Immunoinhibitory checkpoint deficiency in medium andlarge vessel vasculitisHui Zhanga,1, Ryu Watanabea,1, Gerald J. Berryb, Augusto Vaglioc, Yaping Joyce Liaod, Kenneth J. Warringtone,Jörg J. Goronzya, and Cornelia M. Weyanda,2

aDepartment of Medicine, Division of Immunology and Rheumatology, Stanford University School of Medicine, Stanford, CA 94305; bDepartment ofPathology, Stanford University School of Medicine, Stanford, CA 94305; cDivision of Nephrology, University Hospital of Parma, 43100 Parma, Italy;dDepartment of Ophthalmology, Stanford Byers Eye Institute, Stanford University School of Medicine, Stanford, CA 94305; and eDivision of Rheumatology,Mayo Clinic College of Medicine, Rochester, MN 55905

Edited by Tasuku Honjo, Graduate School of Medicine, Kyoto University, Kyoto, Japan, and approved December 19, 2016 (received for review October10, 2016)

Giant cell arteritis (GCA) causes autoimmune inflammation of theaorta and its large branches, resulting in aortic arch syndrome,blindness, and stroke. CD4+ T cells and macrophages form orga-nized granulomatous lesions in the walls of affected arteries, de-stroy the tunica media, and induce ischemic organ damage throughrapid intimal hyperplasia and luminal occlusion. Pathogenic mecha-nisms remain insufficiently understood; specifically, it is unknownwhether the unopposed activation of the immune system is becauseof deficiency of immunoinhibitory checkpoints. Transcriptome anal-ysis of GCA-affected temporal arteries revealed low expression ofthe coinhibitory ligand programmed death ligand-1 (PD-L1) concur-rent with enrichment of the programmed death-1 (PD-1) receptor.Tissue-residing and ex vivo-generated dendritic cells (DC) from GCApatients were PD-L1lo, whereas the majority of vasculitic T cellsexpressed PD-1, suggesting inefficiency of the immunoprotectivePD-1/PD-L1 immune checkpoint. DC–PD-L1 expression correlated in-versely with clinical disease activity. In human artery-SCID chimeras,PD-1 blockade exacerbated vascular inflammation, enriched for PD-1+ effector T cells, and amplified tissue production of multiple T-celleffector cytokines, including IFN-γ, IL-17, and IL-21. Arteries infil-trated by PD-1+ effector T cells developed microvascular neoangio-genesis as well as hyperplasia of the intimal layer, implicating T cellsin the maladaptive behavior of vessel wall endogenous cells. Thus,in GCA, a breakdown of the tissue-protective PD1/PD-L1 checkpointunleashes vasculitic immunity and regulates the pathogenic remod-eling of the inflamed arterial wall.

vasculitis | T cells | immune checkpoint | autoimmunity | PD-1

Giant cell arteritis (GCA) is a granulomatous vasculitis with astringent tissue tropism, named after the multinucleated

giant cells that populate the inflammatory lesions in the arterialwalls. Granulomatous infiltrates composed of CD4+ T cells andmacrophages penetrate from the adventitia into the media anddestroy the lamina elastica interna. T cells with identical T-cellreceptor (TCR) sequences have been isolated from spatiallydistinct lesions (1, 2), highly suggestive for antigen-driven T-cellactivation, yet no singular vasculitogenic antigen has been defined.Lesional T cells provide a spectrum of effector functions, suspi-ciously diverse, and ranging from the production of IFN-γ, IL-17,and IL-9 to IL-21 (3, 4). Similarly, multiple functional macrophagesubsets participate in granuloma formation, spanning from mac-rophages committed to cytokine production (IL-6, IL-1β), to thosereleasing reactive oxygen species, to those providing growth fac-tors (PDGF, FGF) and angiogenic factors (VEGF) (5–7).The wall layers of large and medium vessels have features of

an immunoprivileged niche (8) and the invasion of inflammatorycells in GCA essentially breaks this immune privilege. Underphysiologic conditions endogenous dendritic cells (DC), so-calledvascular DC (vasDC), populate the arterial wall in a vessel-specificdistribution pattern and may protect against immune attack (9).Localized in the adventitial layer, such vasDC are close to the vasavasorum, and are positioned between the vascular access to the

wall and the immunoprivileged tissue site. Their role in controllingthe influx of immune cells into the mural structures remains un-defined. Similarly, understanding how antigen-nonspecific factorsaffect the intensity and the quality of the vasculitogenic immuneresponse could redefine critical pathogenic processes with a majorimpact on immunosuppressive strategies (10).T-cell–dependent immune responses are fine-tuned by a mul-

titude of costimulatory and coinhibitory signals, provided by re-ceptor–ligand interactions that modulate TCR-initiated signalingcascades (11). Such immune checkpoints are crucial for themaintenance of self-tolerance, prevent autoimmune disease, andprotect against collateral tissue damage (12). Conversely, excessiveexpression of immune checkpoint proteins has been associatedwith immune resistance mechanisms, prominently used by tumorcells to escape from antitumor immunity (13). Recent successes incancer immunotherapy have highlighted the importance of in-hibitory immune checkpoints that stop antigen-reactive T cells.Specifically, monoclonal antibodies that block the programmeddeath-1/programmed death ligand-1 (PD-1/PD-L1) pathway haveyielded unprecedented therapeutic benefit in patients with ad-vanced solid tumors (14–16). PD-1 is expressed on activated T andB cells and its engagement by its ligands PD-L1 or PD-L2 disruptskinase activity in the TCR-activation cascade through the phos-phatase SHP2. Resulting immunosuppression involves severalmechanisms, including T-cell apoptosis, T-cell exhaustion, T-cellanergy, T-cell IL-10 production, and Treg induction.

Significance

Antigen recognition by the immune system triggers rapid,specific, and protective responses, which are counterbalancedby inhibitory checkpoints to minimize potentially harmful im-munity. The programmed death-1/ programmed death ligand-1(PD-1/PD-L1) checkpoint is overreactive in cancer patients,curbing antitumor immunity. Whether a failing PD-1/PD-L1checkpoint contributes to spontaneous autoimmune disease inhumans is unknown. Here, we found that in patients with theautoimmune vasculitis giant cell arteritis, antigen-presentingcells provide insufficient negative signaling; unleashing highlyactivated T cells to infiltrate and damage the walls of largearteries. Thus, immunoinhibitory signals protect large arteriesagainst inflammatory attack and checkpoint activation may bea suitable strategy to treat autoimmune vasculitis.

