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Programmed death ligand 1 intracellular interactions with STAT3 and focal adhesion protein Paxillin facilitate lymphatic endothelial cell remodeling

  • Johnathon B. Schafer
    Affiliations
    Department of Medicine, Division of Gastroenterology and Hepatology, University of Colorado School of Medicine, Aurora, CO, USA

    Molecular Biology Graduate Program, University of Colorado School of Medicine, Aurora, CO, USA
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  • Erin D. Lucas
    Affiliations
    Department of Medicine, Division of Gastroenterology and Hepatology, University of Colorado School of Medicine, Aurora, CO, USA

    Immunology Graduate Program, University of Colorado School of Medicine, Aurora, CO, USA
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  • Monika Dzieciettkowska
    Affiliations
    Department of Biochemistry, University of Colorado School of Medicine, Aurora, CO, USA
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  • Tadg Forward
    Affiliations
    Department of Medicine, Division of Gastroenterology and Hepatology, University of Colorado School of Medicine, Aurora, CO, USA
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  • Beth A. Jirón Tamburini
    Correspondence
    Corresponding Author: Beth A Jirón Tamburini.
    Affiliations
    Department of Medicine, Division of Gastroenterology and Hepatology, University of Colorado School of Medicine, Aurora, CO, USA

    Molecular Biology Graduate Program, University of Colorado School of Medicine, Aurora, CO, USA

    Immunology Graduate Program, University of Colorado School of Medicine, Aurora, CO, USA

    Department of Immunology and Microbiology, University of Colorado School of Medicine, Aurora, CO, USA
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Open AccessPublished:November 11, 2022DOI:https://doi.org/10.1016/j.jbc.2022.102694

      Abstract

      Lymphatic endothelial cells (LEC) comprise lymphatic capillaries and vessels that guide immune cells to lymph nodes (LN) and form the subcapsular sinus and cortical and medullary lymphatic structures of the LN. During an active immune response, the lymphatics remodel to accommodate the influx of immune cells from the tissue, but factors involved in remodeling are unclear. Here, we determined that a TSS motif within the cytoplasmic domain of programmed death ligand 1 (PD-L1), expressed by LECs in LN, participates in lymphatic remodeling. Mutation of the TSS motif to AAA does not affect surface expression of PD-L1 but instead causes defects in LN cortical and medullary lymphatic organization following immunostimulant Poly I:C administration in vivo. Supporting this observation, in vitro treatment of the LEC cell line, SVEC4-10, with cytokines TNFα and IFNα significantly impeded SVEC4-10 movement in the presence of the TSS-AAA cytoplasmic mutation. The cellular movement defects coincided with reduced F-actin polymerization, consistent with differences previously found in dendritic cells. Here, in addition to loss of actin polymerization, we define STAT3 and Paxillin as important PD-L1 binding partners. STAT3 and Paxillin were previously demonstrated to be important at focal adhesions for cellular motility. We further demonstrate the PD-L1 TSS-AAA motif mutation reduced the amount of pSTAT3 and Paxillin bound to PD-L1 both before and after exposure to TNFα and IFNα. Together, these findings highlight PD-L1 as an important component of a membrane complex that is involved in cellular motility which leads to defects in lymphatic organization.

      Key words

      Introduction

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      • Li Y.J.
      • Xiong X.X.
      • Liu D.
      • Pan F.
      • Yu S.B.
      • Chen X.Q.
      PD-L1 confers glioblastoma multiforme malignancy via Ras binding and Ras/Erk/EMT activation.
      ,
      • Tu X.
      • Qin B.
      • Zhang Y.
      • Zhang C.
      • Kahila M.
      • Nowsheen S.
      • Yin P.
      • Yuan J.
      • Pei H.
      • Li H.
      • Yu J.
      • Song Z.
      • Zhou Q.
      • Zhao F.
      • Liu J.
      • Zhang C.
      • Dong H.
      • Mutter R.W.
      • Lou Z.
      PD-L1 (B7-H1) Competes with the RNA Exosome to Regulate the DNA Damage Response and Can Be Targeted to Sensitize to Radiation or Chemotherapy.
      ). The protection from type 1 IFN was suggested to be resulting from increased pSTAT3 activation in the absence of PD-L1 resulting in Caspase3/7 activation (
      • Gato-Cañas M.
      • Zuazo M.
      • Arasanz H.
      • Ibañez-Vea M.
      • Lorenzo L.
      • Fernandez-Hinojal G.
      • Vera R.
      • Smerdou C.
      • Martisova E.
      • Arozarena I.
      • Wellbrock C.
      • Llopiz D.
      • Ruiz M.
      • Sarobe P.
      • Breckpot K.
      • Kochan G.
      • Escors D.
      PDL1 Signals through Conserved Sequence Motifs to Overcome Interferon-Mediated Cytotoxicity.
      ). Another report demonstrated defective migration in the absence of PD-L1 caused by interactions between PD-L1 and H-Ras, which led to downstream MEK and ERK phosphorylation(
      • Qiu X.Y.
      • Hu D.X.
      • Chen W.Q.
      • Chen R.Q.
      • Qian S.R.
      • Li C.Y.
      • Li Y.J.
      • Xiong X.X.
      • Liu D.
      • Pan F.
      • Yu S.B.
      • Chen X.Q.
      PD-L1 confers glioblastoma multiforme malignancy via Ras binding and Ras/Erk/EMT activation.
      ).
      In cancer cells, two intracellular domains were identified to regulate PD-L1 in response to type 1 IFN, residues 264-273 and residues 275-281 (
      • Gato-Cañas M.
      • Zuazo M.
      • Arasanz H.
      • Ibañez-Vea M.
      • Lorenzo L.
      • Fernandez-Hinojal G.
      • Vera R.
      • Smerdou C.
      • Martisova E.
      • Arozarena I.
      • Wellbrock C.
      • Llopiz D.
      • Ruiz M.
      • Sarobe P.
      • Breckpot K.
      • Kochan G.
      • Escors D.
      PDL1 Signals through Conserved Sequence Motifs to Overcome Interferon-Mediated Cytotoxicity.
      ). An additional study demonstrated that residues 270-279 of PD-L1 were required to interact with and stabilize messenger RNA (
      • Tu X.
      • Qin B.
      • Zhang Y.
      • Zhang C.
      • Kahila M.
      • Nowsheen S.
      • Yin P.
      • Yuan J.
      • Pei H.
      • Li H.
      • Yu J.
      • Song Z.
      • Zhou Q.
      • Zhao F.
      • Liu J.
      • Zhang C.
      • Dong H.
      • Mutter R.W.
      • Lou Z.
      PD-L1 (B7-H1) Competes with the RNA Exosome to Regulate the DNA Damage Response and Can Be Targeted to Sensitize to Radiation or Chemotherapy.
      ). This data set demonstrated that PD-L1 could act to regulate DNA damage repair enzymes via the mRNA-PD-L1 interaction (
      • Tu X.
      • Qin B.
      • Zhang Y.
      • Zhang C.
      • Kahila M.
      • Nowsheen S.
      • Yin P.
      • Yuan J.
      • Pei H.
      • Li H.
      • Yu J.
      • Song Z.
      • Zhou Q.
      • Zhao F.
      • Liu J.
      • Zhang C.
      • Dong H.
      • Mutter R.W.
      • Lou Z.
      PD-L1 (B7-H1) Competes with the RNA Exosome to Regulate the DNA Damage Response and Can Be Targeted to Sensitize to Radiation or Chemotherapy.
      ). Evaluation of mRNA molecules shown to bind to PD-L1 based on their data did not provide clues regarding why or how PD-L1 may regulate cellular movement. In our previous report(
      • Lucas E.D.
      • Schafer J.B.
      • Matsuda J.
      • Kraus M.
      • Burchill M.A.
      • Tamburini B.A.J.
      PD-L1 Reverse Signaling in Dermal Dendritic Cells Promotes Dendritic Cell Migration Required for Skin Immunity.
      ), we demonstrated that a specific cytoplasmic motif, TSS, is responsible for at least some of the defined intracellular signaling by PD-L1. The function of this motif was demonstrated by mutation of amino acids 277-279 threonine-serine-serine (TSS) to alanine-alanine-alanine (AAA)(
      • Lucas E.D.
      • Schafer J.B.
      • Matsuda J.
      • Kraus M.
      • Burchill M.A.
      • Tamburini B.A.J.
      PD-L1 Reverse Signaling in Dermal Dendritic Cells Promotes Dendritic Cell Migration Required for Skin Immunity.
      ). In dendritic cells, the 3 amino acid mutation in the cytoplasmic domain of PD-L1 caused defective chemokine receptor signaling, loss of ERK phosphorylation, and decreased actin polymerization(
      • Lucas E.D.
      • Schafer J.B.
      • Matsuda J.
      • Kraus M.
      • Burchill M.A.
      • Tamburini B.A.J.
      PD-L1 Reverse Signaling in Dermal Dendritic Cells Promotes Dendritic Cell Migration Required for Skin Immunity.
      ). Loss of the TSS motif in PD-L1 led to defective chemotaxis of DCs, but did not alter surface expression of PD-L1 (
      • Lucas E.D.
      • Schafer J.B.
      • Matsuda J.
      • Kraus M.
      • Burchill M.A.
      • Tamburini B.A.J.
      PD-L1 Reverse Signaling in Dermal Dendritic Cells Promotes Dendritic Cell Migration Required for Skin Immunity.
      ). While PD-L1 expression by LECs and consequences of loss of PD-L1 in LECs has been demonstrated, whether the TSS motif functions similar in LECs as DCs is yet unknown.
      Here we demonstrate that loss of 3 residues within the cytoplasmic domain of PD-L1 significantly impairs LN lymphatic reorganization following polyI:C injection into the footpad of mice. We produced a stable LEC line with constitutive expression of either WT Pdl1 or Pdl1 with the TSS-AAA mutation in the cytoplasmic domain (Pdl1CyMt). We observed a similar growth phenotype and expression of PD-L1 in these cells at steady state. Upon stimulation with either type 1 IFN or TNF alpha we show a significant defect in actin polymerization and cellular movement across a wound. These phenotypic changes appear to be a result of defective intracellular interactions between PD-L1, p-STAT3, and paxillin. Interestingly, p-STAT3 and paxillin were previously reported to form a complex at focal adhesions, which are important for regulating actin polymerization required for cellular movement(
      • Teng T.S.
      • Lin B.
      • Manser E.
      • Ng D.C.H.
      • Cao X.
      Stat3 promotes directional cell migration by regulating Rac1 activity via its activator βPIX.
      ,
      • Silver D.L.
      • Naora H.
      • Liu J.
      • Cheng W.
      • Montell D.J.
      Activated Signal Transducer and Activator of Transcription (STAT) 3.
      ). Together, our data clearly demonstrates that the intracellular domain of PD-L1 contributes to membrane protein interactions that regulate motility and that these interactions are critical for lymphatic remodeling

