Distinct roles of cytolytic effector molecules for antigen-restricted killing by CTL in vivo

Abstract

Cytotoxic T lymphocytes (CTLs) represent one of the front lines of defense for the immune system, killing virus-infected and tumor-transformed cells. CTL use at least two mechanisms to induce apoptosis in their targets, one mediated by perforin and granzymes, and the other triggered by the death ligand, CD95 ligand (CD95L). Here, we used an in vivo cytotoxicity assay to measure specific clearance of antigen-bearing target cells in mice that had previously been immunized with noninfectious cell-associated antigens. We found that perforin was dispensable for efficient clearance of antigen-bearing cells from immunized mice, but only if CD95/CD95L was functional; however, there was a delay in target cell clearance in the absence of perforin. In addition, we observed ∼35% target cell clearance in the absence of both perforin and CD95L, which was only slightly abrogated in the presence of a neutralizing anti-tumor necrosis factor (TNF) antibody. The presence of a dominant negative Fas-associated death domain (FADD) did not block target cell clearance and therefore cannot be attributed to known death receptors. Taken together, these data suggest that perforin- and CD95L-dependent killing are complementary at early time points, each can compensate for the absence of the other at later time points, and that there is an additional component of antigen-restricted CTL killing independent of perforin, CD95L, and TNFα.

Figure 1. CTL-mediated killing is restricted to Ag-bearing targets

A) Splenocytes from Ad5E1-MEC-immunized mice were expanded in vitro and cultured with ³[H]labeled target cells from WT and CD95^−/− mice that were pulsed with E1B_192–200 (Ag) peptide. OVA_257–264 pulsed non-labeled cells were used as negative control. Specific killing was assessed by JAM assay after 4 hours. B) Non-specific “bystander” killing was assessed by JAM assay as described above using ³[H]labeled cells without Ag (bystander) in the presence of non-³[H]labeled Ag-bearing target. Data are expressed as mean +/− s.e.m. (n=3 per group, representative experiment of 3 experiments).

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).