Figure 5.

Antigen-specific killing with TT peptide. Peripheral blood mononuclear cells were harvested from a repeat donor from whom we had previously generated an LCL. This donor had received a scheduled vaccination against tetanus <1 month prior to the experiment shown. PBMCs were cultured in the presence of an immunodominant peptide of the tetanus toxoid for 12 days. CD4⁺ T cells were then isolated by negative selection and cultured overnight with or without autologous LCL-derived exosomes (156 μg/mL) and in the presence or absence of the tetanus toxoid peptide. Neutralizing anti-human FasL antibodies (10 μg/mL) were added to some wells to block interactions between exosome FasL and Fas on the target cells. Apoptosis in activated CD4⁺CD62L^neg T cells was assessed by annexin-V/propidium iodide staining. Two additional experiments with the same donor cells showed the similar trends but were less robust, presumably due to the loss of tetanus-specific T cells in vivo as the time after vaccination increased. (A) Representative contour plots of annexin-V and propidium iodide staining among CD4⁺CD62L^low-gated T cells. (B) Frequency of annexin-V⁺ cells (mean ± SEM of triplicate samples) among CD4⁺CD62L^low T cells. *p < 0.05.