Type 1 IFN and PD-L1 Coordinate Lymphatic Endothelial Cell Expansion and Contraction During an Inflammatory Immune Response

Type 1 IFN and PD-L1 Coordinate Lymphatic Endothelial Cell Expansion and Contraction during an Inflammatory Immune Response This information is current as of September 23, 2021. Erin D. Lucas, Jeffrey M. Finlon, Matthew A. Burchill, Mary K. McCarthy, Thomas E. Morrison, Tonya M. Colpitts and Beth A. Jirón Tamburini J Immunol published online 25 July 2018 http://www.jimmunol.org/content/early/2018/07/26/jimmun Downloaded from ol.1800271

Supplementary http://www.jimmunol.org/content/suppl/2018/07/24/jimmunol.180027 http://www.jimmunol.org/ Material 1.DCSupplemental

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists by guest on September 23, 2021 • Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts Errata An erratum has been published regarding this article. Please see next page or: /content/204/3/726.full.pdf

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published July 27, 2018, doi:10.4049/jimmunol.1800271 The Journal of Immunology

Type 1 IFN and PD-L1 Coordinate Lymphatic Endothelial Cell Expansion and Contraction during an Inﬂammatory Immune Response

Erin D. Lucas,*,† Jeffrey M. Finlon,* Matthew A. Burchill,* Mary K. McCarthy,† Thomas E. Morrison,† Tonya M. Colpitts,‡,x and Beth A. Jiro´n Tamburini*,†

Lymph node (LN) expansion during an immune response is a complex process that involves the relaxation of the fibroblastic network, germinal center formation, and lymphatic vessel growth. These processes require the stromal cell network of the LN to act deliberately to accommodate the influx of immune cells to the LN. The molecular drivers of these processes are not well understood. Therefore, we asked whether the immediate cytokines type 1 IFN produced during viral infection influence the lymphatic network of

the LN in mice. We found that following an IFN-inducing stimulus such as viral infection or polyI:C, programmed cell death ligand 1 Downloaded from (PD-L1) expression is dynamically upregulated on lymphatic endothelial cells (LECs). We found that reception of type 1 IFN by LECs is important for the upregulation of PD-L1 of mouse and human LECs and the inhibition of LEC expansion in the LN. Expression of PD-L1 by LECs is also important for the regulation of LN expansion and contraction after an IFN-inducing stimulus. We demonstrate a direct role for both type 1 IFN and PD-L1 in inhibiting LEC division and in promoting LEC survival. Together, these data reveal a novel mechanism for the coordination of type 1 IFN and PD-L1 in manipulating LEC expansion and

survival during an inﬂammatory immune response. The Journal of Immunology, 2018, 201: 000–000. http://www.jimmunol.org/

ollowing either infection or immunization, the draining endothelial cell growth factor receptor 3 (VEGFR3) interaction lymph node (dLN) must expand to accommodate the in- (3–5, 7–11), alternative signaling mechanisms by which the F ﬂux of lymphocytes and then contract as the immune re- lymphatic endothelium of the lymph node (LN) expand are still sponse resolves and the node returns to homeostasis. We and not well understood. However, some critical events are required others have demonstrated an increase in both the number of for the expansion of the LN and include the recruitment of B cells, lymphatic endothelial cells (LECs) (1–5) and the density of dendritic cells (DCs), and T cells prior to expansion and lymphatic

LECs in the cortical region (6) of the dLN in response to an innate remodeling (1, 3–5, 12, 13). Very little is known about the by guest on September 23, 2021 stimulus. Although factors required for lymphatic vessel growth in contraction of the LN stroma following an immune response. the tissue have been established and include the VEGFC/vascular However, we have demonstrated that the LECs undergo apoptosis during LN contraction (2), and others have noted IFN-g

*Division of Gastroenterology and Hepatology, Department of Medicine, School of production by T cells results in lymphatic regression during Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO 80045; LN contraction (14). †Department of Immunology and Microbiology, School of Medicine, University of Within the LN, LECs differentially express several markers Colorado Anschutz Medical Campus, Aurora, CO 80045; ‡National Emerging Infec- tious Diseases Laboratories, Boston University, Boston, MA 02118; and xDepartment including programmed cell death ligand 1 (PD-L1), ICAM1, of Microbiology, Boston University School of Medicine, Boston, MA 02118 VCAM1, and mucosal addressin cellular adhesion molecule ORCIDs: 0000-0001-7705-2069 (E.D.L.); 0000-0003-3119-8358 (M.K.M.); 0000- (MadCam) (15). Intercellular interactions between LECs and DCs 0003-1991-231X (B.A.J.T.). are established by the interaction of ICAM1, VCAM1, and MHC Received for publication February 26, 2018. Accepted for publication July 9, 2018. class II on stromal cells and Lfa1 and Mac-1 on DCs among others This work was supported by a grant from the National Institutes of Health (R01 (1, 16–19). These interactions are important for LECs and DCs AI121209) (to B.A.J.T.) and a National Institutes of Health “Training in Immunol- to interact to exchange Ags and promote either self-tolerance ogy” grant (T32 AI007405) (to E.D.L.). (15, 20, 21) or promote T cell effector memory (1). However, why E.D.L., B.A.J.T., J.M.F., M.K.M., and T.M.C. performed experiments, E.D.L. and B.A.J.T. analyzed results and made the figures, and E.D.L., B.A.J.T., M.A.B., and the LECs express PD-L1 based on their location in the LN (15) is T.E.M. designed the research and wrote the paper. not well understood. The described primary function of PD-L1 Address correspondence and reprint requests to Dr. Beth A. Jiroń Tamburini, Uni- expression by the lymphatic endothelium is to drive peripheral versity of Colorado Anschutz Medical Campus, School of Medicine, Department of T cell tolerance to self-antigens, nonadjuvanted foreign Ags Medicine, Division of Gastroenterology and Hepatology, Mail Stop B-146, Room P15-10122, 12700 E. 19th Avenue, Aurora, CO 80045. E-mail address: beth. (15, 17, 20–23), and in settings of cancer (24, 25). It has been [email protected] demonstrated that PD-L1 expression by cancer cells induces a The online version of this article contains supplemental material. signaling network that results in increased cellular survival and Abbreviations used in this article: CHIKV, Chikungunya virus; DC, dendritic cell; inhibition of cell division (26–29). However, whether PD-L1 dLN, draining lymph node; GDF10, growth/differentiation factor 10; hLEC, human signaling events occur in LECs has yet to be established. LEC; IFNaR, IFN-a/b receptor; ITGB1, integrin b 1; LEC, lymphatic endothelial cell; LN, lymph node; mLEC, murine LEC; PD-L1, programmed cell death ligand 1; In this study, we reveal that PD-L1 expression by LECs in the LN is PDPN, podoplanin; prox-1 tdt, Prox-1 ERcre Rosa26 STOPflox Tdtomato; RT-qPCR, dynamically regulated during an immune response and that this quantitative RT-PCR; VEGFR3, vascular endothelial cell growth factor receptor 3; regulation is dependent on IFN-a/b receptor (IFNaR) signaling. WT, wild type. Furthermore, we provide evidence that both type 1 IFN and PD-L1 Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 are negative regulators of LEC division during LN expansion. In turn,

