(FADD) Is a Negative Regulator of T-Cell Receptor–Mediated Necroptosis

Fas-associated death domain (FADD) is a negative regulator of T-cell receptor–mediated necroptosis

Stephanie L. Osborn, Gretchen Diehl1, Seong-Ji Han, Ling Xue, Nadia Kurd, Kristina Hsieh, Dragana Cado, Ellen A. Robey, and Astar Winoto2

Cancer Research Laboratory and Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720

Edited by Tak Wah Mak, The Campbell Family Institute for Breast Cancer Research, Ontario Cancer Institute at Princess Margaret Hospital, University Health Network, Toronto, Canada, and approved June 16, 2010 (received for review May 2, 2010) Cell death is an important mechanism to limit uncontrolled T-cell 8, and Fas-associated death domain protein (FADD) in cells expansion during immune responses. Given the role of death- stimulated with TNF in the presence of caspase inhibitors. In ad- − − receptor adapter protein Fas-associated death domain (FADD) in dition, RIP3 / mice suffer from impaired necrosis and reduced apoptosis, it is intriguing that T-cell receptor (TCR)–induced prolifer- innate immunity when infected with vaccinia virus and die pre- ation is blocked in FADD-defective T cells. Necroptosis is an alternate maturely (12). form of death that can be induced by death receptors and is linked to FADD is a 28-kDa adaptor protein that is a critical compo- autophagy. It requires the death domain-containing kinase RIP1 and, nent of the death receptors’ apoptotic signaling pathway (13, 14). in certain instances, RIP3. FADD and its apoptotic partner, Caspase-8, It initiates the formation of a death-inducing signaling complex have also been implicated in necroptosis. To accurately assess the and serves as a docking site for Caspase-8 (15). FADD-deficient role of FADD in mature T-cell proliferation and death, we generated cells exhibit impaired death receptor-mediated apoptosis. In a conditional T-cell–specific FADD knockout mouse strain. The T cells addition, FADD-, Caspase-8-, or c-FLIP–deficient T cells fail to of these mice develop normally, but lack FADD at the mature stage. proliferate when stimulated through the T-cell receptor (TCR) FADD-deficient T cells respond poorly to TCR triggering, exhibit slow complex (13, 16–18). Whereas cell cycle-specific phosphorylation cell cycle entry, and fail to expand over time. We find that pro- of FADD is partially responsible for proper T-cell proliferation grammed necrosis occurs during the late stage of normal T-cell pro- (19–21), the proliferative defect of Caspase-8–deficient T cells or liferation and that this process is greatly amplified in FADD-deficient T cells expressing a truncated FADD transgene (FADDdd) is T cells. Inhibition of necroptosis using an inhibitor of RIP1 kinase associated with excess necroptosis that can be rescued with activity rescues the FADD knockout proliferative defect. However, Necrostatin-1 (8, 22). Necroptosis in these cells, however, is due TCR-induced necroptosis did not appear to require autophagy or in- to hyperautophagy. As FADDdd transgenic mice suffer from volve RIP3. Consistent with their defective CD8 T-cell response, these defective T-cell development (23), mature T cells in these mice mice succumb to Toxoplasma gondii infection more readily than are likely to be abnormal. Abnormalities may also exist for − − − − wild-type mice. We conclude that FADD constitutes a mechanism FADD-deficient mature T cells from FADD / →RAG-1 / to keep TCR-induced programmed necrotic signaling in check during chimeric mice (24), where T-cell development is severely im- early phases