Akt-Dependent Enhanced Migratory Capacity of Th17 Cells from Children with Lupus Nephritis

IL-17–producing CD4⁺CD45RA⁻Foxp3⁻ and Foxp3^low T cells are increased in children with LN. (A) RORγ/γt and IL-17 expression by gated CD4⁺CD45RA⁻Foxp3⁻ T cells was determined by flow cytometry after stimulation of fresh PBMCs with leukocyte activation cocktail. Representative dot plots for one patient with LN and one HC. Data show mean and SD of four children with active LN, four children with inactive LN, and five HCs. (B and C) Quantitative real-time PCR was used to assess the expression of (B) IL-17 mRNA and (C) RORc mRNA relative to GAPDH mRNA in anti-CD3/CD28–activated PBMCs from children with LN (n = 15) and from HCs (n = 15). Data are represented as the mean and SD. (D) PBMCs were activated with anti-CD3/CD28 for 7 d, restimulated with leukocyte activation cocktail, and analyzed by flow cytometry. Representative dot plots of IL-17 expression in the indicated T cell populations are presented. The numbers indicate the percentages of IL-17⁺ cells. Scatter plots depict the data obtained from children with LN (n = 17) and from HCs (n = 17).

mTOR inhibition reduces Stat3 activity and the frequency of IL-17–producing CD4⁺CD45RA⁻Foxp3⁻ and Foxp3^low T cells from children with LN. (A) PBMCs were activated with anti-CD3/CD28 for 7 d in the presence or absence of rapamycin (50 ng/ml), restimulated with leukocyte activation cocktail, and analyzed by flow cytometry. The expression of IL-17 in gated CD4⁺CD45RA⁻Foxp3⁻ and Foxp3^low T cells is shown. The data are representative of 17 children with LN. (B–G) PBMCs from children with LN were activated with anti-CD3/CD28 for 7 d in the presence or absence of rapamycin (Rapa 25 or 50 ng/ml). The phosphorylation of (B) p-Stat3 and (C) p-S6RP in gated CD4⁺CD45RA⁻Foxp3⁻ T cells was analyzed by flow cytometry. Data are represented as the MFI [mean and SD of (B) nine and (C) five children with LN]. (D and E) Representative dot plots of (D) IL-17 and p-S6RP and (E) Foxp3 and IL-17 in gated (D) CD4⁺CD45RA⁻Foxp3⁻ T cells and (E) CD4⁺CD45RA⁻ T cells. (F and G) MFI (mean and SD) of (F) IL-4 and (G) RORγt expression in gated (F) CD4⁺CD45RA⁻ T cells and (G) CD4⁺CD45RA⁻Foxp3⁻ T cells (n = 5). Data are the MFI of specific Ab minus the MFI of matched isotype control. The quadrants are based on the isotype control Abs, and the upper right numbers indicate the percentage of cells in each quadrant.

Enhanced Akt activity in IL-17–producing T cells from lupus patients. (A) Freshly isolated PBMCs from children with active (a) LN (n = 5), inactive (i) LN (n = 5), and HCs (n = 5) were stimulated with PMA and ionomycin for 4 h in the presence of GolgiPlug, and the phosphorylation of Akt in gated CD4⁺CD45RA⁻Foxp3⁻IL-17⁺ T cells was analyzed by flow cytometry. Data are the mean and SD. (B) PBMCs were stimulated with anti-CD3/CD28 for 7 d, restimulated with PMA and ionomycin, rested for 2 h in serum-free medium, and then activated with anti-CD3/CD28 for the indicated times. The histograms of p-Akt expression in gated CD4⁺CD45RA⁻Foxp3⁻IL-17⁺ T cells are representative of four children with LN and four HCs. (C) PBMCs were stimulated with anti-CD3/CD28 for 7 d and restimulated with PMA and ionomycin in the presence of GolgiPlug. (C) p-Akt expression in the indicated T cell populations from children with LN (n = 17), children with NS (n = 5), and HCs (n = 19) was analyzed by flow cytometry (MFI). Data are the MFI of specific Ab minus the MFI of matched isotype control.