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800271 2 TYPE 1 IFN AND PD-L1 COORDINATE LYMPHATIC EXPANSION and in concert with other work (26, 27), we show that PD-L1hi LECs paraffin were obtained from the University of Colorado Pathology Core. are protected from the apoptotic process that ensues during LN About 7- to 10-mm sections were cut using a microtome. Slides were contraction. Therefore, our data outline a role for type 1 IFN and baked for 2 h at 65˚C and then heated in a pressure cooker for 20 min in Tris-EDTA (pH 9) Ag retrieval buffer. Serial sections were stained with PD-L1 in regulating the expansion and contraction of the lymphatic lyve-1 (1:400; Abcam polyclonal) and cleaved caspase-3 (1:100; Cell network in the LN during an inflammatory immune response. Signaling Technology) or with lyve-1 B220-bio (1:100, clone RA3-6B2; BioLegend) and PD-L1 (1:50, clone 10F.9G2; BioLegend). For sections stained with caspase and lyve-1, sections were first stained for caspase and Materials and Methods then the section was blocked with an anti-rabbit IgG before staining with Mice lyve-1. Secondary Abs used were anti-rabbit IgG–FITC and anti-rabbit IgG–DyLight647 (1:200; BioLegend), anti-rat IgG-DyLight647 (1:100; Four- to six-week-old mice were purchased from Charles River Laboratories BioLegend), and streptavidin-PE (1:200; BioLegend). Normal human or Jackson Laboratory, unless otherwise stated, and were bred and housed LNs were purchased, and 7- to 10-mm sections were cut and stained in the University of Colorado Anschutz Medical Campus Animal Barrier with D2-40 (1:50) and PD-L1 (1:300). Secondary Abs used were anti- Facility. Wild type (WT), Prox-1 cre, Rosa26 STOPflox, Ifnar12/2, 2/2 2/2 mouse IgG-DyLight647 and anti-rabbit IgG–FITC. Sections were im- Ifngr1 , and Pdl1 mice were all bred on a C57BL/6 background. All aged using a Nikon Eclipse Ti series fluorescent microscope. Images animal procedures were approved by the Institutional Animal Care and were taken with a Photometrics CoolSNAP DYNO fluorescent camera. Use Committee at the University of Colorado. For image quantification, regions of interest were drawn around the lyve-1–positive regions to designate LECs, and the average mean PolyI:C and viral challenge fluorescence intensity of PD-L1 expression was taken using the Nikon PolyI:C (InvivoGen) and OVA were injected either s.c. or i.p. as described software. For quantification of cleaved caspase-3, sections were stained previously (1, 2). For some experiments, anti-CD40 (Bio X Cell) was also with PD-L1 and positive regions were identified. A serial section was injected. For viral challenge, mice were injected with 1 3 104 PFU per stained for lyve-1 and cleaved caspase-3. Serial images were overlaid, Downloaded from footpad using the vaccinia virus Western Reserve strain (1, 30). For and PD-L1–positive and lyve-1–positive regions were identified as Chikungunya virus (CHIKV) infections, 3- to 4-wk-old mice were injected caspase-3–positive or –negative and quantified. DAPI staining was used with 103 PFU per footpad of the CHIKV strain AF15561 in an animal to ensure the cleaved caspase-3 was nuclear. For all images, Lookup biosafety level 3 laboratory. For dengue virus infection, 6-wk-old mice were Tables were adjusted on each image equally. 4 injected with 10 PFU per footpad of the dengue virus strain DENV2 NGC. LEC sorting and quantitative RT-PCR BrdU treatment Five C57BL/6 mice per group were immunized. Six days later, mice were http://www.jimmunol.org/ Four milligrams of BrdU (BP25081; Fisher BioReagents) was injected into euthanized and dLNs were digested as described above. Cells were stained C57BL/6 mice in 200 ml of PBS i.p. and given at 0.8 mg/ml BrdU in water with CD45-PE (BioLegend) and underwent negative selection using anti- for 1 wk (3). PE microbeads and LS columns (Miltenyi Biotec). LECs were sorted on a FACSAria II (BD Biosciences). RNA was isolated from sorted PD-L1lo and LEC harvesting and staining for flow cytometry PD-L1hi LEC populations with the RNeasy Micro Kit (Qiagen-74004). cDNA was made using the QuantiTect Reverse Transcription Kit LNs were harvested and digested and then stained for flow cytometry (1, 2). (Qiagen-205314). Primers to Cxcl4, Gdf10, Itgb1, and control gene 18s For Ki67, staining cells were stained for surface markers and then fixed were purchased from Qiagen and run on Thermo Fisher Scientific StepOnePlus and permeabilized using the eBioscience Foxp3 Transcription Factor Real-Time PCR machine. Quantification was performed using the d-d Staining Buffer Set (catalog number 00-523). Briefly, cells were fixed and cycle threshold method (35). permeabilized for 30 min at 4˚C in the dark and then stained with Ki67 by guest on September 23, 2021 overnight at 4˚C. Cells were then washed and run on the cytometer. Bone marrow chimeras

For BrdU studies, cells were stained for surface markers and then fixed in 2/2 2/2 137 solution of 4% PFA, 1 mg/ml saponin, and 1% HEPES in HBSS (PFA/saponin WT, Pdl1 ,orIfnar1 were irradiated with 1000 rad using a [ Cs] solution). Cells were incubated in 10% DMSO in PBS/saponin buffer on ice gamma irradiator and rested for 4 h before reconstitution with WT bone for 10 min to enhance staining. Cells were refixed in PFA/saponin solution for marrow. Bone marrow was isolated, and RBCs were lysed prior to i.v. 5 min at room temperature and then digested with 1 mg/ml DNAse for 1 h. transfer and allowed to reconstitute for 10 wk (2). Cells were stained with BrdU FITC (10 mlAbinto45ml PBS/saponin buffer) for 30 min at 4˚C. Results For evaluation of caspase activity by flow cytometry, the CellEvent Caspase-3/7 Green Kit (catalog number C10427; Life Technologies) was Dynamic expression of PD-L1 by LECs used. Briefly, cells were digested as described and then stained for surface At homeostasis, subcapsular and medullary mouse LN LECs have markers. Cell were then stained with the caspase reagent for 30 min at 37˚C been shown to express high levels of PD-L1, whereas cortical LECs and then run on the cytometer. Unless otherwise noted, all flow cytometry Abs were purchased from only express low levels (15). Similarly, in normal human LNs BioLegend. Stromal cells were stained with CD45 APC-Cy7 (1:200) or stained for the lymphatic vasculature with PDPN and PD-L1, we Pacific Blue (1:300) clone 30-F11, podoplanin (PDPN) APC clone 8.1.1 found that LECs express PD-L1 in the subcapsular sinus with (1:200), CD31 PerCP/Cy5.5 clone 390 (1:200), PD-L1 PE or BV421 undetectable staining in the cortex (Fig. 1A, 1B). To determine the (1:200) clone 10F.9G2, Ki67 PE clone SolA15 (1:400; eBioscience), BrdU FITC (clone Bu20A; eBioscience or B44; BDBiosciences), and signals that regulate this low versus high expression of PD-L1 on ICAM PE or Pacific Blue clone 3E2 (1:400; BD Pharmingen). LECs, we asked whether primary LECs derived from hLEC LNs have differential expression of PD-L1. We found hLECs at ho- LEC culture meostasis do not have low or high PD-L1–expressing populations Primary murine LECs (mLECs) (C57-6092; Cell Biologics) and human (Fig. 1C). Based on published findings that IFN-a could regulate LECs (hLECs) (H-6092; Cell Biologics) were cultured in in endothelial PD-L1 expression in other cell lineages (33, 36–38), we asked if cell media (M1168 and H1168; Cell Biologics). Flasks and plates were PD-L1wasregulatedbyIFN-a in vitro. Upon stimulation with coated with Matrigel diluted 1:1000 in media (31). mLECs were treated with rIFN-a [a kind gift from R. Kedl (32)] and rIFN-g (714006; Bio- IFN-a, PD-L1 expression increased both by surface protein ex- Legend) at 250 U/ml (33) for 24 h before cells were harvested. Cells were pression (Fig. 1C) and mRNA (Fig. 1D) in hLECs. Similarly, then stained with aPDPN and aPD-L1 and acquired on a CyAn ADP flow naive primary mLECs express low levels of PD-L1 (Fig. 1E) and cytometer (Dako Products) and analyzed using FlowJo software (Tree do not have bimodal expression of PD-L1. Interestingly, upon Star). hLECs were treated with human IFN-a2 (11100-1; PBL Assay Science ) at 500 U/ml (34) for 24 h and then harvested as above. treatment with polyI:C, pam3CSK, or LPS, mLECs did not upregulate PD-L1, but similarly to hLECs, they did upregulate Fluorescence microscopy PD-L1inresponsetorIFN-a (Fig. 1E, Supplemental Fig. 1A). Popliteal and inguinal LNs from naive or immunized mice were fixed in This suggests that LECs are not upregulating PD-L1 in response formalin and embedded in paraffin. Normal human LNs embedded in to the polyI:C, a TLR3 agonist, even though LECs express The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 1. LEC PD-L1 expression is increased by IFN-a in vitro. (A) Representative image and quantification of a human normal LN stained for PDPN and PD-L1. Original magnification, 3200. Arrows point to cortical lymphatic vessels. Gates were drawn on the subcapsular and cortical LECs, and the geometric mean fluorescence intensity (gMFI) of PD-L1 expression was quantified. Large image magnification, 3100; small image magnification, 3200. Scale bar on large image, 1 mm; scale bar on inset images, 100 mm. (B) Quantification of (A). (C) Representative plot and quantified data showing the regulation of hLEC PD-L1 expression by IFN-a in vitro. Cells were treated with IFN-a for 24 h prior to harvest. (D) RT-qPCR of PD-L1 from hLECs treated with IFN-a in vitro. Cells were treated with IFN-a for 24 h prior to harvest. (E) Representative plot and quantified data showing the regulation of mLEC PD-L1 expression by IFN-a and polyI:C in vitro. Cells were treated with IFN-a or poly I:C for 24 h prior to harvest. (F) Representative image and quantification of Prox-1 tdt murine popliteal LN from naive and 1 d postimmunization with polyI:C stained for lyve-1 and PD-L1. Original magnification, 3200. Scale bar, 250 mm. Gates were drawn on the lyve-1–positive vessels, and the fold increase of PD-L1 expression compared with naive was quantified. All experiments were repeated twice with three to five replicates per group with similar results. Statistical analysis was done using a paired or unpaired t test. *p , 0.05, **p , 0.01, ***p , 0.001.

