Comparison of Fas(Apo-1/CD95)- and perforin-mediated cytotoxicity in primary T lymphocytes

Cytolytic T lymphocytes kill target cells by two independent cytolytic mechanisms. One pathway depends on the polarized secretion of granule-stored proteins including perform and granzymes, causing target cell death through membrane and DNA damage. The second cytolytic effector system relies on the interaction of the Fas ligand (FasL) on the effector cell with its receptor (Fas) on the target cell, leading to apoptotic cell death. Using mixed lymphocyte culture (MLC)-derived primary T lymphocytes of perforin-knockout and gld (with non-functional FasL) mice, the molecular basis of the two killing mechanisms was compared. The activity of both pathways was dependent on extracellular Ca2+. Incubation of MLC-stimulated primary T cells with protein synthesis inhibitors prior to TCR triggering impaired FasL cell surface expression and abolished cytolytic activity, although the cells exhibited an intracellular pool of FasL. The perforin-dependent mechanism induced cell death more rapidly, although both pathways ultimately showed similar killing efficiencies. Both pathways induced comparable levels of DNA degradation, but Fas-induced membrane damage was less pronounced. We conclude that upon TCR triggering FasL may be recruited in part from pre-existing intracellular stores. However, efficient induction of target cell death still depends on the continuous biosynthesis of FasL molecule

Characterization of the non-functional Fas ligand of gld mice

Mice homozygous for either the gld or Ipr mutation develop autoimmune diseases and progressive lymphadenopathy. The Ipr mutation Is characterized by the absence of unctional Fas, whereas gld mice exhibit an inactive FasL due to a point mutation proximal to the extracellular C-terminus. The structural repercussions of this amino acid substitution remain unknown. Here we report that FasL Is expressed at similar levels on the surface of activated T lymphocytes from gld and wild-type mice. Using a polyclonal anti-FasL antibody, Indistinguishable amounts of a 40 kDa protein are detected In both gld and wild-type splenocytes. The molecular model of FasL, based on the known structure of TNF-α, predicts that the Phe→Leu gld mutation is located at the protomer interface which Is close to the FasR Interaction site. We conclude that the gld mutation allows normal FasL biosynthesis, surface expression and ollgomerlzatlon, but induces structural alterations to the Fas binding region leading to the phenotypic changes observe

A role for the src family kinase Fyn in NK cell activation and the formation of the repertoire of Ly49 receptors

NK cell function is regulated by a dual receptor system, which integrates signals from triggering receptors and MHC class I-specific inhibitory receptors. We show here that the src family kinase Fyn is required for efficient, NK cell-mediated lysis of target cells, which lack both self-MHC class I molecules and ligands for NKG2D, an activating NK cell receptor. In contrast, NK cell inhibition by the MHC class I-specific receptor Ly49A was independent of Fyn, suggesting that Fyn is specifically required for NK cell activation via non-MHC receptor(s). Compared to wild type, significantly fewer Fyn-deficient NK cells expressed the inhibitory Ly49A receptor. The presence of a transgenic Ly49A receptor together with its H-2(d) ligand strongly reduced the usage of endogenous Ly49 receptors in Fyn-deficient mice. These data suggest a model in which the repertoire of inhibitory Ly49 receptors is formed under the influenced of Fyn-dependent NK cell activation as well as the respective MHC class I environment. NK cells may acquire Ly49 receptors until they generate sufficient inhibitory signals to balance their activation levels. Such a process would ensure the induction of NK cell self-tolerance

Cytolytic T-cell cytotoxicity is mediated through perforin and Fas lytic pathways

The recent generation of perforin knock-out mice has demonstrated a crucial role for the pore-forming perforin in cytolytic T-lymphocyte (CTL)-mediated cytolysis. Perforin-deficient mice failed to clear lymphocytic choriomeningitis virus in vivo, yet substantial killing activity still remained in perforin-free CTLs in vitro, indicating the presence of (a) further lytic pathway(s). Fas is an apoptosis-signalling receptor molecule on the surface of a number of different cells. Here we report that both perforin-deficient and Fas-ligand-deficient CTLs show impaired lytic activity on all target cells tested. The killing activity was completely abolished when both pathways were inactivated by using target cells from Fas-receptor-deficient lpr mice and perforin-free CTL effector cells. Fas-ligand-based killing activity was triggered upon T-cell receptor occupancy and was directed to the cognate target cell. Thus, two complementary, specific cytotoxic mechanisms are functional in CTLs, one based on the secretion of lytic proteins and one which depends on cell-surface ligand-receptor interaction