      Results

      PD-L1 facilitates lymphatic re-organization following Poly I:C

      We had previously identified a cytoplasmic motif region within murine PD-L1 that contributed to DC chemotaxis(
      • Lucas E.D.
      • Schafer J.B.
      • Matsuda J.
      • Kraus M.
      • Burchill M.A.
      • Tamburini B.A.J.
      PD-L1 Reverse Signaling in Dermal Dendritic Cells Promotes Dendritic Cell Migration Required for Skin Immunity.
      ) and demonstrated that PD-L1 was important for LEC survival(
      • Lucas E.D.
      • Finlon J.M.
      • Burchill M.A.
      • McCarthy M.K.
      • Morrison T.E.
      • Colpitts T.M.
      • Tamburini B.A.J.
      Type 1 IFN and PD-L1 Coordinate Lymphatic Endothelial Cell Expansion and Contraction during an Inflammatory Immune Response.
      ). As DC and LEC movement and survival are important components of LN organization and responsiveness, we asked if there were differences in the LNs WT or Pdl1CyMt mice, in which the cytoplasmic TSS motif is mutated to AAA (Supplemental Figure 1A). We first evaluated LEC subsets by flow cytometry based on transcriptional signature as defined in (
      • Xiang M.
      • Grosso R.A.
      • Takeda A.
      • Pan J.
      • Bekkhus T.
      • Brulois K.
      • Dermadi D.
      • Nordling S.
      • Vanlandewijck M.
      • Jalkanen S.
      • Ulvmar M.H.
      • Butcher E.C.
      A Single-Cell Transcriptional Roadmap of the Mouse and Human Lymph Node Lymphatic Vasculature.
      ) in both WT and Pdl1CyMt mice. We identified LECs as CD45 negative, Podoplanin (PDPN) positive, CD31 positive cells (Supplemental Figure 1B). We further identified cortical/medullary LECs based on Lyve-1 and Mannose Receptor C type 1 (MRC-1) expression, and ceiling and floor LECs based on Intercellular Adhesion Molecule-1 (ICAM1) and Caveolin-1 (CAV1) expression before and after Poly I:C (Figure 1A). We saw that Poly I:C caused the upregulation of PD-L1 on all LEC subsets (Figure 1B) and that there was no difference in upregulation of PD-L1 between WT and the Pdl1CyMt LECs(Supplemental Figure 1C,D). We next compared the number of LECs in each subset and found no differences in any subset except the MRC1 positive LECs which were fewer in frequency and number in the Pdl1CyMt (Figure 1A,C). To evaluate lymphatic organization in the lymph nodes of WT and Pdl1CyMt mice before and after polyI:C, we performed immnostaining for Lymphatic Vessel Hylauronan Receptor 1 (Lyve-1) (Figure 1D). Each LN was sectioned and stained for Lyve-1 (
      • Kong L.-L.
      • Yang N.-Z.
      • Shi L.-H.
      • Zhao G.-H.
      • Zhou W.
      • Ding Q.
      • Wang M.-H.
      • Zhang Y.-S.
      The optimum marker for the detection of lymphatic vessels.
      )(Figure 1D,E). Sections revealed that the lymphatics of naïve LNs look similar between WT and Pdl1CyMt mice. After polyI:C injection we found that in WT mice there was re-organization of both the medullary and cortical LECs as previously demonstrated (
      • Lucas E.D.
      • Finlon J.M.
      • Burchill M.A.
      • McCarthy M.K.
      • Morrison T.E.
      • Colpitts T.M.
      • Tamburini B.A.J.
      Type 1 IFN and PD-L1 Coordinate Lymphatic Endothelial Cell Expansion and Contraction during an Inflammatory Immune Response.
      ,
      • Angeli V.
      • Ginhoux F.
      • Llodrà J.
      • Quemeneur L.
      • Frenette P.S.
      • Skobe M.
      • Jessberger R.
      • Merad M.
      • Randolph G.J.
      B Cell-Driven Lymphangiogenesis in Inflamed Lymph Nodes Enhances Dendritic Cell Mobilization.
      ,
      • Tan K.W.
      • Yeo K.P.
      • Wong F.H.S.
      • Lim H.Y.
      • Khoo K.L.
      • Abastado J.-P.
      • Angeli V.
      Expansion of Cortical and Medullary Sinuses Restrains Lymph Node Hypertrophy during Prolonged Inflammation.
      ). Cortical lymphatics were delineated as lymphatics not connected to the subcapsular sinus (yellow dashed lines). Medullary lymphatics were delineated as lymphatics anatomically located in the medulla of the lymph node and connected to the subcapsular sinus (white dashed lines) (
      • Grigorova I.L.
      • Panteleev M.
      • Cyster J.G.
      Lymph node cortical sinus organization and relationship to lymphocyte egress dynamics and antigen exposure.
      ). The draining LN lymphatic vasculature of Pdl1CyMt mice given polyI:C was largely comprised of lymphatics within the medullary area with minimal occupancy of lymphatics in the cortical area (Figure 1D). Upon quantification, the Pdl1CyMt cortical lymphatics were decreased relative to the medullary lymphatics in the LNs after polyI:C injection into the mouse footpad or flank (Figure 1E). These findings demonstrate that the TSS region within the cytoplasmic domain of PD-L1 is important for regulating lymphatic organization during immune activation with polyI:C.
      Figure thumbnail gr1
      Figure1Mutation of the cytoplasmic domain of PD-L1 reduces MRC1+ LECs and alters lymphatic reorganization following polyI:C. A. Mice were injected with 5ug Poly I:C in 50ul phosphate buffered saline (PBS) in the footpad and flank. Mice were then sacrificed 24 hours later and popliteal as well as inguinal lymph nodes (LN)s were minced, digested and stained for flow cytometry analysis of different LEC subsets. LECs were gated on for CD45-, PDPN+, CD31+ and stained for PD-L1. Subsets were defined as MRC1+ LECs, MRC1-LECs were either ICAM1hi CAV1low (floor LECs) or ICAM1lo CAV1hi (ceiling) B. PD-L1 expression was determined before and after poly I:C. C. Numbers of LEC subsets was compared between WT Pdl1 and Pdl1CyMt were compared. D. LNs were fixed with formalin, embedded in paraffin wax and then sectioned in 7um slices onto glass slides. Sections stained for LYVE-1 to visualize lymphatic endothelial cells (white). Morphological areas were determined as either medullary (white) or cortical (yellow) lymphatics. E. The ratio of lymphatic area of the cortical lymphatics compared to the medullary lymphatics was quantified in LNs of mice 24 hours after Poly I:C. Data shows pooled quantification from two experiments. Students T-test was used to compare groups*=p<.05

      Stable transduction and constitutive expression of Pdl1 and Pdl1CyMt does not impair growth

      In order to determine the contribution of the TSS motif of PD-L1 to reverse signaling in the LECs, we transduced an SVEC4-10 cell line, a cell line that has been previously described to be of lymphatic origin(
      • O'Connell K.
      • Landman G.
      • Farmer E.
      • Edidin M.
      Endothelial cells transformed by SV40 T antigen cause Kaposi's sarcomalike tumors in nude mice.
      ). SVEC4-10 cells were transduced with pBABE-GFP vectors containing either GFP alone (EV), WT Pdl1 tagged with GFP (Pdl1) or Pdl1CyMt tagged with GFP (Pdl1CyMt)(Supplemental Figure 2A). Upon stable transduction with the lentiviral vector containing WT or mutant PD-L1 (Pdl1CyMt), SVEC4-10 cells growth rate (Supplemental Figure 2B) and surface expression of PD-L1 (Supplemental Figure 2C,D) were measured over 8 days (Supplemental Figure 2B,D). Similar to LECs from mice that harbor the Pdl1CyMt mutation (Supplemental Figure 1D) expression of PD-L1 was equivalent between WT and Pdl1CyMt transduced cells and there was no difference in growth rate (Supplemental Figure 2B-D). SVECs normally have extremely low levels of PD-L1, but upregulate PD-L1 following treatment with IFNα, TNFα or both IFNα and TNFα (Supplemental Figure 2E,F). However, our PD-L1 transduced cells constitutively express high levels of PD-L1 compared to endogenous PD-L1 regardless of treatment (Supplemental Figure 2F).

      PD-L1 Cytoplasmic mutation reduces cellular movement in the presence of TNFα and IFNα

      After confirming that the proliferation and surface expression was unaffected by overexpression of Pdl1CyMt compared to WT Pdl1 we tested the ability of these cells to re-organize following disruption of a monolayer (Figure 2A) . We observed that after scratching an SVEC monolayer, transduced SVECs were capable of quickly closing the wound regardless of which Pdl1 construct they contained (Figure 2A,B). These findings align with our in vivo observations where the Pdl1CyMt mutation does not alter LEC structures at homeostasis (Figure 1D). To determine if the cytokines TNFα, IFNα impacted wound closure we treated transduced SVECs with either TNFα, IFNα, or a combination of the two, to mimic the cytokine production by cells in vivo following polyI:C. Cells expressing Pdl1CyMt exhibited a significant delay in wound closure with type 1 IFN (Supplemental Figure 3A,B), TNFα (Supplemental Figure 3A,C) and an even more pronounced defect with a combination of type 1 IFN and TNFα (Figure 2A, C). The pattern of cell movement was observed to be different between cells expressing WT Pdl1 and Pdl1CyMt. The increased number of cells leaving the cell-cell contacts of the scratch edge and migrating independently to the center of the scratch in the Pdl1CyMt cells suggests defects in coordinated cellular movement (Supplemental Figure 3 D,E). During this time period there was no difference in the number of apoptotic cells between WT Pdl1 and Pdl1CyMt (Figure 2D,E and Supplemental Figure 3F,G) and no difference in cell growth between groups over a 5 day period (Supplemental Figure 3H-K). This suggested that PD-L1 was required for LEC remodeling of the scratch when signaling from the inflammatory cytokines, type I IFN and TNFα was active, but not during homeostasis.
      Figure thumbnail gr2
      Figure 2Mutation of PD-L1 significantly impairs SVEC movement but not viability. A. SVEC 4-10 Cells containing EV, WT Pdl1 or Pdl11CyMt vectors were plated in 5-wells per group per treatment, of an Image-Lock 96-well plate, at 1.5e4 cells/well. Cells were allowed to grow to confluence ∼36 hours. Confluent cells were scratched with a Sartorius Woundmaker. Immediately after scratch, media was changed to Serum free MEM media containing either PBS, TNFα (100ng/ml), IFNα (500U/ml), or both TNFα (100ng/ml) and IFNα (500U/ml). Cells were imaged every 2 hours using an IncuCyte. A. Representative images and representative graphs are shown from three independent experiments, 5-7 wells each. B,C. Quantification of scratch width over time after B. PBS, C. IFNα and TNFα at concentrations indicated above. Graph is of representative experiment. Assay was repeated 3 independent times with similar results. P-value for difference in slope of WT Pdl1 vs. Pdl1CyMt best fit line shown. D, E. Number of Active Caspase 3 reagent positive cells to determine the number of apoptotic cells per image over time. No significant differences were found in caspase 3 between WT and PD-L1CyMt. Scale bars are 100μm.

