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Home , Autoimmune disease, Giant cell arteritis

Immunoinhibitory checkpoint deficiency in medium and large vessel

Hui 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 and , Stanford University School of Medicine, Stanford, CA 94305; bDepartment of Pathology, Stanford University School of Medicine, Stanford, CA 94305; cDivision of , 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 October 10, 2016)

Giant cell (GCA) causes autoimmune of the wall and the immunoprivileged tissue site. Their role in controlling and its large branches, resulting in aortic arch syndrome, the influx of immune cells into the mural structures remains un- + blindness, and . CD4 T cells and form orga- defined. Similarly, understanding how -nonspecific factors nized granulomatous lesions in the walls of affected arteries, de- affect the intensity and the quality of the vasculitogenic immune stroy the tunica media, and induce ischemic damage through response could redefine critical pathogenic processes with a major rapid intimal hyperplasia and luminal occlusion. Pathogenic mecha- impact on immunosuppressive strategies (10). nisms remain insufficiently understood; specifically, it is unknown T-cell–dependent immune responses are fine-tuned by a mul- whether the unopposed activation of the is because titude of costimulatory and coinhibitory signals, provided by re- – of deficiency of immunoinhibitory checkpoints. Transcriptome anal- ceptor ligand interactions that modulate TCR-initiated signaling ysis of GCA-affected temporal arteries revealed low expression of cascades (11). Such immune checkpoints are crucial for the the coinhibitory ligand programmed death ligand-1 (PD-L1) concur- maintenance of self-tolerance, prevent autoimmune , and rent with enrichment of the programmed death-1 (PD-1) . protect against collateral tissue damage (12). Conversely, excessive Tissue-residing and ex vivo-generated dendritic cells (DC) from GCA expression of immune checkpoint proteins has been associated patients were PD-L1lo, whereas the majority of vasculitic T cells with immune resistance mechanisms, prominently used by tumor expressed PD-1, suggesting inefficiency of the immunoprotective cells to escape from antitumor immunity (13). Recent successes in PD-1/PD-L1 immune checkpoint. DC–PD-L1 expression correlated in- cancer have highlighted the importance of in- versely with clinical disease activity. In human artery-SCID chimeras, hibitory immune checkpoints that stop antigen-reactive T cells. PD-1 blockade exacerbated vascular inflammation, enriched for PD- Specifically, monoclonal that block the programmed + 1 effector T cells, and amplified tissue production of multiple T-cell death-1/programmed death ligand-1 (PD-1/PD-L1) pathway have effector , including IFN-γ, IL-17, and IL-21. Arteries infil- yielded unprecedented therapeutic benefit in patients with ad- + vanced solid tumors (14–16). PD-1 is expressed on activated T and trated by PD-1 effector T cells developed microvascular neoangio- B cells and its engagement by its ligands PD-L1 or PD-L2 disrupts genesis as well as hyperplasia of the intimal layer, implicating T cells kinase activity in the TCR-activation cascade through the phos- in the maladaptive behavior of vessel wall endogenous cells. Thus, phatase SHP2. Resulting involves several in GCA, a breakdown of the tissue-protective PD1/PD-L1 checkpoint mechanisms, including T-cell , T-cell exhaustion, T-cell unleashes vasculitic immunity and regulates the pathogenic remod- anergy, T-cell IL-10 production, and Treg induction. eling of the inflamed arterial wall.

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

Antigen recognition by the immune system triggers rapid, iant cell arteritis (GCA) is a granulomatous vasculitis with a specific, and protective responses, which are counterbalanced stringent tissue tropism, named after the multinucleated G by inhibitory checkpoints to minimize potentially harmful im- giant cells that populate the inflammatory lesions in the arterial + munity. The programmed death-1/ programmed death ligand-1 walls. Granulomatous infiltrates composed of CD4 T cells and (PD-1/PD-L1) checkpoint is overreactive in cancer patients, macrophages penetrate from the adventitia into the media and curbing antitumor immunity. Whether a failing PD-1/PD-L1 destroy the lamina elastica interna. T cells with identical T-cell checkpoint contributes to spontaneous in receptor (TCR) sequences have been isolated from spatially humans is unknown. Here, we found that in patients with the distinct lesions (1, 2), highly suggestive for antigen-driven T-cell autoimmune vasculitis arteritis, antigen-presenting activation, yet no singular vasculitogenic antigen has been defined. cells provide insufficient negative signaling; unleashing highly Lesional T cells provide a spectrum of effector functions, suspi- activated T cells to infiltrate and damage the walls of large ciously diverse, and ranging from the production of IFN-γ, IL-17, arteries. Thus, immunoinhibitory signals protect large arteries and IL-9 to IL-21 (3, 4). Similarly, multiple functional against inflammatory attack and checkpoint activation may be subsets participate in granuloma formation, spanning from mac- a suitable strategy to treat autoimmune vasculitis. rophages committed to production (IL-6, IL-1β), to those releasing reactive oxygen species, to those providing growth fac- – Author contributions: J.J.G. and C.M.W. designed research; H.Z. and R.W. performed re- tors (PDGF, FGF) and angiogenic factors (VEGF) (5 7). 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., and The wall layers of large and medium vessels have features of C.M.W. analyzed data; Y.J.L. and K.J.W. recruited patients; and J.J.G. and C.M.W. wrote an immunoprivileged niche (8) and the invasion of inflammatory the paper. cells in GCA essentially breaks this immune privilege. Under The authors declare no conflict of interest. physiologic conditions endogenous dendritic cells (DC), so-called This article is a PNAS Direct Submission. vascular DC (vasDC), populate the arterial wall in a vessel-specific 1H.Z. and R.W. contributed equally to this work. distribution pattern and may protect against immune attack (9). 2To whom correspondence should be addressed. Email: [email protected]. Localized in the adventitial layer, such vasDC are close to the vasa This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. vasorum, and are positioned between the vascular access to the 1073/pnas.1616848114/-/DCSupplemental.

