Figure 2. Perforin is dispensable for Ag-dependent killing in vivo and both perforin and CD95/CD95L are required for optimal MHC I-restricted killing of Ag-bearing targets in vivo.

A) Ad5E1-MEC immunized WT and perforin^−/− mice received equal numbers of CFSE^high Ag-pulsed targets and CFSE^med control cells. Twenty hours later clearance of Ag-pulsed target cells was determined in the spleen by flow cytometry, using CFSE^med control cells as internal standard. Data are shown from one naïve and immunized recipient mouse of each strain and are representative of three separate experiments with n=3–4 per group. B) Ad5E1-MEC immunized WT and perforin^−/− mice received equal numbers of CFSE^high Ag-pulsed targets and CFSE^med control cells from indicated mouse strains. Twenty hours later clearance of Ag-pulsed target cells was determined in the spleen by flow cytometry, using CFSE^med control cells as internal standard. C) Four hours prior to transfer of labeled target and control cells, immunized animals were administered neutralizing anti-TNF antibody or isotype control. Specific clearance Ag-bearing wild type and lpr target cells in wild type and perfrin-null recipient mice in the presence or absence of neutralizing anti-TNF was assessed 20 hours after transfer. D) Clearance of FADDdd^lckexpressing targets in immunized WT and perforin^−/− mice was determined. Killing of FADDdd^lck target cells was further dissected according to CD3⁺ targets that expressed FADDdd^lck and CD3- target cells that did not express the transgene. Clearance of non-lck expressing targets cells from FADDdd^lck mice was comparable to WT targets. E) Kinetics of perforin and CD95/CD95L mediated clearance. Immunized WT and perforin^−/− mice received WT or CD95^−/− targets and AG-specific killing was determined at indicated time-points. Data are expressed as mean +/− s.e.m. (n=3 per group, representative experiment of 3 experiments).