      Cells expressing Pdl1CyMt are defective in F-Actin polymerization

      Based on our findings above that Pdl1CyMt LN lymphatics are improperly remodeled after polyI:C and that Pdl1CyMt SVEC cells are defective in wound closure after type 1 IFN and TNFα, we next asked about F-actin levels in SVEC cells after stimulation with TNFα and/or IFNα. To do this, we stained SVEC 4-10 cells with stable expression of Pdl1, Pdl1CyMt or empty vector (EV) with phalloidin conjugated to a fluorophore (F-actin) (Figure 3A). We noticed that while there was no difference in the intensity of F-actin staining or organization between the groups treated with vehicle (PBS), that there was an increase in the intensity and difference in the apparent organization of F-actin upon treatment with IFNα and TNFα with WT Pdl1 that was absent in the cells expressing Pdl1CyMt (Figure 3A). In order to quantify the cytoskeletal differences in actin, we evaluated the F-actin/G-actin ratio. We found that WT Pdl1 and Pdl1CyM transduced SVEC cells have similar levels of F-actin/G-actin without stimulation (Figure 3B, C). Similar to our staining with F-Actin (Figure 3A) we found significant differences in F-actin/G-actin ratio following type 1 IFN. Cells expressing WT Pdl1 had increased levels of F-actin that were absent in the cells expressing Pdl1CyMt (Figure 3B). Furthermore, upon evaluation of the ratio of F-actin to G-actin we found a significant decrease in the ratio in the Pdl1CyMt cells compared to WT Pdl1 expressing cells due to the lack of F-actin polymerization in the Pdl1CyMt cells rather than loss of F-actin (Figure 3C). To demonstrate differences in actin reorganization we imaged transduced WT Pdl1 and Pdl1CyMt cells with a live-cell F-actin probe after wounding and treatment (as in Figure 2A) with vehicle (Movies 1 and 2), with IFNα (Movies 3 and 4), with TNFα (Movies 5 and 6), or after treatment with both IFNα and TNFα (Movies 7 and 8). In the WT Pdl1 and Pdl1CyMt cells treated with vehicle we observed actin reorganization over time as the cells migrated into the scratch (Figure 3D and Movies 1-8). However, in the Pdl1CyMt compared to WT Pdl1 with both IFNα and TNFα, there was both reduced movement and less F-actin reorganization (Figure 3D and Movies 1,2,7,8). These findings demonstrate a significant impairment in actin polymerization and reorganization in the presence of cytokine stimulation which is consistent with ineffective lymphatic remodeling in vivo.
      Figure thumbnail gr3
      Figure 3Actin polymerization in Pdl1CyMt expressing SVEC cells is impaired. A. Cells grown on collagen coated coverslips were treated with either PBS or TNFα (100ng/ml) and IFNα (500U/ml), then fixed and stained with Phalloidin to visualize F-Actin fluorescence (red) and DAPI (blue). B. Cells grown in 6-well plates were treated with IFNα (500U/ml) and lysed. F-actin was pelleted from G-actin using centrifugation and fractions were run on an SDS gel, transferred to a PVDF membrane and probed for Actin via western blot. C. F-actin/G-actin ratio was determined based on band intensity from western blot using 3 independent experiments. D. In conditions identical to the scratch assay, cells were stained with SiR-Actin live cell probe. Cells were imaged every 10 minutes over time after treatment. Frames from the first two hours of imaging were selected every 30 minutes to show changes in cell morphology and actin. Dashed lines indicate cell borders and shape. 2.5x zoomed in images of cells with dashed lines are shown from times 0 and 120 minutes. Statistics were performed using a one-way ANOVA on three independent experiments. Scale bar in A is 100μm and 10μm for inset. Scale bar in D is 50μm and 10μm in zoomed in images of F-actin. *=p<.05, **=p<.01, ***=p<.001

