Gating strategy and IL-17⁺ T cells in RA patients and healthy controls.

<div><p>A) Exemplary gating strategy shown on representative pseudo-colour dot-plots of unstimulated PBMC of a RA patient. Dead cells were excluded using EMA-viability dye. Lymphocytes were gated (FSC vs. SSC), followed by the subgating of CD3⁺ and IL-17⁺ cells. IL-17⁺CD3⁺ T cells and IL-17⁺CD3^- non-T cells were further specified. Frequencies in each subgate are expressed as percentage of their parent population; B-D) Comparative analysis of CD3⁺ lymphocytes in PBMC stimulated with PMA/i, PHA or incubated in RPMI-1640 without stimulation for 20 h in RA patients and healthy controls; B) Percentage of CD3⁺ T cells within the IL-17⁺ lymphocytes; C) Percentage of CD4⁺ and CD8⁺ cells within the IL-17⁺CD3⁺ T cell population, D) Percentage of CD4⁺ cells within CD3⁺ T cells; E-F) Comparative analysis of IL-17 expression in different T cell populations stimulated with PMA/i, PHA, a pool of 23 peptides of MHC-class II-restricted T cell epitopes or incubated in RPMI-1640 without stimulation for 20 h; E) Percentage of IL-17⁺ cells within CD3⁺CD4⁺ Th cells; F) Percentage of IL-17⁺ cells within CD3⁺CD8⁺ Tc cells; G) Percentage of IL-17⁺ cells within CD3⁺CD4⁺CD8⁺ double positive T cells.</p> <p>Data from healthy controls (n = 20) and RA patients (n = 20). *p < 0.05; **p < 0.01, ***p < 0.001,ns = not significant, column represents the mean and bars indicate 95% CI.</p></div

IL-17 expressing B cells.

<div><p>A) Percentage of CD19⁺ B cells within IL-17⁺CD3^- lymphocytes in RA patients (n = 8) and healthy controls (n = 8) in unstimulated PBMC or after stimulation with PMA/i for 20 h; B+C) freshly thawed PBMC or PBMC cultured for 20 h in RPMI-1640 medium with the addition of Brefeldin A for 16h (n = 5 and n = 8, respectively) in RA patients and healthy controls; B) Percentage of CD19⁺ B cells within IL-17⁺CD3^- lymphocytes; C) Percentage of IL-17⁺ cells within CD3^-CD19⁺ B cells; D) Percentage of IgD⁺CD27^- naïve B cells, IgD⁺CD27⁺ non-switched memory B cells and CD24⁺⁺CD38⁺⁺ regulatory B cells of IL-17⁺CD3^-CD19⁺ B cells of freshly thawed PBMC of healthy controls (n=3); E) Percentage of IL-17⁺ cells in EBV-transformed B cell lines without stimulation; F) Representative gel-electrophoresis of amplificates of IL-17A RT-PCR of unstimulated freshly thawed CD4⁺ and CD19⁺ MACS-sorted cells of a healthy donor: CD4⁺ T cells (lane 1), CD19⁺ B cells (lane 2), CD4^-CD19^- cells (lane 3); of a RA patient: CD4⁺ T cells (lane 4), CD19⁺ B cells (lane 5), CD4^-CD19^- cells (lane 6); and of the EBV-transformed B cell lines: Olga (lane 7), AMAI (lane 8); upper level: amplificates of IL-17A, lower level: corresponding control amplificates of β-actin, Marker: Puc 8 mix ladder; The data shown are representative results of RT-PCR of 3 healthy donors and 3 RA patients.</p> <p>*p < 0.05; **p < 0.01, ns = not significant; columns represent the mean and bars indicate 95% CI.</p></div

Influence of in vitro culture on IL-17⁺ cells.

<div><p>A-D) Comparison of freshly thawed cells and cells cultured for 20 h in RPMI-1640 medium with the addition of Brefeldin A for 16 h (n = 5 and n = 8, respectively) in RA patients and healthy controls; A) Percentage of CD3⁺ T cells within IL-17⁺ lymphocytes; B) Percentage of CD4⁺ cells within IL-17⁺CD3⁺ T cells; C) Percentage of CD11b⁺ cells within IL-17⁺CD3^- lymphocytes; D) Percentage of CD56⁺ NK cells within IL-17⁺CD3^- lymphocytes.</p> <p>*p < 0.05, **p < 0.01,***p < 0.001, ns = not significant, columns represent the mean and bars indicate 95% CI.</p></div

IL-17⁺ non-T cells.

<div><p>A-F) Comparative analysis of PBMC stimulated with PMA/i for 20h or incubated in RPMI-1640 medium with the addition of Brefeldin A for 16 h in RA patients and healthy controls; A) Percentage of CD3^- non-T cells within IL-17⁺ lymphocytes (RA n = 20, healthy controls n = 20); B) Percentage of CD56⁺ NK cells within IL-17⁺CD3^- non-T cells; C) Percentage of IL-17⁺ cells within CD3^-CD56⁺ NK cells; D) Percentage of CD14⁺ monocytes in IL-17⁺CD3^- non-T cells within the lymphocyte gate and within the monocyte gate (FSC vs. SSC); E) Percentage of IL-17⁺ cells within CD3^-CD14⁺ monocytes within the monocyte gate; F) Percentage of CD11b⁺ cells within IL-17⁺CD3^- lymphocytes.</p> <p>Data B)-F) PMBC of healthy controls (n = 8) and RA patients (n = 8).</p> <p>*p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant, column represents the mean and bars indicate 95% CI.</p></div