TLR3 in vivo (39) and primary mLECs express TLR3 in vitro STOPflox Tdtomato mice (prox-1 tdt), which express Tdtomato in (Supplemental Fig. 1B). These data demonstrate that type 1 prox-1 cre–positive cells following tamoxifen administration (40) IFN, but not type 1 IFN–inducing TLR agonists, is sufficient to in which prox-1 distinguishes LECs from all other cells in the LN cause the upregulation of PD-L1 by LECs. (41). We then verified that the prox-1–expressing cells were also To address whether PD-L1 expression by LECs changed fol- lyve-1 positive (Fig. 1F, Supplemental Fig. 1C) and quantified lowing type 1 IFN stimulus in vivo, we evaluated PD-L1 expression PD-L1 expression. We confirmed PD-L1 staining was not non- by LECs in the LNs of mice. We used Prox-1 ERcre Rosa26 specific binding of the anti-rat secondary Ab by staining LN 4 TYPE 1 IFN AND PD-L1 COORDINATE LYMPHATIC EXPANSION sections with the anti-rat secondary alone (Supplemental Fig. 1D). this immunization, we know that IFN-g is produced by T cells by Although the Tdtomato does appear dimmer in the center of LNs, day 6 (50) and that this does not result in PD-L1 upregulation this is likely due to the lack of tamoxifen penetrance. (Fig. 2D). To determine if type 1 IFN is required in vivo for PD-L1 We quantified PD-L1 expression on LECs in naive LNs, delineating upregulationbyLECsintheLN,weusedmicedeficientinIFNaR-1 the subcapsular sinus and the cortex (Supplemental Fig. 1C), and (Ifnar2/2). WT and Ifnar2/2 mice were immunized with polyI:C, we confirmed that PD-L1 expression was higher on subcapsular and 1 d after immunization, PD-L1 expression by LN LECs was sinus LECs compared with cortical LECs (Supplemental Fig. 1E). evaluated. Although PD-L1 expression increased on WT LECs, The cortical LECs do appear to express PD-L1, but at lower levels the LECs from Ifnar2/2 mice did not upregulate PD-L1 expression than the subcapsular LECs, as has been previously described (15). in response to immunization (Fig. 2F). To test whether PD-L1 expression increased after an IFN-inducing To determine the role of type 1 IFN in regulating PD-L1 on LECs stimulus, we immunized mice s.c. with polyI:C. We found that 1 d following infection, we infected Ifnar2/2 mice with vaccinia virus following immunization with polyI:C, there was an increase in and evaluated PD-L1 expression on LECs 5 d postinfection. PD-L1 expression on the prox-1 tdt lyve-1–positive vessels of mouse Intriguingly, PD-L1 expression in the Ifnar2/2 hosts was not as LNs (Fig. 1F, Supplemental Fig. 1C). Even after immunization, the high as in WT mice infected with vaccinia virus (Fig. 2F), per- subcapsular LECs expressed higher levels of PD-L1 compared with haps indicating that there are other factors produced during viral the cortical LECs (Supplemental Fig. 1E). This was not because infection that lead to the partial upregulation of PD-L1 on LECs. of differences in IFNaR expression by the LECs as we found no However, there was a significant decrease in PD-L1 expression significant differences in IFNaR expression between the PD-L1hi or following both polyI:C and vaccinia in the Ifnar2/2 hosts 2 2 PD-L1lo LECs (Supplemental Fig. 1F). (Fig. 2G). PD-L1 expression in the naive Ifnar / host was Downloaded from similar to the WT naive host, indicating that type 1 IFN are not PD-L1 upregulation by LECs postinfection or immunization is necessary for the homeostatic bimodal expression of PD-L1 on controlled by type 1 IFN LECs in vivo, which is consistent with published data suggesting To determine if PD-L1 upregulation by LECs in vivo is a normal that lymphotoxin b receptor (Ltßr) expression may be necessary response to viral infection, we infected mice with vaccinia virus for steady-state PD-L1 expression in the LN (15). These data

CHIKV and dengue virus and evaluated PD-L1 expression on dLN demonstrate that type 1 IFN are critical for the upregulation of http://www.jimmunol.org/ LECs by immunofluorescence. We confirmed that in naive mice, PD-L1 on LECs at early time points following both immunization PD-L1 expression was lower in the T cell zone and in the inter- and infection. Because IFN-g treatment can induce PD-L1 ex- follicular ridges by visualization of the B cell follicles (Supplemental pression by LEC (Supplemental Fig. 2D) and other endothelial Fig. 2A) but detectable as previously reported (23) and shown cells in vitro (33, 36, 46–49), we asked if IFN-g was also responsible (Supplemental Fig. 1D). We found a dramatic increase in PD-L1 for the upregulation of PD-L1 in vivo following immunization. One expression on LECs both in the sinus and in the cortex 3–5 d after daypostimmunizationinIFN-g–deficient mice, PD-L1 expression on viral infection (Fig. 2A, Supplemental Fig. 2A). We confirmed these LECs increased despite the inability of LECs to receive a signal from findings using flow cytometry following infection with vaccinia virus, IFN-g (Supplemental Fig. 2E), indicating that IFN-g is not required CHIKV, or immunization with polyI:C. LECs were gated as CD452, for PD-L1 upregulation in response to polyI:C in vivo but is sufficient by guest on September 23, 2021 CD31+, and PDPN+ and then PD-L1 expression was examined in vitro. Collectively, these data indicate that PD-L1 expression on (Supplemental Fig. 2B). Following infection or immunization LN LECs in vivo is regulated by IFNaR signaling following both with polyI:C, PD-L1 expression on LECs increased significantly polyI:C immunization and viral infection. ∼ ( 5-fold) compared with LECs in naive mice (Fig. 2B). To under- lo stand the kinetics of PD-L1 upregulation, we immunized mice PD-L1 LECs divide during LN expansion and evaluated PD-L1 expression over time. We found that PD-L1 The timing in which PD-L1 expression occurs, 0–3 d (Fig. 2D), is upregulation occurred within 3 h postimmunization with‘‘ polyI: prior to the expansion of LECs (3–6 d) (1) after immunization C, in which the peak of expression occurred at 24 h (Fig. 2C, 2D). (Fig. 3A). Therefore, we asked if there was a connection between PD-L1 expression returned to steady-state levels between day 3 PD-L1 expression and LEC division. Mice were immunized as and 6 after polyI:C immunization (Fig. 2D). above with Ag (OVA) and polyI:C with or without anti-CD40 to Importantly, dengue virus, polyI:C, vaccinia virus, and CHIKV elicit LN expansion and LEC division (Fig. 3A, Supplemental are all potent inducers of type 1 IFN (42–45), which is capable of Fig. 3A). To our surprise, by Ki67 staining, only the PD-L1lo inducing PD-L1 expression on both hLECs and mLECs in vitro LECs underwent division at 3–6 d postimmunization (Fig. 3A), (Fig. 1). Although administration of TLR agonists in vitro was not concurrent with the time point in which PD-L1 expression de- sufficient to induce the upregulation of PD-L1, immunization with clined (Fig. 2D). This difference in division between the PD-L1lo other type 1 IFN–inducing TLR agonists LPS and CpG did result and the PD-L1hi LECs was reflected in the number of LECs over in the upregulation of PD-L1 on LECs in vivo. However, immu- the course of the immune response (Fig. 3B). Although there nization with Pam3CSK, a TLR1,2 agonist that does not induce was a dramatic increase in the number and frequency of PD-L1hi type 1 IFN, did not lead to PD-L1 upregulation (Supplemental LECs at 1 d postimmunization, corresponding with the overall Fig. 2C). Indeed, we found a dramatic increase in IFN-b ex- increase in PD-L1 expression, the PD-L1hi LEC subset did not pression and an increase in IFN-a4 expression in the LN at 5 h, divide during LN expansion (3–6 d), whereas the PD-L1lo LEC which decreased, but was still high, at 1 d postimmunization by subset significantly expanded in the days following immunization quantitative RT-PCR (RT-qPCR) (Fig. 2E) after polyI:C immu- (Fig. 3B). These data demonstrate that PD-L1 expression nega- nization. We also evaluated IFN-g expression, as type 2 IFN have tively correlates with the expansion of LECs in response to type 1 also been shown to upregulate PD-L1 (33, 36, 46–49), and found IFN–inducing innate stimuli. that IFN-g expression does increase in the LN at 5 h after polyI:C To investigate how LEC division is controlled following an immunization but not to the extent of IFN-a4andIFN-b and de- innate immune stimulus and whether these two populations of creased by day 1. There was no detectable expression of IFN-a4, LECs, PD-L1hi versus PD-L1lo, activate different transcriptional IFN-b,orIFN-g at3or4dafterimmunization(Fig.2E)when programs, we immunized mice with polyI:C and evaluated the PD-L1 expression also returns to normal levels (Fig. 2D). Following known growth factor receptor associated with LEC division, The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 2. PD-L1 is upregulated following type 1 IFN stimulus. (A) Representative 2003 images and quantification of LNs from naive and day 3–5 vaccinia virus– and CHIKV-infected mice. Sections were stained for Lyve-1 and PD-L1. Fold increase in PD-L1 expression over naive is shown. Gates were drawn around the Lyve-1–positive staining, and the average PD-L1 geometric mean fluorescence intensity (gMFI) was taken and normalized to naive. Large image scale bar, 250 mM; inset image scale bar, 50 mM. (B) Representative plots and quantification of PD-L1 expression on LECs in the dLN following infection with vaccinia and CHIKV virus and immunization with poly I:C. The fold increase in PD-L1 gMFI compared with naive is shown. (C) Rep- resentative flow plots of PD-L1 expression on the dLN LECs following poly I:C immunization. (D) Quantification of (C) showing PD-L1 gMFI on all dLN LECs over time. (E) RT-qPCR showing fold increase in IFN-a4, IFN-b, and IFN-g expression in the dLN 5 h postimmunization with poly I:C. Data are normalized to naive. (F) Representative flow plots of PD-L1 expression in WT and Ifnar2/2 mice, 24 h postimmunization with poly I:C or 5 d postinfection with vaccinia virus. The fold increase in PD-L1 expression on LECs over naive is shown. (G) Quantification of (F). All experiments used two to three replicates per group and were repeated twice with similar results. Statistical analysis was done using an unpaired t test or one-way ANOVA. ***p , 0.001, ****p , 0.0001. 6 TYPE 1 IFN AND PD-L1 COORDINATE LYMPHATIC EXPANSION Downloaded from http://www.jimmunol.org/ FIGURE 3. Only PD-L1lo LECs divide in response to an IFN stimulus. (A) Representative flow plots and quantification of PD-L1 and Ki67 expression on LECs following immunization with poly I:C and anti-CD40. The percentage of PD-L1lo and PD-L1hi LECs that are Ki67 positive is shown. (B) LEC numbers following immunization with poly I:C and anti-CD40. Total LEC, PD-L1lo LEC, and PD-L1hi LEC numbers are shown. (C) RT-qPCR showing the expression fold change in PD-L1lo LECs 6 d postimmunization. All experiments used two to three replicates per group and were repeated twice with similar results. Statistical analysis was done using a one-way ANOVA. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001.