Author contributions: J.J.G. and C.M.W. designed research; H.Z. and R.W. performed re-search; G.J.B. supervised tissue analysis; H.Z., R.W., G.J.B., A.V., Y.J.L., K.J.W., J.J.G., andC.M.W. analyzed data; Y.J.L. and K.J.W. recruited patients; and J.J.G. and C.M.W. wrotethe paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1H.Z. and R.W. contributed equally to this work.2To whom correspondence should be addressed. Email: cweyand@stanford.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1616848114/-/DCSupplemental.

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The contrasting scenario to immune resistance is exuberantimmunity, leading to immune-mediated tissue injury and auto-immune diseases. PD-1 and PD-L1 deficiency have been associ-ated with a lupus-like syndrome (17) and a dilated myocardiopathy(18), respectively. Lack of PD-L1 or PD-1 exacerbates murinediabetes and experimental autoimmune encephalitis (19, 20) andPD-L1 overexpression reduces spinal cord T-cell infiltrates (21).PD-L1−/− antigen-presenting cells (APCs) fail to convert naïveCD4+ T cells into Tregs (22), and PD-1−/− mice are prone toenrich for Th1 and Th17 cells (23).Guided by a transcriptomic signature of GCA-affected arteries

that lacked expression of the inhibitory ligand PD-L1, we haveexplored the role of the PD pathway in regulating the intensityand the functional orientation of vasculitogenic T-cell responses.GCA vascular lesions are occupied by PD-L1lo DCs and PD-1hiT cells. PD-L1lo DCs from GCA patients enhance T-cell acti-vation and proliferative expansion. In a humanized mouse modelof vasculitis, treatment with anti–PD-1 antibodies effectivelyaccelerates the recruitment and retention of T cells and inten-sifies T-cell and macrophage responses in the inflamed artery.Vasculitogeneic T cells produce IFN-γ, IL-17, and IL-21, sus-taining multifunctional effector functions. Accumulation of suchmultifunctional T-cell populations is associated with the rapidoutgrowth of hyperplastic intima, activation of endothelial cells,and the induction of microvascular neoangiogenesis, connectingT cells to disease-relevant remodeling processes in the vascularwall. In essence, a dysfunctional inhibitory immune checkpointexposes the vessel wall to inflammatory attack, formation ofmicrovascular networks, and intimal hyperplasia, ultimatelypromoting ischemic organ damage. Reconstitution of a func-tional PD checkpoint could provide an entirely new strategy totreat medium- and large-vessel vasculitis.

MethodsTissues, Cells, and Antibodies. Temporal arteries were collected from di-agnostic biopsies. A diagnosis of GCA was based on typical histologicalfindings. Temporal arteries were considered negative for GCA if no in-flammatory cells were identified on histology. Artery biopsies from patientswith a diagnosis of polymyalgia rheumatic were excluded, as they can benegative by histology but have altered function of vasDC (24, 25). Normalhuman aorta, temporal, and axillary arteries were donated by organ donors.Patients were enrolled into the protocol if they had a diagnosis of biopsy-positive GCA or anti-cyclic citrullinated peptide-positive rheumatoid arthritis(RA) and had active disease. Patient demographic characteristics are listed inTable 1. Diagnostic biopsies of the lung and the skin of patients with gran-ulomatosis with polyangiitis (GPA), a small-vessel vasculitis, served as controls.Age-matched healthy controls were recruited through the Stanford BloodBank Research Program. A preexisting diagnosis of cancer, autoimmune dis-ease, or chronic viral infection was considered an exclusion criterion.

Peripheral blood mononuclear cells (PBMCs) were isolated from the pe-ripheral blood of patients or healthy donors by density gradient centrifu-gation with Ficoll-Hypaque (Lymphoprep). Total and naïve CD4+ T cells werepurified from PBMC by negative selection using the EasySep human total ornaïve CD4+ T-Cell enrichment kits (Stemcell Technologies).

Antibodies used in the study are listed in Table S1. PCR primers are listed inTable S2.

DC Generation and DC–T-Cell Cultures. Freshly isolated PBMC were seededonto tissue culture plates for 2 h and nonadherent cells were washed away.Adherent cell populations were >95% CD14+ by flow cytometry and werecultured in fresh complete medium supplemented with 50 ng/mL GM-CSFand 50 ng/mL IL-4 to generate monocyte-derived dendritic cells (MoDC),which were matured on day 6 with 100 ng/mL LPS or 100 U/mL IFN-γ. Gene-expression analysis was performed after 8 h, protein expression evaluated 24 hafter stimulation. MoDC (8,000 cells per well) were cocultured with CD4+

T cells (24,000 cells per well) in 96-well round-bottom plates. CD4+CD25+

T cells were quantified by flow cytometry after 48 h. To assess T-cell pro-liferation, CD4+ T cells (24,000 cells per well) were labeled with carboxyfluoresceinsuccinimidyl ester (CFSE) and cocultured with DCs (2,000 or 8,000 cells per well) for5 d. Proliferation rates were analyzed by CFSE dilution, as previously described (26).

Human Artery-Severe Combined Immunodeficiency Mouse Chimeras. NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were purchased from The Jackson

Laboratory and chimeras were generated as previously described (27). Normalhuman temporal or axillary arteries were engrafted subcutaneously into theback of the NSG mice. Six days after the surgery, mice received a single injectionof 100 μg LPS subcutaneously. One to 2 d later, PBMC from GCA patients wereadoptively transferred into the chimeras (1 × 107 cells per mouse). Chimeras wererandomly assigned to two treatment arms. Arm A received 100 μg of anti-humanPD-1 antibody intraperitoneally every other day over 1 wk. Arm B was treatedwith isotype control antibody. Grafts were harvested on days 21–25 and shock-frozen for RNA isolation or embedded into OCT for H&E or immunostaining.

