Figure 7

Time-course analysis of the polyclonal and antigen-specific Treg response to the combined optimized treatment. Unless otherwise indicated in the figure, analysis of the spleen is shown. Seven to 11 weeks-old NOD.Foxp3^EGFP animals (n = 3–6/time point) received the combined optimized treatment (arrows indicate days of immunization with A^g7/2.5 mi tetramer and/or IL-2:mAb complexes and shaded areas inside the graphs indicated the whole duration of the treatment) and were sacrificed on day 1, 3, 5, 7 or 15 after the last injection. Spleen, pancreatic lymph nodes (PaLN) and pancreatic islets were isolated from mice and analyzed by tetramer staining. (A) Tconv (CD4⁺Foxp3⁻2.5 mi⁺) expansion. (B) Antigen-specific Treg (CD4⁺Foxp3⁺2.5 mi⁺) expansion. (C) Polyclonal Foxp3⁺CD4⁺ Treg expansion. Cells in (A,B) and (C) were gated on CD19⁻, CD8⁻, CD11c⁻, F4/80⁻, propidium iodide (PI)⁻ and CD4⁺. Mean ± SEM of two independent experiments is depicted. No A^g7/GPI-specific T cell expansion was observed. (D) Sorted CD4⁺Foxp3⁻ T cells from 11 to 12 weeks-old treated (combined optimized treatment) or from naive NOD.Foxp3^EGFP mice were plated along with total splenocytes from 6–9 weeks-old NOD.SCID mice (as APC) and stimulated with 2.5 mi synthetic peptide. Supernatants were collected after 3 days and IL-10 quantified via ELISA. Data (mean ± SEM) is representative of two independent experiments; each point was collected in triplicate per experiment. ***p < 0.0005.