E970–E979 | PNAS | Published online January 23, 2017 www.pnas.org/cgi/doi/10.1073/pnas.1616848114 Downloaded by guest on September 24, 2021 The contrasting scenario to immune resistance is exuberant Table 1. Clinical characteristics of patients with GCA PNAS PLUS immunity, leading to immune-mediated tissue injury and auto- Parameters Patients (n = 68) immune . PD-1 and PD-L1 deficiency have been associ- ated with a -like syndrome (17) and a dilated myocardiopathy Age (mean ± SD) 72.7 ± 8.1 (18), respectively. Lack of PD-L1 or PD-1 exacerbates murine Female 76.5% and experimental autoimmune (19, 20) and Ethnicity PD-L1 overexpression reduces spinal cord T-cell infiltrates (21). − − Caucasian 86.8% PD-L1 / antigen-presenting cells (APCs) fail to convert naïve + −/− Hispanic 8.8% CD4 T cells into Tregs (22), and PD-1 mice are prone to African American 4.4% enrich for Th1 and Th17 cells (23). 72.1% Guided by a transcriptomic signature of GCA-affected arteries Eye involvement 41.1% that lacked expression of the inhibitory ligand PD-L1, we have explored the role of the PD pathway in regulating the intensity Jaw 23.5% 61.8% and the functional orientation of vasculitogenic T-cell responses. ± ± GCA vascular lesions are occupied by PD-L1lo DCs and PD-1hi Disease duration (mean SD, mo) 15.2 24.0 T cells. PD-L1lo DCs from GCA patients enhance T-cell acti- Disease activity vation and proliferative expansion. In a model High 66.2% of vasculitis, treatment with anti–PD-1 antibodies effectively Moderate 11.8% accelerates the recruitment and retention of T cells and inten- Low 22.1% sifies T-cell and macrophage responses in the inflamed artery. ESR (mean ± SD, mm/h) 44.6 ± 31.1 Vasculitogeneic T cells produce IFN-γ, IL-17, and IL-21, sus- CRP (mean ± SD, mg/dL) 4.2 ± 4.5 taining multifunctional effector functions. Accumulation of such Untreated 35.3% multifunctional T-cell populations is associated with the rapid (mg/d, mean ± SD) 13.4 ± 18.9 outgrowth of hyperplastic intima, activation of endothelial cells, and the induction of microvascular neoangiogenesis, connecting T cells to disease-relevant remodeling processes in the vascular Laboratory and chimeras were generated as previously described (27). Normal wall. In essence, a dysfunctional inhibitory immune checkpoint human temporal or axillary arteries were engrafted subcutaneously into the exposes the vessel wall to inflammatory attack, formation of back of the NSG mice. Six days after the surgery, mice received a single injection microvascular networks, and intimal hyperplasia, ultimately of 100 μg LPS subcutaneously. One to 2 d later, PBMC from GCA patients were promoting ischemic organ damage. Reconstitution of a func- adoptively transferred into the chimeras (1 × 107 cells per mouse). Chimeras were tional PD checkpoint could provide an entirely new strategy to randomly assigned to two treatment arms. Arm A received 100 μg of anti-human treat medium- and large-vessel vasculitis. PD-1 intraperitoneally every other day over 1 wk. Arm B was treated with isotype control antibody. Grafts were harvested on days 21–25 and shock- Methods frozen for RNA isolation or embedded into OCT for H&E or immunostaining. Tissues, Cells, and Antibodies. Temporal arteries were collected from di- agnostic . A diagnosis of GCA was based on typical histological Immunohistochemistry and Immunofluorescence. Tissues were snap-frozen in findings. Temporal arteries were considered negative for GCA if no in- OCT on dry ice and blocks were stored at –80 °C. Ten-micrometer sections flammatory cells were identified on histology. Artery biopsies from patients were air-dried and fixed with acetone at 4 °C for 10 min. Endogenous per-

with a diagnosis of polymyalgia rheumatic were excluded, as they can be oxidases were inactivated with 0.3% H2O2 buffer for 15 min at room tem- negative by histology but have altered function of vasDC (24, 25). Normal perature and nonspecific binding sites were blocked with 1% normal goat human aorta, temporal, and axillary arteries were donated by organ donors. serum for 30 min. Sections were incubated with primary rabbit anti-human Patients were enrolled into the protocol if they had a diagnosis of - CD3, anti-human DC-SIGN, and anti-human PD-L1 antibodies (1:100) at room positive GCA or anti-cyclic citrullinated peptide-positive rheumatoid temperature for 2 h, followed by HRP-conjugated goat anti-rabbit or AP- (RA) and had active disease. Patient demographic characteristics are listed in conjugated goat anti-mouse secondary antibodies for 1 h. Antibody binding Table 1. Diagnostic biopsies of the and the skin of patients with gran- was visualized by 3,3′-diaminobenzidine. Sections were counterstained with + ulomatosis with polyangiitis (GPA), a small-vessel vasculitis, served as controls. hematoxylin. CD3 cells were enumerated in random visual fields distributed Age-matched healthy controls were recruited through the Stanford Blood over the cross-sectional view of the artery. For immunofluorescence staining, Bank Research Program. A preexisting diagnosis of cancer, autoimmune dis- frozen tissues were fixed with cold acetone for 10 min and covered with 1% ease, or chronic viral was considered an exclusion criterion. normal goat serum at room temperature for 30 min. Slides were washed and Peripheral blood mononuclear cells (PBMCs) were isolated from the pe- incubated with anti-CD3 (1:100), anti-PD-1 (1:100), anti-von Willebrand factor ripheral blood of patients or healthy donors by density gradient centrifu- –α α + (vWF; 1:100), and anti -smooth muscle actin ( -SMA; 1:200) antibodies at gation with Ficoll-Hypaque (Lymphoprep). Total and naïve CD4 T cells were 37 °C for 60 min. Bound antibodies were visualized with secondary antibodies purified from PBMC by negative selection using the EasySep human total or + (Alexa Fluor 488 anti-rabbit Ab, Alexa Fluor 546 anti-mouse Ab) at 37 °C for naïve CD4 T-Cell enrichment kits (Stemcell Technologies). 60 min and counterstained with DAPI. Images were taken with an Olympus Antibodies used in the study are listed in Table S1. PCR primers are listed in fluorescence microscopy system. Table S2. Statistics. All data are expressed as mean ± SEM. Statistical analysis was – DC Generation and DC T-Cell Cultures. Freshly isolated PBMC were seeded performed using GraphPad Prism 5.0 and differences were assessed by Stu- onto tissue culture plates for 2 h and nonadherent cells were washed away. dent’s t test or paired Wilcoxon signed-rank test, as indicated. Two-tailed P < > + Adherent cell populations were 95% CD14 by flow cytometry and were 0.05 was considered statistically significant. To adjust for multiple testing cultured in fresh complete medium supplemented with 50 ng/mL GM-CSF and control the false-discovery rate (at level 0.05), the Benjamini–Hochberg and 50 ng/mL IL-4 to generate monocyte-derived dendritic cells (MoDC), procedure (BH step-up procedure) was applied as appropriate. 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 h + Study Approval. All procedures and biospecimen collections were approved after stimulation. MoDC (8,000 cells per well) were cocultured with CD4 + + by the Institutional Review Board at Stanford University and informed T cells (24,000 cells per well) in 96-well round-bottom plates. CD4 CD25 consent was obtained as appropriate. The animal protocol was approved by T cells were quantified by flow cytometry after 48 h. To assess T-cell pro- + the Animal Care and Use Committee at Stanford University. liferation, CD4 T cells (24,000 cells per well) were labeled with carboxyfluorescein succinimidyl ester (CFSE) and cocultured with DCs (2,000 or 8,000 cells per well) for Additional data are available in SI Methods. 5 d. Proliferation rates were analyzed by CFSE dilution, as previously described (26). Results