VegfR3. Following repeated systemic injection of the blocking immunization (Fig. 4A). We found the LECs lacking the IFNaR, VEGFR3 Ab, we did not observe any difference in LEC prolif- even when the hematopoietic compartment was WT, and could not eration after immunization, as assessed by Ki67 staining of LN upregulate PD-L1 (Fig. 4B, 4C). Surprisingly, we saw robust di- by guest on September 23, 2021 LECs between 1 and 7 d postimmunization (Supplemental Fig. 3B). vision in the Ifnar2/2 host 2 d following immunization (Fig. 4B, Therefore, we analyzed expression of other factors known to be 4D), with ∼20% of the LECs dividing, in contrast to the WT mice, involved in endothelial growth or inhibition of growth. To do this, in which very little division (∼5%) occurred at 2 d postimmunization we digested the LN and sorted LECs (1, 2, 51) based on their (Fig. 4D, Supplemental Fig. 3A). As before, only the PD-L1lo expression of PD-L1 (Supplemental Fig. 3C, 3D) 5 d after im- LECs divided following immunization, suggesting that PD-L1lo munization, a time point when we saw robust LEC division. We LEC preference for division is not controlled by type 1 IFN sig- did not observe any difference in VEGFR3/Flt4 expression be- naling. To determine if chronic IFN signaling results in loss of tween PD-L1hi and PD-L1lo LECs (Fig. 3C). We evaluated the LEC division, we used a chronic polyI:C injection model (58) angiostatin platelet factor 4 (PF4/CXCL4), shown to be important (Fig. 4E). Based on the data above, we predicted that chronic IFN in inhibiting angiogenesis (52, 53) by binding to vascular growth signaling would result in sustained upregulation of PD-L1 in the factor receptors (54), and we found an ∼50-fold increase of PF4/ LN. Indeed, when mice were injected with polyI:C every other CXCL4 expression in PD-L1hi LECs (Fig. 3C). We also evaluated day for 1 wk, PD-L1 expression remained high over the course growth/differentiation factor 10 (GDF10), which was shown to be of the week, whereas a single injection of polyI:C resulted in the involved in the growth of CD31+ endothelial cells as well as ax- downregulation of PD-L1 at 4 d following immunization (Fig. 4F). onal sprouting (55). GDF10 was enriched 50- to 100-fold in the When we evaluated the expression of Ki67 by LECs, we again PD-L1lo LECs (Fig. 3C). Finally, we evaluated integrin b 1 (ITGB1) found that Ki67 was produced at high levels at 4 d following a expression owing to its involvement in endothelial growth and single injection of polyI:C, concurrent with the time point in sprouting (56, 57) and found ITGB1 was enriched in the PD-L1lo which PD-L1 expression decreased. Consistent with the loss of LECs ∼2- to 6-fold (Fig. 3C). Together, these data suggest that type 1 IFN signaling inducing dysregulated division in the Ifnar2/2 PD-L1lo LECs have a transcriptional program associated with active chimera (Fig. 4A–C), we found that chronic type 1 IFN signaling cell division, whereas PD-L1hi LECs actively inhibit cell division. signiﬁcantly inhibited the ability of the cells to divide (Fig. 4G). Taken together, these results caused us to conclude that type 1 IFN Early LEC division is inhibited by type 1 IFN resulting from an innate stimulus inhibits LEC division during the Because of the dramatic differences in division we found between time when IFN-a and IFN-b expression is elevated (Fig. 2E). PD-L1hi and PD-L1lo LECs in the LN, the delay in LEC division between days 0 and 3 (Fig. 3A, 3B) and the kinetics of IFN ex- Loss of PD-L1 increases LEC division pression in the LN after polyI:C immunization (Fig. 2E), we asked We have shown above that PD-L1 distinguishes which LECs will if type 1 IFNs were inhibiting LEC division. Using a lethally divide during LN expansion; however, it has been shown that high irradiated Ifnar2/2 mouse reconstituted with WT bone marrow, we levels of PD-L1 can inhibit vascular endothelial cell division through asked again whether the LECs upregulated PD-L1 after polyI:C vascular endothelial cell growth factor regulation in vitro (28). The Journal of Immunology 7 Downloaded from

FIGURE 4. Type 1 IFN inhibits LEC division and promotes PD-L1 upregulation. (A) Schematic of experimental design for (B)–(D). (B) Representative ﬂow plots showing PD-L1 and Ki67 in WT and Ifnar2/2 chimeras 2 d postimmunization. (C) Quantiﬁcation of (A) showing PD-L1 expression on LECs. D A E F G F

( ) Quantification of ( ) showing the percentage of total LECs that are Ki67 positive. ( ) Schematic of experimental design for ( )and( ). ( ) Representative http://www.jimmunol.org/ flow plots and quantification showing PD-L1 expression on LECs 7 d postimmunization. (G) Representative flow plots and quantification displaying Ki67 expression on LECs 4 d postimmunization. All experiments used three replicates per group and were repeated twice with similar results. Statistical analysis was done using an unpaired t test or one-way ANOVA. *p , 0.05, **p , 0.01, ****p , 0.0001.

