Research ArticleInflammation

The microvascular niche instructs T cells in large vessel vasculitis via the VEGF-Jagged1-Notch pathway

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Science Translational Medicine  19 Jul 2017:
Vol. 9, Issue 399, eaal3322
DOI: 10.1126/scitranslmed.aal3322
  • Fig. 1. Adventitial mvECs in GCA arteries express Jagged1.

    Tissue biopsies were collected from temporal arteries and from aortic wall specimens. Arteries affected by GCA had typical transmural granulomatous arteritis (GCA-positive artery and GCA aortitis). Temporal arteries with no inflammatory infiltrates (GCA-negative) served as controls. Nuclei were marked with 4′,6-diamidino-2-phenylindole. Scale bars, 50 μm. (A) Tissue-infiltrating T cells were identified by staining sections from GCA-negative and GCA-positive temporal arteries and from GCA aortitis with mouse anti-human CD3 antibody. Antibody binding was visualized with Alexa Fluor 594–labeled anti-mouse immunoglobulin G (IgG) secondary antibody (red). Representative stains from eight samples each are shown. (B) mRNA was extracted from GCA-positive and GCA-negative temporal arteries and analyzed by reverse transcription polymerase chain reaction (RT-PCR) for the expression of JAG1, DLL1, and DLL4 transcripts. Results (mean ± SEM) from six GCA arteries and six healthy arteries are shown. n.s., not significant. (C and D) Dual-color staining was applied to identify Jagged1 and Delta-like 1 expressed on endothelial cells in GCA-positive, GCA aortitis, and GCA-negative arteries. Tissue sections were double-stained with mouse anti-human CD31 antibody and rabbit anti-human Jagged1 antibody or rabbit anti-human Delta-like 1 antibody. Alexa Fluor 594 anti-mouse IgG (red) and Alexa Fluor 488 anti-rabbit IgG (green) were used as secondary antibodies. Merged images demonstrate colocalization of both markers (yellow). Representative images are from eight samples each.

  • Fig. 2. Circulating VEGF in GCA patients up-regulates microvascular endothelial Jagged1.

    Plasma samples were collected from patients with GCA and age-matched healthy controls. Patients with RA served as disease controls. All data are means ± SEM. (A and B) EC monolayers (HMVECs and HUVECs) were treated with 10% GCA plasma, RA plasma, and control plasma. After 6 hours, mRNA transcripts for JAG1, DLL1, and DLL4 were quantified by RT-PCR (A). After 24 hours, Jagged1 protein was measured by flow cytometry (B). Representative histograms and mean fluorescence intensities (MFIs) corrected by background subtraction from four to six independent experiments. (C) Plasma VEGF concentrations in healthy controls, RA patients, and GCA patients. Each dot represents one individual. (D) Jagged1 protein expression on HMVECs and HUVECs treated with hVEGF (10 ng/ml) for 24 hours. Results are from three to four experiments. (E) HMVECs were treated with GCA plasma ± VEGF receptor inhibitor axitinib (1 μM). Representative histogram of Jagged1 expression and MFIs are from four experiments. (F) HMVECs were treated with GCA plasma ± anti-VEGF blocking antibody (10 μg/ml) for 6 hours. JAG1 transcripts were measured by RT-PCR. Data are from three experiments. (G and H) Slices of human medium-sized arteries were cultured for 5 days with hVEGF (100 ng/ml) or vehicle (G). Alternatively, the arteries were kept in medium containing 30% plasma from healthy donors or from GCA patients in the absence or presence of anti-VEGF antibody (100 μg/ml) (H). Jagged1 protein was visualized by immunohistochemical staining with anti-Jagged1 antibody. Isotype antibody was used as control for binding specificity. Representative images are from five experiments. Scale bars, 20 μm.

  • Fig. 3. Constitutive Notch pathway activation in GCA CD4 T cells.