Human Artery-Severe Combined Mouse Chimeras. NOD. Low Expression of the Inhibitory Ligand PD-L1 in GCA. Vessel-wall INFLAMMATION

+ IMMUNOLOGY AND Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were purchased from The Jackson invasive T cells in GCA are almost exclusively CD4 memory cells

Zhang et al. PNAS | Published online January 23, 2017 | E971 Downloaded by guest on September 24, 2021 that intermingle with highly activated macrophages and giant cells expression on resting and activated T cells, as well as on B cells, (28). T-cell–mediated immune responses are fine-tuned by coun- was indistinguishable between patients and age-matched controls + terbalancing stimulatory and inhibitory signals (e.g., costimulation (Fig. S2). In contrast, GCA CD14 monocytes were PD-L1lo and through CD28-CD80/CD86 and coinhibition through PD-1/PD-L1). this phenotype was maintained after differentiation into DCs (Fig. To assess whether such signals participate in GCA, we quantified 2). In resting and LPS-activated GCA DCs, PD-L1 transcripts PD-L1 and PD-1 transcripts in GCA-affected temporal artery were markedly reduced (Fig. 2A). Flow cytometry confirmed PD- specimens (Fig. 1) that derived from patients who had not yet L1 protein reduction on resting and stimulated DCs (Fig. 2 B and started therapy. Noninflamed, normal aortic wall and C) to about 50% of expression levels in healthy counterparts. PD- noninflamed temporal and axillary arteries (medium artery targets L1 low-expression on GCA DCs was not solely a result of systemic of GCA) served as controls. PD-L1 transcripts were abundant in inflammation; DCs generated from patients with active rheuma- healthy arteries, but expressed at low levels in GCA-affected ar- toid arthritis were identical to control DCs (Fig. 2 B and C). teries (Fig. 1A). Conversely, PD-1 transcripts were essentially ab- To understand why GCA DCs lack PD-L1, they were stimu- sent in healthy vessels, but present at high concentrations in arteries lated with two distinct stimuli known to control PD-L1 expression with GCA (Fig. 1B), reflective of the absence of T cells in normal (30, 31). Both LPS and IFN-γ induced strong up-regulation in the arteries and dense T-cell infiltrates in the vasculitic lesions of GCA. surface density of PD-L1 in healthy DCs. In GCA DCs, responses Immunohistochemical staining assigned PD-L1 expression in the to both stimuli were dampened, particularly INF-γ–dependent noninflamed arteries to vascular DCs, endogenous cells typically induction (Fig. 2 D and E). localized at the media–adventitia border (9) (Fig. 1C). In case of To test whether the PD-L1lo status was related to disease activity granulomatous vasculitis, DC density increases greatly and lesional in GCA, we correlated DC PD-L1 protein levels with the eryth- DCs up-regulate activation markers (e.g., CD83) (24, 29). Despite rocyte sedimentation rate (ESR) and serum C-reactive protein being highly activated, DCs participating in the vasculitic infiltrates concentrations (CRP), two biomarkers of the acute phase response consistently stained negative for PD-L1 (Fig. 1C). The majority of T in this vasculitis (Fig. 2 F and G). PD-L1 surface expression was cells in granulomatous lesions expressed the surface receptor PD-1 particularly low in patients with the highest inflammatory activity. + (Fig. 1 D and E), suggesting selective recruitment/retention of PD-1 The defect was selective for PD-L1. Studies of transcript and + + T cells. Frequencies of PD-1 CD4 T cells in the peripheral blood protein expression for PD-L2, CD80, and CD86 demonstrated that of -treated or untreated GCA patients were 25% the patient-derived DCs were perfectly capable to induce the cos- lower than in healthy controls (Fig. S1), compatible with trapping of timulatoryligandsaswellasPD-L2(Fig.2H–J and Fig. S3). Fur- such cells in the vasculitic lesions. To understand whether PD-1 high thermore, the ability to produce proinflammatory cytokines (IL-1β, expression is a feature of all granulomatous vasculitides, we com- IL-6, TNF-α) was well maintained in patient-derived DC (Fig. S3). pared PD-1 mRNA levels in normal arteries, GCA-affected arteries, These studies identified GCA DCs as PD-L1 low-expressing and in granulomatous tissue lesions from patients with GPA (Fig. cells, enabling them to favor costimulatory over coinhibitory 1F). As expected, the ratio of PD-1/TCR transcripts was low in signals when functioning as APCs. normal. Accumulation of activated T cells in GPA granulomas resulted in higher PD-1/TCR ratios, but such ratios were markedly GCA DCs Are Hyperstimulatory. To examine how PD-L1lo DCs higher in GCA arteries, supporting the concept that vasculitic T cells activate and instruct T cells, we measured DC-induced T-cell + in GCA arteries are preferentially PD-1 . activation and expansion (Fig. 3). Lack of PD-L1 expression In essence, the tissue microenvironment of GCA lacks the in- affected early steps of T-cell activation, measured by the fre- + + hibitory ligand PD-L1 and enriches for PD-1–expressing T cells. quency of CD4 CD25 T cells. As early as 48 h after stimula- tion, PD-L1lo DCs increased the frequency of activated T cells by Selective Defect of PD-L1 Expression in GCA DCs. To examine about 50% (Fig. 3 A and B). To probe the impact of PD-L1lo whether GCA patients have a generalized defect in expressing DCs on T-cell proliferation, GCA DCs and control DCs were PD-L1, we profiled PD-L1–expressing cells in the peripheral cultured with CFSE-labeled CD4 T cells, and 5 d later frequencies + blood and generated MoDCs for functional studies. PD-L1 of dividing CD4 T cells were measured. The effect of PD-L1