of T-cell clonal expansion. pacted. To overcome this issue, we have generated a conditional FADD knockout mouse where T-cell development is normal. apoptosis | RIP1 | RIP3 | Caspase-8 | proliferation Herein we show that FADD does have a role in early cell cycle progression, but the absence of FADD reveals its dominant role he immune response to infection requires a delicate balance to be as a negative regulator of necroptosis during T-cell ex- Tbetween cell expansion and contraction. During the expan- pansion. However, hyperautophagy does not signal the observed sion phase, programmed cell death is held in check by various necroptosis in these FADD knockout cells, nor does it involve mechanisms to allow lymphocytes to proliferate and to counter RIP3. We conclude that FADD constitutes a mechanism to keep fi infection. These mechanisms include up-regulation of prosur- a presently unidenti ed pathway of RIP1-dependent necroptosis vival factors and antiapoptotic proteins. As the immune response in check during early phases of the T-cell immune response. wanes, prodeath signals dominate to cause contraction of the Results responding lymphocytes (1–3). Although apoptosis has long been − − Generation and Phenotypic Analysis of tFADD / Mice. Conditional considered the premier form of programmed cell death, other – fi −/− forms of programmed death have more recently been identified T-cell speci c FADD knockout (tFADD ) mice (on the Materials (4). Despite its reputation as an unregulated, nonspecific event, C57BL/6 background) were generated as described in and Methods (Fig. S1). They are born at the expected Mendelian necrosis can occur in a regulated and programmed fashion under ratios and appear healthy with no gross abnormalities. PCR certain circumstances. This process, termed programmed ne- analysis shows that deletion occurs in the double-negative (DN) crosis or necroptosis (5), is independent from caspases and can − − (CD4 CD8 ) thymocyte population and is complete in the be triggered by the tumor necrosis factor (TNF) death receptors double-positive (DP) (CD4+CD8+) compartment with no de- (e.g., Fas, TNF-R, TRAIL-R) when apoptosis is blocked (6). tectable flox allele (Fig. 1A). Although a residual signal of the More recently, hyperautophagy has been suggested to cause necroptosis as well (7, 8). Necroptosis has also been suggested to contribute to the danger signals that alert the immune system to Author contributions: S.L.O., E.A.R., and A.W. designed research; S.L.O., G.D., S.-J.H., L.X., certain pathogenic invasions (4). N.K., K.H., and D.C. performed research; S.L.O. and A.W. analyzed data; and S.L.O., and The death domain-containing kinase RIP1 is a multifunctional A.W. wrote the paper. serine/threonine kinase known for its functions downstream of the The authors declare no conflict of interest. κ death receptors to induce prosurvival signals such as NF- Band This article is a PNAS Direct Submission. MAPKs (9). RIP1 is also critical for necroptosis as Necrostatin-1, 1Present address: The Kimmel Center for Biology and Medicine of the Skirball Institute, an inhibitor of RIP1 kinase activity, blocks this process (9). More New York University School of Medicine, New York, NY 10016. recently, another member of the RIP1 family, RIP3, has also been 2To whom correspondence should be addressed. E-mail: [email protected]. – shown to be crucial for TNF-induced programmed necrosis (10 This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 12). In this context, RIP3 is found in a complex with RIP1, Caspase- 1073/pnas.1005997107/-/DCSupplemental.

