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Just over twenty years ago, Shige Nagata and colleagues forged an important link between defective apoptosis and autoimmunity when they discovered that loss–offunction mutations in the gene encoding the TNF family cell surface receptor Fas/CD95 formed the genetic basis of the syndrome of lymphoproliferation and autoimmunity in the lpr mouse strain (1). Even before the linkage to Fas mutations, lpr mice had served as a model for human systemic lupus erythematosus (SLE). However, the discovery of mutations in Fas not only provided a molecular explanation for the lpr phenotype, but also inspired investigations that led to the discovery of Fas mutations in human familial autoimmune diseases and spurred research that discovered the molecular mechanisms underlying Fas-induced apoptosis. Recent findings have expanded the role of Fas beyond simply inducing apoptosis and prompted a re-examination of the original premise that autoimmunity in the context of Fas deficiency results simply from defective immune cell death.
Homozygous lpr/lpr mutant mice spontaneously produce a wide variety of autoantibodies to nuclear antigens with a striking resemblance to those found in the sera of patients with SLE (2). Massive lymphadenopathy and splenomegaly develop in these mice, hence the name lymphoproliferation (lpr). The primary cell type accumulating in the lymph nodes and spleen are αβTCR-expressing T cells lacking CD4 and CD8 (termed ‘double negative’ T cells, or DNT) and additionally expressing the CD45 isoform B220. These T cells are oligoclonal but not malignant, and besides being thymus dependent, their origins remain obscure. DNT are unlikely to be the cells that provide help for autoreactive B cells because they are anergic to TCR stimulation and poor producers of cytokines. Reducing the T cell repertoire with a TCR transgene eliminates production of DNT cells but not autoantibody production, showing that DNT cells are not required for autoimmunity in lpr mice (3). Conventional T cells, especially with a memory phenotype, and B cells also accumulate to greater than normal numbers in lpr mice. Background genes are important modifiers of the lpr phenotype, as renal disease and other organ manifestations such as arthritis, vasculitis, and salivary gland and skin inflammation primarily develop in lpr mice back-crossed onto the MRL genetic background (2, 4).
Once Nagata’s group had cloned the mouse Fas locus (5), the identification of Fas mutations in lpr mice using the genetic techniques available at that time was relatively straightforward. As described by Nagata in a 2004 interview, he usually tried to map the loci of genes cloned in the lab to see if there was any relevance to disease (6). Collaborators Nancy Jenkins and Neal Copeland at NIH mapped the mouse Fas locus to a location on chromosome 19, close to where the lpr locus had previously been mapped (7). Rather than proceeding to positional cloning and sequencing, which would likely have taken additional years of effort, the authors made the leap to directly test for Fas expression by northern blotting and immediately hit the genetic ‘jackpot,’ finding that cells from lpr/lpr mice expressed almost no detectable Fas mRNA. Although Watanabe-Fukunaga et al. found alterations in the Fas genomic locus in lpr mice by southern blotting, the exact nature of the genetic lesion was elucidated by Keith Elkon’s group, which showed that Fas transcription was disrupted in the lpr locus by a retrotransposon insertion (8). Watanabe-Fukunaga et al. did solve the mystery of another Fas allele, lprcg, which turned out to be a missense mutation in exon 9, which encodes the death domain. The Fas lprcg mutant protein was non-functional for apoptosis induction. The lprcg allele also had the interesting property of being able to complement the gld (generalized lymphadenopathy) locus with a similar phenotype. This suggested that the gld locus was functionally linked to Fas, and in 1994, Nagata’s group, which had recently cloned the Fas ligand (FasL) gene (9), and a team at Immunex and Duke University identified disabling point mutations in the extracellular domain of FasL as the cause of the gld syndrome (10, 11).
