Research ArticleFibrosis

PD-1 up-regulation on CD4+ T cells promotes pulmonary fibrosis through STAT3-mediated IL-17A and TGF-β1 production

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Science Translational Medicine  26 Sep 2018:
Vol. 10, Issue 460, eaar8356
DOI: 10.1126/scitranslmed.aar8356
  • Fig. 1 PD-1 up-regulation in fibrotic lung disease.

    (A) Baseline PD-1 on total CD4+ T cells (HC, n = 24; IPF = 25). (B) PD-1 expressed on CD4+ T cell subsets (HC, n = 10; TH1, Treg, and TH17, n = 7). (C) Percentage of proliferating CD4+ T cells from the peripheral blood of HC (n = 14) and IPF subjects (n = 20) after TCR stimulation with plate-bound CD3/CD28 antibodies. Corresponding histograms illustrating proliferative capacity for a HC and IPF patient. (D and E) Immunohistochemistry (IHC) assessment for PD-1 and PD-L1 (brown) in healthy and IPF pulmonary biopsies. Increased PD-1 expression on lymphocytes (upper right quadrant, arrows). Increased PD-L1 expression (lower right quadrant, arrows). N = 12. Comparisons between cohorts were performed using an unpaired two-tailed Student’s t test. Multiple group comparisons were performed using a one-way ANOVA with Tukey’s post hoc test. Proliferation data were analyzed using the Mann-Whitney U test. Bars indicate SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. NS, no significance; CFSE, carboxyfluorescein diacetate succinimidyl ester; FMO, fluorescence minus one.

  • Fig. 2 TH17 cells are most likely to secrete TGF-β1 in association with PD-1 up-regulation after ex vivo stimulation.

    Purified CD4+ T cells from the peripheral blood of HC, sarcoidosis, and IPF patients were TCR-stimulated and cultured for 24 hours. (A) Increased active TGF-β1 in the cell culture supernatants of sarcoidosis and IPF CD4+ T cells (HC, n = 9; sarc, n = 6; IPF, n = 8). (B) Frequency (left) and MFI (right) for total CD4+ T cells secreting TGF-β1 (HC, n = 12; sarc, n = 14; IPF, n = 7). (C) PD-1 percentages for total CD4+ T cells expressing TGF-β1 (HC, n = 6; sarc, n = 10; IPF, n = 7) and corresponding histograms. (D) Percentage of Tregs expressing TGF-β1 (HC, n = 14; sarc, n = 13; IPF, n = 7). (E) PD-1 expression on Tregs that are positive for TGF-β1 (HC, n = 8; sarc, n = 10; IPF, n = 7). (F) TH17 cells secreting TGF-β1 (frequency and MFI) in HC (n = 6), sarcoidosis patients according to baseline PD-1 expression (high, n = 8; low, n = 5) and IPF subjects (n = 7). (G) Percentage of TH17 cells that are secreting TGF-β1 and also expressing PD-1 (HC, n = 6; sarc, n = 13; IPF, n = 7). Histograms depict PD-1 expression on TGF-β1+ TH17 cells. (H) Percentage of TGF-β1 secreted by CD4+ T cell subsets. Comparisons between cohorts were performed using an unpaired two-tailed Student’s t test. Multiple group comparisons were performed using a one-way ANOVA with Tukey’s post hoc test. Bars indicate SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. NS, no significance for (A) to (G).

  • Fig. 3 PD-1 blockade normalizes HLF collagen-1 production after coculture with patient T cells.