      PD-L1 intracellular interactions

      To begin to understand what protein-protein interactions with PD-L1 may contribute to differences in actin polymerization we performed mass spectrometry on cellular lysates after an immunoprecipitation of PD-L1. SVECs overexpressing either PD-L1 or PD-L1CyMt were immunoprecipitated with or without sodium vanadate (a phosphatase inhibitor) (Supplemental Figure 4). Immunoprecipitated proteins were assessed by mass spectrometry to determine potential interactions that relied on the TSS domain of PD-L1. Several proteins were identified by mass spectrometry (Table 1 and Supplemetal Table 1). Among those proteins that differed between WT PD-L1 and PD-L1CyMt were Paxillin, a protein involved in cellular focal adhesions(
      • Schaller M.D.
      Paxillin: a focal adhesion-associated adaptor protein.
      ) and STAT3, a protein shown to bind to Paxillin(
      • Silver D.L.
      • Naora H.
      • Liu J.
      • Cheng W.
      • Montell D.J.
      Activated Signal Transducer and Activator of Transcription (STAT) 3.
      ) and have increased tyrosine phosphorylation in the absence of PD-L1 or with the mutation(
      • Lucas E.D.
      • Schafer J.B.
      • Matsuda J.
      • Kraus M.
      • Burchill M.A.
      • Tamburini B.A.J.
      PD-L1 Reverse Signaling in Dermal Dendritic Cells Promotes Dendritic Cell Migration Required for Skin Immunity.
      ,
      • Gato-Cañas M.
      • Zuazo M.
      • Arasanz H.
      • Ibañez-Vea M.
      • Lorenzo L.
      • Fernandez-Hinojal G.
      • Vera R.
      • Smerdou C.
      • Martisova E.
      • Arozarena I.
      • Wellbrock C.
      • Llopiz D.
      • Ruiz M.
      • Sarobe P.
      • Breckpot K.
      • Kochan G.
      • Escors D.
      PDL1 Signals through Conserved Sequence Motifs to Overcome Interferon-Mediated Cytotoxicity.
      )(Table 1). As STAT3 is regulated by phosphorylation we next asked if, in our vanadate treated samples, there were differences in the phosphorylation state of either PD-L1 or proteins bound to PD-L1. Therefore, lysates were enriched for phosphorylated proteins and isolated for mass spectrometry. Only 29 phosphorylated proteins were identified and only 5 were found in the WT sample but not the PD-L1CyMt sample including Krt5, Stat3, Arghap35, Ifih1 and Heatr5b (Table 2). Interestingly, the STAT3 peptide isolated using mass spectrometry contained Serine 727 (Table 3), a modification important in both STAT3 regulation of mitochondrial respiration(
      • Wegrzyn J.
      • Potla R.
      • Chwae Y.J.
      • Sepuri N.B.
      • Zhang Q.
      • Koeck T.
      • Derecka M.
      • Szczepanek K.
      • Szelag M.
      • Gornicka A.
      • Moh A.
      • Moghaddas S.
      • Chen Q.
      • Bobbili S.
      • Cichy J.
      • Dulak J.
      • Baker D.P.
      • Wolfman A.
      • Stuehr D.
      • Hassan M.O.
      • Fu X.Y.
      • Avadhani N.
      • Drake J.I.
      • Fawcett P.
      • Lesnefsky E.J.
      • Larner A.C.
      Function of mitochondrial Stat3 in cellular respiration.
      ,
      • Boengler K.
      • Hilfiker-Kleiner D.
      • Heusch G.
      • Schulz R.
      Inhibition of permeability transition pore opening by mitochondrial STAT3 and its role in myocardial ischemia/reperfusion.
      ,
      • Zhou L.
      • Too H.P.
      Mitochondrial localized STAT3 is involved in NGF induced neurite outgrowth.
      ,
      • Gough D.J.
      • Corlett A.
      • Schlessinger K.
      • Wegrzyn J.
      • Larner A.C.
      • Levy D.E.
      Mitochondrial STAT3 supports Ras-dependent oncogenic transformation.
      ,
      • Valenca-Pereira F.
      • Fang Q.
      • Marie I.J.
      • Giddings E.L.
      • Fortner K.A.
      • Yang R.
      • Villarino A.V.
      • Huang Y.H.
      • Frank D.A.
      • Wen H.
      • Levy D.E.
      • Rincon M.
      IL-6 enhances CD4 cell motility by sustaining mitochondrial Ca(2+) through the noncanonical STAT3 pathway.
      ,
      • Rincon M.
      • Pereira F.
      A New Perspective: Mitochondrial Stat3 as a Regulator for Lymphocyte Function.
      ) and regulation of STAT3 Tyr705 activation of transcription(
      • Wakahara R.
      • Kunimoto H.
      • Tanino K.
      • Kojima H.
      • Inoue A.
      • Shintaku H.
      • Nakajima K.
      Phospho-Ser727 of STAT3 regulates STAT3 activity by enhancing dephosphorylation of phospho-Tyr705 largely through TC45.
      ,
      • Yang J.
      • Kunimoto H.
      • Katayama B.
      • Zhao H.
      • Shiromizu T.
      • Wang L.
      • Ozawa T.
      • Tomonaga T.
      • Tsuruta D.
      • Nakajima K.
      Phospho-Ser727 triggers a multistep inactivation of STAT3 by rapid dissociation of pY705–SH2 through C-terminal tail modulation.
      ). To confirm differences in binding of phosphorylated forms of STAT3 we next performed western blot analysis of both sodium vanadate (inhibitor of threonine phosphatases) and sodium fluoride (inhibitor of serine phosphatases) treated cells following PD-L1 immunoprecipitation. There was not a difference in the total levels put into the immunopreciption of either PD-L1, pSTAT3 Ser727 or Tyr705 between WT PD-L1 and PD-L1CyMt samples (Figure 4A). Evaluation of the immunoprecipitated product revealed STAT3 was pulled down with PD-L1 (Table1, Figure 4B) and that of the pulled down fraction STAT3 was phosphorylated on both Ser727 and Tyr705 regardless of the phosphatase inhibitor used (Table 2, Figure 4B). Quantification of these data confirm that the amount of PD-L1 pulled down was not significantly different between WT and Pdl1CyMt expressing cells (Figure 4C). The amount of phosphorylated pSer727 STAT3 bound to PD-L1 was significantly increased, even in the absence of phosphatase inhibitors, and this interaction was significantly impaired with PD-L1CyMt (Figure 4D). Similar trends were seen when evaluating PD-L1 interactions with pTyr705 STAT3 (Figure 4E). Changes in the interactions with pSTAT3 could be caused by increased total levels of phosphorylated Tyr705 STAT3 following stimulation which has been previously demonstrated in both DCs (
      • Lucas E.D.
      • Schafer J.B.
      • Matsuda J.
      • Kraus M.
      • Burchill M.A.
      • Tamburini B.A.J.
      PD-L1 Reverse Signaling in Dermal Dendritic Cells Promotes Dendritic Cell Migration Required for Skin Immunity.
      ) and cancer cells (
      • Gato-Cañas M.
      • Zuazo M.
      • Arasanz H.
      • Ibañez-Vea M.
      • Lorenzo L.
      • Fernandez-Hinojal G.
      • Vera R.
      • Smerdou C.
      • Martisova E.
      • Arozarena I.
      • Wellbrock C.
      • Llopiz D.
      • Ruiz M.
      • Sarobe P.
      • Breckpot K.
      • Kochan G.
      • Escors D.
      PDL1 Signals through Conserved Sequence Motifs to Overcome Interferon-Mediated Cytotoxicity.
      ) after type 1 IFN. However, when we asked if the STAT3 or pSTAT3 interaction was changed in the presence of IFNα, TNFα or both, we again found a significant reduction in the interaction between pSTAT3 Ser 727 and PD-L1, but not pSTAT3 Tyr705 and PD-L1 (Supplemental Figure 5A, B).
      Table 1Mass-Spectrometry Analysis following Pull-down of GFP Tagged WT PD-L1 and PD-L1CyMt
      Protein NameAlternate IDMolecular WeightNumber of samples identified In WT Pdl1 after IPNumber of samples identified in Pdl1CyMt after IP
      RuvB Like AAA ATPase 2Ruvbl251 kDa50
      Salt Inducible Kinase 2Sik2104 kDa50
      Prolyl 4-Hydroxylase Subunit Alpha 1P4ha161 kDa40
      Glutathione Peroxidase 7Gpx721 kDa40
      PaxillinPxn64 kDa40
      Thyroid Hormone Receptor AlphaThra55 kDa30
      DnaJ Heat Shock Protein Family (Hsp40) Member C7Dnajc756 kDa40
      Perilipin 2Plin247 kDa30
      Solute Carrier Family 25 Member 3Slc25a340 kDa30
      T-cell-specific guanine nucleotide triphosphate-binding protein 1Tgtp147 kDa30
      NOP58 RibonucleoproteinNop5860 kDa30
      Glutamine Rich 1Qrich187 kDa30
      OTU Deubiquitinase 7BOtud7b92 kDa30
      2'-5'-Oligoadenylate Synthetase 2Oas285 kDa30
      Obscurin Like Cytoskeletal Adaptor 1Obsl1198 kDa30
      HDGF Like 2Hdgfl274 kDa30
      TBC1 Domain Family Member 23Tbc1d2376 kDa30
      Rho Gtpase Activating protein 29Arhgap29142 kDa30
      Adenosylhomocysteinase Like 1Ahcyl159 kDa30
      Signal transducer and activator of transcription 3STAT388kDa66
      Programmed cell death 1 ligand 1 (Cd274)PD-L133kDa66
      Rho GTPase Activating Protein 35ARHGAP35170kDa66
      Mass-spectrometry analysis of proteins pulled down by immunoprecipitation (IP) with GFP tagged WT Pdl1 or Pdl1CyMt samples identified proteins bound to both as well as other proteins identified in only WT Pdl1 or Pdl1CyMt samples. Table is curated showing 19 of the most enriched proteins identified as possible preferentially binding to WT PD-L1 as well as other proteins of interest.
      Table 2Phosphorylated Proteins Co-Immunoprecipitated
      Protein NameAlternative IDMolecular WeightNumber Identified in WTNumber Identified in Mut
      Keratin 5Krt562 kDa10
      Signal Transducer and Activator of Transcription 3Stat388 kDa10
      MDA5Ifih1116 kDa10
      Rho GTPase Activating Protein 35Arhgap35170 kDa10
      Heat repeat containing 5bHeatr5b224 kDa10
      ATP Binding Cassette Subfamily F Member 1Abcf195 kDa01
      Cordon-bleu Protein Like-1Cobll1137 kDa01
      COPI Coat Complex Subunit AlphaCopa138 kDa01
      Rho Guanin nucleotide exchange factor 40Arhgef40165 kDa01
      N-Alpha-Acetyltransferase 15, NatA Auxiliary SubunitNaa15101 kDa01
      Drebin 1Dbn177 kDa01
      StriatinStrn86 kDa01
      Scaffold Attachment Factor B2Safb2112 kDa01
      General Transcription Factor IIiGtf2i112 kDa01
      Pleckstrin Homology Like Domain Family B Member 2Phldb2141 kDa01
      GFP was immunoprecipitated from lysates of SVEC4-10 cells expressing either WT Pd-l1 or Pd-l1CyMt following treatment with Sodium Vanadate. Samples were enriched for phosphorylated peptides and analyzed by mass-spectrometry. Phosphorylated proteins identified in samples are shown.
      Table 3Phosphopeptide analysis
      SampleProtein NamePeptide sequence
      WT PD-L1Lima1SDNEETLGRPAQPPNAGESPHSPGVEDAPIAK
      WT PD-L1RptorILDTSSLTQSAPASPTNK
      WT PD-L1Copb2GFQPSRPTAQQEPDGKPASSPVIMASQTTHKEEK
      WT PD-L1Pkn1TDVSNFDEEFTGEAPTLSPPR
      WT PD-L1Lig1NQVVPESDSPVKR
      WT PD-L1Heatr5bGKMVVSIAEDLLR
      WT PD-L1Stat3FICVTPTTCSNTIDLPMSPR
      WT PD-L1Thrap3ASVSDLSPR
      WT PD-L1Thrap3ERSPALKSPLQSVVVR
      WT PD-L1Thrap3HGLTHEELKSPR
      WT PD-L1Thrap3HGLTHEELKSPREPGYK
      WT PD-L1Thrap3IDISPSTFR
      WT PD-L1Hsp90ab1IEDVGSDEEDDSGKDKK
      WT PD-L1AfdnSSPNVANQPPSPGGK
      WT PD-L1Arhgap35TSFSVGSDDELGPIR
      PD-L1CyMtHsph1IESPKLER
      PD-L1CyMtDbn1LSSPVLHR
      PD-L1CyMtGtf2iAQVVMSALPAEDEESSESR
      PD-L1CyMtLig1NQVVPESDSPVKR
      PD-L1CyMtAdd1AAVVTSPPPTTAPHK
      PD-L1CyMtStrnFLESAAADVSDEDEDEDTDGR
      PD-L1CyMtAfdnSSPNVANQPPSPGGK
      PD-L1CyMtArhgef40QISLASETLDSSGDVSPGPR
      PD-L1CyMtBclaf1ADGDWDDQEVLDYFSDKESAK
      PD-L1CyMtBclaf1ELFDYSPPLHK
      PD-L1CyMtBclaf1FHDSEGDDTEETEDYR
      PD-L1CyMtBclaf1KAEGEPQEESPLK
      PD-L1CyMtBclaf1KAEGEPQEESPLKSK
      PD-L1CyMtBclaf1LKELFDYSPPLHK
      PD-L1CyMtBclaf1NTPSQHSHSIQHSPER
      PD-L1CyMtHsp90ab1IEDVGSDEEDDSGK
      PD-L1CyMtHsp90ab1IEDVGSDEEDDSGKDKK
      PD-L1CyMtPkn1TDVSNFDEEFTGEAPTLSPPR
      Specific peptides identified by mass-spectrometry following phospho-peptide enrichment shown from Table 2.
      Figure thumbnail gr4
      Figure 4Immunoprecipitation of PD-L1 followed by probing for STAT3 phosphorylation Sites in the presence of phosphatase inhibitors. A. Input and Unbound Samples for PD-L1 Immunoprecipitation samples from WT Pdl1 or Pdl1CyMt cells. B. Western blots for pSTAT3 Ser 727, pSTAT3 Y705, and PD-L1 following Immunoprecipitation for PD-L1 from WT Pdl1 or Pdl1CyMt cells. C-E. Quantification of PD-L1, pSTAT3 Ser727, or pSTAT3 Tyr705. One-way ANOVA on three Combined experiments performed. **=p<.01
      Previous work has shown pSTAT3 727 was involved in mitochondrial respiration (
      • Macias E.
      • Rao D.
      • Carbajal S.
      • Kiguchi K.
      • Digiovanni J.
      Stat3 Binds to mtDNA and Regulates Mitochondrial Gene Expression in Keratinocytes.
      ,
      • Xu Y.S.
      • Liang J.J.
      • Wang Y.
      • Zhao X.-Z.J.
      • Xu L.
      • Xu Y.-Y.
      • Zou Q.C.
      • Zhang J.M.
      • Tu C.-E.
      • Cui Y.-G.
      • Sun W.-H.
      • Huang C.
      • Yang J.-H.
      • Chin Y.E.
      STAT3 Undergoes Acetylation-dependent Mitochondrial Translocation to Regulate Pyruvate Metabolism.
      ,
      • Carbognin E.
      • Betto R.M.
      • Soriano M.E.
      • Smith A.G.
      • Martello G.
      Stat3 promotes mitochondrial transcription and oxidative respiration during maintenance and induction of naive pluripotency.
      ), however, as we did not see difference in growth rate (Supplemental figure 2B, 3H-K) or apoptosis (Figure 2D,E, Supplemental figure 3F,G). We next asked about STAT3 transcriptional targets. Interestingly, the decreased interaction of PD-L1 with pSTAT3 727 correlated with increased IL-6 production by the Pdl1CyMt cells (Supplemental Figure 5C) suggesting differences in regulation of pSTAT3 Tyr705 transcriptional targets. Finally, as differences in pERK, but not pP38 were demonstrated in dendritic cells in the Pdl1CyMt mice after CCL21 stimulation(
      • Lucas E.D.
      • Schafer J.B.
      • Matsuda J.
      • Kraus M.
      • Burchill M.A.
      • Tamburini B.A.J.
      PD-L1 Reverse Signaling in Dermal Dendritic Cells Promotes Dendritic Cell Migration Required for Skin Immunity.
      ), we next evaluated differences in either ERK or P38. We found no significant differences between WT and CyMT PD-L1 in either ERK, pERK, P38 or pP38 levels after any of the indicated cytokine treatments (Supplemental Figure 5D,E). These data confirm that the TSS motif within PD-L1 cytoplasmic domain is required for interaction with the phosphorylated form, and more specifically serine 727, of STAT3, but not the native form of STAT3.

