Figure 7

Effect of ATG therapy on self antigen-specific T cells in vivo. A, Eight weeks old NOD mice were treated with ATG or isotype IgG along with intraperitoneal injections of β cell antigens NIT1 lysates as described in the Method. A week post treatment, all mice were sacrificed and spleen cells were prepared. Spleen cells (1 × 10⁶/well) were incubated with NIT1 lysates, KLH (un-related antigen) or medium alone. Triplicate wells were set up for each incubation. T cell proliferation was measured by ³H-thymidine incorporation assay as described in the Methods section. Three mice were included in each group. Data shown represent the average of three mice+/−standard deviation (SD). B, Part of the spleen cells were used to isolate CD4+ T cells and the isolated CD4+ T cells (2 × 10⁵/well) were incubated with splenic dendritic cells (2 × 10⁴/well) purified from naive NOD mice in the presence of NIT1 lysates (20 μl/well). Triplicate wells were set up for each incubation. The proliferation of CD4+ T cells was measured by ³H-thymidine incorporation assay. Three mice were included in each group. Data shown represent the average of three mice+/−SD. C, Part of the mixed spleen cells from the above 3 ATG or 3 isotype IgG treated animals were stained with CSFE. Then, the CFSE-labeled spleen cells were intravenously injected into 8 weeks old naive NOD mice (3 mice per group). Four days later, the cells prepared from inguinal and pancreatic lymph nodes were stained with CD4-PerCp. The percentages of the proliferating CFSE-labeled CD4+ T cells (dilution of CFSE) were examined by flow cytometry. D, cytokines IFN-γ and IL-10 in the cultures shown in B were measured by luminex. Data shown represent average values of 3 mice+/−SD. An additional experiment was performed with similar results.