    Peripheral blood mononuclear cells (PBMCs) were freshly isolated from patients with active GCA and age-matched controls, stained with antibodies specific for CD3, CD4, Notch1, activated Notch1, HES, CD45RA, CD25, T-bet, and retinoic acid–related orphan receptor γt (RORγt), and analyzed by multiparametric flow cytometry. All data are means ± SEM. (A) Tissue sections from GCA-positive and GCA-negative arteries were processed as in Fig. 1, and gene expression for NOTCH1 and NOTCH4 was analyzed by RT-PCR. Results are from six GCA/control artery pairs. (B) Notch1 receptor expression was assessed on gated CD3+CD4+ T cells. Expression of activated Notch1 and HES1 was determined on CD3+CD4+Notch1+ T cells. Representative images are from five independent experiments. (C and D) MFIs for Notch1 and HES expression on gated CD4 T cells from 35 GCA patients and 12 age-matched controls. Each dot represents data from an individual patient/control. (E) Correlation of Notch1 expression with HES1 expression in gated CD4 T cells. Each dot represents the data from an individual patient. (F) Phenotypic analysis of gated Notch1+ and Notch1 CD4 T cells for the expression of CD45RA and the activation marker CD25. Representative contour plots from six different GCA patients are shown. (G) Frequencies of T-bet– and RORγt-expressing CD4 T cells in GCA patients and age-matched healthy controls. Representative contour plots and quantitation from 35 patients and 12 controls are shown. (H) Comparison of T-bet and RORγt expression in gated Notch1 and Notch1+ CD4 T cell populations. Representative contour plots are shown.

  • Fig. 4. ECs treated with GCA plasma function as antigen-presenting cells and promote T cell effector functions.

    HMVEC monolayers were conditioned for 24 hours with control or patient-derived plasma, incubated with or without anti-CD3 antibody (1 μg/ml), and cocultured with purified CD4 T cells at an EC to T cell ratio of 1:5. All data are means ± SEM. (A) Scheme of experimental design. (B to F) Expression of the lineage-determining transcription factors T-bet, RORγt, and GATA3 within CD4 T cells was analyzed by flow cytometry after 4 days. Representative histograms and quantitation from 5 to 10 independent experiments are shown. (G) Naïve CD4+CD45RO T cells and memory CD4+CD45RO+ T cells were purified from healthy individuals and cultured on plasma-pretreated HMVECs for 4 days. T-bet and RORγt within CD4 T cells were analyzed by flow cytometry, as described above. Data are from four to six experiments. (H to K) After 6 days of coculture, CD4 T cells were stained for intracellular IFN-γ and IL-17. Representative images and collective data are from six independent experiments. (L) CD4 T cells were isolated from healthy controls or GCA patients. Frequencies of T-bet+ and RORγt+ CD4 T cells were determined after EC–T cell coculture and pretreatment with GCA plasma, as described above. Results are from five to nine independent experiments.

  • Fig. 5. Plasma-conditioned mvECs trigger the Notch signaling pathway in CD4 T cells.

    mvEC monolayers were treated with control or patient-derived plasma, loaded with anti-CD3 antibodies, and overlaid with CD4 T cells, as in Fig. 4A. After 4 days, CD4+T-bet+ and CD4+RORγt+ T cells were measured by flow cytometry. All data are means ± SEM. (A and B) Frequencies of CD4 T cells expressing the Notch target protein HES1 were analyzed by flow cytometry after 24 hours. Representative images and collective MFIs are from five experiments. (C and D) EC–T cell interaction was blocked with anti-Jagged1 antibody or isotype control, and induction of T-bet and RORγt in T cells was assessed by flow cytometry. Data are from six experiments. (E and F) Induction of CD4+T-bet+ and CD4+RORγt+ T cells was quantified after pharmacologic inhibition of the Notch signaling pathway with DAPT (10 μM). Data are from five experiments. (G) The Notch signaling pathway was inhibited with FLI-06 (10 μM), and the effect on T cell differentiation was assessed by flow cytometry for CD4+T-bet+ and CD4+RORγt+ T cells. Data are from five experiments presented as a heat map. (H) The Notch1 receptor was knocked down by transfecting CD4 T cells from healthy subjects and GCA patients with Notch1 siRNA or control siRNA, respectively. T cells were cocultured with pretreated ECs, as described above. Frequencies of T cells acquiring expression of T-bet or RORγt were measured by flow cytometry. Data are from six independent experiments. (I and J) The VEGF receptor inhibitor axitinib (1 μM) and the anti-VEGF antibody (10 μg/ml) were included into the EC–T cell cultures. CD4+T-bet+ and CD4+RORγt+ T cells were quantified by flow cytometry. Data are from five to six independent experiments.

