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

Sudhir Kshirsagar, Magdalena Riedl, Heiko Billing, Burkhard Tönshoff, Shanmugapriya Thangavadivel, Christian Steuber, Hagen Staude, Gottfried Wechselberger and Monika Edelbauer
J Immunol November 15, 2014, 193 (10) 4895-4903; DOI: https://doi.org/10.4049/jimmunol.1400044
Sudhir Kshirsagar
*Department of Pediatrics I, Innsbruck Medical University, A-6020 Innsbruck, Austria;
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Magdalena Riedl
*Department of Pediatrics I, Innsbruck Medical University, A-6020 Innsbruck, Austria;
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Heiko Billing
†University Children’s Hospital, Heidelberg, 69120 Heidelberg, Germany;
‡University Children’s Hospital, 72076 Tuebeingen, Germany;
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Burkhard Tönshoff
†University Children’s Hospital, Heidelberg, 69120 Heidelberg, Germany;
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Shanmugapriya Thangavadivel
§Tyrolean Cancer Research Institute, Innsbruck Medical University, A-6020 Innsbruck, Austria;
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Christian Steuber
¶Klinikum Links der Weser, 28277 Bremen, Germany;
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Hagen Staude
‖Department of Pediatrics, University Children’s Hospital, 18075 Rostock, Germany; and
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Gottfried Wechselberger
#Department of Plastic, Aesthetic, and Reconstructive Surgery, Hospital of the Barmherzige Brüder Salzburg, Paracelsus Medical University, 5020 Salzburg, Austria
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Monika Edelbauer
*Department of Pediatrics I, Innsbruck Medical University, A-6020 Innsbruck, Austria;
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  • FIGURE 1.
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    FIGURE 1.

    Stat3 activity is enhanced in effector T cells from children with LN. The phosphorylation of Stat3 was analyzed by flow cytometry. The cells were pregated on CD4+ cells and subsequently gated based on the expression of CD45RA and Foxp3. (A) Representative histograms and bar graphs show p-Stat3 fluorescence intensity (mean and SD) in fresh CD4+CD45RA−Foxp3− T cells from children with active LN (n = 5), inactive LN (n = 5), and HCs (n = 5). (B) PBMCs from children with LN (n = 17), children with NS (n = 5), and HCs (n = 19) were activated with anti-CD3/CD28 for 7 d. Representative histograms show the expression of p-Stat3 in the indicated T cell populations (left). The data are summarized in box-and-whisker plots (right) (MFI). (C) The cells were stimulated with recombinant human IL-6 for the indicated time periods. Histograms show the p-Stat3 fluorescence intensity in CD4+CD45RA−Foxp3− T cells and are representative of n = 4 for each group. Data are the MFI of specific Ab minus the MFI of matched isotype control.

  • FIGURE 2.
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    FIGURE 2.

    IL-17–producing CD4+CD45RA−Foxp3− and Foxp3low 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).

  • FIGURE 3.
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    FIGURE 3.

    mTOR inhibition reduces Stat3 activity and the frequency of IL-17–producing CD4+CD45RA−Foxp3− and Foxp3low 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 Foxp3low 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.

  • FIGURE 4.
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    FIGURE 4.

    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.

  • FIGURE 5.
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    FIGURE 5.

    Migration of Th17 cells from children with LN is enhanced and Akt-dependent. (A) Th17-polarized CD4+ T cells from children with LN (n = 7) and from HCs (n = 7) were used in transwell migration assays with the indicated concentrations of CCL20 in the lower chamber. The data are represented as the mean and SD. (B) Th17-polarized CD4+ T cells from children with LN (n = 7) were incubated with the Akt inhibitor VIII (Akt inh) at the indicated concentration or with diluent for 30 min prior to addition to the transwells. Migration was evaluated in response to CCL20 (200 ng/ml). The data are expressed as the percentage of migrated Th17 cells relative to the total number of Th17 cells loaded. (C) Transwell migration assays of Th1- or Th2-polarized cells from children with LN (n = 5) and from HCs (n = 5) with CXCL10 (250 ng/ml) or CCL2 (50 ng/ml), respectively, in the lower chamber. Data are the mean and SD. (D) Th1- and (E) Th2-polarized cells from children with LN (n = 5) were treated with the Akt inhibitor (inh) or diluent for 30 min prior to the migration assay. Graphs show the percentage of migrated (D) Th1- and (E) Th2-polarized cells relative to the total number of (D) Th1 or (E) Th2 cells loaded (median and range).*p < 0.05.

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The Journal of Immunology: 193 (10)
The Journal of Immunology
Vol. 193, Issue 10
15 Nov 2014
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Akt-Dependent Enhanced Migratory Capacity of Th17 Cells from Children with Lupus Nephritis
Sudhir Kshirsagar, Magdalena Riedl, Heiko Billing, Burkhard Tönshoff, Shanmugapriya Thangavadivel, Christian Steuber, Hagen Staude, Gottfried Wechselberger, Monika Edelbauer
The Journal of Immunology November 15, 2014, 193 (10) 4895-4903; DOI: 10.4049/jimmunol.1400044

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Akt-Dependent Enhanced Migratory Capacity of Th17 Cells from Children with Lupus Nephritis
Sudhir Kshirsagar, Magdalena Riedl, Heiko Billing, Burkhard Tönshoff, Shanmugapriya Thangavadivel, Christian Steuber, Hagen Staude, Gottfried Wechselberger, Monika Edelbauer
The Journal of Immunology November 15, 2014, 193 (10) 4895-4903; DOI: 10.4049/jimmunol.1400044
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