Figure 1. Activated T cells release GrB into supernatants which cause neuronal toxicity. Peripheral blood mononuclear cells (PBMC) or sorted CD4⁺ or CD8⁺ cells were cultured for 3 days in Iscove’s modified Dulbecco’s Medium +5% human serum. In parallel, these cells were incubated with anti-CD3/CD28 (cells:beads, 10:1) mAb-conjugated magnetic beads to induce polyclonal activation (Ac). Supernatants (sups) were then collected for GrB detection. Both activated CD4⁺ and CD8⁺ T cells released significant high amounts of GrB into the sups as determined by GrB immunoblot (A). Cultured human fetal neurons were treated with culture supernatants (1:10 in Locke’s buffer) from unsorted human T cells. Control cultures were treated with supernatants from unactivated T cells. To determine the role of GrB, activated T cell supernatant (AcT) was firstly immunodepleted with Ab against GrB (GrB-Ab) or control mouse IgG bound to protein A beads before treatment. Neurotoxicity was determined by calculating the percentage of cells positive with trypan blue 48 h later (B). Data represent the mean ± SEM of 3 replicates from 3 independent experiments. Image representative for 3 independent experiments is shown.