Research ArticleIMMUNE REGULATION

Regulatory T cells induce activation rather than suppression of human basophils

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Science Immunology  25 May 2018:
Vol. 3, Issue 23, eaan0829
DOI: 10.1126/sciimmunol.aan0829
  • Fig. 1 Human basophils are refractory to Treg-mediated suppression.

    Basophils were cultured either alone or with IL-3 or cocultured with anti-CD3/anti-CD28–activated Tregs or Tconv cells in the presence of IL-3 at 1:3 ratio for 24 hours. During the last 1 hour of the culture, basophils were stimulated with anti-IgE antibodies. (A to H) Representative plots (A and B) and mean ± SEM (C to H) of data from six independent experiments using cells from different donors showing the expression [mean fluorescence intensity (MFI) and percent positive cells] of CD203c (C), CD13 (D), CD69 (E and F), CD63 (G), and CD107a (H) on the basophils under various experimental conditions. (I) Amount of histamine released under above experimental conditions (mean ± SEM; n = 6). **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns, not significant by one-way ANOVA with Tukey’s multiple comparison test.

  • Fig. 2 Human basophils are unresponsive to contact-dependent or contact-independent inhibition by Tregs.

    (A and B) Expression of HLA-DR and costimulatory molecules CD80 and CD86 (mean ± SEM; n = 4 donors) on resting and IL-3–stimulated basophils from human blood and spleen. (C and D) Percentage of annexin V– and PI-positive basophils that were cultured for 24 hours in medium alone, with IL-3, or with IL-3 plus anti-CD3/anti-CD28–activated Tregs or Tconv. Basophils were also stimulated with anti-IgE during the last 1 hour of the culture, as indicated. Representative plots and mean ± SEM of data from three independent experiments. (E) Expression of TGF-βRII and IL-10Rα (mean ± SEM; n = 6 donors) on human blood basophils. (F) Expression of CD123, CD13, CD69, and CD203c on IL-10Rα+ and IL-10Rα subsets of basophils (mean ± SEM; n = 3 independent experiments using cells from different donors) cultured for 24 hours with IL-3 and various concentrations of IL-10. During the last 1-hour culture, anti-IgE antibodies were added to stimulate basophils. ns, not significant by one-way ANOVA with Tukey’s multiple comparison test (D) or by two-way ANOVA with least significant difference test (F).

  • Fig. 3 Tregs induce activation of resting human basophils.

    Basophils were either cultured for 24 hours alone or cocultured with anti-CD3/anti-CD28–activated Tregs or Tconv. (A and B) Representative plots and mean ± SEM of data from seven independent experiments using cells from different donors showing the expression of CD13, CD203c, and CD69 on the basophils. (C and D) Representative dot plots and mean ± SEM of data from five independent experiments showing the expression of degranulation markers CD63 and CD107a on the basophils. *P < 0.05, **P < 0.01, and ****P < 0.0001; ns, not significant by one-way ANOVA with Tukey’s multiple comparison test.

  • Fig. 4 Induction of basophil cytokines by activated Tregs.

    Basophils were cultured either alone or with anti-CD3/anti-CD28–activated Tregs for 24 hours. (A to C) Supernatants of cocultures were analyzed for the amounts of (pg/ml; mean ± SEM; n = 6 independent experiments using cells from different donors) IL-13 (A), IL-8 (B), and IL-4 (C). *P < 0.05 by two-tailed paired Student’s t test.

  • Fig. 5 LFA-1–ICAM-1 interaction is dispensable for the Treg-induced human basophil activation.

    (A) Expression of ICAM-1 (mean ± SEM; n = 5 donors) on the resting and IL-3–stimulated basophils. (B to D) Basophils were either cultured for 24 hours alone or cocultured with anti-CD3/anti-CD28–activated Tregs with or without blocking mAbs to ICAM-1. (B and C) Representative plots indicating the expression of CD13, CD203c, and CD69 on the basophils. (D) Histograms depicting the expression of CD13, CD203c, and CD69 (mean ± SEM; n = 4 independent experiments using cells from different donors) on the basophils. (E) Expression of CD13, CD203c, and CD69 (mean ± SEM; n = 5 independent experiments using cells from different donors) on the basophils cocultured with Tregs either in direct contact or in transwells. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns, not significant by one-way ANOVA with Tukey’s multiple comparison test.

  • Fig. 6 Soluble mediators released from activated Tregs stimulate basophils.

    Basophils were cultured for 24 hours either alone or with cell-free culture supernatants (SUPs) from unstimulated (U-Tregs) or anti-CD3/anti-CD28–stimulated Tregs (S-Tregs). (A and B) Level of expression of (MFI or percent positive cells) CD13, CD203c, and CD69 (A) and degranulation markers CD63 and CD107a on the basophils (B) (mean ± SEM; n = 3 independent experiments using cells from different donors). *P < 0.05 and **P < 0.01; ns, not significant by one-way ANOVA with Tukey’s multiple comparison test.