13034–13039 | PNAS | July 20, 2010 | vol. 107 | no. 29 www.pnas.org/cgi/doi/10.1073/pnas.1005997107 Downloaded by guest on September 27, 2021 CD4+ and CD8+ T cells is equal to that of littermate controls, suggesting T-cell maturation and migration to the periphery are unaffected (Fig. 1E). This is further supported by a lack of evidence for homeostatically proliferating cells (CD44hi, CD62Lhi) − − in tFADD / mice (Fig. S2C). Further analysis showed that − − tFADD / mice exhibit a drastic increase of B cells in the lymph − − nodes and spleen (Fig. 1E). Whereas control and tFADD / spleens typically maintain similar B-cell percentage proﬁles, the − − absolute number of B cells is higher in tFADD / mice that exhibit splenomegaly. Interestingly, there is also an increase of TER-119+ red blood cells in the spleen but not in the bone − − marrow of tFADD / mice (Fig. 1E). Thus, the enlarged lymph − − nodes and spleen observed in tFADD / mice are due to increased B cells and red blood cells, respectively.

FADD-Deficient T Cells Have Impaired Early Cell Cycle Transition. To understand the nonapoptotic function of FADD in mature T cells, we analyzed the ability of FADD-deficient T cells to proliferate in response to TCR ligation. Similar to published work using − − − − FADD / →RAG1 / chimera mice (13), stimulation with anti- CD3/CD28 antibodies revealed a defect in the ability of FADD- deficient T cells to accumulate in culture over time (Fig. 2A). The effect was most pronounced in the CD8 T-cell subset, as compared with that of the CD4 T-cell subset. Defects were also found when + we stained CD8 FADD-deficient T cells with Ki-67, which detects non-G0 cells, and when we examined cell division using carboxy- fluorescein succinimidyl ester (CFSE) (Fig. 2B and Fig. S3A). In − − − − contrast to T cells from FADD / →RAG1 / chimera mice, − − however, CD4+ FADD-deficient cells from tFADD / mice do proliferate to some extent, showing only a slight delay in Ki-67 expression and cell division. A pulse-chase experiment using concurrent propidium iodide (PI) staining and BrdU labeling also

− − Fig. 1. tFADD / mice exhibit normal T-cell development but have altered peripheral lymphoid organ compartments. (A) Thymocyte and peripheral T-cell − − populations from control (ctrl) and tFADD / (ko) littermates were sorted and analyzedby PCR for the presence of theﬂoxallele(ﬂox) and subsequentdeletion of the FADD exon 1 (del). (B) CD4 and CD8 T cells were sorted and either not

stimulated or stimulated for 24 h with ConA/Phorbol myristate acetate (PMA). IMMUNOLOGY Whole-cell lysates were analyzed for expression of FADD; a nonspeciﬁc(NS) band demonstrates loading equality. (C) The average thymic cellularity of con- − − trol and tFADD / mice with ages ranging from 5 to 12 wk. Each dot represents − − one mouse. (D) CD4/CD8proﬁles of control and tFADD / thymi. (E) Lymphocytes − − from control and tFADD / mice were analyzed for various cell types. (Upper) Lymph nodes were analyzed for B cells (B220) and T cells (TCR). (Lower)Sple- nocytes were analyzed for B cells (B220) and red blood cells (TER-119).

flox allele can be seen in CD8+ mature T cells, Western blot analysis shows that FADD protein is absent in both CD4 and − − CD8 T-cell subsets (Fig. 1B). tFADD / thymi are of equal size and cellularity to wild-type littermate controls (Fig. 1C). The CD4/CD8 profiles, as well as analysis of the DN subsets, of FADD-deficient thymi are also comparable to those of littermate controls (Fig. 1D). Flow analysis of various selection − − markers showed normal positive selection in tFADD / mice. As expected, FADD-deficient thymocytes are resistant to Fas- Fig. 2. FADD-deficient T cells do not proliferate well. (A)Purified peripheral T − − induced apoptosis (Fig. S2A). Older tFADD / mice do not cells were activated by plate-bound anti-CD3 and anti-CD28 antibodies and exhibit any signs of autoimmunity, suggesting that deletion of total cells were counted daily via trypan blue exclusion to obtain the growth −/− autoreactive thymocytes during development is functional. curves. Solid lines, T cells from three separate tFADD mice; dotted lines, wild- − − In contrast to their thymic cellularity, tFADD / mice con- type (wt) T cells from littermate controls. Data are representative of multiple independent experiments(>10) with similar results. (B)Purified control (shaded) sistently possess enlarged lymph nodes and spleens (Fig. S2B; and FADD-deficient (solid lines) T cells were labeled with CFSE, activated with P < 0.05). Whereas the percentage of T cells in the peripheral anti-CD3/CD28 antibodies for the indicated times, and stained for CD4 and CD8. − − organs of tFADD / mice is reduced, the absolute cell number of Data are representative of multiple experiments (>3) with similar results.