+ Fig. 1. PD-L1lo DC and PD-1 T cells in GCA. RNA was extracted from normal aortic wall, noninflamed medium- sized arteries, and from GCA-affected arteries (n = 10 each). In patients with GPA, granulomatous lesions in the 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 sections from temporal arteries were stained with anti–PD-L1 (red) and anti–DC-SIGN (brown) antibodies. (D) Tissue sections from GCA affected temporal arteries were stained with anti–PD-1 (red) and anti-CD3 (green) antibodies. Alexa Fluor 488 anti-rabbit (1:100) and Alexa Fluor 546 anti-mouse (1:100) secondary anti- bodies were used to visualize primary antibody binding. Representative images are shown. (E) Fre- quencies of CD3+PD-1+ T cells were quantified in vascular wall cross-sections. (F) Tissue expression of TCR 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 from 10 different patient samples. *P < 0.05, **P < 0.01, ***P < 0.001. (Original magnification: 600×.)

E972 | www.pnas.org/cgi/doi/10.1073/pnas.1616848114 Zhang et al. Downloaded by guest on September 24, 2021 deficiency was particularly relevant under limiting conditions. In Inhibiting PD-1/PD-L1 Interaction Intensifies Vascular Inflammation. PNAS PLUS cultures containing 2,000 DCs (1 DC:12 T cells), the frequency of To explore whether a defect in PD-L1 expression has impact on + proliferating CD4 T cells more than doubled in the presence of pathogenic immune functions in vasculitis, we made use of a hu- – GCA DCs and remained significantly higher even at a higher DC: man artery NSG mouse model (27). In this model system, human T-cell ratio (Fig. 3 C and D). axillary arteries are engrafted into NSG mice and PBMC from GCA patients or healthy individuals are adoptively transferred To investigate whether the hyperactivation and hyperproliferation + of T cells primed by GCA DCs was directly related to PD-L1 ex- into the chimeras. Patient-derived CD4 T cells and monocytes pression, anti–PD-L1 antibodies were included in the DC:T-cell infiltrate into the human vessel and form intramural infiltrates. If the chimeras are reconstituted with PBMC from non-GCA healthy cultures. Removing a negative signal by blocking the PD-L1/PD1 + E donors, the engrafted human arteries remain free of inflammatory axis increased CD4 T-cell responses by about 30% (Fig. 3 ), infiltrates. To test whether immune checkpoints are involved in the confirming published data (32). Stimulatory effects of blocking vasculitic response, chimeras were treated with a blocking anti– access to PD-L1 were abolished in PD-L1lo GCA DCs (Fig. 3F). + + PD-1 antibody (Fig. 4). Histological and immunohistochemical Frequencies of CD4 CD25 T cells were similar between isotype examination of the explanted arteries confirmed that GCA PBMC, control and anti–PD-L1 cultures, supporting the notion that PD-L1 but not healthy PBMC, are able to induce vasculitis (Fig. 4 A and on GCA DCs no longer participated in delivering negative signals. B). PD-1 checkpoint inhibition enabled very few healthy T cells to Thus, PD-L1 deficiency on GCA DCs has functional impli- enter the vascular wall and no organized infiltrates were formed. + cations, amplifying CD4 T-cell responses. PBMC from GCA patients induced vessel wall inflammation,

Fig. 2. PD-L1lo DCs in GCA. DCs were generated from patients with GCA, patients with RA, and age- matched healthy controls (Con) and stimulated with LPS (100 ng/mL) for 8 h. (A) Relative expression of PD-L1 mRNA measured by quantitative PCR (qPCR). (B) Surface expression of the coinhibitory ligand PD- L1 in activated DC from healthy controls, RA patients and GCA patients quantified by flow cytometry. Rep- resentative histograms are shown. (C) Mean fluores- cence intensities (MFI) of PD-L1 membrane expression from 12 control, GCA and RA samples. (D and E)DCs were stimulated with LPS (100 ng/mL) or IFN-γ (100 U/ mL). Representative histograms (D) and MFIs from six experiments (E). (F and G) Correlation between PD-L1 expression on DC and serum ESR or CRP concentration in individual GCA patients. (H–J) PD-L2 expression was measured by qPCR and flow cytometry. Representative histograms are shown. Results are from six samples. All data are mean ± SEM; *P < 0.05, ***P < 0.001. NS, no

