Research ArticleFibrosis

T follicular helper–like cells contribute to skin fibrosis

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Science Translational Medicine  07 Mar 2018:
Vol. 10, Issue 431, eaaf5307
DOI: 10.1126/scitranslmed.aaf5307
  • Fig. 1 Phenotype of ICOS-expressing inflammatory and TH cells in the skin of patients with SSc.

    (A) Representative hematoxylin and eosin (H&E) staining of systemic sclerosis (SSc) and control (HC) skin samples (magnification, ×20). (B to D) SSc and normal control (HC) skin samples were assessed by TaqMan gene expression assays for the expression of T helper (TH) cell subset characteristic molecules (n = 20 healthy controls and n = 19 patients with SSc). (E) Representative immunohistochemistry (IHC) staining of SSc skin samples with CD4, inducible costimulator (ICOS), and programmed cell death 1 (PD-1) (magnification, ×40). (F) Representative immunofluorescence staining of SSc skin with CD4 (turquoise), ICOS (green), and PD-1 (red; magnification, ×40). Yellow arrows indicate positive cells. (G) Quantitation of CD4, ICOS, PD-1, and CD4+ICOS+ infiltrating T cells in the skin of a patient with SSc by immunofluorescence. (H) Representative immunofluorescence staining of CD4+ICOS+PD-1+ T follicular helper (TFH)–like infiltrating T cells in SSc skin (magnification, ×40). Yellow arrows indicate positive cells. (I) Quantitation of CD4+ICOS+PD-1+ TFH-like T cells in the skin of a patient with SSc (n = 19 patients with SSc). Data are from a cohort of patients with SSc, as described in Tables 1 and 2. P value was determined by unpaired t test (*P < 0.05, **P < 0.005, ***P < 0.0005, and ****P < 0.00005).

  • Fig. 2 Disease correlation and functional impact of ICOS-expressing TFH-like cells on myofibroblast differentiation.

    (A) TaqMan gene expression profile of indicated molecules involved in fibrosis in SSc versus HC. (B) Correlations of the mRSS with cell numbers and serum markers for extracellular matrix turnover and vascular damage in fibrotic skin of patients with SSc. (C) CD4+ICOS+PD-1+CXCR5+ TFH-like cells were differentiated in vitro on plate-bound anti-CD3 with soluble anti-CD28 monoclonal antibodies (MAbs) in the presence of ICOSL-Fc, interleukin-1β (IL-1β), IL-12, and IL-6 for 4 days. (D and E) αSMA+Vim+-differentiated myofibroblasts were determined by flow cytometry after direct coculture assay of in vitro–differentiated TFH-like cells with normal human dermal fibroblasts (NHDFs) at a T cell–to–fibroblast ratio of 1:4 in the presence or absence of suboptimal transforming growth factor–β (TGF-β) and anti-CD3/anti-CD28 antibodies (FMO, fluorescence minus one). All correlations were carried out using Spearman’s correlation analyses (n = 19 patients with SSc). Data represent two independent experiments. P value was determined by unpaired t test (*P < 0.05, ***P < 0.0005, and ****P < 0.00005).

  • Fig. 3 GVHD-SSc skin-infiltrating TFH-like and TH1 cells express ICOS.

    (A) Kinetics of infiltrating T cell– and macrophage-associated genes in graft-versus-host disease (GVHD)–SSc mice (n = 7). Mice were sacrificed on days 7, 14, 21, and 28 after adoptive transfer of splenocytes. The mean gene expression signals were obtained from the skin, and fold differences were calculated in comparison with the syngeneic control group (n = 7). (B) Cells were isolated from murine GVHD-SSc skin and analyzed by flow cytometry to identify T cell subsets. (C) The frequency of specific T cell subsets was examined by flow cytometry. All TH cell lineages were assessed from a parent effector T cell gate (CD44hiCD62Lhi/lo). Subsets were compared with syngeneic controls. Data represents three independent experiments. P value was determined by unpaired t test (**P < 0.005 and ***P < 0.0005).

  • Fig. 4 Anti-ICOS–depleting MAb reduced pathogenic TH subsets and their associated proinflammatory and profibrotic mediators involved in tissue remodeling in GVHD-SSc.