    Purified CD4+ T cells were cultured overnight with or without the presence of anti–PD-1, anti–PD-L1, and anti–PD-L2. The following day, cells were TCR-stimulated and cocultured with purified HLF. (A) Percentage of fibroblasts (CD3CD4CD45) expressing collagen-1 when cultured alone (n = 14) and when cultured with HC (n = 8) or sarcoidosis (n = 12) CD4+ T cells. Collagen-1 assessment in cell culture supernatants from an independent experiment (HLF, n = 10; HC, n = 8; sarc, n = 8). (B) Collagen-1 expression by HLFs when cultured in the presence of purified CD4+ T cells isolated from IPF patients (n = 4). Collagen-1 in cell culture supernatants from an independent experiment (HLF, n = 10; HC, n = 8; IPF, n = 5). (C) Purified sarcoidosis CD4+ T cells from the same patients were cultured with (postblockade) or without (preblockade) the addition of PD-1–blocking antibodies. HLFs were added the following day, and the cells were cocultured at 37°C in 5% CO2 atmosphere. Percentage of fibroblasts expressing collagen-1 at baseline and in cocultures with sarcoidosis CD4+ T cells subsequent to PD-1 blockade [HLF (baseline), n = 14; preblockade, n = 13; postblockade, n = 13]. Collagen-1 expressed by fibroblasts cocultured with CD4+ T cells expressing high or low baseline PD-1 levels, before and after PD-1 blockade (high, n = 9; low, n = 4). (D) Percentage of collagen-1 expressed by HLFs cocultured with IPF CD4+ T cells pre- and postblockade (n = 4). (E) Collagen-1 assessment in coculture supernatants by HLF at baseline (HLF only) and with sarcoidosis CD4+ T cells before and after PD-1 pathway blockade and 24-hour TCR stimulation (n = 8). (F) Coculture lysates were immunoblotted with antibodies against COL1A1, α-SMA, and fibronectin after 24-hour TCR stimulation with and without the addition of PD-1–blocking antibodies. Representative Western blots for three sarcoidosis patients and quantification of results are depicted (baseline, n = 2; preblockade and postblockade, n = 5). Comparisons between cohorts were performed using an unpaired two-tailed Student’s t test. Preblockade and PD-1 pathway blockade cohort comparisons were analyzed using a paired Student’s t test. Multiple group comparisons were performed using a one-way ANOVA with Tukey’s post hoc test. Bars indicate SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. NS, no significance; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

  • Fig. 4 PD-1 blockade reduces IL-17A and TGF-β1 expression.

    Purified CD4+ T cells were cultured overnight with (postblockade) or without (preblockade) the presence of anti–PD-1, PD-L1, and PD-L2. The following day, cells were TCR-stimulated and cocultured with HLF. (A) CD4+IL-17A+ T cells with and without PD-1 blockade (n = 6). Representative FACS plots illustrating CD4+IL-17A+ T cell percentages pre- and postblockade. (B) Percentage of CD4+ T cells from sarcoidosis and (C) IPF patients secreting TGF-β1 pre- and postblockade (sarc, n = 11; IPF, n = 7). (D) Free active TGF-β1 in sarcoidosis coculture supernatants subsequent to PD-1 blockade. (E and F) Sarcoidosis TH17 cells expressing TGF-β1 after PD-1 pathway blockade according to CD4+ T cell baseline PD-1 levels (high PD-1, n = 8; low PD-1, n = 5). (G) Percentage of TH17 cells that are TGF-β1+ before and after PD-1 blockade in IPF patients (n = 7). Comparisons between cohorts were performed using an unpaired two-tailed Student’s t test. Preblockade and PD-1 pathway blockade cohort comparisons were analyzed using a paired Student’s t test. Multiple group comparisons were performed using a one-way ANOVA with Tukey’s post hoc test. Bars indicate SEM. *P < 0.05, **P < 0.01. NS, no significance.

  • Fig. 5 PD-1 blockade reduces STAT3 mRNA and protein.