Osborn et al. PNAS | July 20, 2010 | vol. 107 | no. 29 | 13035 Downloaded by guest on September 27, 2021 showed cell cycle entries of FADD-deficient cells with only a delay FADD-null T cells do exhibit a slower cell cycle kinetic, but this 2-h in progression through the cell cycle (Fig. S3B). lag is unlikely to account for the overt proliferation defect. We thus −/− −/− In T cells from FADD →RAG1 chimera mice, loss of FADD decided to examine the rate of cell death in these T-cell cultures. The − − affects the machinery at all phases of the cell cycle (24). Despite forward/side scatter (FS/SS) profiles of the tFADD / T-cell cultures normal development, phosphorylation mimetic mutant FADD suggested an increase in necrotic death (necrotic cells are FSlo/SShi; fi (S191D) T cells similarly exhibit early cell cycle defects that speci - Fig. 3A). Costaining of Annexin V and PI also pointed to necrotic cally affect the cell cycle master regulator FoxM1 and the Rb protein fi fi −/− death of FADD-de cient T cells following activation. Speci cally, (21). However, T cells from tFADD mice exhibit normal cell cycle − early apoptotic T cells (Annexin V+,PI ) could not be detected in protein profiles. Thus, atypical expression of many cell cycle proteins −/− −/− FADD-deficient cultures, whereas phosphatidyl serine exposure in T cells from FADD →RAG1 chimeras is likely a consequence of abnormal T-cell development in the absence of FADD. only occurred concurrently with compromise of the plasma membrane (Annexin V+,PI+)(Fig. S4A). Measurement of the mito- FADD-Deficient T Cells Die by Necroptosis During Proliferation. Al- chondrial membrane potential further suggests that FADD-deficient − − though FADD-deficient T cells from tFADD / mice are capable of T cells are dying by necrosis. Gating on live cells within the culture, some cell division, they ultimately do not accumulate in culture. we found relatively little loss of 3,3′-dihexyloxacarbocyanine iodide

Fig. 3. TCR-stimulated FADD-deﬁcient T cells die by necrosis and can be rescued by inhibition of RIP1 kinase activity but not by inhibition of hyperautophagy. (A) T cells from control and tFADD−/− littermates were activated with anti-CD3/CD28 antibodies and analyzed by forward scatter (FS) versus side scatter (SS) for evidence of necrotic death. (B) The accumulation of T cells in culture after activation as in A was determined by daily cell counts using trypan blue exclusion. Cultures were also stained for CD4 and CD8 to determine the absolute cell numbers for each T-cell subset. Necrostatin-1 (Nec) was added at 30 μM where − − indicated. (C)(Upper) Activated T cells from control and tFADD / mice were either not treated or treated with Necrostatin-1 and subsequently analyzed at the indicated time points for Ki67, CD4, and CD8 expression. (Lower) Same as the top panels except T cells were labeled with 0.5 μM CFSE preactivation. Shown is the plot for CD4 at day 3 postactivation and CD8 at day 4 postactivation. (D) Puriﬁed T cells were CFSE-labeled, activated as in A, and either not treated (NT) or treated with 0.5 mM 3-MA, and surface-stained for CD8 postharvest. Data are representative of multiple experiments (>3) with similar results.