    (A) Evaluation of the mean clinical disease score during biweekly treatment starting on day 8 with anti-ICOS–depleting MAb (n = 5), anti-ICOS–blocking MAb (n = 5), or isotype control MAb and phosphate-buffered saline (PBS) (n = 10). Black asterisks indicate statistical significance for anti–ICOS-aFuc MAb versus PBS group, red asterisks indicate statistical significance for anti–ICOS-aFuc MAb versus isotype MAb group, and blue asterisks indicate statistical significance for anti–ICOS-aFuc MAb versus anti–ICOS-Tm MAb. (B) H&E assessment of dorsal skin collected from treatment groups at 4 weeks (magnification, ×10). (C) Histological assessment by Masson’s trichrome (MT) staining (magnification, ×10). (D) Quantification of T cell subset frequency after treatment with anti-ICOS MAbs was carried out by flow cytometry. (E) Single-cell suspensions from GVHD-SSc mouse skin of different treatment groups were restimulated in vitro with soluble anti-CD3 MAb for 48 hours on plates coated with αIFN-γ, αIL-21, anti–IL-4, and anti–IL-13 antibodies. The cytokine-producing T cells were quantitated in an enzyme-linked immunospot assay. (F) Total RNA samples isolated from skins of all treatment groups were subjected to TaqMan gene expression analysis against a selected gene set. Data were expressed as fold expression changes compared with syngeneic group. P value for gene expression data was calculated using one-way analysis of variance (ANOVA). (G) Serum samples from each treatment group were assessed for selected molecules using the multiplexed immunoassay profiling platform. Data are graphed on a log2 scale to illustrate the fold change. Data represent three independent experiments. P value was determined by unpaired t test, and the ANOVA model was used to estimate the mean differences in the log2 scale of the protein concentrations (*P < 0.05, **P < 0.005, and ***P < 0.0005). ns, not significant; vWF, von Willebrand factor.

  • Fig. 5 Impact of ICOS+ T cell deletion on profibrotic molecules involved in TH cell–fibroblast cross-talk in GVHD-SSc mice.

    (A) Total skin RNA samples from syngeneic (n = 5), PBS and isotype MAb (n = 10), or anti–ICOS-aFuc MAb (n = 5)–treated GVHD-SSc mice were profiled for MMP-12, IL-21, and IL-21R using TaqMan gene expression. (B) Mean fluorescence intensity (MFI) of IL-21R on CD3CD19CD11cF4/80–gated cells isolated from lesional skin assessed by flow cytometry. (C) MMP-12 expression in supernatants from syngeneic or GVHD-SSc skin fibroblasts cultured for 48 hours was determined by enzyme-linked immunosorbent assay. (D and E) Single-cell suspension was prepared from digested skin of syngeneic and GVHD-SSc mice. Cells were cultured for 12 days with two passages to yield confluent fibroblasts. Fibroblasts isolated from either syngeneic or GVHD-SSc animals were stimulated with recombinant IL-21 for 18 hours, followed by RNA isolation and real-time reverse transcription polymerase chain reaction analysis for IL-21R and MMP-12. Data are representative of two to three independent experiments. P value was determined by unpaired t test (*P < 0.05, **P < 0.005, and ***P < 0.0005).

  • Fig. 6 IL-21 neutralization reduces skin fibrosis in GVHD-SSc mice.

    (A) Live mean clinical score of syngeneic (n = 5) and GVHD-SSc mice treated with either IL-21R–Fc (n = 7) or control protein-Fc (n = 7) three times per week starting at day −1. (B) Histological skin scoring was measured using a rubric assessing tissue inflammation and fibrosis. (C) Histological assessment by H&E and MT staining of dorsal skin (magnification, ×20). (D) Splenic germinal B cells were enumerated by flow cytometry using a CD19+GL-7hiCD95hi gate. (E) TaqMan gene expression assay was conducted on RNA samples isolated from the skins of all treatment groups. Data were expressed as fold expression change relative to the syngeneic group. Data represent two independent experiments. P value was determined by unpaired t test (*P < 0.05, **P < 0.005, and ***P < 0.0005).

  • Table 1 Baseline demographics and clinical characteristics of all analyzed patients.

    Demographics and clinical characteristics are defined according to American College of Rheumatology criteria for systemic sclerosis. mRSS, modified Rodnan skin score; HAQ, health assessment questionnaire; EDAI, European disease activity index; CRP, complement reactive protein; ESR, erythrocyte sedimentation rate; FVC, forced vital capacity; anti-Scl 70, anti-topoisomerase 1 antibody.

    Baseline characteristics (n = 28)
    Mean age (years) ±
    SEM
    47.3 ± 9.74
    n%
    Sex (n = 28)
      Female1968
    Ethnicity (n = 28)
      Asian13.5
      Black310.7
      White2485.7
    Scleroderma subtype (n = 28)
      Diffuse2485.7
      Limited414.3
    Median disease duration
    in years (range)
    4.7 (0.3–16)
    Sclerosis activity score (n = 28)
      mRSS23.0 ± 8.45
      HAQ1.09 ± 0.83
      EDAI4.59 ± 2.07
    Disease manifestation
      Raynaud’s
    phenomenon (n = 28)
    28100
      Vascular digital
    ulcers (n = 28)
    1553.6
      Esophageal
    (dysphagia and
    reflux) (n = 28)
    2485.7
      Elevated acute-phase
    reactants (ESR and
    CRP) (n = 24)
    1458.3
      Cardiopulmonary
    (fibrosis) plain x-ray
    (n = 28)
    828.6
      Restrictive defect
    (FVC <60%
    predicted) (n = 28)
    414.3
    Positive autoantibodies
      Anti–Scl-70 (n = 25)1248.0
      Anti-nuclear (n = 24)2187.5
      Anti-cardiolipin (n = 11)19.09
    Medication (n = 15)
      Immunosuppressant
    (prednisone,
    azathioprine,
    mycophenolate
    mofetil, or
    methotrexate)
    15100
  • Table 2 Baseline demographics for healthy control participants.

