IL-17 production from memory CD4⁺ T cells can be modulated by the expression of IL-1RI and IL-RII. (A) Flow cytometric analysis of IL-1RI up-regulation on sorted IL-1RI⁻ memory CD4⁺ T cells that were incubated for 3 days in the presence or absence of anti-CD3/CD28 antibody-coated beads. (B) Flow cytometric measurement of intracellular IL-17 in sorted IL-1RI⁻ memory CD4⁺ T cells that were incubated for 2 days with anti-CD3/CD28 antibody-coated beads followed by adding IL-1β and/or IL-1R antagonist (IL-1Ra, 125 ng/mL) and culturing for an additional 5 days. Cells were stimulated for 4 hours with PMA and ionomycin before cytokine analysis. Numbers above boxes in dot plots indicate the frequency of IL-17 cells. MFI indicates the mean fluorescent intensity. (C) ELISA of IL-17 production from sorted IL-1RI⁻ memory CD4⁺ T cells that were incubated and treated as in panel B except for PMA and ionomycin stimulation. (D) RT-PCR of IL1RII and IL1RACP expression in sorted and unstimulated IL-1RI⁺ and IL-1RI⁻ memory CD4⁺ T cells. Results are relative to the number of transcripts encoding β-actin. (E) Sorted IL-1RI⁺ memory CD4⁺ T cells were treated for 2 hours with anti–IL-1RII or isotype control antibodies (20 μg/mL) and incubated for 7 days with anti-CD3/CD28 antibody-coated beads in the presence or absence of IL-1β. Intracellular IL-17 and IFN-γ were assessed using flow cytometry after 4 hours of PMA and ionomycin stimulation. Numbers indicate the frequency of cells for each quadrant. (F) ELISA of IL-17 production from sorted IL-1RI⁺ memory CD4⁺ T cells that were treated as in panel E. Data represent 5 (A) and 4 (B,E) independent experiments (1 experiment per donor), mean ± SD of triplicates from 4 independent experiments (C,F) or the mean ± SEM of 5 donors (D).