13036 | www.pnas.org/cgi/doi/10.1073/pnas.1005997107 Osborn et al. Downloaded by guest on September 27, 2021 (DiOC6) in either control or FADD-deficient T cells (Fig. S4B). −/− However, DiOC6 loss is increased in the tFADD culture when the entire culture is analyzed (Fig. S4B), suggesting the mitochondrial membrane is compromised following breakdown of the plasma membrane, a hallmark of necrosis. Recently, a caspase-independent program of cell death that results in a necrotic phenotype has been identified and termed programmed necrosis (25, 26). In particular, necroptosis is a form of programmed necrosis that is dependent upon RIP1 kinase activity for execution. Necrostatin-1 is a specific inhibitor of RIP1 kinase activity (9) and has been used to block necroptosis in various contexts. Using this compound, we found that inhibition of RIP1 kinase activity by Necrostatin-1 fully rescued the inability of FADD-deficient T cells, both CD4 and CD8, to proliferate, as measured by multiple parameters (Fig. 3 B and C). Interestingly, treatment with Necrostatin-1 also consistently increased the accumulation of control T cells in culture versus untreated control T cells at later time points (Fig. 3B). Thus, a basal level of necroptosis occurs in activated T-cell cultures, suggesting that necroptosis may actually be a physiological event in the context of T-cell proliferation and the immune response. FADD(S191D) mice, which express a mutant form of FADD Fig. 4. Necroptosis of FADD-deficient T cells does not induce RIP1 cleavage or that mimics constitutive phosphorylation at serine 191, also ex- a RIP1-RIP3 complex. (A) Whole-cell extracts from activated control and −/− hibit proliferative defects but which can be specifically attributed tFADD T cells at various time points were blotted for RIP1 expression. RIP1c − − to a blockage at the S-phase transition. Unlike tFADD / T cells, denotes cleaved RIP1. (B) Whole-cell extracts were prepared as in A,subjected to RIP3 immunoprecipitations (IP), and subsequently blotted for RIP1 or RIP3. Necrostatin-1 treatment is unable to fully rescue the impaired Coimmunoprecipitation of phosphorylated RIP1 (pRIP1) with RIP3 in L929 cells proliferation of FADD(S191D) T cells (Fig. S5A). This is likely undergoing necroptosis [+ tumor necrosis factor (TNFα) and z-VAD] serves as due to the maintained capacity of FADD(S191D) T cells to a positive control. The asterisk denotes a nonspecific band that we were unable undergo death by apoptosis in response to impaired S-phase to distinguish as either a nonspecific protein or an unmodified RIP1 that was progression. Thus, necroptosis appears to occur uninhibited in pulled down nonspecifically with control IgG as previously reported by others the absence of FADD during T-cell expansion and is the main (44). (Lower) Whole-cell extracts (WCE) were immunoblotted for RIP1 or RIP3 cause of proliferative failure of FADD-deficient T cells. expression. Data are representative of several experiments with similar results.

Necroptosis of Proliferating FADD-Deficient T Cells Is Not Due to Hyperautophagic Signaling, RIP3 Signaling, or Failure of Caspase-8 to Recently, RIP3, another RIP kinase family member, has been − − fi Cleave RIP1. To understand the mechanism of tFADD / T-cell identi ed as a critical partner of RIP1 during TNF-R1-induced necroptosis, we analyzed various pathways or molecules previously necroptosis, and this association induces phosphorylation of both identified to be involved in necroptotic signaling. FADD has pre- proteins (10, 32). Although T cells die by necroptosis through viously been linked to ATG5, a component of the autophagy RIP1, RIP3 does not appear to be involved in this context. First, pathway (8, 27, 28), and T cells expressing a truncated form of the whole-cell extracts of L929 cells undergoing necroptosis show FADD protein (FADDdd) exhibit hyperautophagy and subsequent phosphorylation of RIP1, which is not present in untreated L929 necroptotic death (8). We analyzed the role of autophagy in the or T-cell extracts (Fig. 4B Lower). Second, coimmunoprecipita- − − necroptotic phenotype of the tFADD / T cells by using 3-methyl- tion experiments fail to show a phospho-RIP1 associating with