Immunohistochemistry and Immunofluorescence. Tissues were snap-frozen inOCT on dry ice and blocks were stored at –80 °C. Ten-micrometer sectionswere air-dried and fixed with acetone at 4 °C for 10 min. Endogenous per-oxidases were inactivated with 0.3% H2O2 buffer for 15 min at room tem-perature and nonspecific binding sites were blocked with 1% normal goatserum for 30 min. Sections were incubated with primary rabbit anti-humanCD3, anti-human DC-SIGN, and anti-human PD-L1 antibodies (1:100) at roomtemperature for 2 h, followed by HRP-conjugated goat anti-rabbit or AP-conjugated goat anti-mouse secondary antibodies for 1 h. Antibody bindingwas visualized by 3,3′-diaminobenzidine. Sections were counterstained withhematoxylin. CD3+ cells were enumerated in random visual fields distributedover the cross-sectional view of the artery. For immunofluorescence staining,frozen tissues were fixed with cold acetone for 10 min and covered with 1%normal goat serum at room temperature for 30 min. Slides were washed andincubated with anti-CD3 (1:100), anti-PD-1 (1:100), anti-von Willebrand factor(vWF; 1:100), and anti–α-smooth muscle actin (α-SMA; 1:200) antibodies at37 °C for 60 min. Bound antibodies were visualized with secondary antibodies(Alexa Fluor 488 anti-rabbit Ab, Alexa Fluor 546 anti-mouse Ab) at 37 °C for60 min and counterstained with DAPI. Images were taken with an Olympusfluorescence microscopy system.

Statistics. All data are expressed as mean ± SEM. Statistical analysis wasperformed using GraphPad Prism 5.0 and differences were assessed by Stu-dent’s t test or paired Wilcoxon signed-rank test, as indicated. Two-tailed P <0.05 was considered statistically significant. To adjust for multiple testingand control the false-discovery rate (at level 0.05), the Benjamini–Hochbergprocedure (BH step-up procedure) was applied as appropriate.

Study Approval. All procedures and biospecimen collections were approvedby the Institutional Review Board at Stanford University and informedconsent was obtained as appropriate. The animal protocol was approved bythe Animal Care and Use Committee at Stanford University.

Additional data are available in SI Methods.

ResultsLow Expression of the Inhibitory Ligand PD-L1 in GCA. Vessel-wallinvasive T cells in GCA are almost exclusively CD4+ memory cells

Table 1. Clinical characteristics of patients with GCA

Parameters Patients (n = 68)

Age (mean ± SD) 72.7 ± 8.1Female 76.5%Ethnicity

Caucasian 86.8%Hispanic 8.8%African American 4.4%

Headaches 72.1%Eye involvement 41.1%Jaw claudication 23.5%Polymyalgia rheumatica 61.8%Disease duration (mean ± SD, mo) 15.2 ± 24.0Disease activity

High 66.2%Moderate 11.8%Low 22.1%

ESR (mean ± SD, mm/h) 44.6 ± 31.1CRP (mean ± SD, mg/dL) 4.2 ± 4.5Untreated 35.3%Prednisone (mg/d, mean ± SD) 13.4 ± 18.9

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that intermingle with highly activated macrophages and giant cells(28). T-cell–mediated immune responses are fine-tuned by coun-terbalancing stimulatory and inhibitory signals (e.g., costimulationthrough CD28-CD80/CD86 and coinhibition through PD-1/PD-L1).To assess whether such signals participate in GCA, we quantifiedPD-L1 and PD-1 transcripts in GCA-affected temporal arteryspecimens (Fig. 1) that derived from patients who had not yetstarted corticosteroid therapy. Noninflamed, normal aortic wall andnoninflamed temporal and axillary arteries (medium artery targetsof GCA) served as controls. PD-L1 transcripts were abundant inhealthy arteries, but expressed at low levels in GCA-affected ar-teries (Fig. 1A). Conversely, PD-1 transcripts were essentially ab-sent in healthy vessels, but present at high concentrations in arterieswith GCA (Fig. 1B), reflective of the absence of T cells in normalarteries and dense T-cell infiltrates in the vasculitic lesions of GCA.Immunohistochemical staining assigned PD-L1 expression in the

noninflamed arteries to vascular DCs, endogenous cells typicallylocalized at the media–adventitia border (9) (Fig. 1C). In case ofgranulomatous vasculitis, DC density increases greatly and lesionalDCs up-regulate activation markers (e.g., CD83) (24, 29). Despitebeing highly activated, DCs participating in the vasculitic infiltratesconsistently stained negative for PD-L1 (Fig. 1C). The majority of Tcells in granulomatous lesions expressed the surface receptor PD-1(Fig. 1D and E), suggesting selective recruitment/retention of PD-1+

T cells. Frequencies of PD-1+ CD4+ T cells in the peripheral bloodof glucocorticoid-treated or untreated GCA patients were 25%lower than in healthy controls (Fig. S1), compatible with trapping ofsuch cells in the vasculitic lesions. To understand whether PD-1 highexpression is a feature of all granulomatous vasculitides, we com-pared PD-1 mRNA levels in normal arteries, GCA-affected arteries,and in granulomatous tissue lesions from patients with GPA (Fig.1F). As expected, the ratio of PD-1/TCR transcripts was low innormal. Accumulation of activated T cells in GPA granulomasresulted in higher PD-1/TCR ratios, but such ratios were markedlyhigher in GCA arteries, supporting the concept that vasculitic T cellsin GCA arteries are preferentially PD-1+.In essence, the tissue microenvironment of GCA lacks the in-

hibitory ligand PD-L1 and enriches for PD-1–expressing T cells.

Selective Defect of PD-L1 Expression in GCA DCs. To examinewhether GCA patients have a generalized defect in expressingPD-L1, we profiled PD-L1–expressing cells in the peripheralblood and generated MoDCs for functional studies. PD-L1

expression on resting and activated T cells, as well as on B cells,was indistinguishable between patients and age-matched controls(Fig. S2). In contrast, GCA CD14+ monocytes were PD-L1lo andthis phenotype was maintained after differentiation into DCs (Fig.2). In resting and LPS-activated GCA DCs, PD-L1 transcriptswere markedly reduced (Fig. 2A). Flow cytometry confirmed PD-L1 protein reduction on resting and stimulated DCs (Fig. 2 B andC) to about 50% of expression levels in healthy counterparts. PD-L1 low-expression on GCA DCs was not solely a result of systemicinflammation; DCs generated from patients with active rheuma-toid arthritis were identical to control DCs (Fig. 2 B and C).To understand why GCA DCs lack PD-L1, they were stimu-