significant difference. INFLAMMATION IMMUNOLOGY AND

Zhang et al. PNAS | Published online January 23, 2017 | E973 Downloaded by guest on September 24, 2021 Fig. 3. PD-L1lo DCs from GCA patients are hyper- stimulatory and insensitive to PD-L1 blockade. DCs were generated from GCA patients and healthy con- trols (Con), stimulated with LPS for 24 h. Their stimu- latory capacity was probed by coculturing them with + CD4 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 determined through CFSE dilution. (A) Activated CD4+CD25+ T cells quantified by flow cytometry after 48 h. Fluo- rescence minus one (FMO) controls superimposed as + gray areas. (B) Percentage of activated CD4 T cells + in six GCA-control pairs. (C) Proliferation of CD4 T cells was measured by flow cytometry after 5 d of coculture. Representative histograms of CSFE ex- pression. The number of DCs per culture is indicated. + (D) Frequencies of proliferating CD4 T cells when cocultured with either control or patient-derived DCs. Results from six control-patient pairs. (E and F) Control and GCA DCs were cocultured with purified + CD4 T cells in the presence of anti–PD-L1 antibodies or isotype control. CD4+ T cells cocultured without DCs served as control. Frequencies of activated CD4+ + CD25 T cells in six to eight independent experiments were measured after 48 h by flow cytometry. All data are mean ± SEM; **P < 0.01, ***P < 0.001.

with a significant increase in the density of the intramural T-cell age-determining transcription factors for the Th2 lineage, was infiltrate in anti–PD-1–treated chimeras. Tissue expression of similar in treated and untreated arteries (Fig. 5 C–G). In in vitro TCR mRNA doubled after PD-1 blockade, in line with the his- experiments, we confirmed that anti–PD-1 antibody was sufficient tologic results (Fig. 4C). to shift the lineage commitment of T cells toward Th1 and Th17 Anti–PD-1–enhanced T-cell recruitment/retention had marked differentiation (Fig. S4). In the inflamed arteries, gene expression effects on the intensity of vessel wall inflammation. Gene-expression for IFN-γ, IL-17A, and IL-21 were increased, clearly biasing the profiling revealed robust up-regulation of inflammatory cytokines— T-cell infiltrate toward proinflammatory effector functions (Fig. 5 including IL-1β,IL-6,andTNF-α, which originate mostly in macro- D–F). Stable production of FoxP3, GATA3, and IL-4 transcripts phages—and DC that participate in the granulomatous lesions (Fig. supported the notion that removal of negative signaling enabled 4D). Notably, PD pathway blockade enhanced tissue transcript levels selected T cells to infiltrate into the tissue lesions (Fig. 5 C and G). of four cytokines involved in T-cell activation and proliferation, in- Analysis of receptor expression demonstrated enrich- cluding IL-7, IL-15, IL-23p19, and IL-27p28 (Fig. 4E). Thus, T cells ment of CXCR3, CCR6, and CXCR5, predominantly found on were recruited into a T-cell tropic microenvironment, providing ideal Th1, Th17, and follicular helper T cells, respectively. Anti–PD-1 conditions for unopposed T-cell expansion. PD-1 blockade had no treatment resulted in preferential expression of the memory marker effect on the antiinflammatory cytokine IL-10 (Fig. 4F). CCR5 and disadvantaged the naïve marker CCR7, suggestive for a In essence, inhibiting PD-1/PD-L1 interaction intensifies shift in the balance between naïve and memory T cells (Fig. 5H). T-cell accumulation in the vessel wall and profoundly enhances These experiments yielded unexpected results, demonstrating tissue inflammation. Notably, PD-1 blockade is insufficient to that the lack of inhibitory signaling lead to redistribution of convert healthy alloreactive T cells into vasculitogenic T cells. lesional T cells, favoring IFN-γ–,IL-17–, and IL-21–producing + + + effector T cells. Enrichment for CXCR3 ,CCR6 , and CXCR5 PD-1 Checkpoint Inhibition Selects for Proinflammatory T Cells. cells is compatible with a survival advantage for proinflammatory Inhibiting negative immune checkpoints should lead to unselected effector cells in the otherwise immunoprivileged tissue niche. T-cell activation, restricted only by the preferential expression of the PD-1 receptor on memory and recently stimulated T cells. PD-1 Checkpoint Inhibition Aggravates the Maladaptive Remodeling Alternatively, the absence of a PD-1–derived negative signal could of the Inflamed Arterial Wall. In GCA, effector T cells contribute lead to biased T-cell recruitment and retention. We first analyzed to a number of disease-relevant processes, most importantly, + whether anti–PD-1 treatment enriched for PD-1 T cells in the vessel wall restructuring (33). Inflamed arteries typically have tissue infiltrates (Fig. 5A). Whereas only a small subset of lesional thinning of the medial layer and intimal thickening, often T cells expressed PD-1 in the vehicle-treated arteries, checkpoint leading to luminal occlusion. Several growth factors, including + inhibition clearly resulted in preferential recruitment of PD-1 T PDGF and FGF, have been implicated in driving myofibroblast cells (Fig. 5B). Eliminating inhibitory signaling shifted the T-cell migration and proliferation (34, 35). Intimal hyperplasia is con- population toward T-bet and RAR-related orphan receptor C sistently associated with marked neoangiogenesis (6, 36), creating (RORC)-expressing cells, whereas GATA3 expression, the line- microvascular networks to support recruitment of inflammatory

E974 | www.pnas.org/cgi/doi/10.1073/pnas.1616848114 Zhang et al. Downloaded by guest on September 24, 2021 PNAS PLUS

Fig. 4. Blocking of the PD-1/PD-L1 axis aggravates vascular inflammation. Sections of normal medium- sized arteries were engrafted into NSG mice and 7 d later the human-artery NSG chimeras were recon- stituted with PBMC from GCA patients or age- matched healthy controls (Healthy PBMC). Mice were subsequently treated with anti–PD-1 antibodies or control IgG (100 μg, i.p.) given three times over 1 wk. Human arteries were explanted and processed for immunohistochemistry or RNA extraction. Relative gene expression was measured by RT-PCR. (A) Tissue sections from the explanted arteries were stained with H&E (Upper) or anti-CD3 antibodies (Lower)asin Fig. 1. (Original magnification: 200×.) (B) The density of 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 tissues are 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-15 are statistically significant with a false-discovery rate of less than 0.05.

