Research ArticleHELPER T CELLS

Human “TH9” cells are a subpopulation of PPAR-γ+ TH2 cells

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Science Immunology  18 Jan 2019:
Vol. 4, Issue 31, eaat5943
DOI: 10.1126/sciimmunol.aat5943
  • Fig. 1 IL-9–expressing TH cells are highly enriched in CCR4+/CCR8+ effector memory TH cells.

    (A) Naïve, central memory (TCM), and effector memory (TEM) TH cells were sorted from PBMCs of healthy donors according to the expression of CCR7, CD45RA, and CD25 and activated with αCD3/CD2/CD28 beads. The production of IL-9 and IFN-γ was assessed by flow cytometry at the indicated time points. (B) Effector memory TH cells were repeatedly activated with αCD3/2/28 beads, and IL-9 production was measured at the indicated time points by flow cytometry. (C) CD4+/CD25 T cells were sorted according to the expression of the indicated chemokine receptors into positive (black bars) and negative (white bars) populations and activated with αCD3/CD2/CD28 beads. On day 2, production of IFN-γ, IL-13, IL-17, and IL-9 was simultaneously analyzed by flow cytometry (IL-9single+ = IL-9+/IFN-γ/IL-13/IL-17 TH cells). ns, not significant. (D) Representative plots of CXCR3-, CCR4-, CCR8-, and CCR6-positive and negative memory T cells, stimulated as in (C). (E) CCR4 and CCR8 expression in freshly isolated CXCR3/CCR6/CD25 memory TH cells. (F to H) CCR4+/CCR8 and CCR4+/CCR8+ CD25 memory TH cells were sorted from healthy donors and activated with αCD3/CD2/CD28 beads. Production of cytokines was assessed on day 2 (F) or day 6 (G) as in (C). (H) Representative example of cytokine expression in CCR4+/CCR8+ CD25 memory TH cells before and at different time points after activation. Data are representative of independent experiments with at least four donors (B, G, and H) or at least six donors (A and C to F) and presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Statistics: One-way ANOVA, followed by Tukey multiple comparison test (A), or paired two-tailed t test (C, F, and G).

  • Fig. 2 IL-9–expressing TH cell clones express high levels of TH2 cytokines in the resting state.

    Single cells from memory TH cell subsets were isolated from healthy donors according to the expression of chemokine receptors and used to generate TH cell clones: CXCR3+/CCR4/CCR6 (“CXCR3+,” enriched for TH1), CXCR3/CCR4+/CCR6+ (“CCR6+,” enriched for TH17), CXCR3/CCR4+/CCR6 (“CCR4+,” enriched for TH2), and CXCR3/CCR4+/CCR6/CCR8+ (“CCR4+/CCR8+,” enriched for “TH9”). (A) TH cell clones were analyzed in the resting state (left graph) and at 18 hours after activation with αCD3/CD2/CD28 beads (right graph) for their expression of IL-9. (B) TH cell clones were analyzed in the resting state for their expression of IL-4, IL-5, and IL-13. (C) Time course analysis of IL-9 expression in TH cell subset clones after activation with αCD3/CD2/CD28 beads as measured by flow cytometry. (D) Cytokine production of unselected TH cell subset clones in the resting state (y axis) compared with IL-9 production 18 hours after activation (x axis) as measured by flow cytometry. Each dot represents a single TH cell clone. (E) Representative examples of cytokine profiles of TH cell subset clones in the resting state and at 18 hours after activation. Data are representative of independent experiments with at least four (C) or six (A, B, D, and E) donors and presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Statistics: One-way ANOVA, followed by Tukey multiple comparison test (A and B, left), or unpaired t test (B, middle and right).

  • Fig. 3 Activation-dependent expression of IL-9 and TH2 cytokines gives rise to transient “TH9” phenotype.

    (A to C) Freshly isolated PBMCs (A) or IL-9+ TH2 clones (B and C) were stimulated with αCD3, αCD3/CD28, or αCD3/CD2/CD28 beads. IL-9 expression and viability were analyzed by flow cytometry 2 days after activation in TH cells (A) or in TH cell clones (B and C). (D) TH cell clones were stimulated at different bead/T cell ratios and analyzed by flow cytometry 2 days after activation. (E) Example of activation-dependent IL-9, IL-4, IL-5, and IL-13 expression in a representative IL-9+ TH2 clone before and at different time points after activation with αCD3/CD2/CD28. (F) Aggregate data of cytokine production by IL-9+ TH2 clones before and at different time points after activation with αCD3/CD2/CD28 beads. Analysis as in (B). (G and H) IL-9 and TH2 cytokine expression kinetics after repeated activation of a representative IL-9+ TH2 clone, as analyzed at the indicated time points by flow cytometry (G) or in cell culture supernatants (H). Data are representative of independent experiments with clones from four donors (A to F) or one representative clone (G and H) and presented as means ± SD. *P < 0.05, **P < 0.01, ****P < 0.0001. Statistics: One-way ANOVA, followed by Tukey multiple comparison test.