lated with two distinct stimuli known to control PD-L1 expression(30, 31). Both LPS and IFN-γ induced strong up-regulation in thesurface density of PD-L1 in healthy DCs. In GCA DCs, responsesto both stimuli were dampened, particularly INF-γ–dependentinduction (Fig. 2 D and E).To test whether the PD-L1lo status was related to disease activity

in GCA, we correlated DC PD-L1 protein levels with the eryth-rocyte sedimentation rate (ESR) and serum C-reactive proteinconcentrations (CRP), two biomarkers of the acute phase responsein this vasculitis (Fig. 2 F and G). PD-L1 surface expression wasparticularly low in patients with the highest inflammatory activity.The defect was selective for PD-L1. Studies of transcript and

protein expression for PD-L2, CD80, and CD86 demonstrated thatthe patient-derived DCs were perfectly capable to induce the cos-timulatory ligands as well as PD-L2 (Fig. 2 H–J and Fig. S3). Fur-thermore, the ability to produce proinflammatory cytokines (IL-1β,IL-6, TNF-α) was well maintained in patient-derived DC (Fig. S3).These studies identified GCA DCs as PD-L1 low-expressing

cells, enabling them to favor costimulatory over coinhibitorysignals when functioning as APCs.

GCA DCs Are Hyperstimulatory. To examine how PD-L1lo DCsactivate and instruct T cells, we measured DC-induced T-cellactivation and expansion (Fig. 3). Lack of PD-L1 expressionaffected early steps of T-cell activation, measured by the fre-quency of CD4+ CD25+ T cells. As early as 48 h after stimula-tion, PD-L1lo DCs increased the frequency of activated T cells byabout 50% (Fig. 3 A and B). To probe the impact of PD-L1lo

DCs on T-cell proliferation, GCA DCs and control DCs werecultured with CFSE-labeled CD4 T cells, and 5 d later frequenciesof dividing CD4+ T cells were measured. The effect of PD-L1

Fig. 1. PD-L1lo DC and PD-1+ T cells in GCA. RNA wasextracted from normal aortic wall, noninflamedmedium-sized arteries, and from GCA-affected arteries (n = 10each). In patients with GPA, granulomatous lesions inthe lung and in the skin were examined (n = 10). (A)Expression of PD-L1 transcripts and (B) PD-1 tran-scripts was quantified by RT-PCR. (C) Tissue sectionsfrom temporal arteries were stained with anti–PD-L1(red) and anti–DC-SIGN (brown) antibodies. (D) Tissuesections from GCA affected temporal arteries werestained with anti–PD-1 (red) and anti-CD3 (green)antibodies. Alexa Fluor 488 anti-rabbit (1:100) andAlexa Fluor 546 anti-mouse (1:100) secondary anti-bodies were used to visualize primary antibodybinding. Representative images are shown. (E) Fre-quencies of CD3+PD-1+ T cells were quantified invascular wall cross-sections. (F) Tissue expression ofTCR and PD-1 was assessed in nonvasculitic and vas-culitis-affected tissues (GCA or GPA). Ratios of PD-1/TCR are presented. Data are mean ± SEM from10 different patient samples. *P < 0.05, **P < 0.01,***P < 0.001. (Original magnification: 600×.)

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deficiency was particularly relevant under limiting conditions. Incultures containing 2,000 DCs (1 DC:12 T cells), the frequency ofproliferating CD4+ T cells more than doubled in the presence ofGCA DCs and remained significantly higher even at a higher DC:T-cell ratio (Fig. 3 C and D).To investigate whether the hyperactivation and hyperproliferation

of T cells primed by GCA DCs was directly related to PD-L1 ex-pression, anti–PD-L1 antibodies were included in the DC:T-cellcultures. Removing a negative signal by blocking the PD-L1/PD1axis increased CD4+ T-cell responses by about 30% (Fig. 3E),confirming published data (32). Stimulatory effects of blockingaccess to PD-L1 were abolished in PD-L1lo GCA DCs (Fig. 3F).Frequencies of CD4+CD25+ T cells were similar between isotypecontrol and anti–PD-L1 cultures, supporting the notion that PD-L1on GCA DCs no longer participated in delivering negative signals.Thus, PD-L1 deficiency on GCA DCs has functional impli-

cations, amplifying CD4+ T-cell responses.

Inhibiting PD-1/PD-L1 Interaction Intensifies Vascular Inflammation.To explore whether a defect in PD-L1 expression has impact onpathogenic immune functions in vasculitis, we made use of a hu-man artery–NSG mouse model (27). In this model system, humanaxillary arteries are engrafted into NSG mice and PBMC fromGCA patients or healthy individuals are adoptively transferredinto the chimeras. Patient-derived CD4+ T cells and monocytesinfiltrate into the human vessel and form intramural infiltrates. Ifthe chimeras are reconstituted with PBMC from non-GCA healthydonors, the engrafted human arteries remain free of inflammatoryinfiltrates. To test whether immune checkpoints are involved in thevasculitic response, chimeras were treated with a blocking anti–PD-1 antibody (Fig. 4). Histological and immunohistochemicalexamination of the explanted arteries confirmed that GCA PBMC,but not healthy PBMC, are able to induce vasculitis (Fig. 4 A andB). PD-1 checkpoint inhibition enabled very few healthy T cells toenter the vascular wall and no organized infiltrates were formed.PBMC from GCA patients induced vessel wall inflammation,

Fig. 2. PD-L1lo DCs in GCA. DCs were generatedfrom patients with GCA, patients with RA, and age-matched healthy controls (Con) and stimulated withLPS (100 ng/mL) for 8 h. (A) Relative expression ofPD-L1 mRNA measured by quantitative PCR (qPCR).(B) Surface expression of the coinhibitory ligand PD-L1 in activated DC from healthy controls, RA patientsand GCA patients quantified by flow cytometry. Rep-resentative histograms are shown. (C) Mean fluores-cence intensities (MFI) of PD-L1 membrane expressionfrom 12 control, GCA and RA samples. (D and E) DCswere stimulated with LPS (100 ng/mL) or IFN-γ (100 U/mL). Representative histograms (D) and MFIs from sixexperiments (E). (F and G) Correlation between PD-L1expression on DC and serum ESR or CRP concentrationin individual GCA patients. (H–J) PD-L2 expression wasmeasured by qPCR and flow cytometry. Representativehistograms are shown. Results are from six samples. Alldata are mean ± SEM; *P < 0.05, ***P < 0.001. NS, nosignificant difference.

