Figure 4. Allogeneic T cell anergy induction requires two stimulations by lowGM-DC.

A. Allogeneic T cells were stimulated twice (for 5 days and then for another 3 days) with either immature lowGM-DC or immature highGM+IL-4-DC or mature highGM+IL-4-DC and then restimulated a third time for another 3 days with IL-2 or the indicated antibodies or LPS-matured DC from the original allotype (matDC allo) or a third party allotype (matDC 3rd) or cells were left without stimulation (control) for another 3 days. B. Then [³H]-thymidine ([³H]-Th.) was added overnight to measure proliferation. The data are representative of 4 independent experiments. C. Supernatants from the cultures shown in Figure 4B were tested for their content of IL-2, IFN-γ, or IL-10 by cytokine bead array (CBA). The relative mean fluorescence values of the FACS analysis are shown. The data from one experiment shown are representative for 3 independent experiments. Values in bar graphs represent the mean±standard deviation error bars of triplicate cultures from one experiment. D. CFSE-labeled T cells were restimulated by the indicated DC type for 4 days and then stained for intracellular expression of Foxp3. Cells were measured by flow cytometry. Gating was performed on CFSE⁺ cells and is plotted against Foxp3 or isotype. CFSE^low cells represent allo-responsive proliferated cells.