Because we see selective division of PD-L1lo LECs after IFN We then evaluated whether there were differences in the factors expression declines, we asked if PD-L1 was also contributing to involved in endothelial growth and/or inhibition we had seen the control of LEC division. We first asked if complete loss of between PD-L1hi and PD-L1lo LECs in the Pdl12/2 mice. Thus, PD-L1 led to aberrant LEC division. We used Pdl12/2 mice to we immunized mice and 5 d later digested the LNs and sorted determine if LEC division after immunization was affected when LECs. Although we found no significant differences in either by guest on September 23, 2021 PD-L1 was absent. We used both total Pdl12/2 mice and Pdl12/2 GDF10 or ITGB, we saw decreased expression of the angiostatin mice lethally irradiated and reconstituted with WT bone marrow CXCL4 (Fig. 5E). As CXCL4 has been shown to negatively to eliminate any contribution of PD-L1 coming from hematopoi- regulate angiogenesis (52–54), a reduction in CXCL4 expression etic cells, such as macrophages and DCs. We found that there was may be one mechanism by which LEC division increases in the significantly more LEC division in the total Pdl12/2 host and in Pdl12/2 chimeras. These data demonstrate a role for PD-L1 in the chimeric Pdl12/2 host compared with each WT counterpart at regulating LEC growth, perhaps through the inhibition of CXCL4 5 d postimmunization (Fig. 5A). This increase in division corre- during the process of LN expansion after an immune stimulus. sponded with a greater fold increase in total LEC number in the lo Pdl12/2 host than the WT host independent of whether it was a PD-L1 LECs undergo apoptosis during LN contraction complete Pdl12/2 or bone marrow chimera (Fig. 5B). When we Other reports outline a role for PD-L1 in promoting cancer cell asked if loss of PD-L1 could initiate LEC division earlier, we survival (26, 27, 29) via domains in the cytoplasmic tail of PD-L1 found that unlike the Ifnar2/2 chimeras, in the Pdl12/2 chimera, (26, 29). Our previous findings suggest that LEC apoptosis occurs there was neither an increase in LEC division at 2 d post- during LN contraction (2). Thus, we asked whether PD-L1 ex- immunization (Fig. 5A) nor an increase in division in the Pdl12/2 pression was promoting LEC survival of specific subsets of LECs. mice during chronic polyI:C immunization (Supplemental Fig. Therefore, we used a pulse–chase experiment in which immedi- 4B), indicating that the role of PD-L1 in regulating division is ately following immunization with polyI:C and anti-CD40, BrdU independent and secondary to type 1 IFN. Importantly, we did not was given to mice for 1 wk to allow the BrdU to incorporate into observe any differences in total LN cell number, CD4 and CD8 DNA during DNA replication (Fig. 6A). After 1 wk of BrdU, mice T cell number, or B cell number between the WT and the Pdl12/2 were given normal water for an additional 2 wk to evaluate the host or Pdl12/2 chimera (Supplemental Fig. 4C), indicating that loss of cells that incorporated BrdU and thus which cells un- the differences observed in LEC division or number were not derwent LEC death (Fig. 6A). At 1 wk postimmunization, we simply due to increased LN size or cellularity. observed BrdU incorporation in the PD-L1lo LECs but not in the To evaluate whether LEC division was dysregulated in the PD-L1hi LECs, confirming our Ki67 results that PD-L1–expressing population of LECs that normally express PD-L1, we used ICAM1 LECs do not undergo division (Fig. 6B). To evaluate which pop- as a marker of subcapsular LECs, as described previously (15) ulations underwent contraction, we evaluated the loss of BrdU+ (Supplemental Fig. 2B). The expression of ICAM1 does not or BrdU2 cells. When we quantified the number of PD-L1lo LECs change in the Pdl12/2 chimera compared with WT (Fig. 5C). We between 1 and 3 wk, we found a significant loss in PD-L1lo BrdU+ found that in the WT hosts, very few of the ICAM1+ LECs divided LECs (Fig. 6C), whereas we saw similar numbers of PD-L1hi during LN expansion; strikingly, a substantial number of ICAM1+ LECs between week 1 and week 3 (Fig. 6D). These data suggest LECs underwent division in the Pdl12/2 chimera (Fig. 5C, 5D). that not only do the PD-L1lo LECs divide in response to an 8 TYPE 1 IFN AND PD-L1 COORDINATE LYMPHATIC EXPANSION Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 5. Loss of PD-L1 promotes LEC division after immunization. (A) Representative flow plots and quantification showing Ki67 expression in WT and Pdl12/2 chimeras and the total Pdl12/2 2 and 5 d postimmunization. p = 0.0001, day 5 WT compared with day 5 Pdl12/2 chimeras. p = 0.008, day 5 WT compared with total day 5 Pdl12/2.(B) The fold increase in total LEC number over naive. (C) Representative flow plots displaying Ki67 expression by ICAM1 expression in WT and Pdl12/2 chimeras. (D) Quantification of (C) showing the number of Ki67+, ICAM1hi LECs. (E) RT-qPCR showing the expression fold change in Pdl12/2 LECs 6 d postimmunization. All experiments used three replicates per group and were repeated twice with similar results. Statistical analysis was done using a one-way ANOVA followed by Bonferroni posttest. *p , 0.05, ***p , 0.001. immune stimulus but they are also the cells that contract during the number of caspase-3+ cells that were either lyve-1+PD-L1hi or the resolution of the immune response. lyve-1+PD-L1lo (yellowarrows,Fig.6G).Toensureweonly To directly determine if low PD-L1 expression marks LECs that evaluated caspase-3+ LECs, only cells in which caspase-3 over- undergo caspase-mediated apoptosis during LN contraction, we lappedwithDAPIinlyve-1+ vessels were counted. Further, immunized mice and 2 wk later asked which LECs expressed we used sections stained without the primary caspase Ab to caspase-3/7 using caspase flow reagent (Fig. 6E). Consistent with distinguish caspase staining from nonspecific staining (Supplemental the above data (Fig. 6C, 6D), PD-L1 expression inversely corre- Fig. 4A). White arrows indicate caspase-3+ cells that are not lyve-1 lated with the expression of caspase-3/7 by LECs (Fig. 6E, 6F). positive. The number of caspase-positive PD-L1lo LECs per LN was We also evaluated explanted LNs from mice immunized 10 d prior significantly higher than the number of caspase-positive PD-L1hi to evaluate caspase staining by immunofluorescence. We stained serial LECs, indicating that PD-L1lo LECs undergo apoptosis at a higher sections with lyve-1 and cleaved caspase-3 or PD-L1 and quantified frequency than PD-L1hi LECs. Together, these data demonstrate that The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 6. PD-L1+ LECs are protected from apoptosis. (A) Schematic of experimental design for (B)–(D). (B) Representative flow plots showing PD-L1 expression and BrdU incorporation on LECs. (C) Quantification of (B) showing the number of BrdU+ PD-L1lo LECs. (D) Quantification of (B) showing the number of PD-L1hi LECs. (E) Representative flow plot displaying PD-L1 and cleaved caspase-3/7 expression on LECs. (F) Quantification of (E) displaying the percentage of PD-L1lo and PD-L1hi LECs that are cleaved caspase-3/7 positive. (G) Quantification and representative (Figure legend continues) 10 TYPE 1 IFN AND PD-L1 COORDINATE LYMPHATIC EXPANSION during an acute, inflammatory immune response, the PD-L1lo LECs Ags normally presented by LECs to T cells to maintain peripheral divide and then undergo apoptosis during LN expansion and tolerance (15, 17, 20, 21, 25, 59). contraction, respectively, whereas the PD-L1hi LECs neither We provide ample evidence in this study that both loss of PD-L1 divide nor die during an immune response. and IFNaR results in increased LEC division in a Pdl12/2 chimeric mouse model, the intact Pdl12/2 mouse, and in an Ifnar2/2 chimeric PD-L1 protects LECs from apoptosis mouse (Figs. 4, 5). We also suggest that PD-L1 signaling may lead We next asked if loss of PD-L1 increased LEC apoptosis dur- to downstream CXCL4 activation as CXCL4 expression is de- ing an immune response at times other than LN contraction, as creased in the Pdl12/2 mice. Because CXCL4 has been shown to we saw only a low frequency of PD-L1–expressing LECs that inhibit endothelial cell division, we predict that loss of CXCL4 underwent apoptosis during LN contraction. To address this, results, at least in part, in the increased LEC division we see in weimmunizedWTmice,and5dafterimmunization,we the Pdl12/2. These data are intriguing as others have shown that evaluated caspase-3/7–positive LECs (Fig. 7A). Following our CXCL4 is also involved in neutrophil recruitment during inflam- normal digestion and staining protocol, we observed ∼40% mation (60) and that neutrophils contribute to inflammatory lym- of LECs were caspase-3/7–positive with the majority being phangiogenesis through VEGFD (61). Although GDF10 and lo PD-L1 (∼30%) (Fig. 7B). To evaluate if PD-L1 was acting ITGB1 are not changed in the Pdl12/2, they may still be impor- to protect LECs from death, we performed the same procedure in tant for differences between PD-L1hi and PD-L1lo LECs in the 2/2 Pdl1 mice and found a higher mean fluorescence intensity of WT mouse. We further show that repeated polyI:C injection and 2/2 caspase-3/7 in the Pdl1 LECs (Fig. 7C), indicating the level chronic IFN results in maintained upregulation of PD-L1 and in a of caspase-3/7 activity per cell is higher in LECs when PD-L1 is loss of LEC division at the 3- to 4-d time point during which we Downloaded from absent. Remarkably, we found a significant increase in the per- and others have described LEC division to occur (1, 4, 5). This centage (Fig. 7D) and number (Fig. 7E) of caspase-positive LECs loss of LEC division may occur as a result of IFN-mediated in- 2/2 in the Pdl1 mice compared with WT mice, indicating that hibition, increased PD-L1 inhibition, or both. Interestingly, the PD-L1 is protective against apoptosis. This is consistent with loss of LEC division during chronic polyI:C injection never re- published data demonstrating a prosurvival effect of PD-L1 sig- covers. Not only do these data predict that IFN stimulation and

naling in tumor cells (26, 27, 29). These data support the conclusion PD-L1 upregulation inhibit LEC division, but they also predict http://www.jimmunol.org/ that there are distinct populations of LECs in the LN that undergo that chronic IFN signaling is likely to have detrimental effects to division, apoptosis, and survival during LN expansion and con- the normal immune response. This is borne out in the literature in traction. Thus, we propose a model in which type 1 IFN and PD-L1 models in which chronic IFN signaling dramatically reduces im- independently inhibit LEC division and PD-L1 expression promotes munity and promotes increased PD-1 expression by T cells and LEC survival (Fig. 7F). PD-L1 expression by DCs (62–71). We acknowledge that there are likely other cells participating in the process of immune dysreg- Discussion ulation during chronic IFN signaling; however, it does appear that The data presented in this study support a model in which PD-L1 in addition to these other cell types, LEC proliferation during LN expression by LN LECs regulates the expansion, contraction, expansion does not occur normally in situations of chronic IFN by guest on September 23, 2021 and survival of LECs in the LN. Some of the factors needed for stimulation. LEC expansion in the LN during an immune response have been Finally, we found that high levels of PD-L1 expression protected identified and include production of VEGF-C as well as DC, T cell, LECs from undergoing apoptosis regardless of the timing after and B cell recruitment (4, 5). We find that PD-L1 expression immunization. These data indicate that PD-L1 expression also not only regulates LEC division and survival, but that PD-L1 is promotes LEC survival, consistent with a described phenomenon in dynamically regulated by type 1 IFN. Furthermore, we find that cancer cells (26, 27, 29), in which it appears that there are sig- type 1 IFN signaling also regulates LEC division in a pathway naling motifs in the C-terminal cytoplasmic domain of PD-L1 that independent of PD-L1. Our data uncover a novel mechanism by confer protection against multiple inducers of apoptosis (27, 29). which PD-L1 drives LEC survival and inhibits LEC division, The findings we outline in this study are important as they dem- explaining the described phenomenon of specific cortical LEC onstrate that reverse signaling of PD-L1 is not an aberrant process expansion following an immune stimulus (6).Thus, PD-L1 ex- that occurs only in mutated cancer cells but is instead a process pression by LECs is intertwined with the innate immune re- that regulates how the LN expands and contracts in response sponse following an immune challenge. Inhibition of LEC to immunization or infection. With this in mind, we predict that division and PD-L1 regulation by type 1 IFN may be a critical these findings and future studies may provide an understanding of first signal for the LECs to pause, perhaps allowing for the consequences resulting from the inhibition or promotion of PD-L1 relaxation and spreading of the fibroblastic reticular cell net- reverse signaling in settings of cancer or chronic infection. This is work, which also requires DC–fibroblastic reticular cell inter- important as we provide evidence that PD-L1 expression and actions (13) before initiating lymphatic expansion to draw in regulation by type 1 IFN also occurs in normal human LNs and large numbers of tissue-resident immune cells. The immediate primary hLECs (Fig. 1). upregulation of PD-L1 may be important for preventing an The role of PD-1 and CD80 expression by hematopoietic cells active immune response against self-antigens or nonadjuvanted in PD-L1 reverse signaling also needs to be considered. Current

