Figure 5. Lamp-1 externalization and GZMB-Tom degranulation during CTL activation.

OT1 CTL from B6 (WT) and from GZMB-Tom-KI/KI mice were prepared from LN CD8 T cells by antigenic triggering and expanded with IL-2 as described in Materials and Methods. To measure Lamp-1 externalization, 10⁵ CTL were stimulated for 2h at 37°C with RMA-S target cells pre-loaded with the relevant OVA peptide (Rel P) at different concentrations (10⁻⁶–10⁻¹² M) or Irrelevant peptide (Irr P) at 10⁻⁶ M. Effector to target ratio was 3/1. a-Lamp-1 Ab was added to the activation medium (see Materials and Methods). FACS analysis was performed on cells gated as CD8 positive (CTL). One experiment with concordant duplicates is shown, and is representative of at least 3 experiments. A: Overlays of the analysis are represented for Lamp-1 versus GZMB-Tom for WT and GZMB-Tom-KI/KI OT1 CTL. B and C: Quantification of the experiments as % CTL positive for lamp-1 mAb uptake (B) and CTL tdTom Mean Fluorescence Intensity (MFI) (C). D: Overlays of the analysis are represented for all cells including CTL and RMA-S cells and are plotted as CD8 versus GZMB-Tom fluorescence. Quantification of the experiments gated on the RMA-S target cells as % cells positive for tdTomato (E) and tdTom MFI (F). Statistics are shown for values of Lamp-1 externalization for CTL incubated with RMA-S targets pre-loaded with different concentrations of relevant peptide versus irrelevant peptide (C), as well as for acquisition of tdTomato fluorescence as % (E) and as MFI (F) by RMA-S targets pre-loaded with different concentrations of relevant peptide versus irrelevant peptide. P<0.01 (**); P<0.05 (*); P>0.05 (NS) (see Materials and Methods).