Jagan R. Muppidi

Ligand-independent redistribution of Fas (CD95) into lipid rafts mediates clonotypic T cell death

Clonotypic elimination of activated T cells through Fas–Fas ligand (CD95–CD95L) interactions is one mechanism of peripheral self-tolerance. T cell receptor (TCR) stimuli trigger FasL synthesis but also sensitize activated T cells to Fas-mediated apoptosis through an unknown mechanism. Here we show that TCR restimulation of activated human CD4+ T cells resulted in Fas translocation into lipid raft microdomains before binding FasL, rendering these cells sensitive to apoptosis after stimulation with bivalent antibody or FasL. Disruption of lipid rafts reduced sensitivity to Fas-mediated apoptosis after TCR restimulation. Thus, the redistribution of Fas and other tumor necrosis factor family receptors into and out of lipid rafts may dynamically regulate the efficiency and outcomes of signaling by these receptors.

SPOTS signaling protein oligomeric transduction structures are early mediators of death receptor–induced apoptosis at the plasma membrane

Fas (CD95, APO-1, TNFRSF6) is a TNF receptor superfamily member that directly triggers apoptosis and contributes to the maintenance of lymphocyte homeostasis and prevention of autoimmunity. Although FADD and caspase-8 have been identified as key intracellular mediators of Fas signaling, it is not clear how recruitment of these proteins to the Fas death domain leads to activation of caspase-8 in the receptor signaling complex. We have used high-resolution confocal microscopy and live cell imaging to study the sequelae of early events in Fas signaling. These studies have revealed a new stage of Fas signaling in which receptor ligation leads to the formation of surface receptor oligomers that we term signaling protein oligomerization transduction structures (SPOTS). Formation of SPOTS depends on the presence of an intact Fas death domain and FADD but is independent of caspase activity. Analysis of cells expressing Fas mutations from patients with the autoimmune lymphoproliferative syndrome (ALPS) reveals that formation of SPOTS can be disrupted by distinct mechanisms in ALPS.

Measurement of Apoptosis and Other Forms of Cell Death

As programmed cell death (PCD) or apoptosis has emerged as an important regulator of development and homeostasis in multicellular organisms, methods to quantify apoptosis and to distinguish it from necrosis have been developed. This unit presents a set of assays for these purposes, many of which are technically very simple and ideally suited to the study of hematopoietic cells. The first basic protocol allows the qualitative and quantitative assessment of apoptosis in lymphocyte cell cultures using light or fluorescent microscopy. Three protocols follow that are designed to detect nuclear DNA fragmentation and support protocols describe methods to radiolabel the DNA and cytoplasm of the cells to be tested. Techniques that quantitate apoptotic cells using flow cytometry are then described and support protocols provide methods for priming T cell clones and freshly isolated lymph node cells, respectively, for T cell receptor (TCR)‐induced apoptosis. Quantitative detection of DNA fragmentation in apoptotic cells is also described. TdT‐mediated dUTP‐biotin nick end‐labeling (TUNEL) methods are provided for the detection of apoptotic cells, along with procedures for the flow cytometric quantitation of apoptotic cells using TUNEL, and TUNEL, staining of tissue sections to identify apoptotic cells. Since much remains incompletely understood about the molecular pathways of programmed death, and it is probably best to perform more than one of the basic protocols to confirm an observation of apoptotic cell death.

Homotypic FADD interactions through a conserved RXDLL motif are required for death receptor-induced apoptosis

Death receptors in the TNF receptor superfamily signal for apoptosis via the ordered recruitment of FADD and caspase-8 to a death-inducing signaling complex (DISC). However, the nature of the protein–protein interactions in the signaling complex is not well defined. Here we show that FADD self-associates through a conserved RXDLL motif in the death effector domain (DED). Despite exhibiting similar binding to both Fas and caspase-8 and preserved overall secondary structure, FADD RDXLL motif mutants cannot reconstitute FasL- or TRAIL-induced apoptosis and fail to recruit caspase-8 into the DISC of reconstituted FADD-deficient cells. Abolishing self-association can transform FADD into a dominant-negative mutant that interferes with Fas-induced apoptosis and formation of microscopically visible receptor oligomers. These findings suggest that lateral interactions among adapter molecules are required for death receptor apoptosis signaling and implicate self-association into oligomeric assemblies as a key function of death receptor adapter proteins in initiating apoptosis.

Cutting Edge: Rac GTPases Sensitize Activated T Cells to Die via Fas

In activated CD4+ T cells, TCR restimulation triggers apoptosis that depends on interactions between the death receptor Fas and its ligand, FasL. This process, termed restimulation-induced cell death (RICD), is a mechanism of peripheral immune tolerance. TCR signaling sensitizes activated T cells to Fas-mediated apoptosis, but what pathways mediate this process are not known. In this study we identify the Rho GTPases Rac1 and Rac2 as essential components in restimulation-induced cell death. RNA interference-mediated knockdown of Rac GTPases greatly reduced Fas-dependent, TCR-induced apoptosis. The ability of Rac1 to sensitize T cells to Fas-induced apoptosis correlated with Rac-mediated cytoskeletal reorganization, dephosphorylation of the ERM (ezrin/radixin/moesin) family of cytoskeletal linker proteins, and the translocation of Fas to lipid raft microdomains. In primary activated CD4+ T cells, Rac1 and Rac2 were independently required for maximal TCR-induced apoptosis. Activating Rac signaling may be a novel way to sensitize chronically stimulated lymphocytes to Fas-induced apoptosis, an important goal in the treatment of autoimmune diseases.

Death receptor signaling and autoimmunity.

In recent years, it has become clear that self-nonself discrimination by the immune system is driven not so much by the specificities of the antigen receptors themselves, but by ligand-receptor systems that sense the presence of foreign pathogens (toll-like receptors) and those that regulate the balance between cellular proliferation and programmed cell death (tumor necrosis factor [TNF] family ligands and receptors). Interestingly, these two receptor families share a number of common signaling pathways, mediated by the cytoplasmic proteins containing death domains and TRAF domains, which trigger the complementary processes of programmed cell death and inflammation. Both humans and mice with genetic defects in the TNF-receptor family member Fas accumulate abnormal lymphocytes and develop systemic autoimmunity. These findings high-lighted the importance of this TNF-receptor family member in the homeostasis of the immune system. In particular, the Fas receptor has been shown to be important in immunoreceptor-mediated apoptosis of activated T and B lymphocytes. Six members of the TNF-receptor superfamily share a common signaling domain with Fas, termed the death domain, that directly links these receptors to the apoptotic machinery of the cell, and, collectively, these receptors have been designated as “death receptors”. We are currently investigating a number of important unresolved issues in this field, including: (1) how susceptibility to apoptosis through death receptors is regulated, (2) how Fas and related death receptors function in the maintenance of self-tolerance and homeostasis in the major cell types of the immune system, and (3) recently described nonapoptotic lymphocyte activation signals that use components of death receptor signaling.