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with a significant increase in the density of the intramural T-cellinfiltrate in anti–PD-1–treated chimeras. Tissue expression ofTCR mRNA doubled after PD-1 blockade, in line with the his-tologic results (Fig. 4C).Anti–PD-1–enhanced T-cell recruitment/retention had marked

effects on the intensity of vessel wall inflammation. Gene-expressionprofiling revealed robust up-regulation of inflammatory cytokines—including IL-1β, IL-6, and TNF-α, which originate mostly in macro-phages—and DC that participate in the granulomatous lesions (Fig.4D). Notably, PD pathway blockade enhanced tissue transcript levelsof four cytokines involved in T-cell activation and proliferation, in-cluding IL-7, IL-15, IL-23p19, and IL-27p28 (Fig. 4E). Thus, T cellswere recruited into a T-cell tropic microenvironment, providing idealconditions for unopposed T-cell expansion. PD-1 blockade had noeffect on the antiinflammatory cytokine IL-10 (Fig. 4F).In essence, inhibiting PD-1/PD-L1 interaction intensifies

T-cell accumulation in the vessel wall and profoundly enhancestissue inflammation. Notably, PD-1 blockade is insufficient toconvert healthy alloreactive T cells into vasculitogenic T cells.

PD-1 Checkpoint Inhibition Selects for Proinflammatory T Cells.Inhibiting negative immune checkpoints should lead to unselectedT-cell activation, restricted only by the preferential expression ofthe PD-1 receptor on memory and recently stimulated T cells.Alternatively, the absence of a PD-1–derived negative signal couldlead to biased T-cell recruitment and retention. We first analyzedwhether anti–PD-1 treatment enriched for PD-1+ T cells in thetissue infiltrates (Fig. 5A). Whereas only a small subset of lesionalT cells expressed PD-1 in the vehicle-treated arteries, checkpointinhibition clearly resulted in preferential recruitment of PD-1+ Tcells (Fig. 5B). Eliminating inhibitory signaling shifted the T-cellpopulation toward T-bet and RAR-related orphan receptor C(RORC)-expressing cells, whereas GATA3 expression, the line-

age-determining transcription factors for the Th2 lineage, wassimilar in treated and untreated arteries (Fig. 5 C–G). In in vitroexperiments, we confirmed that anti–PD-1 antibody was sufficientto shift the lineage commitment of T cells toward Th1 and Th17differentiation (Fig. S4). In the inflamed arteries, gene expressionfor IFN-γ, IL-17A, and IL-21 were increased, clearly biasing theT-cell infiltrate toward proinflammatory effector functions (Fig. 5D–F). Stable production of FoxP3, GATA3, and IL-4 transcriptssupported the notion that removal of negative signaling enabledselected T cells to infiltrate into the tissue lesions (Fig. 5 C andG).Analysis of chemokine receptor expression demonstrated enrich-ment of CXCR3, CCR6, and CXCR5, predominantly found onTh1, Th17, and follicular helper T cells, respectively. Anti–PD-1treatment resulted in preferential expression of the memory markerCCR5 and disadvantaged the naïve marker CCR7, suggestive for ashift in the balance between naïve and memory T cells (Fig. 5H).These experiments yielded unexpected results, demonstrating

that the lack of inhibitory signaling lead to redistribution oflesional T cells, favoring IFN-γ–, IL-17–, and IL-21–producingeffector T cells. Enrichment for CXCR3+, CCR6+, and CXCR5+

cells is compatible with a survival advantage for proinflammatoryeffector cells in the otherwise immunoprivileged tissue niche.

PD-1 Checkpoint Inhibition Aggravates the Maladaptive Remodelingof the Inflamed Arterial Wall. In GCA, effector T cells contributeto a number of disease-relevant processes, most importantly,vessel wall restructuring (33). Inflamed arteries typically havethinning of the medial layer and intimal thickening, oftenleading to luminal occlusion. Several growth factors, includingPDGF and FGF, have been implicated in driving myofibroblastmigration and proliferation (34, 35). Intimal hyperplasia is con-sistently associated with marked neoangiogenesis (6, 36), creatingmicrovascular networks to support recruitment of inflammatory

Fig. 3. PD-L1lo DCs from GCA patients are hyper-stimulatory and insensitive to PD-L1 blockade. DCswere generated from GCA patients and healthy con-trols (Con), stimulated with LPS for 24 h. Their stimu-latory capacity was probed by coculturing them withCD4+ T cells purified from healthy donors. T-cell acti-vation was quantified by the frequency of CD4+CD25+

T cells and T-cell proliferation was determinedthrough CFSE dilution. (A) Activated CD4+CD25+

T cells quantified by flow cytometry after 48 h. Fluo-rescence minus one (FMO) controls superimposed asgray areas. (B) Percentage of activated CD4+ T cellsin six GCA-control pairs. (C) Proliferation of CD4+ Tcells was measured by flow cytometry after 5 d ofcoculture. Representative histograms of CSFE ex-pression. The number of DCs per culture is indicated.(D) Frequencies of proliferating CD4+ T cells whencocultured with either control or patient-derivedDCs. Results from six control-patient pairs. (E and F)Control and GCA DCs were cocultured with purifiedCD4+ T cells in the presence of anti–PD-L1 antibodiesor isotype control. CD4+ T cells cocultured withoutDCs served as control. Frequencies of activated CD4+

CD25+ T cells in six to eight independent experimentswere measured after 48 h by flow cytometry. Alldata are mean ± SEM; **P < 0.01, ***P < 0.001.

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cells and supplying oxygen and nutrients for outgrowing myo-fibroblasts. Ultimately, the remodeling of the arterial wallcauses luminal compromise and gives rise to the ischemiccomplications of GCA, including blindness, stroke, and aorticarch syndrome (37). We explored whether the enrichment ofPD-1+ T cells in the mural infiltrates has consequences for theoutgrowth of new microvessels and for the expansion of theintimal layer. We measured the thickness of the tunica intima inexplanted arteries from control- and anti–PD-1–treated chi-meras (Fig. 6 A and B). Checkpoint inhibition led to rapidexpansion of the intimal layer. In PD-1–blocked grafts, theintima was multilayered and densely packed (Fig. 6A), doublingin thickness (Fig. 6B). In control-treated arteries, the intimaconsisted of a single-cell layer of vWF+ endothelial cells andtwo to three layers of α-SMC+ myofibroblasts. In contrast, thehyperplastic intima in anti–PD-1–treated arteries was assem-bled from six to eight layers of α-SMC+ myofibroblasts, coveredby large, partially vWF+α-SMC+ endothelial cells (Fig. 6C).