images of an inguinal LN stained with lyve-1, PD-L1, cleaved caspase-3, and DAPI. Magnification on large image, 320; magnification on inset images, 340. Scale bar on large images, 250 mm; scale bar on inset images, 10 mm. Yellow arrows indicate cleaved caspase-3–positive LECs; white arrows indicate cleaved caspase-3–positive cells that are not LECs. Quantification displays the number of cleaved caspase-3–positive PD-L1+ and PD-L12 LECs per LN section. All experiments used three replicates per group. BrdU staining was repeated four times, caspase flow staining was repeated twice, and caspase immunofluorescence was repeated four times with four LNs per group with similar results. Statistical analysis was done using a paired or unpaired t test. **p , 0.01, ****p , 0.0001. The Journal of Immunology 11 Downloaded from http://www.jimmunol.org/

FIGURE 7. Loss of PD-L1 increases LEC apoptosis following immunization. (A) Schematic of experiment design for (B)–(E). (B) Representative flow by guest on September 23, 2021 plot and quantification of caspase-3/7 staining on WT LECs 5 d postimmunization (dpi). (C) Representative flow plot and geometric mean fluorescence intensity (gMFI) quantification of caspase-3/7 staining on WT and Pdl12/2 LECs 5 dpi. (D) Quantification displaying percentage of LECs positive for caspase-3/7 expression in WT and Pdl12/2 LECs 5 dpi. (E) Quantification displaying of number of caspase-3/7–positive LEC expression in WT and Pdl12/2 LECs 5 dpi. (F) Model of type 1 IFN and PD-L1 regulation of division and survival of LECs. All experiments used three replicates per group and were repeated twice with similar results. Statistical analysis was done using a one-way ANOVA, followed by Bonferroni posttest. *p , 0.05, **p , 0.01. literature suggests that the survival benefit of PD-L1 expression may studies are aimed at evaluating the downstream mechanisms by be independent of binding to PD-1 or CD80, as cells that express which PD-L1 signals into the LEC as well as a role for PD-L1hi PD-L1 survive better in cultures even in the absence of cells LECs in coordinating the immune response via interactions with expressing PD-1 or CD80 (26, 27, 29). However, it is still unclear migratory DCs as they enter the LN. whether CD80 or PD-1 may contribute. As such, an in vitro study with vascular endothelial cells found that PD-L1 ex- Acknowledgments pression could limit vascular endothelial cell division and that 2/2 suppression of CD80 increased VEGFR2 expression, thereby We thank Dr. Dong (Mayo Clinic, Rochester, MN) for the Pdl1 mice, Dr. Lenz (University of Colorado Anschutz Medical Campus) for the promoting division (28). 2 2 2 2 Ifngr / and Ifnar / mice, and Dr. Kedl (University of Colorado In summary, these studies outline a role for PD-L1 in regulating Anschutz Medical Campus) for the recombinant mouse IFN-a. the LEC response to an immune stimulus. We have identified a major role for type 1 IFN in the upregulation of PD-L1 on LECs and in the inhibition of LEC division when IFN-ab expression is high. Disclosures These data corroborate new findings that type 1 IFN can inhibit The authors have no financial conflicts of interest. lymphatic growth during infection (72). We also identified a novel role for PD-L1 reverse signaling into the LEC that prevents LEC References expansion postinfection or postimmunization. We show that loss 1. Tamburini, B. A., M. A. Burchill, and R. M. Kedl. 2014. Antigen capture and of PD-L1 results in lymphatic expansion after IFN levels decrease archiving by lymphatic endothelial cells following vaccination or viral infection. in the LN, potentially so that tissue-resident cells can infiltrate the Nat. Commun. 5: 3989. LN when it is primed to do so. Finally, we provide evidence that 2.Kedl,R.M.,R.S.Lindsay,J.M.Finlon,E.D.Lucas,R.S.Friedman,and B. A. J. Tamburini. 2017. Migratory dendritic cells acquire and present lym- PD-L1 expression leads to an LEC state that confers protection phatic endothelial cell-archived antigens during lymph node contraction. against apoptosis and inhibits division. Taken together, these Nat. Commun. 8: 2034. 3. Webster, B., E. H. Ekland, L. M. Agle, S. Chyou, R. Ruggieri, and T. T. Lu. studies identify a novel role for IFN-a and PD-L1 in regulating 2006. Regulation of lymph node vascular growth by dendritic cells. J. Exp. Med. LEC division during the initiation of the immune response. Future 203: 1903–1913. 12 TYPE 1 IFN AND PD-L1 COORDINATE LYMPHATIC EXPANSION

4. Chyou, S., F. Benahmed, J. Chen, V. Kumar, S. Tian, M. Lipp, and T. T. Lu. 27.Azuma,T.,S.Yao,G.Zhu,A.S.Flies,S.J.Flies,andL.Chen.2008.B7-H1 2011. Coordinated regulation of lymph node vascular-stromal growth first by is a ubiquitous antiapoptotic receptor on cancer cells. Blood 111: 3635– CD11c+ cells and then by T and B cells. J. Immunol. 187: 5558–5567. 3643. 5. Angeli, V., F. Ginhoux, J. Llodra`, L. Quemeneur, P. S. Frenette, M. Skobe, 28. Jin, Y., S. K. Chauhan, J. El Annan, P. T. Sage, A. H. Sharpe, and R. Dana. 2011. R. Jessberger, M. Merad, and G. J. Randolph. 2006. B cell-driven lym- A novel function for programmed death ligand-1 regulation of angiogenesis. phangiogenesis in inflamed lymph nodes enhances dendritic cell mobilization. [Published erratum appears in 2012 Am. J. Pathol. 180: 1324.] Am. J. Pathol. Immunity 24: 203–215. 178: 1922–1929. 6.Tan,K.W.,K.P.Yeo,F.H.Wong,H.Y.Lim,K.L.Khoo,J.P.Abastado, 29. Gato-Canãs, M., M. Zuazo, H. Arasanz, M. Iban˜ez-Vea, L. Lorenzo, and V. Angeli. 2012. Expansion of cortical and medullary sinuses restrains G. Fernandez-Hinojal, R. Vera, C. Smerdou, E. Martisova, I. Arozarena, et al. lymph node hypertrophy during prolonged inflammation. J. Immunol. 188: 2017. PDL1 signals through conserved sequence motifs to overcome interferon- 4065–4080. mediated cytotoxicity. Cell Rep. 20: 1818–1829. 7. Kaipainen, A., J. Korhonen, T. Mustonen, V. W. van Hinsbergh, G. H. Fang, 30. Norbury, C. C., D. Malide, J. S. Gibbs, J. R. Bennink, and J. W. Yewdell. 2002. D. Dumont, M. Breitman, and K. Alitalo. 1995. Expression of the fms-like Visualizing priming of virus-specific CD8+ T cells by infected dendritic cells tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during in vivo. Nat. Immunol. 3: 265–271. development. Proc. Natl. Acad. Sci. USA 92: 3566–3570. 31. Lyons, T. R., V. F. Borges, C. B. Betts, Q. Guo, P. Kapoor, H. A. Martinson, S. Jindal, 8. Enholm, B., L. Jussila, M. Karkkainen, and K. Alitalo. 1998. Vascular en- and P. Schedin. 2014. Cyclooxygenase-2-dependent lymphangiogenesis promotes dothelial growth factor-C: a growth factor for lymphatic and blood vascular nodal metastasis of postpartum breast cancer. J. Clin. Invest. 124: 3901–3912. endothelial cells. Trends Cardiovasc. Med. 8: 292–297. 32. McWilliams, J. A., P. J. Sanchez, C. Haluszczak, L. Gapin, and R. M. Kedl. 9. Lymboussaki,A.,T.A.Partanen,B.Olofsson, J. Thomas-Crusells, C. D. M. Fletcher, 2010. Multiple innate signaling pathways cooperate with CD40 to induce potent, R. M. W. de Waal, A. Kaipainen, and K. Alitalo. 1998. Expression of the vascular CD70-dependent cellular immunity. Vaccine 28: 1468–1476. endothelial growth factor C receptor VEGFR-3 in lymphatic endothelium of the 33. Mu¨hlbauer, M., M. Fleck, C. Schu¨tz, T. Weiss, M. Froh, C. Blank, J. Scho¨lmerich, skin and in vascular tumors. Am. J. Pathol. 153: 395–403. and C. Hellerbrand. 2006. PD-L1 is induced in hepatocytes by viral infection and 10. Rutkowski, J. M., J. E. Ihm, S. T. Lee, W. W. Kilarski, V. I. Greenwood, by interferon-alpha and -gamma and mediates T cell apoptosis. J. Hepatol. 45: M. C. Pasquier, A. Quazzola, D. Trono, J. A. Hubbell, and M. A. Swartz. 2013. 520–528. VEGFR-3 neutralization inhibits ovarian lymphangiogenesis, follicle matu- 34. Sharara, A. I., D. J. Perkins, M. A. Misukonis, S. U. Chan, J. A. Dominitz, and ration, and murine pregnancy. Am. J. Pathol. 183: 1596–1607. J. B. Weinberg. 1997. Interferon (IFN)-alpha activation of human blood Downloaded from 11. Gu¨ç, E., P. S. Briquez, D. Foretay, M. A. Fankhauser, J. A. Hubbell, mononuclear cells in vitro and in vivo for nitric oxide synthase (NOS) W. W. Kilarski, and M. A. Swartz. 2017. Local induction of lymphangiogenesis type 2 mRNA and protein expression: possible relationship of induced with engineered fibrin-binding VEGF-C promotes wound healing by increasing NOS2 to the anti-hepatitis C effects of IFN-alpha in vivo. J. Exp. Med. immune cell trafficking and matrix remodeling. Biomaterials 131: 160–175. 186: 1495–1502. 12. Acton, S. E., A. J. Farrugia, J. L. Astarita, D. Mouraõ-Sa´, R. P. Jenkins, E. Nye, 35. Livak, K. J., and T. D. Schmittgen. 2001. Analysis of relative gene expression S. Hooper, J. van Blijswijk, N. C. Rogers, K. J. Snelgrove, et al. 2014. Dendritic data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. cells control fibroblastic reticular network tension and lymph node expansion. Methods 25: 402–408.