    N/A, not applicable.

    Baseline characteristics (n = 20)
    Mean age (years) ±
    SEM
    41.0 ± 11.6
    n%
    Sex (n = 20)
      Female20100
    Ethnicity (n = 20)
      AsianN/AN/A
      BlackN/AN/A
      WhiteN/AN/A
    Disease manifestation
      N/A (weight reduction surgery)20100
  • Table 3 Correlation of mRNA transcript signature for TFH-like phenotypic molecules in fibrotic skin of patients with systemic sclerosis.
    mRNA transcript
    combination
    Correlation analysis
    Pr
    PDCD-1 versus ICOS0.00820.54
    PDCD-1 versus CXCR50.0250.45
    ICOS versus CXCR56.25 × 10−50.77
  • Table 4 Association profile of the frequency of TFH-like phenotypic marker expression in fibrotic skin-infiltrating cells determined by immunofluorescence.
    Immunofluorescence
    stain combination
    Correlation analysis
    Pr
    PD-1 versus ICOS2.30 × 10−60.89
    PD-1 versus CD43.05 × 10−50.83
    ICOS versus CD46.27× 10−50.81
  • Table 5 Correlation of mRNA transcript signature for TH2 and TH1 phenotypic molecules in fibrotic skin of patients with systemic sclerosis.
    TH2 mRNA transcript
    combination
    Correlation analysis
    Pr
    CRTH2 versus CXCR47.34 × 10−70.88
    CRTH2 versus CD40.0013−0.68
    CXCR4 versus CD40.00059−0.71
    TH1 mRNA transcript
    combination
    Correlation analysis
    Pr
    CXCR3 versus IFN-γ0.2−0.3
    CXCR3 versus CD40.014−0.55
    IFN-γ versus CD40.00450.62

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/431/eaaf5307/DC1

    Materials and Methods

    Fig. S1. Skin transcription factor profiling and correlational analysis of infiltrating T cells and molecules related to disease activity.

    Fig. S2. Evaluation of cellular subsets in the fibrotic skin of mice with GVHD-induced skin fibrosis.

    Fig. S3. Anti–ICOS-aFuc MAb elicits efficient depletion of ICOS-expressing T cells.

    Fig. S4. Anti-ICOS treatment blocks B cell functional response.

    Fig. S5. Skin-infiltrating T cells express both proinflammatory and profibrotic cytokines.

    Fig. S6. Anti-ICOS treatment reduced the systemic levels of molecules involved in lymphocyte function, migration, and fibrosis.

    Table S1. Correlative comparison of mRNA transcript signature for TH1, TH2, and TFH phenotypic molecules in fibrotic skin of patients with SSc.

    Table S2. Summary of binding affinity for parental and therapeutic antibody constructs.

    Table S3. Designated probes used for screening human SSc and murine GVHD-SSc genes.

    Table S4. Primary data.

    References (6264)

  • Supplementary Material for:

    T follicular helper–like cells contribute to skin fibrosis

    Devon K. Taylor,* Nanette Mittereder, Ellen Kuta, Tracy Delaney, Timothy Burwell, Karma Dacosta, Weiguang Zhao, Lily I. Cheng, Charles Brown, Anmarie Boutrin, Xiang Guo, Wendy I. White, Jie Zhu, Huifang Dong, Michael A. Bowen, Jia Lin, Changshou Gao, Li Yu, Madhu Ramaswamy, Marie-Claude Gaudreau, Rob Woods, Ronald Herbst, Gianluca Carlesso*

    *Corresponding author. Email: taylord{at}medimmune.com (D.K.T.); carlessog{at}medimmune.com(G.C.)

    Published 7 March 2018, Sci. Transl. Med. 10, eaaf5307 (2018)
    DOI: 10.1126/scitranslmed.aaf5307

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Skin transcription factor profiling and correlational analysis of infiltrating T cells and molecules related to disease activity.
    • Fig. S2. Evaluation of cellular subsets in the fibrotic skin of mice with GVHD-induced skin fibrosis.
    • Fig. S3. Anti–ICOS-aFuc MAb elicits efficient depletion of ICOS-expressing T cells.
    • Fig. S4. Anti-ICOS treatment blocks B cell functional response.
    • Fig. S5. Skin-infiltrating T cells express both proinflammatory and profibrotic cytokines.
    • Fig. S6. Anti-ICOS treatment reduced the systemic levels of molecules involved in lymphocyte function, migration, and fibrosis.
    • Table S1. Correlative comparison of mRNA transcript signature for TH1, TH2, and TFH phenotypic molecules in fibrotic skin of patients with SSc.
    • Table S2. Summary of binding affinity for parental and therapeutic antibody constructs.
    • Table S3. Designated probes used for screening human SSc and murine GVHD-SSc genes.
    • References (6264)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S4 (Microsoft Excel format). Primary data.

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