Small vascular lumina appeared in the proximal media, and alsoin the vasa vasorum-containing adventitia (Fig. 6D). Anti–PD-1treatment resulted in the brisk formation of new vWF+α-SMC+

microvessels. Enumeration in control and anti–PD-1–treatedarterial grafts yielded a 100% increase in the number of micro-vessels after checkpoint inhibition (Fig. 6E). Removing negativesignaling through the PD pathway was associated with endo-thelial activation, revealed by the robust induction of intercel-lular adhesion molecule (ICAM), vascular cell adhesionmolecule (VCAM), CD31, vWF, and VE-cadherin (Fig. 6F).All markers increased by up to fourfold, when vehicle-treatedand anti–PD-1–treated arteries were compared. Endothelialcells expanded in size and in the intensity of vWF expression(Fig. 6C), compatible with acute endothelial cell activation.In essence, the PD1/PD-L1–regulated inhibitory immune

checkpoint has a major impact on the remodeling of the vesselwall in T-cell–induced vasculitis, affecting the process of neo-angiogenesis, as well as intimal hyperplasia.

Fig. 4. Blocking of the PD-1/PD-L1 axis aggravatesvascular inflammation. Sections of normal medium-sized arteries were engrafted into NSG mice and 7 dlater the human-artery NSG chimeras were recon-stituted with PBMC from GCA patients or age-matched healthy controls (Healthy PBMC). Mice weresubsequently treated with anti–PD-1 antibodies orcontrol IgG (100 μg, i.p.) given three times over 1 wk.Human arteries were explanted and processed forimmunohistochemistry or RNA extraction. Relativegene expression was measured by RT-PCR. (A) Tissuesections from the explanted arteries were stainedwith H&E (Upper) or anti-CD3 antibodies (Lower) as inFig. 1. (Original magnification: 200×.) (B) The densityof the vessel wall infiltrate was evaluated by enu-merating CD3+ T cells in 10 high-powered fields. Re-sults from eight tissues are presented as mean ± SEM.(C) T-cell accumulation in the vessel wall was de-termined by the expression of TCR gene transcripts.(D–F) Transcriptome analysis for the indicated cyto-kines in control and anti–PD-1–treated arteries mea-sured by RT-PCR. Gene expression data from 10 tissuesare presented as mean ± SEM; *P < 0.05, **P < 0.01.After adjustment for multiple testing using the Ben-jamini–Hochberg method, the comparisons of TCR,IL-1β, IL-6, TNF-α, IL-23p19, ILP27p28, IL-7, and IL-15are statistically significant with a false-discovery rateof less than 0.05.

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DiscussionPatients with the autoimmune vasculopathy GCA, a disease ofthe aorta and its major branches, have a spontaneous loss of theimmunoinhibitory molecule PD-L1, which has devastating con-sequences for the course of their vasculitis. Ordinarily an im-

mune privileged niche, the wall layers of GCA-affected arteriesare occupied by PD-L1lo, CD80hi, CD86hi DCs, and CD4+

T cells arranged in granulomatous infiltrates. In the absenceof negative signaling, PD-1+ T cells find a safe haven in anotherwise inaccessible tissue niche and sustain rampant yet

Fig. 5. PD-1 blockade selects for tissue-infiltrating Tcells with proinflammatory functions. NSG mice car-rying human arteries were reconstituted with GCAPBMC and treated with anti–PD-1 antibody or con-trol Ig as in Fig. 4. Inflamed arteries were explantedand processed for gene-expression analysis by RT-PCR or embedded for immunohistochemically anal-ysis. (A) Tissue sections were double-stained withanti-CD3 (green) and anti–PD-1 (red) antibodies.Merged pictures show PD-1+ T cells. (Original mag-nification: 600×.) (B) Percentages of CD3+PD-1+ cellswere enumerated in cross-sections from seven iso-type and anti–PD-1–treated arteries. (C–G) Tissuetranscriptome analysis for lineage-determiningtranscription factors and T-cell effector molecules.Markers related to the same T-cell lineage areboxed. Data from 10 different tissues are presentedas mean ± SEM. (H) Tissue transcriptome analysis forchemokine receptor genes. Results from 10 tissuegrafts in each treatment arm are shown as a heatmap. *P < 0.05, **P < 0.01, ***P < 0.001. After ad-justment for multiple testing using the Benjamini–Hochberg method, the comparisons of T-bet, IFN-γ,RORC, IL-17A, and IL-21 are statistically significantwith a false-discovery rate of less than 0.05.

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selective inflammatory activity, affecting DCs, macrophages,endothelial cells, and vascular stromal cells. A functionally im-portant outcome is the induction of intimal hyperplasia, the ul-timate disease pathway causing blindness, stroke, and aortic archsyndrome. The deficiency of the immune-inhibitory check-point implicates antigen-nonspecific processes in GCA patho-genesis and opens the door to advanced immunomodulatorytherapy and the attempt to reestablish the immunoprivilege ofthe arterial wall.Physiologically, the coinhibitory ligand PD-L1 has limited ex-

pression in normal tissues, but is promptly up-regulated on APCsundergoing activation to jumpstart a negative feedback looppreventing T-cell overactivity and inflammatory tissue injury.Accordingly, PD-L1−/− mice are different from CTLA-4−/− mice,which develop spontaneous diffuse inflammatory infiltrates innormal organs (38). Mice lacking PD-L1 or PD-1 are highlysusceptible to autoimmune disease, specifically when challengedwith autoantigens (17, 20, 39). A unique feature of GCA is itsstringent tissue tropism. The susceptible tissue niche is guardedby vasDC, strategically positioned outside of the media (33).Such vasDC display a vessel-specific Toll-like receptor (TLR)expression pattern, enabling them to respond in a vessel-specificway to danger signals (9). The closeness of vasDC to the vasavasorum network allows them access to circulating pathogen-and damage-associated molecular patterns and monitoring oforganism-wide danger signals. One possibility is that GCA’sbreakdown of the arterial immunoprivilege is a direct conse-quence of PD-L1 loss, because vasDCs in healthy arteries arePD-L1+, even under resting conditions (Fig. 1).PD-1+ T cells are highly enriched in vasculitic lesions (Fig.