  • Fig. 4 “ TH9” priming of naïve CD4+ T cells in vitro induces a transient “TH9” phenotype in TH2 cells.

    (A and B) Naïve CD4+ T cells isolated from PBMCs of healthy donors were primed under “TH9”-polarizing conditions (IL-4 + TGF-β) and analyzed for the expression of IFN-γ, IL-17, IL-13, and IL-9 by flow cytometry at the indicated time points after initiation of polarization. Aggregate data are shown in (A), and representative examples of IL-9, IL-13, and IL-5 expression under initial “TH9” priming are shown in (B). (C) Naïve CD4+ T cells were subjected to three rounds of in vitro priming under continuous “TH9” priming conditions. Expression of cytokines was measured during the third round of priming at the indicated time points by flow cytometry. (D to F) Naïve CD4+ T cells were primed under TH0, TH2 (IL-4), or “TH9” (IL-4 + TGF-β) priming conditions, and expression of cytokines (D) or transcription factors (E and F) was measured by RT-PCR on days 3 and 7 (D and E) or by flow cytometry on day 7 (F). Data are representative of independent experiments with two (C), three (F), or at least five (A, B, D, and E) donors and presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Statistics: One-way ANOVA, followed by Tukey multiple comparison test (D), or paired two-tailed t test (E).

  • Fig. 5 TGF-β and IL-4 induce the expression of the transcription factor PPAR-γ in IL-9+ TH2 cells.

    (A to C) TH1, TH17, TH2, and IL-9+ TH2 clones were subjected to RNA-seq analysis in resting state. (A) Clones were selected on the basis of cytokine secretion, as measured by bead-based immunoassay 2 days after activation. (B) Transcription factor expression at resting state in TH1, TH17, TH2, and IL-9+ TH2 clones, as determined by RNA-seq. (C) Principal components (PC) analysis of transcriptomes of clones. (D to G) Representative TH1, TH2, and IL-9+ TH2 clones were subjected to RNA-seq analysis in resting state and at different time points after activation. (D) Gene expression in resting state was correlated with IL9 expression after activation for all clones and for every gene. A ranked list of the top correlating genes (Pearson’s linear correlation coefficient) is shown. TH2-related genes are highlighted. (E) Correlation of PPARG expression at resting state (0 hours) and IL9 at 4 and 12 hours after activation. (F) PPARG expression at resting state (0 hours) in TH1, TH17, TH2, and IL-9+ TH2 clones. (G) Expression time course of PPARG and IL9 in IL-9+ TH2 clones as measured by RNA-seq. (H) PPARG expression was measured in ex vivo–isolated TH1 (CXCR3+), TH2 (CCR4+/CCR8), and IL-9+ TH2 (CCR4+/CCR8+) memory TH cells in the resting state and at 4 hours after activation with αCD3/CD2/CD28 by RT-PCR. (I and J) Naïve TH cells were primed under TH0-, TH2-, or “TH9”-polarizing conditions. PPARG expression was analyzed during (I) initial priming or (J) at day 10 (resting) and 12 hours after activation with αCD3/CD2/CD28. Data are representative of independent experiments with one (A to G), six (H), or five (I and J) donors and presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Statistics: One-way ANOVA, followed by pairwise comparison using Tukey multiple comparison test (A, B, F, and H to J), or Pearson’s linear correlation coefficients (D and E).

  • Fig. 6 PPAR-γ regulates IL-9 expression in TH2 cells.

    (A) In vitro–primed TH1, TH2, and “TH9” cells or (B) TH1, TH17, TH2, and IL-9+ TH2 clones were incubated for 48 hours with either the PPAR-γ antagonist GW9662 or solvent (DMF) as control. Cytokine production was then measured by flow cytometry in resting cells or in cells activated with αCD3/CD2/CD28 for 18 hours (as indicated). (C) IL-9+ TH2 clones were pretreated and activated as in (B), and cytokine production was measured by flow cytometry at the indicated time points after activation. (D and E) IL-9+ TH2 clones were transfected with PPARG or control siRNA and activated with αCD3/CD2/CD28. After 1 day, cytokines were measured by flow cytometry. Aggregate data in (D) and example FACS plots in (E). Data are representative of independent experiments with at least two (A, D, and E), three (C), or five donors (B) and presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Statistics: One-way ANOVA, followed by Tukey multiple comparison test (A and B), or paired two-tailed t test (C and D).

  • Fig. 7 IL-9–expressing PPAR-γ+ TH2 cells infiltrate acute allergic skin inflammation.