cells and supplying oxygen and nutrients for outgrowing myo- Small vascular lumina appeared in the proximal media, and also fibroblasts. Ultimately, the remodeling of the arterial wall in the -containing adventitia (Fig. 6D). Anti–PD-1 + + causes luminal compromise and gives rise to the ischemic treatment resulted in the brisk formation of new vWF α-SMC complications of GCA, including blindness, stroke, and aortic microvessels. Enumeration in control and anti–PD-1–treated arch syndrome (37). We explored whether the enrichment of arterial grafts yielded a 100% increase in the number of micro- + PD-1 T cells in the mural infiltrates has consequences for the vessels after checkpoint inhibition (Fig. 6E). Removing negative outgrowth of new microvessels and for the expansion of the signaling through the PD pathway was associated with endo- intimal layer. We measured the thickness of the tunica intima in thelial activation, revealed by the robust induction of intercel- explanted arteries from control- and anti–PD-1–treated chi- lular adhesion molecule (ICAM), vascular meras (Fig. 6 A and B). Checkpoint inhibition led to rapid molecule (VCAM), CD31, vWF, and VE-cadherin (Fig. 6F). expansion of the intimal layer. In PD-1–blocked grafts, the All markers increased by up to fourfold, when vehicle-treated intima was multilayered and densely packed (Fig. 6A), doubling and anti–PD-1–treated arteries were compared. Endothelial in thickness (Fig. 6B). In control-treated arteries, the intima cells expanded in size and in the intensity of vWF expression + consisted of a single-cell layer of vWF endothelial cells and (Fig. 6C), compatible with acute endothelial cell activation. + twotothreelayersofα-SMC myofibroblasts. In contrast, the In essence, the PD1/PD-L1–regulated inhibitory immune hyperplastic intima in anti–PD-1–treated arteries was assem- checkpoint has a major impact on the remodeling of the vessel +

bled from six to eight layers of α-SMC myofibroblasts, covered wall in T-cell–induced vasculitis, affecting the process of neo- INFLAMMATION

+ + IMMUNOLOGY AND by large, partially vWF α-SMC endothelial cells (Fig. 6C). angiogenesis, as well as intimal hyperplasia.

Zhang et al. PNAS | Published online January 23, 2017 | E975 Downloaded by guest on September 24, 2021 Fig. 5. PD-1 blockade selects for tissue-infiltrating T cells with proinflammatory functions. NSG mice car- rying human arteries were reconstituted with GCA PBMC and treated with anti–PD-1 antibody or con- trol Ig as in Fig. 4. Inflamed arteries were explanted and processed for gene-expression analysis by RT- PCR or embedded for immunohistochemically anal- ysis. (A) Tissue sections were double-stained with anti-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 cells were enumerated in cross-sections from seven iso- type and anti–PD-1–treated arteries. (C–G)Tissue transcriptome analysis for lineage-determining transcription factors and T-cell effector molecules. Markers related to the same T-cell lineage are boxed. Data from 10 different tissues are presented as mean ± SEM. (H) Tissue transcriptome analysis for chemokine receptor genes. Results from 10 tissue grafts in each treatment arm are shown as a heat map. *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 significant with a false-discovery rate of less than 0.05.

Discussion mune privileged niche, the wall layers of GCA-affected arteries + Patients with the autoimmune vasculopathy GCA, a disease of are occupied by PD-L1lo,CD80hi,CD86hi DCs, and CD4 the aorta and its major branches, have a spontaneous loss of the T cells arranged in granulomatous infiltrates. In the absence + immunoinhibitory molecule PD-L1, which has devastating con- of negative signaling, PD-1 T cells find a safe haven in an sequences for the course of their vasculitis. Ordinarily an im- otherwise inaccessible tissue niche and sustain rampant yet