1E). Notably, the frequency of PD-1+ T cells in the peripheralblood of GCA patients is reduced, compatible with the conceptthat inflamed arteries act as a sink for these powerful effector Tcells. In line with recent observations that vasculitic lesionspersist despite glucocorticoid therapy, frequencies of CD4+PD-1+cells were similarly reduced in treated and untreated patients.A reduction of circulating PD-1–expressing cells contrasts withdata reported in small-vessel vasculitis (40). Patients with gran-ulomatosis with polyangiitis have higher frequencies of circulat-ing PD1+ T cells, whereas renal lesions do not enrich PD-1+ Tcells, suggesting fundamentally different roles of such T cells indifferent disease categories. In transcriptome studies, we con-firmed that enrichment of PD-1 in the tissue lesions occurs to amuch higher degree in GCA than in GPA (Fig. 1F). Much aboutthe role of PD-1+ T cells in the immune system has been learnedfrom the clinical application of PD pathway inhibitors in cancerpatients (14). Immune checkpoint inhibitors have emerged as apowerful approach to activating therapeutic antitumor immunityand have provided valuable insights into the process of main-taining self-tolerance in humans. Durable antitumor responseshave been induced in patients treated with anti–PD-1 antibodies,particularly in solid tumors with high immunogenicity (14). Re-moving a break in the immune system, however, comes with aprice; many of the patients treated with checkpoint inhibitorsdevelop immune-related adverse events that manifest with diffuseand tissue injurious inflammation of the gut, skin, endocrineglands, liver, and lung, but potentially of any organ system (16).Patients treated with antibodies blocking checkpoint signalinghave been reported to develop GCA (41). Thus, patients treatedwith PD pathway blockers should be monitored for inflammatorydisease in their large arteries.Unblocked PD-1+ T cells in tumor patients elegantly destroy

cancer cells, probably through a plethora of mechanisms (42).We interrogated effector functions of PD-1+ T cells in a human-ized mouse model, in which GCA T cells, B cells, and monocytesinduce vasculitis in engrafted human arteries. Blocking PD-1 ac-cess resulted in massive enrichment of PD-1+ T cells in the arterialwall. PD-1 is induced as a late marker of T-cell activation and theintense expression of PD-1 on tissue-residing T cells argues for awidespread and acute immune activation (43). A broad spectrumof inflammatory mediators was up-regulated in the treated

chimeras, including markers related to DCs and macrophagefunction (IL-1β, IL-6, TNF-α, IL-23p19, IL-27p28), as well asstromal cell function (IL-7, IL-15). The process was selective, asexpression of the antiinflammatory mediator IL-10 was un-affected. PD-1 blockade also imposed selectivity on T-cell effectorfunctions, biasing the infiltrate toward T-bet and RORC expres-sion and away from FoxP3 and GATA-3. Transcriptome analysisindicated multiplicity of T-cell effector molecules, ranging fromIFN-γ to IL-17 to IL-21. All of these T-cell effector molecules havebeen detected in GCA vascular lesions (3, 44). Notably, PD-1checkpoint inhibition in this model system was not able to simplyconvert healthy alloreactive T cells into wall-infiltrating T cells (Fig.4), indicating that T cells from GCA patients are particularly sus-ceptible to the unleashing effect of anti–PD-1 antibodies.The PD-1 pathway has been implicated in two major mecha-

nisms thwarting self-reactive T cells, inducing immunosuppres-sive Tregs, and directly inhibiting effector T cells (45). Data onTreg function in GCA are sparse, but a recent study has providedevidence that GCA patients are lacking a potent immunoreg-ulatory CD8 Treg population (26), compatible with PD-L1deficiency shaping a vasculitogenic T-cell repertoire. When an-alyzing disease-relevant effector T cells, PD-1 appeared to beexpressed on multiple T-cell lineages (Fig. 5), with PD-1 block-ade allowing entrance and survival of IFN-γ, IL-17, and IL-21producers, but disfavoring IL-4+ and FoxP3+ cells. These find-ings suggest selectivity in how PD-1–mediated signaling inter-feres with the TCR-dependent signaling cascade. PD-1 ligationon T cells is believed to preferentially attenuate the PI3K/AKTpathway (46, 47), leading to suppression of the mammaliantarget of rapamycin-dependent signaling knot, which is criticallyinvolved in lineage commitment, regulation of bioenergetics, andT-cell expansion (48, 49). In the absence of sufficient PD-1 sig-naling, a scenario exemplified in the PD-L1–deficient GCA lesions,T cells gain proliferative potential, differentiate into metabolicallyhyperactive effector cells, and commit to proinflammatory cytokineproduction.PD-1+ T cells in vasculitic lesions have profound impact on

stromal and endothelial cells in the vessel wall. We observedthree processes that were accelerated in the inflamed arteriesafter checkpoint inhibition (Fig. 6): (i) formation of micro-vessels, which are believed to sprout off the adventitial vasavasorum network and invade into more proximal parts of thevessel wall; (ii) endothelial activation; and (iii) size expansion ofthe intimal layer. In temporal artery biopsies from GCA patients,neovessel formation and intimal thickening have been correlatedwith IFN-γ tissue levels (36), thus implicating T-cell effectorfunctions with wall remodeling. Whether this is a direct or in-direct consequence of T cells guiding the behavior of endothelialcells and myofibroblasts remains to be investigated. Conceivably,unopposed PD-1+ T cells could activate macrophages and DCs,which in turn could modify the functioning of nonimmune cellsin the tissue microenvironment. The expansion of α-SMA+ cellsforming a lumen-compromising neotissue was a fast process thatoccurred in only 1 wk. Although multiple mechanisms contributeto intimal hyperplasia, the chimeric mouse model allows elimi-nating some and exploring others. Chimeric mice have no accessto human bone marrow, thus bone-marrow-derived stem cellscannot play a role in promoting intimal outgrowth. All precursorcells for the expanding myofibroblasts occupying the hyperplasticintima must be part of the human vessel wall that is engraftedinto the animals. Gene-expression studies demonstrated up-regulation of the transcription factor TWIST in explants fromanti–PD-1–treated chimeras. TWIST has been implicated in en-dothelial-mesenchymal transition (50, 51), but how T cells couldachieve regulatory control of intimal hyperplasia remains unexplored.Our data do not suggest that PD-1+ T cells have signs of im-

mune exhaustion (52, 53); in contrast, they appear strongly ac-tivated and activate surrounding immune and stromal cells. In arecent study, the exhaustion marker PD-1 was associated withbeneficial suppression of the “nonexhausted” T-cell state drivenby costimulation and the balance between costimulation/exhaustion