      PD-L1-Paxillin interactions facilitate paxillin organization and cellular structure

      Paxillin is a focal adhesion protein that has been demonstrated to interact with pSTAT3 at the membrane to facilitate cell movement (
      • Silver D.L.
      • Naora H.
      • Liu J.
      • Cheng W.
      • Montell D.J.
      Activated Signal Transducer and Activator of Transcription (STAT) 3.
      ). Therefore, we next confirmed the mass spectrometry data demonstrating a lack of interaction with Paxillin and PD-L1, when PD-L1 contained the CyMt mutation (Table 1, Figure 5, Supplemental Figure 4). Indeed, we found that Paxillin binding to PD-L1 was reduced in the Pdl1CyMt mutant cells (Figure 5A). The defect in actin polymerization we detected in cells (Figure 3), and the disorganization of the lymphatics in the LN of Pdl1CyMt mice (Figure 1), suggested that these differences may be compounded by the inflammatory cytokines IFNα and TNFα. Therefore, we next asked if Paxillin interactions were impaired after treatment with either IFNα, TNFα or both. We found that, indeed, after treatment of IFNα and TNFα that Paxillin bound to PD-L1 was still reduced in the Pdl1CyMt cells, compared to WT, both following short (30 minutes) or overnight exposure to cytokines (Figure5A, B and Supplemental Fig 6A,B)(
      • Lucas E.D.
      • Schafer J.B.
      • Matsuda J.
      • Kraus M.
      • Burchill M.A.
      • Tamburini B.A.J.
      PD-L1 Reverse Signaling in Dermal Dendritic Cells Promotes Dendritic Cell Migration Required for Skin Immunity.
      ,
      • Gato-Cañas M.
      • Zuazo M.
      • Arasanz H.
      • Ibañez-Vea M.
      • Lorenzo L.
      • Fernandez-Hinojal G.
      • Vera R.
      • Smerdou C.
      • Martisova E.
      • Arozarena I.
      • Wellbrock C.
      • Llopiz D.
      • Ruiz M.
      • Sarobe P.
      • Breckpot K.
      • Kochan G.
      • Escors D.
      PDL1 Signals through Conserved Sequence Motifs to Overcome Interferon-Mediated Cytotoxicity.
      ). However, the diminished levels of paxillin pulled down with PD-L1 were not caused by changes in the level of total protein within the cells as Paxillin amounts were similar between WT PD-L1 and PD-L1CyMt cells (Figure 5A). To determine if the differences in binding changed the localization of PD-L1 or Paxillin after treatment we performed immunofluorescence in non-transduced (NTD) SVECs as well as WT Pdl1 and Pdl1CyMt transduced cells after IFNα and TNFα (Figure 5C). We saw that in the NTD cells, as shown in Supplemental Figure 2E PD-L1 levels were minimal with no treatment, but following IFNα and TNFα, PD-L1 was upregulated and a portion of the endogenous PD-L1 localized to similar areas as paxillin (Figure 5C). In the WT Pdl1 transduced cells PD-L1 and Paxillin localization was similar to the NTD cells (Figure 5C). In the Pdl1CyMt, the Paxillin appeared localized to the cell body instead of at focal adhesions, and the PD-L1 appeared disorganized following IFNα and TNFα (Figure 5C). We next asked if Paxillin could form proper focal adhesions connected to the actin cytoskeleton in the Pdl1CyMt cells. Similar to Figure 3 and 5C, we found that in the Pdl1CyMt cells Paxillin mediated focal adhesions were disrupted and F-actin was disorganized during wound healing assay conditions (Figure 5D). The Pdl1CyMt expressing cells exhibited significant changes in morphology, a similar phenotype to either migrating adherent cells lacking STAT3 or defective cell spreading seen in paxillin null cells(
      • Teng T.S.
      • Lin B.
      • Manser E.
      • Ng D.C.H.
      • Cao X.
      Stat3 promotes directional cell migration by regulating Rac1 activity via its activator βPIX.
      ,
      • Wade R.
      • Bohl J.
      • Vande Pol S.
      Paxillin null embryonic stem cells are impaired in cell spreading and tyrosine phosphorylation of focal adhesion kinase.
      ). To quantify this phenotype we measured cell length, width, perimeter area and circularity. We identified significant differences in circularity with or without cytokine treatment and significant differences in area, width and length to width ratio only after cytokine treatment (Figure 5D-F). Taken together, these findings demonstrate that the TSS motif within the cytoplasmic tail of PD-L1 is necessary for cell morphology and cellular motility. These may be a result of the observed loss of interactions between PD-L1 and pSTAT3 and/or Paxillin required for proper focal adhesion formation.
      Figure thumbnail gr5
      Figure 5Immunoprecipitation of PD-L1 after treatment with PBS, IFNα (500U/mL), TNFα (100ng/mL), or both for 30 minutes. A. Western Blot probing for co-IP of Paxillin with WT PD-L1 and Pd-l1CyMt. after Stimulation. B. Quantification of Paxillin coimmunoprecipitated with PD-L1 as well as in input, normalized to loading control. C. Non-transduced SVEC cells in addition to WT Pdl1 and Pdl1CyMt were treated during a wound closure assay on gelatin coated coverslips. Cells were stained for Paxillin and PD-L1. Inserts show PD-L1 organization at areas of paxillin marked focal adhesions. Arrows show other areas of PD-L1 and Paxillin colocalization. D. Representative Images showing Paxillin and F-actin representing defective cell spreading. E,F. Quantification of cell morphological properties during wound closure assay (length, width, ratio, perimeter, area, circularity). Two-Way ANOVA performed on group analysis, for western blot, quantification shows combined separate experiments, for cell morphology analysis, representative quantification shown for one of three independent experiments performed. Scale bar is 10μm for all images in C and D. P=0.10-0.05 P-value shown *=p<.05, **=p<.01, ***=p<.001, ****=p<.0001