Nature 514: 498–502. 36. Eppihimer, M. J., J. Gunn, G. J. Freeman, E. A. Greenﬁeld, T. Chernova, http://www.jimmunol.org/ 13. Astarita, J. L., V. Cremasco, J. Fu, M. C. Darnell, J. R. Peck, J. M. Nieves- J. Erickson, and J. P. Leonard. 2002. Expression and regulation of the PD-L1 Bonilla, K. Song, Y. Kondo, M. C. Woodruff, A. Gogineni, et al. 2015. The immunoinhibitory molecule on microvascular endothelial cells. Microcirculation CLEC-2-podoplanin axis controls the contractility of ﬁbroblastic reticular cells 9: 133–145. and lymph node microarchitecture. Nat. Immunol. 16: 75–84. 37. Schreiner, B., M. Mitsdoerffer, B. C. Kieseier, L. Chen, H. P. Hartung, 14. Kataru, R. P., H. Kim, C. Jang, D. K. Choi, B. I. Koh, M. Kim, S. Gollamudi, M. Weller, and H. Wiendl. 2004. Interferon-beta enhances monocyte and den- Y. K. Kim, S. H. Lee, and G. Y. Koh. 2011. T lymphocytes negatively regulate dritic cell expression of B7-H1 (PD-L1), a strong inhibitor of autologous T-cell lymph node lymphatic vessel formation. Immunity 34: 96–107. activation: relevance for the immune modulatory effect in multiple sclerosis. 15. Cohen, J. N., E. F. Tewalt, S. J. Rouhani, E. L. Buonomo, A. N. Bruce, X. Xu, J. Neuroimmunol. 155: 172–182. S. Bekiranov, Y. X. Fu, and V. H. Engelhard. 2014. Tolerogenic properties of 38. Staples, K. J., B. Nicholas, R. T. McKendry, C. M. Spalluto, J. C. Wallington, lymphatic endothelial cells are controlled by the lymph node microenvironment. C. W. Bragg, E. C. Robinson, K. Martin, R. Djukanovic´, and T. M. Wilkinson. PLoS One 9: e87740. 2015. Viral infection of human lung macrophages increases PDL1 expression via

16. Podgrabinska, S., O. Kamalu, L. Mayer, M. Shimaoka, H. Snoeck, IFNb. PLoS One 10: e0121527. by guest on September 23, 2021 G. J. Randolph, and M. Skobe. 2009. Inflamed lymphatic endothelium sup- 39. Heng, T. S., and M. W. Painter, Immunological Genome Project Consortium. presses dendritic cell maturation and function via Mac-1/ICAM-1-dependent 2008. The immunological genome project: networks of gene expression in mechanism. J. Immunol. 183: 1767–1779. immune cells. Nat. Immunol. 9: 1091–1094. 17. Rouhani, S. J., J. D. Eccles, P. Riccardi, J. D. Peske, E. F. Tewalt, J. N. Cohen, 40. Bianchi, R., A. Teijeira, S. T. Proulx, A. J. Christiansen, C. D. Seidel, T. Ru¨licke, R. Liblau, T. Ma¨kinen, and V. H. Engelhard. 2015. Roles of lymphatic endo- T. Ma¨kinen, R. Ha¨gerling, C. Halin, and M. Detmar. 2015. A transgenic thelial cells expressing peripheral tissue antigens in CD4 T-cell tolerance in- Prox1-Cre-tdTomato reporter mouse for lymphatic vessel research. PLoS One duction. Nat. Commun. 6: 6771. 10: e0122976. 18. Teijeira, A., A. Rouzaut, and I. Melero. 2013. Initial afferent lymphatic vessels 41. Wigle, J. T., and G. Oliver. 1999. Prox1 function is required for the development controlling outbound leukocyte traffic from skin to lymph nodes. Front. Immunol. of the murine lymphatic system. Cell 98: 769–778. 4: 433. 42. Fox, J. M., and M. S. Diamond. 2016. Immune-mediated protection and path- 19. Petrova, T. V., and G. Y. Koh. 2018. Organ-specific lymphatic vasculature: from ogenesis of chikungunya virus. J. Immunol. 197: 4210–4218. development to pathophysiology. J. Exp. Med. 215: 35–49. 43. van den Broek, M. F., U. Mu¨ller, S. Huang, M. Aguet, and R. M. Zinkernagel. 20. Cohen, J. N., C. J. Guidi, E. F. Tewalt, H. Qiao, S. J. Rouhani, A. Ruddell, 1995. Antiviral defense in mice lacking both alpha/beta and gamma interferon A. G. Farr, K. S. Tung, and V. H. Engelhard. 2010. Lymph node-resident receptors. J. Virol. 69: 4792–4796. lymphatic endothelial cells mediate peripheral tolerance via Aire-independent 44. Matsumoto, M., and T. Seya. 2008. TLR3: interferon induction by double- direct antigen presentation. J. Exp. Med. 207: 681–688. stranded RNA including poly(I:C). Adv. Drug Deliv. Rev. 60: 805–812. 21. Dubrot, J., F. V. Duraes, L. Potin, F. Capotosti, D. Brighouse, T. Suter, 45. Urcuqui-Inchima, S., J. Cabrera, and A. L. Haenni. 2017. Interplay between S. LeibundGut-Landmann, N. Garbi, W. Reith, M. A. Swartz, and S. Hugues. dengue virus and Toll-like receptors, RIG-I/MDA5 and microRNAs: implica- 2014. Lymph node stromal cells acquire peptide-MHCII complexes from den- tions for pathogenesis. Antiviral Res. 147: 47–57. dritic cells and induce antigen-specific CD4+ T cell tolerance. J. Exp. Med. 211: 46. Gong, A. Y., R. Zhou, G. Hu, X. Li, P. L. Splinter, S. P. O’Hara, N. F. LaRusso, 1153–1166. G. A. Soukup, H. Dong, and X. M. Chen. 2009. MicroRNA-513 regulates B7-H1 22. Baptista, A. P., R. Roozendaal, R. M. Reijmers, J. J. Koning, W. W. Unger, translation and is involved in IFN-gamma-induced B7-H1 expression in M. Greuter, E. D. Keuning, R. Molenaar, G. Goverse, M. M. Sneeboer, et al. cholangiocytes. J. Immunol. 182: 1325–1333. 2014. Lymph node stromal cells constrain immunity via MHC class II self-antigen 47. Seo, S. K., D. I. Seo, W. S. Park, W. K. Jung, D. S. Lee, S. G. Park, J. S. Choi, presentation. eLife 3: e04433. M. S. Kang, Y. H. Choi, I. Choi, et al. 2014. Attenuation of IFN-g-induced 23. Hirosue, S., E. Vokali, V. R. Raghavan, M. Rincon-Restrepo, A. W. Lund, B7-H1 expression by 15-deoxy-delta(12,14)-prostaglandin J2 via downregula- P. Corthe´sy-Henrioud, F. Capotosti, C. Halin Winter, S. Hugues, and M. A. Swartz. tion of the Jak/STAT/IRF-1 signaling pathway. Life Sci. 112: 82–89. 2014. Steady-state antigen scavenging, cross-presentation, and CD8+ T cell priming: 48. Lee, S. J., B. C. Jang, S. W. Lee, Y. I. Yang, S. I. Suh, Y. M. Park, S. Oh, a new role for lymphatic endothelial cells. J. Immunol. 192: 5002–5011. J. G. Shin, S. Yao, L. Chen, and I. H. Choi. 2006. Interferon regulatory factor-1 is 24. Dieterich, L. C., K. Ikenberg, T. Cetintas, K. Kapaklikaya, C. Hutmacher, and prerequisite to the constitutive expression and IFN-gamma-induced upregulation M. Detmar. 2017. Tumor-associated lymphatic vessels upregulate PDL1 to in- of B7-H1 (CD274). FEBS Lett. 580: 755–762. hibit t-cell activation. Front. Immunol. 8: 66. 49. Lee, S. K., S. H. Seo, B. S. Kim, C. D. Kim, J. H. Lee, J. S. Kang, P. J. Maeng, 25. Lund, A. W., F. V. Duraes, S. Hirosue, V. R. Raghavan, C. Nembrini, and J. S. Lim. 2005. IFN-gamma regulates the expression of B7-H1 in dermal S. N. Thomas, A. Issa, S. Hugues, and M. A. Swartz. 2012. VEGF-C promotes fibroblast cells. J. Dermatol. Sci. 40: 95–103. immune tolerance in B16 melanomas and cross-presentation of tumor antigen by 50. Ahonen, C. L., C. L. Doxsee, S. M. McGurran, T. R. Riter, W. F. Wade, lymph node lymphatics. Cell Rep. 1: 191–199. R. J. Barth, J. P. Vasilakos, R. J. Noelle, and R. M. Kedl. 2004. Combined TLR 26. Ghebeh, H., C. Lehe, E. Barhoush, K. Al-Romaih, A. Tulbah, M. Al-Alwan, and CD40 triggering induces potent CD8+ T cell expansion with variable S. F. Hendrayani, P. Manogaran, A. Alaiya, T. Al-Tweigeri, et al. 2010. Doxo- dependence on type I IFN. J. Exp. Med. 199: 775–784. rubicin downregulates cell surface B7-H1 expression and upregulates its nuclear 51. Kedl, R. M., and B. A. Tamburini. 2015. Antigen archiving by lymph node expression in breast cancer cells: role of B7-H1 as an anti-apoptotic molecule. stroma: a novel function for the lymphatic endothelium. Eur. J. Immunol. 45: Breast Cancer Res. 12: R48. 2721–2729. The Journal of Immunology 13

