Anthony C. Cruz

The Fas-FADD death domain complex structure reveals the basis of DISC assembly and disease mutations

The death-inducing signaling complex (DISC) formed by the death receptor Fas, the adaptor protein FADD and caspase-8 mediates the extrinsic apoptotic program. Mutations in Fas that disrupt the DISC cause autoimmune lymphoproliferative syndrome (ALPS). Here we show that the Fas–FADD death domain (DD) complex forms an asymmetric oligomeric structure composed of 5–7 Fas DD and 5 FADD DD, whose interfaces harbor ALPS-associated mutations. Structure-based mutations disrupt the Fas–FADD interaction in vitro and in living cells; the severity of a mutation correlates with the number of occurrences of a particular interaction in the structure. The highly oligomeric structure explains the requirement for hexameric or membrane-bound FasL in Fas signaling. It also predicts strong dominant negative effects from Fas mutations, which are confirmed by signaling assays. The structure optimally positions the FADD death effector domain (DED) to interact with the caspase-8 DED for caspase recruitment and higher-order aggregation.

Concerted action of wild-type and mutant TNF receptors enhances inflammation in TNF receptor 1-associated periodic fever syndrome

TNF, acting through p55 tumor necrosis factor receptor 1 (TNFR1), contributes to the pathogenesis of many inflammatory diseases. TNFR-associated periodic syndrome (TRAPS, OMIM 142680) is an autosomal dominant autoinflammatory disorder characterized by prolonged attacks of fevers, peritonitis, and soft tissue inflammation. TRAPS is caused by missense mutations in the extracellular domain of TNFR1 that affect receptor folding and trafficking. These mutations lead to loss of normal function rather than gain of function, and thus the pathogenesis of TRAPS is an enigma. Here we show that mutant TNFR1 accumulates intracellularly in peripheral blood mononuclear cells of TRAPS patients and in multiple cell types from two independent lines of knockin mice harboring TRAPS-associated TNFR1 mutations. Mutant TNFR1 did not function as a surface receptor for TNF but rather enhanced activation of MAPKs and secretion of proinflammatory cytokines upon stimulation with LPS. Enhanced inflammation depended on autocrine TNF secretion and WT TNFR1 in mouse and human myeloid cells but not in fibroblasts. Heterozygous TNFR1-mutant mice were hypersensitive to LPS-induced septic shock, whereas homozygous TNFR1-mutant mice resembled TNFR1-deficient mice and were resistant to septic shock. Thus WT and mutant TNFR1 act in concert from distinct cellular locations to potentiate inflammation in TRAPS. These findings establish a mechanism of pathogenesis in autosomal dominant diseases where full expression of the disease phenotype depends on functional cooperation between WT and mutant proteins and also may explain partial responses of TRAPS patients to TNF blockade.

Memory T cell–driven differentiation of naive cells impairs adoptive immunotherapy

Adoptive cell transfer (ACT) of purified naive, stem cell memory, and central memory T cell subsets results in superior persistence and antitumor immunity compared with ACT of populations containing more-differentiated effector memory and effector T cells. Despite a clear advantage of the less-differentiated populations, the majority of ACT trials utilize unfractionated T cell subsets. Here, we have challenged the notion that the mere presence of less-differentiated T cells in starting populations used to generate therapeutic T cells is sufficient to convey their desirable attributes. Using both mouse and human cells, we identified a T cell-T cell interaction whereby antigen-experienced subsets directly promote the phenotypic, functional, and metabolic differentiation of naive T cells. This process led to the loss of less-differentiated T cell subsets and resulted in impaired cellular persistence and tumor regression in mouse models following ACT. The T memory-induced conversion of naive T cells was mediated by a nonapoptotic Fas signal, resulting in Akt-driven cellular differentiation. Thus, induction of Fas signaling enhanced T cell differentiation and impaired antitumor immunity, while Fas signaling blockade preserved the antitumor efficacy of naive cells within mixed populations. These findings reveal that T cell subsets can synchronize their differentiation state in a process similar to quorum sensing in unicellular organisms and suggest that disruption of this quorum-like behavior among T cells has potential to enhance T cell-based immunotherapies.

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.

The TNF-Family Ligand TL1A and Its Receptor DR3 Promote T Cell–Mediated Allergic Immunopathology by Enhancing Differentiation and Pathogenicity of IL-9–Producing T Cells

The TNF family cytokine TL1A (Tnfsf15) costimulates T cells and type 2 innate lymphocytes (ILC2) through its receptor DR3 (Tnfrsf25). DR3-deficient mice have reduced T cell accumulation at the site of inflammation and reduced ILC2-dependent immune responses in a number of models of autoimmune and allergic diseases. In allergic lung disease models, immunopathology and local Th2 and ILC2 accumulation is reduced in DR3-deficient mice despite normal systemic priming of Th2 responses and generation of T cells secreting IL-13 and IL-4, prompting the question of whether TL1A promotes the development of other T cell subsets that secrete cytokines to drive allergic disease. In this study, we find that TL1A potently promotes generation of murine T cells producing IL-9 (Th9) by signaling through DR3 in a cell-intrinsic manner. TL1A enhances Th9 differentiation through an IL-2 and STAT5-dependent mechanism, unlike the TNF-family member OX40, which promotes Th9 through IL-4 and STAT6. Th9 differentiated in the presence of TL1A are more pathogenic, and endogenous TL1A signaling through DR3 on T cells is required for maximal pathology and IL-9 production in allergic lung inflammation. Taken together, these data identify TL1A–DR3 interactions as a novel pathway that promotes Th9 differentiation and pathogenicity. TL1A may be a potential therapeutic target in diseases dependent on IL-9.

