1. Induction of antigen-specific regulatory T lymphocytes by human dendritic cells expressing the glucocorticoid-induced leucine zipper
by Haifa Hamdi, Véronique Godot, Marie-Christine Maillot, Maria Victoria Prejean, Nicolas Cohen, Roman Krzysiek, François M. Lemoine, Weiping Zou, and Dominique Emilie
Blood
Volume 110(1):
July 1, 2007
©2007 by American Society of Hematology

2. Expression of CD25hi and FOXP3 by CD4+ T lymphocytes stimulated with DEX-treated DCs. (A-B) DCs were treated with DEX, pulsed with PPD, and used to stimulate CD4+ T lymphocytes.
Expression of CD25hi and FOXP3 by CD4+ T lymphocytes stimulated with DEX-treated DCs. (A-B) DCs were treated with DEX, pulsed with PPD, and used to stimulate CD4+ T lymphocytes. The fraction of CD25hiFOXP3+ cells among CD4+ T lymphocytes was then determined. Results representative of 5 experiments with DEX (10−7 M) are shown in panel A, and means ± SEM of 3 experiments with various concentrations of DEX are shown in panel B. (C) DCs were treated with various concentrations of DEX and production of GILZ was analyzed by Western blot. Quantification of actin was used as an internal control. MW indicates molecular weight markers. One representative of 3 experiments is shown on the top of the panel. The ratio between the intensities of GILZ and actin expressions in 3 experiments (mean ± SEM) is shown on the bottom of the panel.
Haifa Hamdi et al. Blood 2007;110:
©2007 by American Society of Hematology

3. Induction of Tregs by GILZ-expressing DCs
Induction of Tregs by GILZ-expressing DCs. (A-C) DCs were nucleofected with siC or siGILZ and treated or not treated with DEX (10−7 M).
Induction of Tregs by GILZ-expressing DCs. (A-C) DCs were nucleofected with siC or siGILZ and treated or not treated with DEX (10−7 M). (D-F) DCs were nucleofected with the control vector (pCTRL, ▩) or the GILZ-encoding vector (pGILZ, −). (A,D) Production of GILZ was analyzed by immunoprecipitation. Quantification of actin was used as an internal control. (B-C,E-F) DCs were pulsed with PPD and used to stimulate autologous CD4+ T lymphocytes. Expression of Treg markers was determined by flow cytometry. Staining with a control mAb is shown as a dotted line in panels C and F. All results are representative of 3 to 5 experiments. MW indicates molecular weight markers.
Haifa Hamdi et al. Blood 2007;110:
©2007 by American Society of Hematology

4. Suppressive functions of CD4+ T lymphocytes stimulated with GILZ-expressing DCs. (A-B) Inhibition of GILZ expression and induction of Tregs.
Suppressive functions of CD4+ T lymphocytes stimulated with GILZ-expressing DCs. (A-B) Inhibition of GILZ expression and induction of Tregs. DCs, nucleofected with siC or siGILZ and treated or not treated with DEX (10−7 M), were pulsed with PPD and used to stimulate CD4+ T lymphocytes, which were then added in various numbers to CFSE-labeled PBMCs stimulated with PPD. Proliferation of CD4+ responders was assessed by flow cytometry. Means ± SEM of 7 experiments are shown in (A), and a representative experiment with 5 × 104 CD4+ T lymphocytes added to PBMCs is shown in (B). *P < .05. (C) Induction of Tregs by GILZ-transfected DCs. DCs were nucleofected with pCTRL or pGILZ, pulsed with PPD, and used to stimulate CD4+ T lymphocytes; the suppressive function of these lymphocytes was tested on the PPD-induced proliferation of CFSE-labeled CD4+ responders. Results shown are representative of 7 experiments. Percentages represent the fraction of CFSElo CD4+ T lymphocytes cells among CD3+CD4+ cells.
Haifa Hamdi et al. Blood 2007;110:
©2007 by American Society of Hematology

5. GILZ-transfected DCs stimulate the expression of Treg markers by antigen-specific CD4+ T lymphocytes.
GILZ-transfected DCs stimulate the expression of Treg markers by antigen-specific CD4+ T lymphocytes. Control and GILZ-transfected DCs were pulsed with PPD and used to stimulate CFSE-labeled CD4+ T lymphocytes. (A-B) Expression of Treg markers by CFSEhi and CFSElo CD4+ T lymphocytes was determined. A typical of 3 independent experiments is shown. Control mAb staining obtained with GILZ-transfected DCs is shown as a dotted line in panel B. (C) Expression of IL-10 and FOXP3 by CFSElo CD4+ cells was determined. A typical example of 2 independent experiments is shown.
Haifa Hamdi et al. Blood 2007;110:
©2007 by American Society of Hematology

6. Antigen specificity of the Tregs induced by GILZ-transfected DCs
Antigen specificity of the Tregs induced by GILZ-transfected DCs. (A-B) Control and GILZ-transfected DCs were pulsed with either PPD or CMV (first stimulation) and used to stimulate CD4+ T lymphocytes, which were then added to CFSE-labeled PBMCs stimulated ...
Antigen specificity of the Tregs induced by GILZ-transfected DCs. (A-B) Control and GILZ-transfected DCs were pulsed with either PPD or CMV (first stimulation) and used to stimulate CD4+ T lymphocytes, which were then added to CFSE-labeled PBMCs stimulated with either PPD or CMV (second stimulation). Proliferation of CFSE-labeled CD4+ responders (A) and CD8+ responders (B) was assessed by flow cytometry. Results are representative of 4 experiments.
Haifa Hamdi et al. Blood 2007;110:
©2007 by American Society of Hematology

7. Role of IL-10 in the induction of Tregs and in their suppressive function.
Role of IL-10 in the induction of Tregs and in their suppressive function. Control and GILZ-transfected DCs were pulsed with PPD and used to stimulate CD4+ T lymphocytes, which were then added to CFSE-labeled PBMCs stimulated with PPD. Proliferation of CD4+ responders was assessed by flow cytometry. (A) IL-10 is involved in the induction of Tregs. An anti–IL-10 mAb or its isotype control was added to the coculture of DCs and CD4+ T lymphocytes. (B) Production of IL-10 by Tregs is involved in their suppressive function. An anti–IL-10 mAb or its isotype control was added to the coculture of CD4+ T lymphocytes and responders. Results are representative of 3 experiments.
Haifa Hamdi et al. Blood 2007;110:
©2007 by American Society of Hematology

8. GILZ in APCs from GC-treated patients modulates the production of cytokines and chemokines and induces Tregs.
GILZ in APCs from GC-treated patients modulates the production of cytokines and chemokines and induces Tregs. APCs were collected before (■) and 48 hours after (▩) the initiation of GC treatment and they were nucleofected with siC or siGILZ. (A) APCs were stimulated with LPS and their production of CCL5, TNFα, and IL-10 was determined by ELISA (mean ± SEM). (B-D) APCs were pulsed with PPD and used to activate CD4+ T lymphocytes. (B) Expression of IL-10 by CFSElo CD4+ T lymphocytes was determined by flow cytometry. Control mAb staining obtained with APCs nucleofected with siC is shown as a dotted line. (C-D) CD4+ T lymphocytes were added in various numbers to CFSE-labeled PBMCs. Proliferation of CD4+ responders was assessed by flow cytometry. Means ± SEM from 3 experiments are shown in (A) and (C), and a representative experiment is shown in (B) and (D). CD4+ T lymphocytes (5 × 104) were added to PBMCs in panel D. *P < .05 using a Wilcoxon test.
Haifa Hamdi et al. Blood 2007;110:
©2007 by American Society of Hematology