      Discussion

      In this manuscript we identify a mutation in PD-L1 that affects lymphatic organization in the lymph nodes of mice injected with polyI:C. The flow cytometry data confirms that PD-L1 expression is dramatically upregulated following polyI:C on all LEC subsets within 24 hours after injection as we previously showed (
      • Lucas E.D.
      • Finlon J.M.
      • Burchill M.A.
      • McCarthy M.K.
      • Morrison T.E.
      • Colpitts T.M.
      • Tamburini B.A.J.
      Type 1 IFN and PD-L1 Coordinate Lymphatic Endothelial Cell Expansion and Contraction during an Inflammatory Immune Response.
      ). Mutation of three residues in the cytoplasmic domain of PD-L1 (TSS-AAA) causes reorganization of the cortical/medullary LECs based on LN anatomy and results in fewer of the transcriptionally defined Ptx3/Marco LECs (
      • Xiang M.
      • Grosso R.A.
      • Takeda A.
      • Pan J.
      • Bekkhus T.
      • Brulois K.
      • Dermadi D.
      • Nordling S.
      • Vanlandewijck M.
      • Jalkanen S.
      • Ulvmar M.H.
      • Butcher E.C.
      A Single-Cell Transcriptional Roadmap of the Mouse and Human Lymph Node Lymphatic Vasculature.
      ). Unfortunately, it is difficult to distinguish the cortical LECs, defined anatomically, by their transcriptional profile. This may be due to the infrequency of cortical LECs in the lymph node or because the transcriptional profile is not different between medullary and cortical LECs (
      • Xiang M.
      • Grosso R.A.
      • Takeda A.
      • Pan J.
      • Bekkhus T.
      • Brulois K.
      • Dermadi D.
      • Nordling S.
      • Vanlandewijck M.
      • Jalkanen S.
      • Ulvmar M.H.
      • Butcher E.C.
      A Single-Cell Transcriptional Roadmap of the Mouse and Human Lymph Node Lymphatic Vasculature.
      ,
      • Sibler E.
      • He Y.
      • Ducoli L.
      • Keller N.
      • Fujimoto N.
      • Dieterich L.C.
      • Detmar M.
      Single-Cell Transcriptional Heterogeneity of Lymphatic Endothelial Cells in Normal and Inflamed Murine Lymph Nodes.
      ). Regardless, the differences we find in vivo in the different LEC subsets upon PD-L1 upregulation suggests PD-L1 has an important role in regulating LECs. We aimed to determine if PD-L1 upregulation caused by cytokines such as type 1 IFN (
      • Lucas E.D.
      • Finlon J.M.
      • Burchill M.A.
      • McCarthy M.K.
      • Morrison T.E.
      • Colpitts T.M.
      • Tamburini B.A.J.
      Type 1 IFN and PD-L1 Coordinate Lymphatic Endothelial Cell Expansion and Contraction during an Inflammatory Immune Response.
      ) defined the differences in the Pdl1CyMt LECs or if the cytokines themselves were affecting the LECs independent of the coincident PD-L1 upregulation. To do this we transduced either WT or mutant PD-L1 into the SVEC lymphatic cell line to induce constitutive expression that did not change with cytokine treatment (Supplemental Figure 2F). Based on the data presented herein, it appears that it is not the induction of PD-L1 that is significant, but instead the binding partners of PD-L1 that impart differences in the lymphatic endothelial cells upon cytokine exposure.
      PD-L1 reverse signaling has been studied in multiple cell types and each cell type PD-L1 has been shown to be involved in a variety of cell signaling pathways and mechanisms. Loss of PD-L1 reverse signaling in multiple cells types results in increased STAT3 Tyr705 phosphorylation. This increased Tyr705 phosphorylation is associated with caspase mediated cell death in response to IFNβ in cancer cells (
      • Gato-Cañas M.
      • Zuazo M.
      • Arasanz H.
      • Ibañez-Vea M.
      • Lorenzo L.
      • Fernandez-Hinojal G.
      • Vera R.
      • Smerdou C.
      • Martisova E.
      • Arozarena I.
      • Wellbrock C.
      • Llopiz D.
      • Ruiz M.
      • Sarobe P.
      • Breckpot K.
      • Kochan G.
      • Escors D.
      PDL1 Signals through Conserved Sequence Motifs to Overcome Interferon-Mediated Cytotoxicity.
      ); in T-cells, promotes Th17 responses (
      • Diskin B.
      • Adam S.
      • Cassini M.F.
      • Sanchez G.
      • Liria M.
      • Aykut B.
      • Buttar C.
      • Li E.
      • Sundberg B.
      • Salas R.D.
      • Chen R.
      • Wang J.
      • Kim M.
      • Farooq M.S.
      • Nguy S.
      • Fedele C.
      • Tang K.H.
      • Chen T.
      • Wang W.
      • Hundeyin M.
      • Rossi J.A.K.
      • Kurz E.
      • Haq M.I.U.
      • Karlen J.
      • Kruger E.
      • Sekendiz Z.
      • Wu D.
      • Shadaloey S.A.A.
      • Baptiste G.
      • Werba G.
      • Selvaraj S.
      • Loomis C.
      • Wong K.-K.
      • Leinwand J.
      • Miller G.
      PD-L1 engagement on T cells promotes self-tolerance and suppression of neighboring macrophages and effector T cells in cancer.
      ); and has been reported in DCs(
      • Lucas E.D.
      • Schafer J.B.
      • Matsuda J.
      • Kraus M.
      • Burchill M.A.
      • Tamburini B.A.J.
      PD-L1 Reverse Signaling in Dermal Dendritic Cells Promotes Dendritic Cell Migration Required for Skin Immunity.
      ), but the consequence of which is currently unknown. Many of these mechanisms only occur as a response to inflammatory cytokines such as type I and type II interferon, IL-6, TNFα, and those produced by TLR agonists (
      • Lucas E.D.
      • Schafer J.B.
      • Matsuda J.
      • Kraus M.
      • Burchill M.A.
      • Tamburini B.A.J.
      PD-L1 Reverse Signaling in Dermal Dendritic Cells Promotes Dendritic Cell Migration Required for Skin Immunity.
      ,
      • Lucas E.D.
      • Finlon J.M.
      • Burchill M.A.
      • McCarthy M.K.
      • Morrison T.E.
      • Colpitts T.M.
      • Tamburini B.A.J.
      Type 1 IFN and PD-L1 Coordinate Lymphatic Endothelial Cell Expansion and Contraction during an Inflammatory Immune Response.
      ,
      • Gato-Cañas M.
      • Zuazo M.
      • Arasanz H.
      • Ibañez-Vea M.
      • Lorenzo L.
      • Fernandez-Hinojal G.
      • Vera R.
      • Smerdou C.
      • Martisova E.
      • Arozarena I.
      • Wellbrock C.
      • Llopiz D.
      • Ruiz M.
      • Sarobe P.
      • Breckpot K.
      • Kochan G.
      • Escors D.
      PDL1 Signals through Conserved Sequence Motifs to Overcome Interferon-Mediated Cytotoxicity.
      ,
      • Diskin B.
      • Adam S.
      • Cassini M.F.
      • Sanchez G.
      • Liria M.
      • Aykut B.
      • Buttar C.
      • Li E.
      • Sundberg B.
      • Salas R.D.
      • Chen R.
      • Wang J.
      • Kim M.
      • Farooq M.S.
      • Nguy S.
      • Fedele C.
      • Tang K.H.
      • Chen T.
      • Wang W.
      • Hundeyin M.
      • Rossi J.A.K.
      • Kurz E.
      • Haq M.I.U.
      • Karlen J.
      • Kruger E.
      • Sekendiz Z.
      • Wu D.
      • Shadaloey S.A.A.
      • Baptiste G.
      • Werba G.
      • Selvaraj S.
      • Loomis C.
      • Wong K.-K.
      • Leinwand J.
      • Miller G.
      PD-L1 engagement on T cells promotes self-tolerance and suppression of neighboring macrophages and effector T cells in cancer.
      ). These cytokines elicit a number of signaling pathways, but overlap in STAT3 Tyr705 phosphorylation(
      • De Simone V.
      • Franzè E.
      • Ronchetti G.
      • Colantoni A.
      • Fantini M.C.
      • Di Fusco D.
      • Sica G.S.
      • Sileri P.
      • Macdonald T.T.
      • Pallone F.
      • Monteleone G.
      • Stolfi C.
      Th17-type cytokines, IL-6 and TNF-α synergistically activate STAT3 and NF-kB to promote colorectal cancer cell growth.
      ,
      • Huynh J.
      • Chand A.
      • Gough D.
      • Ernst M.
      Therapeutically exploiting STAT3 activity in cancer — using tissue repair as a road map.
      ), perhaps suggesting why loss of PD-L1 reverse signaling affects so many pathways and cell types in different ways. Here we show that, in a cell line (SVEC) derived from murine LECs, that PD-L1 can form complexes with STAT3, and that the TSS domain of PD-L1 specifically affects the phosphorylation state of STAT3 both within and outside of this complex. STAT3, a transcription factor and protein involved in regulating cellular respiration and focal adhesions (
      • Teng T.S.
      • Lin B.
      • Manser E.
      • Ng D.C.H.
      • Cao X.
      Stat3 promotes directional cell migration by regulating Rac1 activity via its activator βPIX.
      ,
      • Silver D.L.
      • Naora H.
      • Liu J.
      • Cheng W.
      • Montell D.J.
      Activated Signal Transducer and Activator of Transcription (STAT) 3.
      ,
      • Wegrzyn J.
      • Potla R.
      • Chwae Y.J.
      • Sepuri N.B.
      • Zhang Q.
      • Koeck T.
      • Derecka M.
      • Szczepanek K.
      • Szelag M.
      • Gornicka A.
      • Moh A.
      • Moghaddas S.
      • Chen Q.
      • Bobbili S.
      • Cichy J.
      • Dulak J.
      • Baker D.P.
      • Wolfman A.
      • Stuehr D.
      • Hassan M.O.
      • Fu X.Y.
      • Avadhani N.
      • Drake J.I.
      • Fawcett P.
      • Lesnefsky E.J.
      • Larner A.C.
      Function of mitochondrial Stat3 in cellular respiration.
      ,
      • Boengler K.
      • Hilfiker-Kleiner D.
      • Heusch G.
      • Schulz R.
      Inhibition of permeability transition pore opening by mitochondrial STAT3 and its role in myocardial ischemia/reperfusion.
      ,
      • Zhou L.
      • Too H.P.
      Mitochondrial localized STAT3 is involved in NGF induced neurite outgrowth.
      ,
      • Gough D.J.
      • Corlett A.
      • Schlessinger K.
      • Wegrzyn J.
      • Larner A.C.
      • Levy D.E.
      Mitochondrial STAT3 supports Ras-dependent oncogenic transformation.
      ,
      • Valenca-Pereira F.
      • Fang Q.
      • Marie I.J.
      • Giddings E.L.
      • Fortner K.A.
      • Yang R.
      • Villarino A.V.
      • Huang Y.H.
      • Frank D.A.
      • Wen H.
      • Levy D.E.
      • Rincon M.
      IL-6 enhances CD4 cell motility by sustaining mitochondrial Ca(2+) through the noncanonical STAT3 pathway.
      ,
      • Rincon M.
      • Pereira F.
      A New Perspective: Mitochondrial Stat3 as a Regulator for Lymphocyte Function.
      ,
      • Wakahara R.
      • Kunimoto H.
      • Tanino K.
      • Kojima H.
      • Inoue A.
      • Shintaku H.
      • Nakajima K.
      Phospho-Ser727 of STAT3 regulates STAT3 activity by enhancing dephosphorylation of phospho-Tyr705 largely through TC45.
      ,
      • Yang J.
      • Kunimoto H.
      • Katayama B.
      • Zhao H.
      • Shiromizu T.
      • Wang L.
      • Ozawa T.
      • Tomonaga T.
      • Tsuruta D.
      • Nakajima K.
      Phospho-Ser727 triggers a multistep inactivation of STAT3 by rapid dissociation of pY705–SH2 through C-terminal tail modulation.
      ,
      • Balic J.J.
      • Albargy H.
      • Luu K.
      • Kirby F.J.
      • Jayasekara W.S.N.
      • Mansell F.
      • Garama D.J.
      • De Nardo D.
      • Baschuk N.
      • Louis C.
      • Humphries F.
      • Fitzgerald K.
      • Latz E.
      • Gough D.J.
      • Mansell A.
      STAT3 serine phosphorylation is required for TLR4 metabolic reprogramming and IL-1β expression.
      ,
      • Sakaguchi M.
      • Oka M.
      • Iwasaki T.
      • Fukami Y.
      • Nishigori C.
      Role and Regulation of STAT3 Phosphorylation at Ser727 in Melanocytes and Melanoma Cells.
      ) has multiple different impacts on cell phenotype. As we have shown PD-L1 interactions with STAT3, this may explain the multiple phenotypic outcomes seen in the absence of PD-L1 reverse signaling. One possible mechanism by which PD-L1 regulates STAT3 activity is via the regulation of the STAT3 phosphorylation state. Given our data, pSer727 appears to be more readily and dynamically bound to PD-L1 compared to pTyr705. pSer727 is thought to be more important for cellular metabolism at the mitochondria(
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      IL-6 enhances CD4 cell motility by sustaining mitochondrial Ca(2+) through the noncanonical STAT3 pathway.
      ,
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      STAT3 serine phosphorylation is required for TLR4 metabolic reprogramming and IL-1β expression.
      ) suggesting loss of STAT3 pSer727 interactions with PD-L1 could influence mitochondrial functions. Multiple studies have also demonstrated the STAT3 pSer727 can regulate pTyr705 levels by de-stabilizing STAT3 homodimers and limiting transcription(
      • Wakahara R.
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      Phospho-Ser727 of STAT3 regulates STAT3 activity by enhancing dephosphorylation of phospho-Tyr705 largely through TC45.
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      Phospho-Ser727 triggers a multistep inactivation of STAT3 by rapid dissociation of pY705–SH2 through C-terminal tail modulation.
      ). Our studies might suggest that increased pSer727 STAT3 bound to the PD-L1CyMt is sequestering the pSer727 and allowing for increased activation of pTyr705. Loss of these interactions in the PD-L1CyMt would thus increase pTyr705 STAT3 in favor of STAT3 mediated transcription and lead to increased IL6 production (Supplemental Figure 4C). Our findings would suggest that PD-L1/pSTAT3/Paxillin complexes regulate focal adhesions and manipulation of these complexes could alter the balance of the different roles for STAT3. The exact mechanism of PD-L1 regulation of pSTAT3 is still not clear, however, these studies highlight the importance of PD-L1/STAT3 interactions to the cellular response to cytokines.
      We also demonstrate the ability of PD-L1 to form complexes with Paxillin in addition to or together with, pSTAT3. Another report has demonstrated the capacity of pSTAT3 to interact with Paxillin at focal adhesions which promotes cellular movement(
      • Silver D.L.
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      Activated Signal Transducer and Activator of Transcription (STAT) 3.
      ). As our PD-L1 TSS-AAA mutation seems to both disrupt the amount of STAT3 phosphorylation as well as Paxillin levels bound to PD-L1, it seems likely that Paxillin and pSTAT3 interactions with PD-L1 are important for phospho-STAT3/Paxillin complex formation which coordinates cellular movement. Indeed, we demonstrated altered SVEC cell movement and LN lymphatic vessel disorganization in the presence of inflammatory cytokines and that cellular morphology is significantly impaired. These data suggest that the mechanisms of PD-L1/STAT3/Paxillin to coordinate cell movement in the presence of inflammatory cytokines is important for in vivo immune responses. Indeed, in our previous paper(
      • Lucas E.D.
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      PD-L1 Reverse Signaling in Dermal Dendritic Cells Promotes Dendritic Cell Migration Required for Skin Immunity.
      ) we demonstrated significant impairment of T cell responses in our mouse model of Pdl1CyMt. While we attributed these differences to DCs, which also have defective actin polymerization and migration, it is now clear that loss of PD-L1 reverse signaling could also impact lymphatic re-organization, during an immune response, that may contribute to defective DC migration.
      Several other potential PD-L1 binding partners are of particular interest based on studies of PD-L1 reverse signaling(
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      PD-L1 Reverse Signaling in Dermal Dendritic Cells Promotes Dendritic Cell Migration Required for Skin Immunity.
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      ). These include Arghap35, a Rho GTPase activating protein, and Ifih1, also known as MDA5, which is important for sensing double strand RNA and affecting type 1 IFN responses (Table 2). Arhgap35 (Table 2) and Arhgap29 (Table 1) and Arhgap5 (Supplemental Table 1) are of particular interest as Rho GTPases are well described to be involved in actin where RhoA is important for protrusions of the lamellipodia(
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      ) which are critical for cell migration. Other proteins were identified as bound to PD-L1 by mass spectrometry that could be of consequence. One of which is ADP ribosylation factor4 (ARF4), a member of the ARF family proteins (Supplemental Table1). ARF family proteins generally regulate endocytic vesicle trafficking from the golgi, but downstream or alternative functions have been observed in migration, actin organization, and paxillin localization(
      • Kondo A.
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      ARF1 Mediates Paxillin Recruitment to Focal Adhesions and Potentiates Rho-stimulated Stress Fiber Formation in Intact and Permeabilized Swiss 3T3 Fibroblasts.
      ,
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      Paxillin-ARF GAP signaling and the cytoskeleton.
      ). We also found additional ARF regulatory proteins bound to PD-L1 by mass spectrometry, including, ADP-ribosylation factor GTPase-activating proteins 1,2,3 (ARFGAP1, ARFGAP2, ARFGAP3), ARF GTPase-activating protein GIT1 and GIT2 (GIT1, GIT2), Arf-GAP with coiled-coil, ANK repeat and PH domain-containing protein (ACAP2) Arf-GAP with Rho-GAP domain, ANK repeat and PH domain-containing protein 1 (ARAP1), and Brefeldin A-inhibited guanine nucleotide-exchange protein 2 (ARFGEF2) (Supplemental Table 1)(
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      The Role of ARF Family Proteins and Their Regulators and Effectors in Cancer Progression: A Therapeutic Perspective.
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      BIG2-ARF1-RhoA-mDia1 Signaling Regulates Dendritic Golgi Polarization in Hippocampal Neurons.
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      PTK6 Inhibits Down-regulation of EGF Receptor through Phosphorylation of ARAP1.
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      GIT1 Paxillin-binding Domain Is a Four-helix Bundle, and It Binds to Both Paxillin LD2 and LD4 Motifs.
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      An ADP-Ribosylation Factor GTPase-activating Protein Git2-short/KIAA0148 Is Involved in Subcellular Localization of Paxillin and Actin Cytoskeletal Organization.
      ). Intriguingly, GIT2 was demonstrated to bind to paxillin in another report (
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      ). PIK3CB and PIK3C3, subunits of the PI3K, were also pulled out of the mass spectrometry data (Supplemental Table 1). PI3K has been shown to be important for focal adhesions and cellular spreading in some cell types (
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      PI3K/Akt signalling is required for the attachment and spreading, and growth in vivo of metastatic scirrhous gastric carcinoma.
      ). MDA5 interaction with PD-L1 is also interesting as many of the differences we observe are following inflammatory cytokine (IFN and TNF) exposure which are regulated in part by the MDA5/RigI innate sensing pathway (
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      mda-5: An interferon-inducible putative RNA helicase with double-stranded RNA-dependent ATPase activity and melanoma growth-suppressive properties.
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      ) which is critical for sensing double strand RNA during the immune response. We do not yet understand how these other interactions are involved in: regulating protection from cell death in cancer cells (
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      PD-L1 (B7-H1) Competes with the RNA Exosome to Regulate the DNA Damage Response and Can Be Targeted to Sensitize to Radiation or Chemotherapy.
      ), promoting Th17 skewing (
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      ) of T cells or cellular migration of DCs (
      • Lucas E.D.
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      PD-L1 Reverse Signaling in Dermal Dendritic Cells Promotes Dendritic Cell Migration Required for Skin Immunity.
      ) and LECs. Furthermore, it is possible that PD-L1 is part of a larger membrane bound complex that contains multiple trans-membrane or effector proteins.
      As we begin to dissect the domains of PD-L1, the binding partners, and the functional consequences, use of this TSS-AAA mutation in vitro and in vivo will be important to delineate which alterations in the immune response are a consequence of PD-L1 forward and/or reverse signaling. Since PD-L1 is expressed by LECs, how alterations in reverse signaling may impact the immune response is critical as LECs utilize PD-L1 to promote peripheral tolerance via interaction with PD-1 on T cells(
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      Cousin, N., Cap, S., Dihr, M., Tacconi, C., Detmar, M., and Dieterich, L. C. (2020) Lymphatic PD-L1 expression restricts tumor-specific CD8+ T cell responses. Cold Spring Harbor Laboratory