−/− IMMUNOLOGY adenine (3-MA), an inhibitor that blocks autophagosome formation RIP3 in activated tFADD T cells at various time points (Fig. (29). We found that this inhibitor was unable to rescue the death of 4B). In contrast, a slower-migrating phosphorylated RIP1 band − − activated tFADD / T cells (Fig. 3D). In fact, 3-MA was toxic to was coimmunoprecipitated with anti-RIP3 antibodies in L929 primary T-cell cultures at the published concentrations (0.5–1mM), cells undergoing necroptosis, as shown previously (10, 11). Thus, as evidenced by the reduced number of wild-type T cells (Fig. 3D TCR-induced necroptosis appears to be RIP3-independent. and Fig. S5B).If titratedtolesstoxicranges (0.1 mM), 3-MA was still −/− incapable of rescuing FADD-deficient T-cell proliferation (Fig. tFADD Mice Are More Susceptible to Toxoplasma gondii Infection. S5C). Addition of 3-MA post-TCR activation at 48 h also failed to To address the consequence of the lack of FADD and un- − − rescue tFADD / T-cell proliferation. In contrast, a similar con- controlled T-cell necroptosis in an immune response setting, we centration of 3-MA (0.5 mM) could rescue z-VAD-induced auto- performed Toxoplasma gondii infection studies. T. gondii is an phagic cell death of L929 cells (30) (Fig. S5D). These data suggest intracellular parasite that causes a robust CD8 T-cell–dependent that if hyperautophagy does ensue in the process of FADD-deficient immune response (33), and CD8-deficient mice (on the C57BL/6 T-cell death, it is likely a consequence of the necroptotic program background) die within 20–25 d postinfection (34). We found rather than the cause. Hyperautophagy as a consequence of nec- that the absence of FADD renders mice more susceptible to roptosis was previously observed by others (9), but is in contrast to chronic T. gondii infection. Whereas infection of wild-type a recent publication that suggests 3-MA can rescue the proliferation C57BL/6 mice with a relatively low dose of parasite led to the death of 50% of mice at 50 d postinfection (Fig. 5A Top), 100% defect observed in T cells expressing FADDdd (8). − − Caspase-8 has been shown to cleave and inactivate RIP1 during of tFADD / mice died by 31 d postinfection. At the same time, − − apoptosis, but this mechanism is also unlikely to be responsible for tFADD / mice also exhibit more weight loss than the control keeping RIP1-mediated necroptosis in check in actively pro- group (Fig. 5A Bottom). We subsequently analyzed the serum liferating T cells (31). We did not detect an increase in the incidence from mice at days 8 and 14 postinfection and performed cytokine − − of cleaved RIP1 in activated control versus tFADD / T-cell cul- profile studies using the cytokine bead array assay. Interestingly, − − tures at early time points (Fig. 4A). Therefore, a failure of RIP1 the tFADD / serum levels of TNF, IL-6, IL-10, MCP-1, and IL- cleavage by Caspase-8 cannot explain the necroptotic phenotype of 12 were normal (Fig. S6). In contrast, γ-IFN was significantly − − − − tFADD / T cells. elevated in the infected tFADD / mice at day 14 postinfection

Osborn et al. PNAS | July 20, 2010 | vol. 107 | no. 29 | 13037 Downloaded by guest on September 27, 2021 extent, as demonstrated by cell cycle entry of FADD-deficient T cells, although their cycling is slower than wild-type T cells. However, accumulation is not restored, and this is particularly − − evident for CD8 tFADD / T cells. In contrast, FADD(S194D) T cells still succumb to apoptotic cell death in response to strong S-phase impairment and are insensitive to Necrostatin-1 rescue. Necroptosis is a specific form of programmed necrosis that is executed by RIP1 kinase activity and has been shown to be physi- ologically relevant in certain human pathologies (5). Although the role of necroptosis in the immune system is less clear, it has been suggested to act as a danger signal to the immune system in certain contexts (4). Furthermore, necroptosis may constitute a checks- and-balances type of mechanism for instances in which apoptosis is ineffective for various reasons. We show here that FADD-deficient T cells fail to proliferate primarily due to an increased rate of programmed necrosis and that inhibition of RIP1 kinase activity by Necrostatin-1 is able to fully rescue the proliferative defect of these T cells. This suggests that FADD is normally a negative regulator of RIP1-dependent necroptosis during the T-cell expansion phase. This is in contrast to Jurkat T cells where, in the absence of Cas- pase-8, stimulation of the death receptors, specifically Fas, TNF- R1, and TRAIL-R, leads to necroptosis (6) and FADD is specifically required for the Fas- and TRAIL-R-mediated process (6).