    (A) IL9 expression was analyzed by RT-PCR in NS and in lesional skin of acute or chronic psoriasis (aPS and cPS), acute or chronic atopic dermatitis (aAD and cAD), and acute allergic contact dermatitis (aACD). (B) IL9 levels were determined in lesional skin of AD and aACD by RT-PCR and correlated with severity of disease. PGA, physician global assessment. (C) IL9 levels in aACD skin before and at indicated time points after elicitation. (D and E) T cells were isolated from lesional skin of cPS, cAD, and aACD. TH cell expression of IL-9, IFN-γ, IL-17, and IL-13 was simultaneously determined by flow cytometry. (F) TH cells were isolated from aACD, and cytokine profiles were determined as in (D) either directly or 5 days after in vitro culture. (G) IL-9+ TH2 cell lines were generated from aACD by short-term ex vivo culture, reactivated with αCD3/CD2/CD28, and subjected to time course analysis of cytokine profiles by flow cytometry. (H) TH cell clones were generated from aACD, and cytokine profiles were analyzed in the resting state or at 18 hours after activation by flow cytometry. Each dot represents a TH cell clone. (I) PPARG expression in NS and in lesional skin of AD and aACD. (J) Skin sections from NS, AD, and aACD were immunohistochemically stained for PPAR-γ, and infiltrating PPAR-γ+ cells were quantified by digital image analysis. (K and L) Representative pictures of immunofluorescence for PPAR-γ and CD3 (K) or CD4 (L) in lesional skin of aACD. Scale bars, 50 μm. Open arrows, PPAR-γ+/CD3+ cells (K) or PPAR-γ+/CD4+ cells (L). Closed arrows, PPAR-γ+/CD3 cells (K) or PPAR-γ+/CD4 cells (L). DAPI, 4′,6-diamidino-2-phenylindole. (M) PPARG expression in representative TH cell clones generated as in (H). (N) IL-9+ TH2 cell lines generated from aACD were incubated for 48 hours with the PPAR-γ antagonist GW9662 or solvent (DMF) as control. Cells were then activated with αCD3/CD2/CD28 for 18 hours, and cytokine production was measured by flow cytometry. Data are representative of independent experiments with at least two (H and M), three (B, F, and G), four (C to E, K, and L), five (A), or seven (I, J, and N) donors and presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Statistics: One-way ANOVA, followed by Tukey multiple comparison test.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/31/eaat5943/DC1

    Fig. S1. Identification of human TH cell subsets by chemokine receptor expression.

    Fig. S2. Activation-dependent expression of cytokines in TH2 clones.

    Fig. S3. Cytokine profile after reactivation of in vitro–primed “TH9” cells.

    Fig. S4. Correlation values of all genes with IL-9 in TH1, TH2, and IL-9+ TH2 clones.

    Fig. S5. PPARG levels during first and second rounds of in vitro priming of naïve TH cells.

    Fig. S6. IL-7Rα and CRLF2 form a functional TSLP-R on IL-9+ TH2 cells.

    Fig. S7. IL-6Rα is highly expressed on IL-9+ TH2 cells, and IL-6 promotes IL-9 expression in polarizing “TH9” cells.

    Fig. S8. Effect of PPAR-γ inhibition by GW9662 or RNA interference on cytokine production in human TH cells.

    Fig. S9. PPAR-γ agonists do not promote IL-9 or TH2 cytokine expression in IL-9+ TH2 cells.

    Fig. S10. Cytokine profiles of TH cells from human inflammatory skin disease.

    Fig. S11. PPAR-γ immunohistochemistry in AD and aACD and PPAR-γ antagonism in skin TH cell lines.

    Fig. S12. Gating strategies for flow cytometry analysis.

    Table S1. Raw data file.

    Table S2. Antibodies used in this study.

    Table S3. RT-PCR primers used in this study.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Identification of human TH cell subsets by chemokine receptor expression.
    • Fig. S2. Activation-dependent expression of cytokines in TH2 clones.
    • Fig. S3. Cytokine profile after reactivation of in vitro–primed “TH9” cells.
    • Fig. S4. Correlation values of all genes with IL-9 in TH1, TH2, and IL-9+ TH2 clones.
    • Fig. S5. PPARG levels during first and second rounds of in vitro priming of naïve TH cells.
    • Fig. S6. IL-7Rα and CRLF2 form a functional TSLP-R on IL-9+ TH2 cells.
    • Fig. S7. IL-6Rα is highly expressed on IL-9+ TH2 cells, and IL-6 promotes IL-9 expression in polarizing “TH9” cells.
    • Fig. S8. Effect of PPAR-γ inhibition by GW9662 or RNA interference on cytokine production in human TH cells.
    • Fig. S9. PPAR-γ agonists do not promote IL-9 or TH2 cytokine expression in IL-9+ TH2 cells.
    • Fig. S10. Cytokine profiles of TH cells from human inflammatory skin disease.
    • Fig. S11. PPAR-γ immunohistochemistry in AD and aACD and PPAR-γ antagonism in skin TH cell lines.
    • Fig. S12. Gating strategies for flow cytometry analysis.
    • Legend for table S1
    • Table S2. Antibodies used in this study.
    • Table S3. RT-PCR primers used in this study.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Raw data file.

    Files in this Data Supplement:

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