52. Bikfalvi, A. 2004. Platelet factor 4: an inhibitor of angiogenesis. Semin. Thromb. 62. Hallam, D. M., and L. E. Maroun. 1998. Anti-gamma interferon can prevent the Hemost. 30: 379–385. premature death of trisomy 16 mouse cortical neurons in culture. Neurosci. Lett. 53. Perollet, C., Z. C. Han, C. Savona, J. P. Caen, and A. Bikfalvi. 1998. Platelet 252: 17–20. factor 4 modulates fibroblast growth factor 2 (FGF-2) activity and inhibits FGF-2 63. Lee, H. C., K. L. Tan, P. S. Cheah, and K. H. Ling. 2016. Potential role of dimerization. Blood 91: 3289–3299. JAK-STAT signaling pathway in the neurogenic-to-gliogenic shift in down 54. Bikfalvi, A. 2004. Recent developments in the inhibition of angiogenesis: syndrome brain. Neural Plast. 2016: 7434191. examples from studies on platelet factor-4 and the VEGF/VEGFR system. 64. Ling, K. H., C. A. Hewitt, K. L. Tan, P. S. Cheah, S. Vidyadaran, M. I. Lai, Biochem. Pharmacol. 68: 1017–1021. H. C. Lee, K. Simpson, L. Hyde, M. A. Pritchard, et al. 2014. Functional 55. Li, S., E. H. Nie, Y. Yin, L. I. Benowitz, S. Tung, H. V. Vinters, F. R. Bahjat, transcriptome analysis of the postnatal brain of the Ts1Cje mouse model for M. P. Stenzel-Poore, R. Kawaguchi, G. Coppola, and S. T. Carmichael. 2015. Down syndrome reveals global disruption of interferon-related molecular GDF10 is a signal for axonal sprouting and functional recovery after stroke. Nat. networks. BMC Genomics 15: 624. Neurosci. 18: 1737–1745. 65. Maroun, L. E. 1979. Interferon effect on ribosomal ribonucleic acid related to 56. Malan,D.,D.Wenzel,A.Schmidt,C.Geisen,A.Raible,B.Bo¨lck, B. K. Fleischmann, chromosome 21 ploidy. Biochem. J. 179: 221–225. and W. Bloch. 2010. Endothelial beta1 integrins regulate sprouting and network for- 66. Maroun, L. E. 1996. Interferon action and chromosome 21 trisomy (Down mation during vascular development. Development 137: 993–1002. syndrome): 15 years later. J. Theor. Biol. 181: 41–46. 57. Tanjore, H., E. M. Zeisberg, B. Gerami-Naini, and R. Kalluri. 2008. Beta1 67. Maroun, L. E., T. N. Heffernan, and D. M. Hallam. 2000. Partial IFN-alpha/beta integrin expression on endothelial cells is required for angiogenesis but not for and IFN-gamma receptor knockout trisomy 16 mouse fetuses show improved vasculogenesis. Dev. Dyn. 237: 75–82. growth and cultured neuron viability. J. Interferon Cytokine Res. 20: 197–203. 58. Pietras, E. M., R. Lakshminarasimhan, J. M. Techner, S. Fong, J. Flach, 68. Tan, Y. H., E. L. Schneider, J. Tischfield, C. J. Epstein, and F. H. Ruddle. 1974. M. Binnewies, and E. Passegue´. 2014. Re-entry into quiescence protects he- Human chromosome 21 dosage: effect on the expression of the interferon in- matopoietic stem cells from the killing effect of chronic exposure to type I duced antiviral state. Science 186: 61–63. interferons. J. Exp. Med. 211: 245–262. 69. Weil, J., L. B. Epstein, and C. J. Epstein. 1980. Synthesis of interferon-induced 59. Tewalt, E. F., J. N. Cohen, S. J. Rouhani, C. J. Guidi, H. Qiao, S. P. Fahl, polypeptides in normal and chromosome 21-aneuploid human fibroblasts: rela- M. R. Conaway, T. P. Bender, K. S. Tung, A. T. Vella, et al. 2012. Lymphatic tionship to relative sensitivities in antiviral assays. J. Interferon Res. 1: 111–124. endothelial cells induce tolerance via PD-L1 and lack of costimulation leading to 70. Snell, L. M., T. L. McGaha, and D. G. Brooks. 2017. Type I interferon in chronic high-level PD-1 expression on CD8 T cells. Blood 120: 4772–4782. virus infection and cancer. Trends Immunol. 38: 542–557. Downloaded from 60. Wetterholm, E., J. Linders, M. Merza, S. Regner, and H. Thorlacius. 2016. 71. Ye, B., X. Liu, X. Li, H. Kong, L. Tian, and Y. Chen. 2015. T-cell exhaustion in Platelet-derived CXCL4 regulates neutrophil infiltration and tissue damage in chronic hepatitis B infection: current knowledge and clinical significance. Cell severe acute pancreatitis. Transl. Res. 176: 105–118. Death Dis. 6: e1694. 61. Tan, K. W., S. Z. Chong, F. H. Wong, M. Evrard, S. M. Tan, J. Keeble, 72. Loo, C. P., N. A. Nelson, R. S. Lane, J. L. Booth, S. C. Loprinzi Hardin, D. M. Kemeny, L. G. Ng, J. P. Abastado, and V. Angeli. 2013. Neutrophils A. Thomas, M. K. Slifka, J. C. Nolz, and A. W. Lund. 2017. Lymphatic vessels contribute to inflammatory lymphangiogenesis by increasing VEGF-A bioavailability balance viral dissemination and immune activation following cutaneous viral and secreting VEGF-D. Blood 122: 3666–3677. infection. Cell Rep. 20: 3176–3187. http://www.jimmunol.org/ by guest on September 23, 2021 726 The Journal of Immunology

Corrections

Lucas, E. D., J. M. Finlon, M. A. Burchill, M. K. McCarthy, T. E. Morrison, T. M. Colpitts, and B. A. J. Tamburini. 2018. Type 1 IFN and PD-L1 coordinate lymphatic endothelial cell expansion and contraction during an inﬂammatory immune response. J. Immunol. 201: 1735–1747.

In the ﬂow cytometry histogram plot panel in Fig. 2F, in the ﬁrst two panels (polyI:C) the naive mice were both Ifnar2/2, and in the second two panels (Vaccinia) the naive mice were both WT. This was a mistake. Although these data were collected at the same time and are presented next to the same controls for reference, the naive samples for the Ifnar2/2 should have been placed in the Ifnar2/2 panels, and the naive samples for the WT should have been placed in the WT panels.

In the corrected version, we have placed the WT naive sample in the WT histograms and the Ifnar2/2 naive sample in the Ifnar2/2 histograms. The data and conclusions in the article are not affected. The ﬁgure legend was correct as published and is shown below for reference. The ﬁgure has also been corrected online, which now differs from the print version as originally published.