig (10 mg/ml) protein or IgG2a isotype control (10mg/ml).
Supernatants were collected from days 4–7. IL-17A concentration in supernatants was determined by ELISA, graph depicts mean 6 SEM values (*p = 0.0118, **p ,0.0083). (D) Human PBMCs from RR-MS patients were isolated and directly ex vivo (re)stimulated for 4 h with anti-CD3 and anti-CD28 Abs and brefeldin A in the
presence or absence of B7-H1-Ig. Subsequently, surface staining for CD3 and CD4 and intracellular IL-17A and IFN-g staining were performed. Graphs summarize data
from a total of 13 patients and show percentages of cytokine-producing cells within the CD4+ T cell population. Before and after plots demonstrate cytokine-producing cells
in individual patients. *p = 0.0159.
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duction in CD4+ T cells by flow cytometry. Importantly, B7-H1-Igsignificantly suppressed IL-17A (p = 0.015), but not IFN-g(p = 0.16), production by effector T cells (Fig. 5D). We thereforeconclude that also in humans, B7-H1-Ig might be used success-fully to control effector TH17 responses.
DiscussionOur study provides the first evidence, to our knowledge, thatB7-H1 nearly abolishes TH17 lineage differentiation of murineand human CD4+ T cells and hence controls T cell–mediated CNSautoimmunity.Several lines of evidence point to a novel PD-1–independent
effect of B7-H1-Ig during TH17 differentiation. First, we blockedinteraction of B7-H1-Ig with murine or human PD-1 using a PD-1binding Ab and found no interference with the anti-TH17 effect.Second, the completely preserved effect of B7-H1-Ig on TH17differentiation using PD-1KO T cells provides unequivocal evi-dence that, at least in the murine system, this effect is not medi-ated via PD-1. Although the presence of a non-PD receptor forB7-H1 based on molecular modeling and functional mappinghas been suggested before (12, 39), our study now provides clearexperimental evidence of non–PD-1–mediated effects by B7-H1.Recently, B7.1 was identified as another receptor for B7-H1 onT cells (40), but in our setup, involvement of B7.1 could also beexperimentally excluded. Together, our data suggest that block-ade of TH17 differentiation by B7-H1-Ig is mediated via a so farunrecognized B7-H1 receptor. We are fully aware that wecannot rule out the possibility that in human T cells B7-H1 mightregulate TH17 lineage differentiation via a novel receptor andPD-1 combined. However, the fact that presence of a blockingAb against PD-1 on human T cells did not affect their respon-siveness toward B7-H1-Ig strongly suggests that this novel re-ceptor also plays a dominant role in control of human TH17differentiation.Interestingly, B7-H1 did not interfere with TH1 differentiation,
indicating that B7-H1 does not generally interfere with effectorT cell generation. This result was unexpected, because severalstudies proposed that this pathway primarily affects generation ofIFN-g–producing TH1 cells (41–44). Another aspect that arguesagainst a general interference with T cell responses by B7-H1 isthe selective promoting effect of B7-H1-Ig on murine TGF-b–mediated inducible Treg (iTreg) differentiation, in line with otherstudies (30, 45, 46). However, it has been reported that inductionof iTreg generation and function by B7-H1 is mediated via PD-1(30, 46). Hence although our data set provides clear evidence forthe presence of a novel non–PD-1 receptor executing B7-H1effects on TH17 differentiation, other studies complementarilyillustrate that under certain conditions such as presence of steady-state dendritic cells or under suboptimal stimulatory conditions,B7-H1-PD-1 interaction is also involved in the modulation ofT cell differentiation. Our study clearly extends current knowledgeby providing evidence of a reciprocal influence of B7-H1 on TH17versus Treg differentiation without affecting TH1 and TH2 differ-entiation, which is not mediated via PD-1.Concerning the molecular mechanisms of B7-H1-Ig–mediated
modulation of T cell differentiation, we made several interestingobservations. First, B7-H1-Ig suppressed RORgt expression inT cells during TH17 differentiation, thus indicating that B7-H1-Igindeed interferes with TH17 differentiation rather than merelysuppressing IL-17A production. Second, B7-H1-Ig interfered withearly expression of IRF-4 during TH17 differentiation, which isparticularly interesting, because interference with IRF-4 notonly results in impaired RORgt and consequently IL-17A ex-pression, but also reciprocally enhanced Foxp3 expression (37,