  • Fig. 7 Tregs induce human basophil activation by an IL-3–dependent mechanism.

    Basophils were either cultured for 24 hours alone or cocultured with anti-CD3/anti-CD28–stimulated Tregs in the presence of isotype control mAbs or blocking mAbs to IL-3R. (A) Representative plots of CD13, CD203c, CD69, and FcεRI expression on basophils. (B) Histograms (mean ± SEM) depicting the expression of CD13 (n = 8 independent experiments using cells from different donors), CD203c, FcεRI, and CD69 (n = 11 independent experiments) on the basophils. (C) Amounts of secretion of IL-13 and IL-8 (mean ± SEM; n = 5 independent experiments using cells from different donors) by basophils. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by one-way ANOVA with Tukey’s multiple comparison test.

  • Fig. 8 Tregs induce activation of basophils by STAT5 pathway.

    (A and B) Basophils were either cultured for 6 hours alone or with IL-3 or cocultured with anti-CD3/anti-CD28–stimulated Tregs. (A) Representative plots of phosphorylated STAT5 (pSTAT5) expression in basophils (A) and mean ± SEM of pSTAT5-positive basophils (B) from four independent experiments using cells from different donors. (C and D) Basophils were pretreated with STAT5 inhibitor (STAT5 inh) or DMSO, followed by coculture with Tregs for 24 hours. Expression of CD13, CD203c, and CD69 on the basophils (C) and the amounts of IL-13 and IL-8 in the culture supernatants (D) (mean ± SEM; n = 4 independent experiments). *P < 0.05, **P < 0.01, and ****P < 0.0001; ns, not significant by one-way ANOVA with Tukey’s multiple comparison test.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/3/23/eaan0829/DC1

    Materials and Methods

    Fig. S1. Expression of HLA-DR, CD80, and CD86 on human DCs.

    Fig. S2. Expression of TGF-βRII and IL-10Rα on human splenic basophils.

    Fig. S3. Effect of IL-10 on the phenotype of human DCs.

    Fig. S4. Anti-CD3 and anti-CD28 mAbs do not alter the phenotype of basophils.

    Fig. S5. Blocking of ICAM-1 on DCs inhibits DC-mediated CD4+ T cell proliferation.

    Fig. S6. ICOS-ICOSL interaction is dispensable for the Treg-mediated human basophil activation.

    Fig. S7. Representative dot plots depicting the absence of CD4+ T cells in the lower chambers of transwells.

    Fig. S8. IL-3 secretion by human Tregs.

    Fig. S9. Dose-dependent effects of IL-3 on the phenotype of human basophils.

    Fig. S10. Tregs license basophils to undergo activation by degranulation signals.

    Fig. S11. The phenotypic and functional features of isolated human Tregs.

    Fig. S12. The positive controls for annexin V and PI staining.

    Table S1. Raw data from figure graphs (Excel).

  • Supplementary Materials

    Supplementary Material for:

    Regulatory T cells induce activation rather than suppression of human basophils

    Meenu Sharma, Mrinmoy Das, Emmanuel Stephen-Victor, Caroline Galeotti, Anupama Karnam, Mohan S. Maddur, Patrick Bruneval, Srini V. Kaveri, Jagadeesh Bayry*

    *Corresponding author. Email: jagadeesh.bayry{at}crc.jussieu.fr

    Published 25 May 2018, Sci. Immunol. 3, eaan0829 (2017)
    DOI: 10.1126/sciimmunol.aan0829

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Expression of HLA-DR, CD80, and CD86 on human DCs.
    • Fig. S2. Expression of TGF-βRII and IL-10Rα on human splenic basophils.
    • Fig. S3. Effect of IL-10 on the phenotype of human DCs.
    • Fig. S4. Anti-CD3 and anti-CD28 mAbs do not alter the phenotype of basophils.
    • Fig. S5. Blocking of ICAM-1 on DCs inhibits DC-mediated CD4+ T cell proliferation.
    • Fig. S6. ICOS-ICOSL interaction is dispensable for the Treg-mediated human basophil activation.
    • Fig. S7. Representative dot plots depicting the absence of CD4+ T cells in the lower chambers of transwells.
    • Fig. S8. IL-3 secretion by human Tregs.
    • Fig. S9. Dose-dependent effects of IL-3 on the phenotype of human basophils.
    • Fig. S10. Tregs license basophils to undergo activation by degranulation signals.
    • Fig. S11. The phenotypic and functional features of isolated human Tregs.
    • Fig. S12. The positive controls for annexin V and PI staining.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1. Raw data from figure graphs (Excel).

    Files in this Data Supplement:

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