    Purified CD4+ T cells from HC and sarcoidosis peripheral blood mononuclear cells (PBMCs) were TCR-stimulated for 24 hours at 37°C in 5% CO2. STAT3 protein expression was assessed using flow cytometry. (A) Percentage and MFI for CD4+ T cells expressing STAT3 (HC, n = 4; sarc, n = 8). (B) STAT3 gene expression was assessed using quantitative reverse transcription polymerase chain reaction (PCR). (HC, n = 6; high PD-1, n = 6; low PD-1, n = 4; IPF, n = 4). (C) STAT3 mRNA expression from sarcoidosis and IPF CD4+ T cells after PD-1 pathway blockade (n = 8). (D) CD4+ T cells were cultured overnight with or without the pSTAT3 inhibitor STATTIC. The following day, cells were TCR-stimulated in the presence of HLFs and cultured at 37°C in 5% CO2. Percentage of collagen-1 produced by fibroblasts that have been cultured in the presence of sarcoidosis CD4+ T cells without STATTIC (gray bar) and with STATTIC (green bar) (HLF, n = 14; sarcoidosis without STATTIC, n = 5; STATTIC, n = 5). Histograms illustrate percentage of collagen produced by fibroblasts before and after STAT3 inhibition. (E) Percentage of CD4+ T cells producing IL-17A before and after STATTIC inhibition and representative histograms. Comparisons between cohorts were performed using an unpaired two-tailed Student’s t test. Preblockade and PD-1 pathway blockade cohort comparisons were analyzed using a paired Student’s t test. Multiple group comparisons were performed using a one-way ANOVA with Tukey’s post hoc test. Bars indicate SEM. *P < 0.05, **P < 0.01. NS, no significance.

  • Fig. 6 TH17 gene expression profiles in IPF.

    (A) Hierarchical clustering of IPF discovery [University of Chicago (N = 45) (GSE27957)] and (B) validation cohort [Imperial College London (N = 55) (GSE93606)] based on a TH17 signature in PBMCs (discovery cohort) and whole blood (validation cohort) by GeneChip microarrays. Two major clusters of IPF patients were identified. Distinctions are present in transplant-free survival (TFS) over the course of 3.5 years post-diagnosis and overall survival between clusters in both the discovery and validation cohorts, respectively. (C) TFS for clusters 1 and 2 from the discovery cohort and (D) validation cohort based on the expression of STAT3.

  • Fig. 7 Bleomycin-induced lung fibrotic murine model reveals reduced lung fibrosis in PD-1 null mice or after PD-L1 blockade.

    (A) PD-1 percentages and MFI on CD4+ T cells from single-cell lung suspensions 21 days after bleomycin treatment. Matching histograms depicting PD-1 on CD4+ T cells. (B) Percentage and MFI for lung-derived CD4+PD-1+ T cells after TCR stimulation with plate-bound anti-CD3/CD28 antibodies after bleomycin treatment (PBS, n = 4; bleomycin, n = 8). Representative histograms showing percentage of PD-1 on CD4+ T cells after TCR stimulation. (C) CD4+ T cell proliferation subsequent to 5-day TCR stimulation and corresponding histograms (PBS, n = 3; bleomycin, n = 5). (D) Percentage of TH17 cells secreting TGF-β1 (PBS, n = 4; bleomycin, n = 8). Representative FACS plots illustrating percentage of TGF-β1 in a PBS- and bleomycin-treated mouse. (E) TGF-β1 percentages expressed by Tregs in PBS-treated (n = 4) and bleomycin-treated (n = 8) mice. Flow cytometry plots demonstrating percentage of TGF-β1 expressed by Tregs. (F) Percentage of TGF-β1 secreted by CD4+ T cell subsets. (G) Representative images for H&E- and trichrome-stained lungs with Ashcroft scoring 14 days after bleomycin injury from WT and PD-1 null mice. Scale bar, 100 μm. n = 4 in each cohort. Lungs were processed for measuring hydroxyproline levels by HPLC (n = 4 in each bleomycin cohort; n = 2 in NS cohort). (H) Trichrome-stained lungs and quantification of collagen content after isotype or anti–PD-L1 treatment 4 days after bleomycin administration to mice, as well as weight differences for isotype and anti–PD-L1 bleomycin-treated mice (n = 7). (I) Percentage of CD4+PD-1+ T cells by flow cytometry after anti–PD-L1 antibody administration to bleomycin-treated mice (n = 5). (J) Percentage of phosphorylated STAT3 at Tyr705 in CD4+ T cells from single-cell lung suspensions of bleomycin-treated mice after isotype and anti–PD-L1 antibody treatment (n = 10 and n = 12, respectively). (K) Linear correlation between PD-1 and pSTAT3 (Y705) after bleomycin administration. Comparisons between cohorts were performed using an unpaired two-tailed Student’s t test. Preblockade and PD-1 pathway blockade cohort comparisons were analyzed using a paired Student’s t test. Multiple group comparisons were performed using a one-way ANOVA with Tukey’s post hoc test. Proliferation data were analyzed using the Mann-Whitney U test. PD-1/pSTAT3 correlation analyzed using Spearman correlation. Bars indicate SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. NS, no significance.

