Fig. 2

Residual perforin bound to CTLs is deactivated. a Binding of WT-GFP-PRF to EL4 (CD8⁻) and CTL (CD8⁺) cells, at concentrations that are sub-lytic to CTLs, as assessed by flow cytometry. At 4 °C and in the presence of Ca²⁺ (−EGTA), both cell types bind perforin (GFP⁺), but the CTLs at a lower level than EL4 cells. At 37 °C and in the presence of Ca²⁺ (−EGTA), WT-GFP-PRF is lytic to the EL4 cells, so no significant CD8⁻ population remains under those conditions (asterisks). Upon subsequent Ca²⁺ chelation by 2 mM EGTA, non-porating perforin assemblies (WT-GFP-PRF at 4 °C) are removed from the cell membranes (GFP⁻). However, Ca²⁺ chelation does not affect WT-GFP-PRF bound to CTLs at 37 °C (GFP⁺, dagger), despite WT-GFP-PRF being apparently non-porating (see Fig. 1c). b Binding of non-lytic TMH1-GFP-PRF to EL4 (CD8⁻) and CTL (CD8⁺) cells, as assessed by flow cytometry. In the presence of Ca²⁺ (−EGTA), TMH1-GFP-PRF binds to both cell types, but less to CTLs, as observed for WT-GFP-PRF. Upon subsequent Ca²⁺ chelation by 2 mM EGTA (+EGTA), TMH1-GFP-PRF is invariably removed from the cell membranes, leaving only GFP⁻ populations (both at 4 °C and 37 °C). c Quantification of flow cytometry results, with MFI of WT-GFP-PRF and TMH-GFP-PRF signals, normalized to the respective MFIs for the CD8⁻ population at 4 °C. EGTA exposure drastically reduces the GFP MFI for all cases at 4 °C and for (non-lytic) TMH1-GFP-PRF at 37 °C. By contrast, WT-GFP-PRF is EGTA resistant once bound to CD8⁺ cells at 37 °C. Note that the low GFP MPI at 37 °C in the first panel (CD8⁻, WT-GFP-PRF) is due to the majority of the CD8⁻ cells being killed and thus not yielding a GFP signal in the flow cytometer anymore. The TMH1-GFP-PRF data (−EGTA) allow for a quantitative comparison between binding to CD8⁺ and CD8⁻ cells, showing that the CTLs (CD8⁺) bind less than half as much perforin as the target cells (CD8⁻). Each column represents a mean (±s.e.m.) of three independent flow cytometry experiments. Source data for c are provided as a Source Data file.