Although Watanabe-Fukunaga et al. speculated that Fas mutations identified in lpr syndrome may also cause human autoimmune conditions, no human diseases linked to either Fas mutations or defective Fas-mediated apoptosis had so far been described. Just at that time however, clinical investigators were evaluating patients who turned out to have an immune disorder remarkably similar to that seen in lpr mice. The findings linking the lpr phenotype to Fas mutations undoubtedly accelerated the discovery of Fas mutations responsible for human disease. At the National Institutes of Health’s Clinical Center in Maryland, virologist Steven Straus was referred a number of patients with chronic lymphadenopathy for evaluation of possible EBV infection as a factor in their disease. Characterization of these patients in collaboration with Mike Sneller, a fellow in Warren Strober’s group at NIH, revealed a significant pattern of autoimmunity, primarily autoantibody-mediated hemolytic anemia, thrombocytopenia, and splenomegaly and lymphadenopathy with underlying polyclonal expansion of B cells and αβTCR-positive T cells lacking CD4 and CD8 (12). In this paper, the authors presciently speculated about the similarities between these patients and lpr and gld mice.
The discovery linking Fas to the lpr mouse phenotype led to discussions at NIH about the possibility of Fas mutations and apoptosis defects in this syndrome with immunologist Mike Lenardo, a discoverer of the phenomenon of TCR-induced apoptosis, who demonstrated an apoptosis defect in the patient cells. The team was joined by geneticist and clinical immunologist Jennifer Puck, who showed that missense mutations in Fas coding sequences clustering in the death domain were inherited in an autosomal dominant fashion in five independent families with this disorder, which was named the Autoimmune Lymphoproliferative Syndrome, or ALPS (13). Expression of the mutant protein blocked Fas-induced apoptosis in normal cells, and T cells from ALPS patients also failed to undergo death due to restimulation through the TCR (Restimulation-Induced Cell Death, or RICD) (13). These data confirmed findings about the role of Fas in RICD in CD4+ T cells made around the same time (14–16). Independently, Frederic Rieux-Laucat and colleagues in Paris (17) and Keith Elkon’s group in New York (18) identified Fas mutations in similar groups of patients, some of whom had been described in the 1960’s as having Canale-Smith syndrome, with many of the same clinical features as what became known as ALPS. Cohorts of patients with ALPS were subsequently described around the world (19–21). Today, there are likely to be at least 500 families, about 80% of whom have Fas mutations, with the clinical syndrome of ALPS fitting recently revised diagnostic criteria (22). While most Fas mutations in ALPS are inherited, some patients with a similar clinical syndrome but without germline Fas mutations were found to have somatic point mutations in Fas that likely underlie their disease (23). Mutations found in other non-Fas-related genes in ALPS patients have further shed light on apoptosis signaling pathways (22). Ironically, despite lpr mice serving as a model for systemic lupus in humans, investigation of mutations or polymorphisms in Fas in patients with SLE and unbiased whole genome association studies of SLE susceptibility loci have not yielded evidence of common or rare genetic variants in Fas that drive susceptibility to lupus. Although its usefulness as a single-gene model of autoimmunity was not diminished, these findings made it clear that lpr is a mouse model for ALPS, rather than lupus.
The discoveries that Fas mutations can cause genetic autoimmune disease in both mouse and man triggered intensive study of this receptor and the molecular basis of transmembrane signaling initiated by Fas. Within the next four years, the essential components of the “Death-inducing Signaling Complex” (DISC) that are recruited to the Fas death domain (24) were elucidated. The adaptor protein FADD and the cysteinyl aspartic protease, caspase-8, were found to be essential components required for Fas-induced apoptosis (25–27). Aggregation in the Fas DISC activates caspase-8, which in turn catalyzes the cleavage of downstream or “effector” caspase-3, activation of which results in irreversible cell death. In some cells, amplification of the cell death signal through the mitochondria is also required (28). Fas and FasL can form homotrimers, but a number of lines of evidence suggest that formation of receptor oligomers beyond the 3:3 complex of Fas with FasL is critical for effective activation of Fas apoptotic signaling. Only membrane-bound FasL is capable of triggering active downstream receptor complexes and soluble FasL cannot induce Fas-induced apoptosis in vitro or in vivo (29, 30). The crystal structure of Fas and FADD consists of five receptor oligomers, suggesting at least a dimer of Fas trimers as the minimal active signaling complex (31), and microscopically visible clusters of receptors are seen after receptor ligation (32, 33). Fas clustering and efficient signaling is supported by receptor localization in lipid rafts, which is mediated by palmitoylation of a membrane-proximal cysteine (34–36), and pre-association of receptor chains is mediated by a separate domain from ligand binding (37).