  • Table 1 Demographics of IPF and age-matched control populations.

    C, Caucasian.

    Healthy controlIPF
    Number533
    Sex (female, male)0, 57, 26
    Age (year), median
    (minimum,
    maximum)
    61 (44, 68)66 (49, 80)
    Race5 C33 C
  • Table 2 Demographics of peripheral blood sarcoidosis, IPF, and control populations.

    AA, African-American; H, Hispanic.

    Healthy controlSarcoidosisIPF
    Number324633
    Sex (female,
    male)
    19, 1332, 147, 26
    Age (year),
    median
    (minimum,
    maximum)
    44 (25, 68)54 (30, 75)66 (49, 80)
    Race4 AA; 28 C17 AA; 28 C; 1 H33 C

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/460/eaar8356/DC1

    Materials and Methods

    Fig. S1. PD-1 up-regulation in fibrotic lung disease.

    Fig. S2. CD4+ T cells secreting LAP/TGF-β1.

    Fig. S3. Increased Tregs in sarcoidosis.

    Fig. S4. Increased IL-6 expression in sarcoidosis CD4+ T cells after TCR stimulation.

    Fig. S5. TGF-β1 secretion by CD4+ T cell subsets.

    Fig. S6. Increased intracellular TGF-β1 expression by CD4+IL-17A+ T cells.

    Fig. S7. Collagen-1 production by fibroblasts cocultured with sarcoidosis CD4+ T cells with high and low PD-1 baseline expression.

    Fig. S8. Fibrosis markers in cocultured supernatants.

    Fig. S9. CD4+ T cells secreting LAP/TGF-β1.

    Fig. S10. STATTIC specificity in CD4+ T cells.

    Fig. S11. Reduced RORC expression after PD-1 blockade.

    Fig. S12. IL-12 expression in IPF patients in the discovery cohort.

    Fig. S13. Reduced IL-23R expression in PD-1 null bleomycin-treated mice.

    Table S1. HLF and sarcoidosis PD-1+CD4+ T cell coculture demonstrates increased collagen-1 synthesis.

    Table S2. Primary data.

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. PD-1 up-regulation in fibrotic lung disease.
    • Fig. S2. CD4+ T cells secreting LAP/TGF-β1.
    • Fig. S3. Increased Tregs in sarcoidosis.
    • Fig. S4. Increased IL-6 expression in sarcoidosis CD4+ T cells after TCR stimulation.
    • Fig. S5. TGF-β1 secretion by CD4+ T cell subsets.
    • Fig. S6. Increased intracellular TGF-β1 expression by CD4+IL-17A+ T cells.
    • Fig. S7. Collagen-1 production by fibroblasts cocultured with sarcoidosis CD4+ T cells with high and low PD-1 baseline expression.
    • Fig. S8. Fibrosis markers in cocultured supernatants.
    • Fig. S9. CD4+ T cells secreting LAP/TGF-β1.
    • Fig. S10. STATTIC specificity in CD4+ T cells.
    • Fig. S11. Reduced RORC expression after PD-1 blockade.
    • Fig. S12. IL-12 expression in IPF patients in the discovery cohort.
    • Fig. S13. Reduced IL-23R expression in PD-1 null bleomycin-treated mice.
    • Table S1. HLF and sarcoidosis PD-1+CD4+ T cell coculture demonstrates increased collagen-1 synthesis.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S2 (Microsoft Excel format). Primary data.

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