Both GM-CSF produced by mobilized progenitors and recombinant GM-CSF promote Treg expansion. (A) The expression of GM-CSF mRNA was measured in freshly sorted Lin⁻c-kit⁺Sca-1⁺ double positive HSCs and HPCs by real-time PCR. All data were normalized to GAPDH mRNA levels and expressed as fold increases relative to control values obtained from HSCs. Data are shown as the means (± SEM) of 3 experiments performed in triplicate. (B) For FACS analysis of the intracellular content of GM-CSF, sorted HSCs and HPCs were immediately fixed and permeabilized and subsequently stained with specific PE-labeled anti–GM-CSF antibody. Values represent the percentages of GM-CSF–producing cells. Data are from 1 representative experiment of 3. (C) Enhanced thymidine incorporation by Treg resulting from the coculture of CD4⁺CD25⁺-sorted Treg with HPCs and APCs at a 1:1:1 ratio, calculated as stimulation index. Cells at a 1:1:1 HPC:CD4⁺CD25⁺:APC cell ratio, 25 000 cells of each subset per well, were stimulated with the anti-CD3 mAb (clone 145-2C11) at 10 μg/mL for 96 hours, in the presence or absence of neutralizing anti-IL10R (clone 1B1.2), anti-TGFβ1 (clone 2G7), anti-IL2, (clone 1A12), and anti–GM-CSF (2 μg/mL, clone MP122E9; R&D Systems Europe, Lille, France) antibodies or control isotypes. Proliferation was measured by ³H-thymidine incorporation, 1 μCi/well, during the last 8 hours of culture.³  The stimulation index was calculated as the ratio of cpm in triplicate wells [a-b/b] where a and b stand for (CD25⁺ + HPCs + APCs) and (CD25⁺ + APCs), respectively. Results are from 1 representative experiment of 3. *P = .005, using nonparametric Mann-Whitney test. (D) CD4⁺CD25⁺ cells (2.5 × 10⁴ per well) were cultured with APCs at a 1:1 ratio in the presence of anti-CD3 mAb (clone 145-2C11) at 10 μg/mL for 96 hours and of increasing concentrations of recombinant GM-CSF. Proliferation was measured and stimulation index calculated as in panel C. Results are expressed as means (± SEM) of quadruplicate determinations in 1 representative experiment of 4. P < .02, 1-way ANOVA. (E) Stimulation of Treg expansion by 1 ng/mL GM-CSF, performed as in panel D, in the presence or absence of neutralizing anti-IL2 antibody (2 μg/mL). Results are expressed as means (± SEM) of triplicate determinations in 1 representative experiment of 3. N.S. (F) CD4⁺CD25⁺Foxp3^hi cells (1.25 × 10⁵ cells at day 0) cocultured with HPCs (ratio 1:1) together with anti-CD3/CD28–coated microbeads (8 beads per cell; Invitrogen, Cergy Pontoise, France) in complete medium supplemented with 100 U/mL IL2 (R&D Systems) during 5 days in presence (■) or absence (□) of 2 ng/mL GM-CSF were numerated, after magnetic bead removal, by measuring CD4, CD25, and Foxp3 cell expression by flow cytometry. Results are expressed as means (± SEM) of 4 experiments. P = .028, using nonparametric Mann-Whitney test. (G) GM-CSF Rα chain expression was measured by FACS in purified CD4⁺CD25⁺ Treg at 0 hour (dotted line), and after 65 hours of activation with coated anti-CD3 (10 μg/mL) and soluble anti-CD28 (5 μg/mL) in the presence (thick line) or absence (thin line) of 2 ng/mL GM-CSF, with or without (shadowed histogram) anti-CD116 primary antibody and FITC-anti–rabbit Fab'2. Results shown are from 1 representative experiment of 3.