      ,
      • Garnier L.
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      Tumor-Associated Lymphatic Vessel Features and Immunomodulatory Functions.
      ,
      • Jiang C.
      • Cao S.
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      • Jiang L.
      • Sun T.
      PD-1 and PD-L1 correlated gene expression profiles and their association with clinical outcomes of breast cancer.
      ) as well as control LEC proliferation and viability during polyI:C injection(
      • Lucas E.D.
      • Schafer J.B.
      • Matsuda J.
      • Kraus M.
      • Burchill M.A.
      • Tamburini B.A.J.
      PD-L1 Reverse Signaling in Dermal Dendritic Cells Promotes Dendritic Cell Migration Required for Skin Immunity.
      ,
      • Lucas E.D.
      • Finlon J.M.
      • Burchill M.A.
      • McCarthy M.K.
      • Morrison T.E.
      • Colpitts T.M.
      • Tamburini B.A.J.
      Type 1 IFN and PD-L1 Coordinate Lymphatic Endothelial Cell Expansion and Contraction during an Inflammatory Immune Response.
      ). These new findings, which demonstrate PD-L1 interactions can control cell movement, it will be important to understand how PD-L1 reverse versus forward signaling impacts the immune response.

      Experimental Procedures

      Mice

      6-8 week old male or female C57BL/6 or Pdl1CyMt mice were used in experiments. No differences between male and female mice were detected. Mice were bred in house or purchased through the NIH NCI at Charles River. All animal studies performed were approved by the Institutional Review Board and Institutional Animal Care and Use Committee at the University of Colorado Anschutz Medical Campus.

      LN dissection for Flow Cytometry

      Mice were injected with Poly I:C (5ug/site) (Invitrogen) into both footpads and flank. Popliteal and inguinal LNs from mice after 24 hours or naïve mice and LNs were digested as previously described (
      • Lucas E.D.
      • Finlon J.M.
      • Burchill M.A.
      • McCarthy M.K.
      • Morrison T.E.
      • Colpitts T.M.
      • Tamburini B.A.J.
      Type 1 IFN and PD-L1 Coordinate Lymphatic Endothelial Cell Expansion and Contraction during an Inflammatory Immune Response.
      ). Once in single cell suspension, cells were stained with antibodies Caveolin-1 (1:200 Cell Signaling 3238S), ICAM-AF488 (1:600 Biolegend Clone YN1/1.7.4), Podoplanin-PE (1:200 Biolegend Clone 8.1.1), CD31 PerCP-cy5.5 (1:200 Biolegend Clone 390), CD206 PE-Cy7 (MRC-1) (1:100 Biolegend Clone C068C2), Lyve-1-APC (1:50 R&D Clone 223322), CD45-BV510 (1:300 Biolegend Clone 30-F11), PD-L1-BV421 (1:200 Biolegend Clone 10F.9G2) 30min 4C and washed 2x with FACS. Samples were then stained with the secondary biotinylated anti-rabbit (1:100 Jackson Immunoresearch 111-066-003) 30min 4C then washed 2xwith FACS, then with the Streptavidin APC/Cy7 (1:200 Biolegend 405208) for 30min 4C and washed 2x with FACS. For flow cytometry of SVECs, cells were treated with either PBS as mock, PBS, IFNα (Biolegend 752802), TNFα (Peprotech315-01A-20ug), or both IFNα and TNFα overnight. Cells were then trypsinized and mixed into a single cell suspension. Cells were then stained with PD-L1-PE (Biolegend 10F.9G2) for 30 minutes 4 degrees C, and analyzed by flow, similar to LN cells.
      Samples were filtered and run on a BD LSR canto II flow cytometer using DIVA software (BD Biosciences) and analyzed with FlowJo software (Treestar).

      LN dissection and staining

      Immunization and dissection performed similar to preparation for flow cytometry. LNs were removed and fixed in 16% buffered formalin phosphate and embedded in paraffin. 7mm sections were cut and placed on slides. Prior to staining Slides were heated in 60 degrees celsius oven for 2 hours to melt off paraffin wax. Slides were then rehydrated by washing with xylene twice for 10 minutes, then briefly washed in containers containing ethanol (EtOH) at 100%, 95%, 80%, then 75% before washing 3x briefly in de-ionized water. Slides then underwent antigen retrieval with Antigen retrieval buffer pH 9 (AR900250ML- Perkin Elmer) in pressure cooker on high for 15 minutes. Slides were then blocked with 5% normal donkey serum and 5% normal goat serum in 2.5% fetal bovine serum (FBS) in phosphate buffered saline (PBS) (blocking buffer). Slides were then stained with Hamster anti-podoplanin (PDPN) (1:100 Biolegend Clone 8.1.1) and rabbit anti- Lymphatic Vessel Hylauronan Receptor 1 (Lyve-1)-APC (R&D 223322 1:200) diluted in blocking buffer for 2 hours room temperature. Following primary antibody staining slides were washed a with 2.5%FBS in PBS 3 x 5 minutes and then stained in blocking buffer with a Donkey anti-hamster Dylight 647 (1:500 Biolegend 405505). Slides were then washed and mounted in Vecta Shield mounting media containing DAPI (Vector Laboratories H-1200). Sections were imaged using a Nikon Eclipse Ti series fluorescent microscope. Images were taken with a Photometrics CoolSNAP DYNO fluorescent camera. For image quantification, regions of interest were drawn around the lyve-1–positive regions to designate LECs within the either the cortical and medullary areas as defined by anatomical morphology.

      Cell Lines (creation and culturing)

      SVEC4-10 cells were purchased from (ATCC-CRL-2161). Cells were grown in Dulbeccos Modified Eagle Medium (DMEM) high glucose (4.5mg/mL) (Gibco) with 10% FBS (Atlanta biologicals S12450) as well as additives (1:100 each of: HEPES Corning 25-060-CI, Sodium Pyruvate Corning 25-000-CI, Nonessential Amino Acids Corning 25-025-CI, L-Glutamine Corning 25-005-CI, Penicillin-Streptomycin Sigma P4333-100ML) as well as 1.75μL of 2-mercaptoethanol (FisherChemical BP176-100).