The murine equivalent of the A181E TACI mutation associated with common variable immunodeficiency severely impairs B-cell function

TNFRSF13B, which encodes TACI (transmembrane activator and calcium-modulator and cyclophilin ligand interactor), is mutated in 10% of patients with common variable immune deficiency (CVID). One of the 2 most common TACI mutations in CVID, A181E, introduces a negative charge into the transmembrane domain. To define the consequence of the A181E mutation on TACI function, we studied the effect of its murine equivalent, mTACI A144E, on TACI signaling in transfected cells and on TACI function in transgenic mice. The mTACI A144E mutant, like its human TACI A181E counterpart, was expressed on the surface of 293T transfectants and was able to bind ligand, but exhibited impaired constitutive and ligand-induced NF kappaB signaling. In addition, constitutive and ligand-induced clustering of the intracellular domain was deficient for A144E as measured by fluorescence resonance energy transfer. Transgenic mice expressing the A144E mutant on TACI(-/-) background had low serum IgA levels and significantly impaired antibody responses to the type II T-independent antigen TNP-Ficoll. B cells from A144E transgenic mice were impaired in their capacity to proliferate and secrete IgG1 and IgA in response to TACI ligation. These results suggest that mTACI A144E mutation and its human counterpart, A181E, disrupt TACI signaling and impair TACI-dependent B-cell functions.

Many Checkpoints on the Road to Cell Death:Regulation of Fas–FasL Interactions and Fas Signaling in Peripheral Immune Responses

Interactions between the TNF-family receptor Fas (CD95) and Fas Ligand (FasL, CD178) can efficiently induce apoptosis and are critical for the maintenance of immunological self-tolerance. FasL is kept under strict control by transcriptional and posttranslational regulation. Surface FasL can be cleaved by metalloproteases, resulting in shed extracellular domains, and FasL can also traffic to secretory lysosomes. Each form of FasL has distinct biological functions. Fas is more ubiquitously expressed, but its apoptosis-inducing function is regulated by a number of mechanisms including submembrane localization, efficiency of receptor signaling complex assembly and activation, and bcl-2 family members in some circumstances. When apoptosis is not induced, Fas-FasL interactions can also trigger a number of activating and proinflammatory signals. Harnessing the apoptosis-inducing potential of Fas for therapy of cancer and autoimmune disease has been actively pursued, and despite a number of unexpected side-effects that result from manipulating Fas-FasL interactions, this remains a worthy goal.

Structural Basis and Functional Role of Intramembrane Trimerization of the Fas/CD95 Death Receptor.

Fas (CD95, Apo-1, or TNFRSF6) is a prototypical apoptosis-inducing death receptor in the tumor necrosis factor receptor (TNFR) superfamily. While the extracellular domains of TNFRs form trimeric complexes with their ligands and the intracellular domains engage in higher-order oligomerization, the role of the transmembrane (TM) domains is unknown. We determined the NMR structures of mouse and human Fas TM domains in bicelles that mimic lipid bilayers. Surprisingly, these domains use proline motifs to create optimal packing in homotrimer assembly distinct from classical trimeric coiled-coils in solution. Cancer-associated and structure-based mutations in Fas TM disrupt trimerization in vitro and reduce apoptosis induction in vivo, indicating the essential role of intramembrane trimerization in receptor activity. Our data suggest that the structures represent the signaling-active conformation of Fas TM, which appears to be different from the pre-ligand conformation. Analysis of other TNFR sequences suggests proline-containing sequences as common motifs for receptor TM trimerization.

Specific elimination of effector memory CD4+ T cells due to enhanced Fas signaling complex formation and association with lipid raft microdomains

Elimination of autoreactive CD4+ T cells through the death receptor Fas/CD95 is an important mechanism of immunological self-tolerance. Fas deficiency results in systemic autoimmunity, yet does not affect the kinetics of T-cell responses to acute antigen exposure or infection. Here we show that Fas and TCR-induced apoptosis are largely restricted to CD4+ T cells with an effector memory phenotype (effector memory T cells (TEM)), whereas central memory and activated naïve CD4+ T cells are relatively resistant to both. Sensitivity of TEM to Fas-induced apoptosis depends on enrichment of Fas in lipid raft microdomains, and is linked to more efficient formation of the Fas death-inducing signaling complex. These results explain how Fas can cull T cells reactive against self-antigens without affecting acute immune responses. This work also identifies Fas-induced apoptosis as a possible immunotherapeutic strategy to eliminate TEM linked to the pathogenesis of a number of autoimmune diseases.

Fas/CD95 prevents autoimmunity independently of lipid raft localization and efficient apoptosis induction

Mutations affecting the apoptosis-inducing function of the Fas/CD95 TNF-family receptor result in autoimmune and lymphoproliferative disease. However, Fas can also costimulate T-cell activation and promote tumour cell growth and metastasis. Palmitoylation at a membrane proximal cysteine residue enables Fas to localize to lipid raft microdomains and induce apoptosis in cell lines. Here, we show that a palmitoylation-defective Fas C194V mutant is defective in inducing apoptosis in primary mouse T cells, B cells and dendritic cells, while retaining the ability to enhance naive T-cell differentiation. Despite inability to efficiently induce cell death, the Fas C194V receptor prevents the lymphoaccumulation and autoimmunity that develops in Fas-deficient mice. These findings indicate that induction of apoptosis through Fas is dependent on receptor palmitoylation in primary immune cells, and Fas may prevent autoimmunity by mechanisms other than inducing apoptosis.