Figure 3

Antigen-stimulated T-cell death is dependent on disease severity and is mediated by B cells. CD19⁺ B cells were depleted from pooled splenic cells of severely, moderately, and mildly arthritic mice (see Table 1 for groupings), leaving enriched populations of T cells. B-depleted cells from each group were cultured for 7 days with cII_257-273 peptide and cocultured with purified B cells (1:1 ratio) from each group. Apoptosis was measured by Annexin V-based three-color flow cytometry as described in Materials and methods. Control T-cell apoptosis in parallel cultures without antigen ranged from 5.3% to 22.5%, followed a similar pattern between T-cell groups as shown above, was not stimulated by B cells, and was significantly lower than antigen-stimulated apoptosis for every combination. Data shown are mean ± standard error for triplicate samples. Statistical comparisons by Student t tests within groups are marked as ^§mild T versus severe T, ^¥mild T versus moderate T, and ^¶moderate T versus severe T. Comparisons of B cell-mediated death are made to the corresponding culture without B cells and are designated with an asterisk. One symbol = P < 0.05, two symbols = P < 0.02, three symbols = P < 0.01. AnnV, Annexin V; PI, propidium iodide.