38). Moreover, it has been demonstrated that abrogation ofIRF-4 signaling does not affect IL-22 production. In our hands,we observed that B7-H1-Ig: 1) repressed IRF-4 and RORgt in-duction under TH17-inducing conditions, 2) resulted in enhancedFoxp3 expression under TH17-inducing conditions, and 3) differ-entially affected IL-17A and IL-22 production. Although formalproof of the functional link between B7-H1-Ig–mediated TH17suppression and IRF-4 is lacking as targeted restriction of IRF-4expression would generally interfere with TH17 differentiation andthus preclude assessment of B7-H1-Ig–mediated effects, severallines of evidence strongly support the notion that B7-H1-Ig indeedexerts its striking effects during TH17 differentiation by repressionof IRF4 induction via the novel receptor.We observed augmented TH17 differentiation when using
CD4+ T cells from MS patients compared with healthy controlsubjects, which illustrates the enhanced susceptibility of T cellsfrom autoimmune-prone patients to develop into effector T cells.Importantly, B7-H1-Ig was still capable of strongly affecting denovo TH17 differentiation, highlighting the potential therapeuticrelevance of an external B7-H1 signal to control important stepsduring T cell–mediated autoimmunity. Because this was also thecase in the presence of a PD-1–blocking Ab, it can be speculatedthat a novel B7-H1 receptor exists also in humans that is func-tionally homologue with the mouse novel B7-H1 receptor.Following this line, we addressed whether in vivo application of
B7-H1-Ig during the priming phase of EAE has the capacity tocontrol CNS autoimmunity. Indeed, B7-H1-Ig was capable ofcontrolling T cell–mediated CNS autoimmunity, indicating thatthe novel receptor is operative in vivo. In strong support of ourin vitro experiments, control of disease severity in the active EAEmodel was accompanied by a selective reduction in peripheral andCNS local IL-17A production, but not IFN-g production, indi-cating that B7-H1-Ig–mediated selective control of TH17 responsesis also operative in vivo in the context of CNS autoimmunity.Notably, B7-H1-treatment was also efficient after the primingphase, because restimulation of encephalitogenic T cells in thepresence of B7-H1-Ig was effective to ameliorate CNS autoim-munity in the adoptive transfer EAE, and this was clearly inde-pendent from PD-1 on T cells. Finally, and in accordance withthese results, we demonstrate that B7-H1-Ig was even capable ofcontrolling IL-17A production during restimulation of fully dif-ferentiated human effector T cells, thus indicating that also inhumans this pathway might be exploited for control of alreadyongoing TH17-mediated CNS autoimmunity.So far there has been no experimental evidence that a signal
provided by B7-H1 exerts protective effects in animal models ofautoimmunity, although in vivo application of B7-H1 fusion pro-teins has been shown to protect from liver ischemia and reperfusioninjury, as well as from transplant rejection (47, 48). Hence our datademonstrate for the first time, to our knowledge, that in vivo de-livery of a B7-H1 signal might be useful as therapeutic approachfor treatment of autoimmunity.Taken together, this study provides evidence for the existence of
a clinically relevant novel B7-H1 non–PD-1–mediated “anti-TH17”immune-regulatory pathway. It can be concluded that: 1) B7-H1-Ig is capable of interfering with autoimmunity in vivo at leastpartly via a novel receptor; 2) human T cells are also responsivetoward B7-H1-Ig–mediated suppressive effects; and 3) this ap-proach might even be useful for alleviation of already ongoingT cell–mediated autoimmunity, that is, which is the situationclinicians are regularly handling.We therefore propose that targeted signaling via B7-H1 in vivo
might be exploited for control of T cell–mediated autoimmunity ina variety of diseases including MS.
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AcknowledgmentsWe are grateful to the MS patients and healthy volunteers for donating
blood for this study.
DisclosuresH.W. has received funding for travel and speaker honoraria from Bayer
Schering Pharma, Biogen Idec/Elan Corporation, Sanofi-Aventis, Merck
Serono, and Teva Pharmaceutical Industries Ltd.; serves and has served
as a consultant for Merck Serono, Medac, Inc., Sanofi-Aventis/Teva Phar-
maceutical Industries Ltd., Biogen Idec, Bayer Schering Pharma, Novartis,
and Novo Nordisk; and receives research support from Bayer Schering
Pharma, Biogen Idec/Elan corporation, Sanofi-Aventis, Merck Serono,
and Novo Nordisk. L.K. has received honoraria for lecturing and travel
expenses for attendingmeetings, and has received financial research support
from Novartis, CSL Behring, Merck Serono, Genzyme, and the Deutsche
Forschungsgemeinschaft. The other authors have no financial conflicts of
interest.
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