E976 | www.pnas.org/cgi/doi/10.1073/pnas.1616848114 Zhang et al. Downloaded by guest on September 24, 2021 selective inflammatory activity, affecting DCs, macrophages, chimeras, including markers related to DCs and macrophage PNAS PLUS endothelial cells, and vascular stromal cells. A functionally im- function (IL-1β, IL-6, TNF-α, IL-23p19, IL-27p28), as well as portant outcome is the induction of intimal hyperplasia, the ul- function (IL-7, IL-15). The process was selective, as timate disease pathway causing blindness, stroke, and aortic arch expression of the antiinflammatory mediator IL-10 was un- syndrome. The deficiency of the immune-inhibitory check- affected. PD-1 blockade also imposed selectivity on T-cell effector point implicates antigen-nonspecific processes in GCA patho- functions, biasing the infiltrate toward T-bet and RORC expres- genesis and opens the door to advanced immunomodulatory sion and away from FoxP3 and GATA-3. Transcriptome analysis therapy and the attempt to reestablish the immunoprivilege of indicated multiplicity of T-cell effector molecules, ranging from the arterial wall. IFN-γ to IL-17 to IL-21. All of these T-cell effector molecules have Physiologically, the coinhibitory ligand PD-L1 has limited ex- been detected in GCA vascular lesions (3, 44). Notably, PD-1 pression in normal tissues, but is promptly up-regulated on APCs checkpoint inhibition in this model system was not able to simply undergoing activation to jumpstart a negative feedback loop convert healthy alloreactive T cells into wall-infiltrating T cells (Fig. preventing T-cell overactivity and inflammatory tissue injury. 4), indicating that T cells from GCA patients are particularly sus- − − − − Accordingly, PD-L1 / mice are different from CTLA-4 / mice, ceptible to the unleashing effect of anti–PD-1 antibodies. which develop spontaneous diffuse inflammatory infiltrates in The PD-1 pathway has been implicated in two major mecha- normal organs (38). Mice lacking PD-L1 or PD-1 are highly nisms thwarting self-reactive T cells, inducing immunosuppres- susceptible to autoimmune disease, specifically when challenged sive Tregs, and directly inhibiting effector T cells (45). Data on with autoantigens (17, 20, 39). A unique feature of GCA is its Treg function in GCA are sparse, but a recent study has provided stringent tissue tropism. The susceptible tissue niche is guarded evidence that GCA patients are lacking a potent immunoreg- by vasDC, strategically positioned outside of the media (33). ulatory CD8 Treg population (26), compatible with PD-L1 Such vasDC display a vessel-specific Toll-like receptor (TLR) deficiency shaping a vasculitogenic T-cell repertoire. When an- expression pattern, enabling them to respond in a vessel-specific alyzing disease-relevant effector T cells, PD-1 appeared to be way to danger signals (9). The closeness of vasDC to the vasa expressed on multiple T-cell lineages (Fig. 5), with PD-1 block- vasorum network allows them access to circulating pathogen- ade allowing entrance and survival of IFN-γ, IL-17, and IL-21 + + and damage-associated molecular patterns and monitoring of producers, but disfavoring IL-4 and FoxP3 cells. These find- organism-wide danger signals. One possibility is that GCA’s ings suggest selectivity in how PD-1–mediated signaling inter- breakdown of the arterial immunoprivilege is a direct conse- feres with the TCR-dependent signaling cascade. PD-1 ligation quence of PD-L1 loss, because vasDCs in healthy arteries are on T cells is believed to preferentially attenuate the PI3K/AKT + PD-L1 , even under resting conditions (Fig. 1). pathway (46, 47), leading to suppression of the mammalian + PD-1 T cells are highly enriched in vasculitic lesions (Fig. target of rapamycin-dependent signaling knot, which is critically + 1E). Notably, the frequency of PD-1 T cells in the peripheral involved in lineage commitment, regulation of bioenergetics, and blood of GCA patients is reduced, compatible with the concept T-cell expansion (48, 49). In the absence of sufficient PD-1 sig- that inflamed arteries act as a sink for these powerful effector T naling, a scenario exemplified in the PD-L1–deficient GCA lesions, cells. In line with recent observations that vasculitic lesions T cells gain proliferative potential, differentiate into metabolically + + persist despite glucocorticoid therapy, frequencies of CD4 PD-1 hyperactive effector cells, and commit to proinflammatory cytokine cells were similarly reduced in treated and untreated patients. production. + A reduction of circulating PD-1–expressing cells contrasts with PD-1 T cells in vasculitic lesions have profound impact on data reported in small-vessel vasculitis (40). Patients with gran- stromal and endothelial cells in the vessel wall. We observed ulomatosis with polyangiitis have higher frequencies of circulat- three processes that were accelerated in the inflamed arteries + + ing PD1 T cells, whereas renal lesions do not enrich PD-1 T after checkpoint inhibition (Fig. 6): (i) formation of micro- cells, suggesting fundamentally different roles of such T cells in vessels, which are believed to sprout off the adventitial vasa different disease categories. In transcriptome studies, we con- vasorum network and invade into more proximal parts of the firmed that enrichment of PD-1 in the tissue lesions occurs to a vessel wall; (ii) endothelial activation; and (iii) size expansion of much higher degree in GCA than in GPA (Fig. 1F). Much about the intimal layer. In temporal artery biopsies from GCA patients, + the role of PD-1 T cells in the immune system has been learned neovessel formation and intimal thickening have been correlated from the clinical application of PD pathway inhibitors in cancer with IFN-γ tissue levels (36), thus implicating T-cell effector patients (14). Immune checkpoint inhibitors have emerged as a functions with wall remodeling. Whether this is a direct or in- powerful approach to activating therapeutic antitumor immunity direct consequence of T cells guiding the behavior of endothelial and have provided valuable insights into the process of main- cells and myofibroblasts remains to be investigated. Conceivably, + taining self-tolerance in humans. Durable antitumor responses unopposed PD-1 T cells could activate macrophages and DCs, have been induced in patients treated with anti–PD-1 antibodies, which in turn could modify the functioning of nonimmune cells + particularly in solid tumors with high immunogenicity (14). Re- in the tissue microenvironment. The expansion of α-SMA cells moving a break in the immune system, however, comes with a forming a lumen-compromising neotissue was a fast process that price; many of the patients treated with checkpoint inhibitors occurred in only 1 wk. Although multiple mechanisms contribute develop immune-related adverse events that manifest with diffuse to intimal hyperplasia, the chimeric mouse model allows elimi- and tissue injurious inflammation of the gut, skin, endocrine nating some and exploring others. Chimeric mice have no access glands, , and lung, but potentially of any organ system (16). to human bone marrow, thus bone-marrow-derived stem cells Patients treated with antibodies blocking checkpoint signaling cannot play a role in promoting intimal outgrowth. All precursor have been reported to develop GCA (41). Thus, patients treated cells for the expanding myofibroblasts occupying the hyperplastic with PD pathway blockers should be monitored for inflammatory intima must be part of the human vessel wall that is engrafted disease in their large arteries. into the animals. Gene-expression studies demonstrated up- + Unblocked PD-1 T cells in tumor patients elegantly destroy regulation of the TWIST in explants from cancer cells, probably through a plethora of mechanisms (42). anti–PD-1–treated chimeras. TWIST has been implicated in en- + We interrogated effector functions of PD-1 T cells in a human- dothelial-mesenchymal transition (50, 51), but how T cells could ized mouse model, in which GCA T cells, B cells, and monocytes achieve regulatory control of intimal hyperplasia remains unexplored. + induce vasculitis in engrafted human arteries. Blocking PD-1 ac- Our data do not suggest that PD-1 T cells have signs of im- + cess resulted in massive enrichment of PD-1 T cells in the arterial mune exhaustion (52, 53); in contrast, they appear strongly ac- wall. PD-1 is induced as a late marker of T-cell activation and the tivated and activate surrounding immune and stromal cells. In a intense expression of PD-1 on tissue-residing T cells argues for a recent study, the exhaustion marker PD-1 was associated with

widespread and acute immune activation (43). A broad spectrum beneficial suppression of the “nonexhausted” T-cell state driven INFLAMMATION of inflammatory mediators was up-regulated in the treated by costimulation and the balance between costimulation/exhaustion IMMUNOLOGY AND