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signatures was predictive of clinical outcomes in infectious versusautoimmune diseases (54). Indeed, inducing T-cell exhaustionwas proposed as a therapeutic strategy in autoimmunity (55). Inthe current study, PD-1+ T cells were critical perpetrators ofautoimmune functions and the lack of PD-L1 expression onAPCs translated into a detriment for the patient, suggesting thatdifferent disease states may require a different balance of cos-timulatory and coinhibitory signals.An important question raised by the present work is the

mechanism leading to PD-L1 low-expression in GCA DC. NeitherT cells nor B cells were affected by this defect, but monocytesshared the PD-L1lo phenotype. PD-L1 expression is considered tobe regulated by the inflammatory milieu, with cytokines inducingPD-L1 and PD-L2 expression. Type I and type II IFNs and TNF-αhave all been implicated in driving up PD-L1 (56–59). For humanmonocytes and DCs, TLR ligands are potent PD-L1 inducers.Patient-derived DC had low responses to the TLR4 ligand LPS,but were even less responsive to IFN-γ. IFN-γ is a key cytokines inthe vasculitic lesions of GCA, literally exposing the DC to a IFN-γ–high environment (60). We considered the possibility that low-expression of PD-L1 is an age-dependent factor, reflective ofvascular aging, but neither in healthy arteries nor in GCA patientswas the donor age predictive for the level of PD-L1 expression.More detailed studies of PD-L1 transcription in patient-derivedand control DC are needed to understand the molecular under-pinnings of this defect.Data presented here have considerable implications for the

understanding and the management of medium- and large-vesselvasculitis. First, the granulomatous nature of the infiltrates hasfostered models proposing specific antigens driving disease. Noconclusive evidence for such antigens has been provided anddata presented here favor a critical role for antigen-nonspecificimmune regulation, and emphasize the protective nature of abalanced immune system. Second, guided by the observation that

DC in healthy arteries—even in the resting state—express PD-L1, we propose that negative signaling through PD-1 plays acritical role in establishing and maintaining the immune privilegeof the arterial wall. In that model, loss of immunoinhibitorysignaling would make the tissue site susceptible to immune in-vasion. The strong enrichment of PD-1+ T cells in the vasculiticlesions of GCA supports a disease-critical role of these effectorcells, which definitely have no signs of exhaustion but appear tobe strongly activated. Granulomatous tissue lesion in GPA pa-tients do not have a similar signature (Fig. 1F), arguing againstthe inflammatory environment present in every tissue infiltrateas the sole reason for PD-1 up-regulation. Expression of PD-1,rather than assignment to a single functional lineage, appears tobe the common denominator of vasculitogenic T cells in GCA.Third, this report implicates T cells in the process of intimalhyperplasia, a critical event in patients developing blindness,stroke, or aortic arch syndrome. Taken together, these obser-vations should encourage rethinking the therapeutic approachesin GCA. Chronic glucocorticoid therapy fails to remove thevasculitic infiltrates (61, 62), with Th1-committed effector T cellsparticularly resistant to treatment. Additionally, targeting indi-vidual inflammatory markers will unlikely provide a curative in-tervention for a multipronged immune attack, which involvesa multitude of effector functions. Repairing the PD-1/PD-L1pathway to reestablish immunoinhibitory signals and revive aprotective “molecular shield” may allow reconstitution of theartery’s immune privilege. Eliminating/blocking the relativelysmall population of PD-1+ T cells may be an equally effectiveapproach to protect the vessel wall. Finally, understanding thespontaneous deficiency of PD-L1 in GCA DC would have directimpact on the field of cancer immunotherapy, in which avoid-ance of PD-L1 up-regulation on cancer cells represents a majortherapeutic strategy to undermine immune resistance. The pre-sent work unifies efforts in inflammatory vessel disease and in

Fig. 6. PD-1 blockade aggravates maladaptiveremodeling of the inflamed arterial wall. NSG micewere engrafted with human arteries, reconstitutedwith GCA PBMC, and treated with anti–PD-1 orcontrol Ig as in Fig. 4, and the thickness of the in-timal layer was measured in cross-sections ofexplanted arteries. (A) H&E stains of cross-sections ofexplanted arteries. The intima-media border, iden-tified through the lamina elastica interna, is in-dicated. (Original magnification: 200×.) (B) Thethickness of the intimal layer was measured in anti–PD-1 and control-Ig–treated arteries. Results fromfour independent experiments are shown. (C) Tissuesections were double-stained with anti-vWF (green)and anti–α-SMA (red) antibodies. Representativeimages of the intima layer are shown. (D) Tissuesections were double-stained with anti-vWF (green)and anti–α-SMA (red) antibodies. Representativeimages of the adventitial layer are shown. (E)Numbers of microvessels were enumerated in 10high-powered fields. Results from four independentexperiments are shown. (F) Transcript levels formarkers of endothelial activation and of myofibro-blasts were measured in 10 tissues by RT-PCR andare shownas a heatmap. Data aremean± SEM; *P< 0.05,**P < 0.01.

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cancer immunotherapy to optimize immunostimulatory andimmunoinhibitory signals for disease management.

ACKNOWLEDGMENTS. This work was supported by the National Institutesof Health Grants R01 AR042527, R01 HL117913, R01 AI108906, and P01

HL129941 (to C.M.W.), and R01 AI108891, R01 AG045779, U19 AI057229,U19 AI057266, and I01 BX001669 (to J.J.G.). R.W. received fellowship supportfrom the Govenar Discovery Fund. The content is solely the responsibility ofthe authors and does not necessarily represent the official views of theNational Institutes of Health.

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Zhang et al. PNAS | Published online January 23, 2017 | E979

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