Research ArticleT CELL DIFFERENTIATION

The TNFRSF members CD27 and OX40 coordinately limit TH17 differentiation in regulatory T cells

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Science Immunology  21 Dec 2018:
Vol. 3, Issue 30, eaau2042
DOI: 10.1126/sciimmunol.aau2042
  • Fig. 1 Skin-resident Tregs differentiate toward IL-17–producing cells during TH17-mediated tissue inflammation.

    (A) Percentage of RORγt-expressing Tregs (gated on Live CD45+CD4+FOXP3+ cells) in the colon, skin, and SDLNs of WT mice as quantified by flow cytometry. Results are from one experiment with n = 6 mice. (B) Tregs and Teffs were sort purified from normal healthy skin of FOXP3-GFP mice, and gene expression was quantified by whole-transcriptome RNA-seq. Heat maps of cytokines (top), transcription factors and cytokine receptors (middle), and Treg-specific genes (bottom) are shown. (C and D) The percentage of (C) RORγt- and (D) IL-17A–expressing Tregs in the skin of WT mice on days 0, 3, and 6 (d0, d3, and d6, respectively) after cutaneous C. albicans infection was quantified by flow cytometry. Results are from two replicate experiments with n = 2 to 6 mice per group. Data are means ± SEM. P values are determined using one-way ANOVA. **P < 0.01 and ****P < 0.0001.

  • Fig. 2 The TNFR family members CD27 and OX40 are preferentially expressed by skin-resident Tregs.

    Representative flow cytometry plots and quantification of percentages and MFI of (A) CD27 and (B) OX40 expression on Tregs and Teffs in healthy murine skin. Populations are pregated on Live CD45+CD4+ cells. Results are pooled from three independent experiments with n = 9 to 10 mice per experiment. Data are means ± SEM. P values are determined using paired Student’s t test. ****P < 0.0001.

  • Fig. 3 CD27 and OX40 signaling attenuate TH17 differentiation in Tregs in vitro.

    Tregs were sort purified from FOXP3-GFP reporter mice and cultured with anti-CD3/anti-CD28–coated Dynabeads under either TH0- or TH17-polarizing conditions in the presence or absence of FcCD70 and/or an agonistic anti-OX40 monoclonal antibody (OX86). On day 6, Tregs were restimulated with PMA and ionomycin. Representative flow cytometry plots of (A) IL-17A, (B) IL-17F, (C) IFN-γ, (D) CCR6, (E) RORγt, (F) FOXP3, and (G) CD25 after gating on Live+CD4+FOXP3+ cells are shown and either percentages (A to D) or MFIs (F and G) are quantified. Data are representative of two to three independent experiments with technical replicates and graphs depict means ± SEM. P values are determined using one-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001.

  • Fig. 4 CD27 and OX40 attenuate TH17 differentiation in Tregs in a cell-intrinsic manner in vivo.

    (A) WT CD45.1 Tregs, CD27−/− or OX40−/− CD45.2 Tregs, and WT CD45.1/CD45.2 Teffs were sort purified and cotransferred at a 1:1:2 ratio into RAG−/− recipients. Fourteen days later, mice were infected with C. albicans, and skin and SDLNs were harvested 7 days after infection. (B) Representative flow cytometry plots and quantification of percentages of WT (CD45.1) and CD27−/− (CD45.2) Tregs in the SDLNs after gating on Live+CD4+TCRβ+FOXP3+ cells. (C to E) Representative plots and quantification of (C) RORγt, (D) IL-17A, and (E) IFN-γ expression by WT or CD27−/− Tregs in the SDLNs. (F to I) Representative plots and quantification of (F) percentages of WT (CD45.1) and OX40−/− (CD45.2) Tregs and expression of (G) RORγt, (H) IL-17A, and (I) IFN-γ expression in Live+CD4+TCRβ+FOXP3+ cells in the SDLNs. Data are compiled from two independent experiments with n = 11 to 13 mice per group. Graphs depict mean ± SEM. P values are determined using a paired Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, P > 0.05.

  • Fig. 5 Signaling through CD27 and OX40 synergize to attenuate TH17 differentiation in Tregs in vivo.