Zhang et al. PNAS | Published online January 23, 2017 | E977 Downloaded by guest on September 24, 2021 Fig. 6. PD-1 blockade aggravates maladaptive remodeling of the inflamed arterial wall. NSG mice were engrafted with human arteries, reconstituted with GCA PBMC, and treated with anti–PD-1 or control Ig as in Fig. 4, and the thickness of the in- timal layer was measured in cross-sections of explanted arteries. (A) H&E stains of cross-sections of explanted arteries. The intima-media border, iden- tified through the lamina elastica interna, is in- dicated. (Original magnification: 200×.) (B)The thickness of the intimal layer was measured in anti– PD-1 and control-Ig–treated arteries. Results from four independent experiments are shown. (C) Tissue sections were double-stained with anti-vWF (green) and anti–α-SMA (red) antibodies. Representative images of the intima layer are shown. (D) Tissue sections were double-stained with anti-vWF (green) and anti–α-SMA (red) antibodies. Representative images of the adventitial layer are shown. (E) Numbers of microvessels were enumerated in 10 high-powered fields. Results from four independent experiments are shown. (F) Transcript levels for markers of endothelial activation and of myofibro- blasts were measured in 10 tissues by RT-PCR and are shown as a heat map. Data are mean ± SEM; *P < 0.05, **P < 0.01.

signatures was predictive of clinical outcomes in infectious versus DC in healthy arteries—even in the resting state—express PD- autoimmune diseases (54). Indeed, inducing T-cell exhaustion L1, we propose that negative signaling through PD-1 plays a was proposed as a therapeutic strategy in autoimmunity (55). In critical role in establishing and maintaining the immune privilege + the current study, PD-1 T cells were critical perpetrators of of the arterial wall. In that model, loss of immunoinhibitory autoimmune functions and the lack of PD-L1 expression on signaling would make the tissue site susceptible to immune in- + APCs translated into a detriment for the patient, suggesting that vasion. The strong enrichment of PD-1 T cells in the vasculitic different disease states may require a different balance of cos- lesions of GCA supports a disease-critical role of these effector timulatory and coinhibitory signals. cells, which definitely have no signs of exhaustion but appear to An important question raised by the present work is the be strongly activated. Granulomatous tissue lesion in GPA pa- mechanism leading to PD-L1 low-expression in GCA DC. Neither tients do not have a similar signature (Fig. 1F), arguing against T cells nor B cells were affected by this defect, but monocytes the inflammatory environment present in every tissue infiltrate shared the PD-L1lo phenotype. PD-L1 expression is considered to as the sole reason for PD-1 up-regulation. Expression of PD-1, be regulated by the inflammatory milieu, with cytokines inducing rather than assignment to a single functional lineage, appears to PD-L1 and PD-L2 expression. Type I and type II IFNs and TNF-α be the common denominator of vasculitogenic T cells in GCA. have all been implicated in driving up PD-L1 (56–59). For human Third, this report implicates T cells in the process of intimal monocytes and DCs, TLR ligands are potent PD-L1 inducers. hyperplasia, a critical event in patients developing blindness, Patient-derived DC had low responses to the TLR4 ligand LPS, stroke, or aortic arch syndrome. Taken together, these obser- butwereevenlessresponsivetoIFN-γ.IFN-γ is a key cytokines in vations should encourage rethinking the therapeutic approaches the vasculitic lesions of GCA, literally exposing the DC to a IFN- in GCA. Chronic glucocorticoid therapy fails to remove the γ–high environment (60). We considered the possibility that low- vasculitic infiltrates (61, 62), with Th1-committed effector T cells expression of PD-L1 is an age-dependent factor, reflective of particularly resistant to treatment. Additionally, targeting indi- vascular aging, but neither in healthy arteries nor in GCA patients vidual inflammatory markers will unlikely provide a curative in- was the donor age predictive for the level of PD-L1 expression. tervention for a multipronged immune attack, which involves More detailed studies of PD-L1 transcription in patient-derived a multitude of effector functions. Repairing the PD-1/PD-L1 and control DC are needed to understand the molecular under- pathway to reestablish immunoinhibitory signals and revive a pinnings of this defect. protective “molecular shield” may allow reconstitution of the Data presented here have considerable implications for the artery’s immune privilege. Eliminating/blocking the relatively + understanding and the management of medium- and large-vessel small population of PD-1 T cells may be an equally effective vasculitis. First, the granulomatous nature of the infiltrates has approach to protect the vessel wall. Finally, understanding the fostered models proposing specific driving disease. No spontaneous deficiency of PD-L1 in GCA DC would have direct conclusive evidence for such antigens has been provided and impact on the field of cancer immunotherapy, in which avoid- data presented here favor a critical role for antigen-nonspecific ance of PD-L1 up-regulation on cancer cells represents a major immune regulation, and emphasize the protective nature of a therapeutic strategy to undermine immune resistance. The pre- balanced immune system. Second, guided by the observation that sent work unifies efforts in inflammatory vessel disease and in

E978 | www.pnas.org/cgi/doi/10.1073/pnas.1616848114 Zhang et al. Downloaded by guest on September 24, 2021 cancer immunotherapy to optimize immunostimulatory and HL129941 (to C.M.W.), and R01 AI108891, R01 AG045779, U19 AI057229, PNAS PLUS immunoinhibitory signals for disease management. U19 AI057266, and I01 BX001669 (to J.J.G.). R.W. received fellowship support from the Govenar Discovery Fund. The content is solely the responsibility of ACKNOWLEDGMENTS. This work was supported by the National Institutes the authors and does not necessarily represent the official views of the of Health Grants R01 AR042527, R01 HL117913, R01 AI108906, and P01 National Institutes of Health.

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