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. 2016 Mar 2;7:67. doi: 10.3389/fimmu.2016.00067

Figure 4.

Figure 4

Induction of apoptosis of bystander CD4+ T cells. CD4+ T cells isolated from female NOD mice previously immunized with WTGAD65 peptide and expanded in vitro served as bystander CD4+ T cells. (A) These cells were CFSE stained and cocultured with APCs loaded with WTGAD65 peptide (left column) showing basal mortality rate measured by Annexin V binding (vertical axis). cCD4+ T cells generated with CCGAD65 peptide were added to this coculture (right column). A control well was added in which the cCD4+ T cells were replaced with the same number of unlabeled CD4+ T cells generated with WTGAD65 (middle column). Error bars represent 1 SD. Statistical significance was calculated with one-way ANOVA test, P < 0.0001 followed by Dunnett’s multiple comparison test as indicated, *P < 0.05. (B) Same CFSE-stained cell line was used in this setting in coculture with APCs and cCD4+ T cells generated with CCGAD65 in the presence of the indicated peptide and mortality was measured by Annexin V expression (vertical axis). Hatched column 4 shows the effect of FasL antibody addition to the coculture, cross-hatched column 5 shows addition of GZB inhibitor, and black column 6 shows combination of both blockers. Error bars represent 1 SD. ****P < 0.0001 (One-way ANOVA test and Dunnett’s multiple comparison test). Data are representative of three independent experiments.