    (A) Model of cutaneous C. albicans infection. WT or CD27−/−OX40−/− DKO CD45.2 Tregs were sort purified and adoptively transferred with CD45.1 Teffs into RAG−/− recipients. On day 14 after transfer, mice were infected cutaneously with C. albicans. (B) Representative histology of dorsal skin 7 days after infection. Scale bars, 100 μm. (C) Representative clinical images of dorsal skin inflammation 7 days after infection. (D) Quantification of disease based on blinded histologic scoring of epidermal hyperplasia and mononuclear cell infiltrate. (E) Absolute numbers of SDLNs cells as measured by a hemocytometer. (F and G) Quantification of percentages and absolute numbers of CD45.2+ (Tregs) and CD45.1+ (Teff) cells (gated on Live CD4+TCRβ+) in (F) the skin and (G) SDLNs by flow cytometry. WT or DKO Treg expression of (H and I) RORγt, (J and K) IL-17A, and (L and M) IFN-γ (gated on CD4+CD45.2+FOXP3+ cells) in the skin and SDLNs as quantified by flow cytometry. Data are combined from three independent experiments with n = 13 to 14 mice per group, and graphs depict mean ± SEM. P values are determined using unpaired Student’s t test. *P < 0.05; ***P < 0.001; ****P < 0.0001; ns, P > 0.05.

  • Fig. 6 CD27 expression inversely correlates with Treg production of IL-17 in diseased human skin.

    (A) Representative flow cytometry plots of CD27 on Tregs and Teffs (gated on Live CD45+CD3+CD4+) within healthy human skin and blood. Percentages and MFI of CD27 on skin-resident cells are quantified, and results are pooled from n = 21 healthy human skin biopsies. (B) Treg IL-17 production in healthy or lesional psoriatic (PSO) skin was quantified by flow cytometry after PMA/ionomycin restimulation [n = 22 (healthy) and n = 26 (PSO)]. (C) Representative plot and quantification of CD27 expression on Tregs and Teffs in psoriatic skin are shown. (D) Correlation between Treg IL-17 production and CD27 MFI in lesional psoriatic skin was quantified by flow cytometry. Lines represent paired data from the same sample (n = 10). Results are pooled from 10 patients with psoriasis. (E) Treg IL-17 production in biopsies from paired nonlesional (NL-HS) and lesional (L-HS) skin of patients with HS was quantified by flow cytometry (gated on Live CD45+CD3+CD4+FOXP3+ cells). Lines represent paired data from a single patient (n = 8). (F) Representative plot and quantification of CD27 expression on Tregs and Teffs in lesional HS skin. (G) Correlation between Treg IL-17 production and CD27 MFI in lesional skin biopsies from patients with HS as quantified by flow cytometry. Lines represent paired data from the same sample (n = 20). P values are determined using either an unpaired (A and B) or a paired (D to G) Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/3/30/eaau2042/DC1

    Fig. S1. Human skin-resident Treg expression of TH17-associated genes.

    Fig. S2. CD27 and OX40 coexpression on Tregs.

    Fig. S3. Deletion of CD27 or OX40 on Tregs results in increased IL-17 expression in vitro.

    Fig. S4. CD27 signaling suppresses Treg expression of TH17-associated genes.

    Fig. S5. OX40 is important for Treg accumulation in adoptive transfer model before C. albicans infection.

    Fig. S6. Deletion of either CD27 or OX40 in Tregs has minimal effect on skin inflammation.

    Fig. S7. OX40 is preferentially expressed on Tregs in healthy human skin.

    Fig. S8. Percentages and MFI of FOXP3 in Tregs in human disease.

    Fig. S9. Gating strategy for murine and human Tregs and Teffs.

    Table S1. Effects of CD27 engagement on TH17 cytokines.

    Table S2. Effects of CD27 engagement on signature Treg genes.

    Table S3. Antibodies.

    Table S4. Raw data.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Human skin-resident Treg expression of TH17-associated genes.
    • Fig. S2. CD27 and OX40 coexpression on Tregs.
    • Fig. S3. Deletion of CD27 or OX40 on Tregs results in increased IL-17 expression in vitro.
    • Fig. S4. CD27 signaling suppresses Treg expression of TH17-associated genes.
    • Fig. S5. OX40 is important for Treg accumulation in adoptive transfer model before C. albicans infection.
    • Fig. S6. Deletion of either CD27 or OX40 in Tregs has minimal effect on skin inflammation.
    • Fig. S7. OX40 is preferentially expressed on Tregs in healthy human skin.
    • Fig. S8. Percentages and MFI of FOXP3 in Tregs in human disease.
    • Fig. S9. Gating strategy for murine and human Tregs and Teffs.
    • Table S1. Effects of CD27 engagement on TH17 cytokines.
    • Table S2. Effects of CD27 engagement on signature Treg genes.
    • Table S3. Antibodies.

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    Other Supplementary Material for this manuscript includes the following:

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

    Files in this Data Supplement:

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