      Scratch assay

      A 96 Well ImageLock plate (Sartorius 4806) was coated with gelatin based coating solution (Cell Biologics 6950) for 30 minutes at 37 degrees C. Cells were seeded onto gelatin coated wells at 1.5e4 cells/well, grown to a confluent monolayer, in DMEM with 10% FBS, 4.5g/ml glucose supplemented then scratched using a 96-well Woundmaker (Sartorius). Following the scratch, media was immediately changed to serum free media containing Incucyte Caspase-3/7 Red Dye for Apoptosis (Sartorius 4707) with no supplements and either PBS, IFNα (Biolegend 752802), TNFα (Peprotech315-01A-20ug), or both IFNα and TNFα. Images were taken every 2 hours with both phase and red on an Incucyte (Sartorius) to observe both wound closure and apoptosis. For immunofluorescent staining, cells were treated similarly after being grown on gelatin coated glass coverslips in a 6-well plate. The cover slips were then scratched with a sterile pipette tip before fixation and staining at above indicated timepoints.

      Cytoskeletal G:F Actin ratio

      The F-actin/G-actin ratios were determined using Cytoskeleton kit (Cytoskeleton-BK037) using manufacturer’s protocol using secondary antibody anti-mouse IRDye 680 (LI-COR Biosciences-926-68070) diluted 1:20,000 for 1 hour at RT. Membranes were then washed 3x and imaged on Bio-Rad ChemiDoc MP Imaging System.

      Immunofluorescence

      22mmx22mm Glass Coverslips (VWR-16004-302) were sterilized with 70% EtOH until use. Coverslips were then washed 1x with PBS before coating with Collagen based coating solution (Cell Biologics-6950) for 30 minutes at 37C in 6-well plates. Cells were then seeded at 0.3eˆ6 cells per well and grown on collagen coated coverslips, until 80% confluency. Cells were either scratched or left unscratched then treated overnight with either PBS, IFNα (500U/mL), TNFα (100ng/ml) or both overnight. Then coverslips were stained. For F-actin, Cytoskeleton F-actin Visualization kit was used (Cytoskeleton-BK005) with or without Paxillin (Thermo Scientific Clone 5H11 1:50) with secondary Goat-Anti Mouse AF633 (1:500 Invitrogen A21126) following Cytoskeleton manufacturer’s instructions. 5% Goat serum in 2.5%FBS/PBS was used as blocking buffer. For PD-L1 staining, cells were stained with PD-L1-PE (1:200 Biolegend clone 10F.9G2) with 24G2 block prior to fixation. Cells were then washed and fixed/stained for paxillin similar to the Cytoskeleton kit’s instructions (4% paraformaldehyde, 3% TritonX-100 in PBS used as fix and perm buffers). Cells were visualized on the Nikon eclipse Ti Series fluorescent microscope and images were captured using the Photometrix CoolSNAP DYNO. Cell measurements were taken in Adobe Photoshop using the measure tool and object selection tool.

      Live Cell F-actin Microscopy

      Cells were grown in 6-well plates to confluence. Then cell monolayer was scratched with a sterile P1000 pipette tip. Media was immediately changed to treatment (similar to scratch assay treatment) that additionally contained the live cell F-actin probe (SiR-actin) from Cytoskeleton (CY-SC006) at 100nM (1:10,000). Next day cells were imaged on Olympus IX83 live cell apparatus, using the 10x magnification for plastic tissue culture plates. Individual 10x images were captured in the Cy5 channel every 10 minutes to visualize F-actin reorganization and cellular movement across the scratch.

      Immunoprecipitation

      Cells were grown to 90% confluency then treated overnight with either Sodium Fluoride (1mM), Sodium Vanadate (0.5mM), TNFα (100ng/mL), IFNα (500U/mL) overnight. Cells were lysed using lysis buffer comprised of 90% m-PER (Thermo Scientific 78503) with the added Sodium Fluoride (50mM Final) Sodium Vanadate (1mM Final) and Complete Protease Inhibitor cocktail (1x Final Sigma Aldrich 11873580001). Cells were lysed for 10 minutes on ice with vortexing every 5 minutes. Then lysate was clarified with a 15 minute spin at 20,000xg. Lysate protein concentration was determined with BCA, and equivalent protein amounts were added to new tubes, diluted to equivalent concentrations with lysis buffer. GST tagged GFP nanobody was made in house using vector pGEX6P1-GFP-Nanobody which was a gift from Kazuhisa Nakayama (Addgene plasmid #61838 ; http://n2t.net/addgene:61838 ; RRID: Addgene_61838). GST Beads for immunoprecipitation was purchased from GoldBio (Cat No. G-250-5). 75μl of 50% Beads/PBS were isolated per approximately 1mg of cell lysate.
      Sample was incubated overnight on rotator with 75μl of 50% Beads/PBS per 1mg of cell lystate with 10μl of glutathione S Transferase (GST) tagged anti-green fluorescent protein (GFP) nanobody (5mg/mL). Beads were then washed 4x in Tris buffered saline with 0.1% tween-20 (TBST). Sample was eluted by adding 25μl of 4x Laemmli with 10% 2-mercaptoethanol, followed by heating in heat block ∼90 C for 10 minutes. Sample was then either analyzed by mass-spectrometry as described or by western blot, by running sample on Tris Glycine acrylamide gels (10%).

      Mass Spectrometry

      Global bottom-up LC-MS/MS analysis.

      Experimental Design and Rationale

      All samples were processed in a blinded fashion and no data points were excluded. N=6 samples per cell type were loaded onto a 1.5 mm thick NuPAGE Bis-Tris 4−12% gradient gel (Invitrogen). The BenchMark™ Protein Ladder (Invitrogen) was used as a protein molecular mass marker. The electrophoretic run was performed by using MES SDS running buffer, in an X-Cell II mini gel system (Invitrogen) at 200 V, 120 mA, 25 W per gel for 30 minutes. The gel was stained using SimplyBlue™ SafeStain (Invitrogen, Carlsbad, CA) stain and de-stained with water according to the manufacturer’s protocol. Each lane of the gel was divided into 4 equal-sized bands, and proteins in the gel were digested as follows. Gel pieces were de-stained in 200 μL of 25 mM ammonium bicarbonate in 50 % v/v acetonitrile for 15 min and washed with 200 μL of 50% (v/v) acetonitrile. Disulfide bonds in proteins were reduced by incubation in 10 mM dithiothreitol (DTT) at 60 °C for 30 min and cysteine residues were alkylated with 20 mM iodoacetamide (IAA) in the dark at room temperature for 45 min. Gel pieces were subsequently washed with 100 μL of distilled water followed by addition of 100 mL of acetonitrile and dried on SpeedVac (Savant ThermoFisher). 100 ng of trypsin was added to each sample and allowed to rehydrate the gel plugs at 4 °C for 45 min and then incubated at 37 °C overnight. The tryptic mixtures were acidified with formic acid up to a final concentration of 1%. Peptides were extracted two times from the gel plugs using 1% formic acid in 50% acetonitrile. The collected extractions were pooled with the initial digestion supernatant and dried on SpeedVac (Savant ThermoFisher). Samples were desalted on Thermo Scientific Pierce C18 Tip.

      Phosphopeptide enrichment

      Phosphopeptide enrichment was performed on n=3 samples per group using Hight- Select Fe-NTA Phosphopeptide Enrichment Kit according to the manufacturer’s instructions and supplied buffers. The dry phosphopeptides were resuspended in 200 μl of binding/wash buffer and incubated with Fe–nitrilotriacetic acid beads for 30 min at room temperature. Three 200 μl washes with binding/wash buffer were perform. Phosphopeptides bound to the Fe–nitrilotriacetic acid beads were eluted twice with 100 μl of elution buffer. The eluent was dry immediately in a SpeedVac concentrator.

      Analysis of peptides

      A 20 μl of each sample was loaded onto individual Evotips for desalting and then washed with 20 μL 0.1% FA followed by the addition of 100 μL storage solvent (0.1% FA) to keep the Evotips wet until analysis. The Evosep One system (Evosep, Odense, Denmark) was used to separate peptides on a Pepsep column, (150 um inter diameter, 15 cm) packed with ReproSil C18 1.9 um, 120A resin. The system was coupled to the timsTOF Pro mass spectrometer (Bruker Daltonics, Bremen, Germany) via the nano-electrospray ion source (Captive Spray, Bruker Daltonics). The mass spectrometer was operated in PASEF mode. The ramp time was set to 100 ms and 10 PASEF MS/MS scans per topN acquisition cycle were acquired. MS and MS/MS spectra were recorded from m/z 100 to 1700. The ion mobility was scanned from 0.7 to 1.50 Vs/cm2. Precursors for data-dependent acquisition were isolated within ± 1 Th and fragmented with an ion mobility-dependent collision energy, which was linearly increased from 20 to 59 eV in positive mode. Low-abundance precursor ions with an intensity above a threshold of 500 counts but below a target value of 20000 counts were repeatedly scheduled and otherwise dynamically excluded for 0.4 min.

      Database Searching and Protein Identification

      MS/MS spectra were extracted from raw data files and converted into .mgf files using MS Convert (ProteoWizard, Ver. 3.0). Peptide spectral matching was performed with Mascot (Ver. 2.5) against the Uniprot mouse database. Mass tolerances were +/- 15 ppm for parent ions, and +/- 0.4 Da for fragment ions. Trypsin specificity was used, allowing for 1 missed cleavage. Met oxidation, protein N-terminal acetylation, peptide N-terminal pyroglutamic acid formation and and Phospho (STY) were set as variable modifications with Cys carbamidomethylation set as a fixed modification.
      Scaffold (version 4.9, Proteome Software, Portland, OR, USA) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability as specified by the Peptide Prophet algorithm. Protein identifications were accepted if they could be established at greater than 99.0% probability and contained at least two identified unique peptides.

      Statistical Analysis

      Data was analyzed using Prism9 (GraphPad). Data was either analyzed by T-Test or One-way ANOVA when multiple comparisons were required. Wound closure assays were analyzed by generating linear best-fit lines and determining P-value for differences in slope. Each experiment was performed with 3-7 replicates and at least 2-3 times with similar results.

      Data Availability

      All data are contained within the manuscript except Tables 1 and 3 which are mass spectrometry data. Mass spectrometry data has been deposited at: Center for Computation Mass Spectrometry (CCMS). Follow the instructions contained within the url to view the data: https://massive.ucsd.edu/ProteoSAFe/private-dataset.jsp?task=f20bd28b30434e42ae527a0223f08c9e

      Supporting Information

      This article contains supporting information (Supplemental Figures 1-6 and Movies 1-8).

      Conflict of Interest

      The authors declare that they have no conflicts of interest with the contents of this article.

      Acknowledgements

      We would like to thank Matthew Burchill and Uma Kantheti for their critical reading of this manuscript. We would also like to that Dr. Rytis Prekeris for gifting us the anti-GFP nanobody construct. All shRNA/ORF/CRISPR constructs (or lentiviral suspensions thereof) were purchased from Functional Genomics Facility (Denver, CO) which is supported by the Cancer Center Support Grant (P30CA046934). Incucyte data was supported by the Cell Technologies Shared Resource also supported by (P30CA046934). We thank Veronica Wessells for tissue mounting and sectioning.

      Supplementary data

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