  • Fig. 6. Endothelial Jagged1 induces T effector cells via the Notch1-mTORC1 pathway.

    HMVECs were preconditioned with the indicated plasma, loaded with anti-CD3 antibody, and overlaid with CD4 T cells, as in Fig. 4A. Intracellular expression of pS6, T-bet, or RORγt was analyzed by flow cytometry. All data are means ± SEM. (A and B) pS6 expression in CD4 T cells was measured after 24 hours. Representative histograms and collective MFIs are from three to six independent experiments. (C and D) Effect of the Notch1 signaling inhibitor DAPT (10 μM) or vehicle on the induction of pS6. Data are from six experiments. (E to H) Effect of the mTORC1 inhibitor rapamycin (RAPA; 50 nM) or the AKT inhibitor VIII (AKT Inh; 5 μM) on the induction of T-bet+ and RORγt+ CD4 T cells quantified after 4 days. Data are from five to six experiments. (I) Healthy CD4 T cells were transfected with Raptor siRNA or control siRNA and analyzed for RPTOR mRNA expression 12 hours later by quantitative PCR (qPCR). Data are from five independent experiments. (J) CD4 T cells from healthy donors were transfected with Raptor siRNA or control siRNA, respectively, and cocultured with pretreated ECs, as described above. Data are from six experiments. (K and L) Expression of pS6 in CD4 T cells was measured in freshly isolated PBMCs from GCA patients and healthy donors. Representative histograms and data are from five control-patient pairs. (M to O) Expression of Notch1, pS6, T-bet, and RORγt in CD4 T cells was determined by multiparametric flow cytometry in freshly isolated PBMCs of GCA patients. Each dot represents an individual patient.

  • Fig. 7. VEGF amplifies vasculitogenic T cell responses.

    Pairs of NSG mice were engrafted with human axillary arteries, reconstituted with PBMCs from patients with GCA, and treated with hVEGF (2 μg per mouse) or vehicle. Alternatively, the chimeras were treated with axitinib by daily injection of the inhibitor (25 mg/kg) for eight consecutive days. Artery grafts were explanted on day 15 after transplantation and processed for immunohistochemistry or tissue transcriptome analysis by RT-PCR. All data are means ± SEM. (A and B) Tissue gene expression of JAG1, NOTCH1, TRB, and CD68 mRNA in different treatment arms. TRB mRNA identified tissue-infiltrating T cells; CD68 mRNA is derived from tissue-infiltrating macrophages. Data are from 8 to 11 different artery grafts. (C) Vessel wall–infiltrating CD3 T cells were visualized by immunostaining. Representative images are from eight grafts. Scale bars, 20 μm. (D) Activated Notch1 in vessel wall–resident CD3 T cells visualized by immunofluorescence staining. Representative images and quantitation from six to seven grafts. Scale bars, 50 μm. DAPI, 4′,6-diamidino-2-phenylindole. (E) Expression of Jagged1 protein in human CD31+ mvECs visualized by dual-color immunofluorescence staining. Representative images and quantitation are from six to seven grafts. Scale bars, 50 μm. (F to J) Tissue transcriptome analysis for lineage-determining transcription factors (TBX21, RORC, GATA3, and FOXP3) and innate and adaptive cytokines (TNF, IL6, IFNG, IL17A, and IL4) quantified by qPCR. Data are from 8 to 12 grafts. (K) IFN-γ expression in vessel wall–infiltrated CD3+ T cells visualized by dual-color immunofluorescence staining. Representative images and quantitation are from six to seven grafts. Scale bars, 50 μm.