Despite their essential role in Fas apoptotic signaling, analysis of the in vivo role of FADD and caspase-8 has revealed additional unexpected functions. FADD- and caspase-8-deficient mice were found to be embryonically lethal and revealed that these molecules were also required for efficient hematopoietic and T cell development (38–40). Patients homozygous for a hypomorphic mutation in caspase-8 had reduced Fas-induced apoptosis in their T cells, but also significant immunodeficiency and T cell activation defects distinct from those of Fas-deficient ALPS patients (41). A role for FADD and caspase-8 in preventing programmed necrosis has recently provided an explanation for these apparently paradoxical functions (42–45), as FADD- or caspase-8- deficient cells may default into programmed necrosis and become eliminated more easily when Fas or other activating stimuli are given.
Apart from preventing programmed necrosis, Fas can deliver signals that oppose cell death in a number of contexts (46). Fas can costimulate T cell activation (47, 48), and non-apoptotic Fas signaling contributes to liver regeneration in partial hepatectomy models (49, 50). Fas is expressed on most primary T cells after activation, but only a small fraction of T cells, primarily those with an effector memory phenotype, are highly susceptible to Fas-induced apoptosis (51, 52). It has become clear that the intrinsic cell death machinery, regulated primarily by the bcl-2 family of proteins, particularly the pro-apoptotic BH3 domain-containing protein bim, controls apoptosis independently of Fas during thymic negative selection and the clonal contraction of T cells after acute antigenic stimulation (53, 54). Fas-FasL interactions are only required for the elimination of T cells responding to repeatedly administered antigens such as occurs during chronic infections (14, 55, 56). The non-apoptotic pathway may be the dominant function in Fas-expressing tumor cells where Fas is highly expressed, and loss of Fas in hepatic and ovarian tumors can result in tumor regression in mouse models (57). These findings brought the understanding of Fas and apoptosis signaling full circle from its initial discovery as a receptor for Abs that induce apoptosis in tumor cells (58).
Even with all these advances over the past two decades, a number of fundamental questions about how Fas prevents autoimmunity still remain to be addressed. Although the apoptotic defect in Fas-deficient cells is easy to demonstrate in vitro in activated T cells, it is not clear that this defect is responsible for the loss of self-tolerance that results in autoimmunity in Fas deficiency. Experiments with mice lacking Fas expression specifically in T cells, B cells, or dendritic cells revealed that autoimmunity can result from deletion of Fas in any of these compartments (59, 60), though deletion of Fas in any single cell lineage could not reproduce the syndrome of complete Fas deficiency. Less is known about how Fas signaling is regulated in B cells and other cell types. In another of their many contributions to the understanding of the biology of Fas/CD95, Nagata’s group discovered that anti-Fas Abs cause lethal hepatic necrosis due to engagement of Fas on hepatocytes, limiting the therapeutic use of anti-Fas Abs to eliminate autoreactive immune cells (61). Thus, understanding how cells control whether they die, survive, or proliferate after Fas engagement, and what other signals influence this cell fate decision, remains important for the design of immunotherapies that target this remarkable receptor (62).
We would like to thank Mike Lenardo and Mike Sneller for discussions about the history of the discovery of ALPS and Fas mutations in humans, and Vera Siegel for proofreading the manuscript.