  • Table 1. Clinical characteristics of patient cohorts.

    ESR, erythrocyte sedimentation rate; CRP, C-reactive protein.

    ParametersPatients
    with GCA
    Patients
    with RA
    Number of patients8054
    Age (years) (mean ± SD)69.63 ± 7.9553.16 ± 16.35
    Female77.5%79.63%
    Headaches70%
    Eye involvement31.25%
    Jaw claudication26.25%
    Polymyalgia rheumatica60%
    Aortic/large vessel involvement43.75%
    Prednisone (mg/day) (mean ± SD)7.49 ± 11.29
    Disease duration (months)
    (mean ± SD)
    14.36 ± 12.4014.88 ± 11.48
    ESR (mm/hour) (mean ± SD)51.43 ± 35.3924.63 ± 19.53
    CRP (mg/dl) (mean ± SD)6.25 ± 7.481.72 ± 4.47
    Untreated patients32.5%16.67%

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/399/eaal3322/DC1

    Fig. S1. IL-6 but not VEGF drives endothelial Delta-like 1 expression.

    Fig. S2. Activation of ECs in response to plasma treatment.

    Fig. S3. Infrequent expression of Notch1 on CD8 T cells.

    Fig. S4. Low expression of Notch4 on CD4 T cells.

    Fig. S5. Lack of memory CD4 T cell expansion in GCA.

    Fig. S6. Kinetics and durability of Jagged1 induction in ECs.

    Fig. S7. Plasma-pretreated HUVECs induce effector T cells.

    Fig. S8. IL-6–stimulated ECs fail to induce effector T cells.

    Fig. S9. VEGF fails to inhibit the activation of human CD4 T cells.

    Fig. S10. Efficiency of Notch1 knockdown by siRNA transfection.

    Fig. S11. Induction of T effector cells by plasma-treated HUVECs is Jagged1-dependent.

    Fig. S12. RA plasma fails to induce T cell stimulatory capacity of ECs.

    Fig. S13. Notch1 receptor expression on CD4 T cells.

    Fig. S14. T cell differentiation induced by endothelial Jagged1 is mTORC1-dependent.

    Table S1. Individual subject–level data.

  • Supplementary Material for:

    The microvascular niche instructs T cells in large vessel vasculitis via the VEGF-Jagged1-Notch pathway

    Zhenke Wen, Yi Shen, Gerald Berry, Farhad Shahram, Yinyin Li, Ryu Watanabe, Yaping Joyce Liao, Jörg J. Goronzy, Cornelia M. Weyand*

    *Corresponding author. Email: cweyand{at}stanford.edu

    Published 19 July 2017, Sci. Transl. Med. 9, eaal3322 (2017)
    DOI: 10.1126/scitranslmed.aal3322

    This PDF file includes:

    • Fig. S1. IL-6 but not VEGF drives endothelial Delta-like 1 expression.
    • Fig. S2. Activation of ECs in response to plasma treatment.
    • Fig. S3. Infrequent expression of Notch1 on CD8 T cells.
    • Fig. S4. Low expression of Notch4 on CD4 T cells.
    • Fig. S5. Lack of memory CD4 T cell expansion in GCA.
    • Fig. S6. Kinetics and durability of Jagged1 induction in ECs.
    • Fig. S7. Plasma-pretreated HUVECs induce effector T cells.
    • Fig. S8. IL-6–stimulated ECs fail to induce effector T cells.
    • Fig. S9. VEGF fails to inhibit the activation of human CD4 T cells.
    • Fig. S10. Efficiency of Notch1 knockdown by siRNA transfection.
    • Fig. S11. Induction of T effector cells by plasma-treated HUVECs is Jagged1-dependent.
    • Fig. S12. RA plasma fails to induce T cell stimulatory capacity of ECs.
    • Fig. S13. Notch1 receptor expression on CD4 T cells.
    • Fig. S14. T cell differentiation induced by endothelial Jagged1 is mTORC1-dependent.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Individual subject–level data.

    [Download Table S1]

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