− − Caspase-8 and FADD have been proposed to be involved in Fig. 5. tFADD / mice exhibit decreased survival in response to T. gondii in- −/− a complex that inhibits hyperautophagy during T-cell pro- fection. (A) tFADD mice and their littermate controls were infected in- liferation, thereby keeping necroptosis in check (8). T cells from traperitoneally with T. gondii. Survival data (top panel) and relative body weight loss (bottom panel) were compiled over a 50-d period. (B) Serum γ-IFN transgenic mice expressing a truncated form of FADD (FADDdd) − − levels of control or tFADD / Toxoplasma-infected mice at day 14 post- exhibit proliferative defects that can be reversed by inhibition of infection. Cytokine levels were determined by the cytokine bead array assay autophagy. In contrast, autophagy inhibitors cannot rescue −/− and using the corresponding standards. tFADD T-cell proliferation. We speculate that the differences − − in phenotypes between FADDdd and tFADD / T cells can be attributed to abnormal T-cell development in FADDdd mice (23) (Fig. 5B). This may be due to increased γ-IFN production by NK which produces an unusual population of mature T cells. Fur- cells and antigen-presenting cells in response to the necrotizing T thermore, the FADDdd protein is not truly a dominant-negative cells and uncontrolled parasite replication during these later protein, as previously thought. The level of transgenic FADDdd stages of the immune response (35–37). Thus, these data suggest protein is at least 100-fold that of endogenous FADD protein that programmed necrosis in activated FADD-deficient T cells expression (40); expression of FADDdd at physiological levels might trigger increased levels of γ-IFN, to promote the immune failed to suppress Fas-induced apoptosis (40). It is not clear response, as seen in other systems (4, 11), but these cytokines are whether the defective proliferation of Caspase-8–deficient T cells not sufficient to protect the mice from infection as there is a lack can be rescued by an autophagic inhibitor or not. However, there − − of functional CD8+ T cells. Thus, an enhancement of the im- are several phenotypic differences between tFADD / and T-cell– − − mune response by necroptosis is context-dependent. specific Caspase-8 knockout (tcaspase-8 / ) mice. Whereas T-cell − − development is also normal in tcaspase-8 / mice (22, 41), old − − Discussion tcaspase-8 / mice suffer from lymphoproliferative and lym- FADD has long been known to be an important mediator of phoinfiltrative immune disorder, with many of them dying within lymphocyte proliferation in addition to its classical role in death 1 y (41). The phenotypic discrepancies between the Caspase-8 and receptor-mediated apoptosis. However, the mechanism by which FADD models suggest that Caspase-8 and FADD may not act in FADD exerts its proliferative function has been elusive. Work the same proliferative/anti-death pathways. done by our lab, as well as others, has pointed to a link between Recent work demonstrates that in certain cell types, RIP3 works FADD and cell cycle regulation (19–21, 24, 38, 39). Using in a phosphorylation-dependent manner with RIP1 to induce a FADD knockout model that allows for proper T-cell de- necroptosis signaled through TNF-R1 (10–12). However, in velopment, we now show that although FADD does have a role FADD-deficient T cells undergoing necroptosis, RIP3 does not in early cell cycle transition, the dominant function of FADD is appear to associate with RIP1. Thus, T-cell necroptosis is de- inhibition of programmed necrosis during T-cell proliferation. pendent upon RIP1 but not RIP3. Furthermore, we are so far − − We have previously shown that FADD phosphorylation occurs unable to block necroptosis in tFADD / T cells by neutralizing upon entry into the cell cycle and that altered phosphorylation of Fas, TNF-R1, and TRAIL-R signaling alone or in combination. FADD leads to cell cycle defects (20, 21, 39). The phenotype of Our data suggest TCR-induced necroptosis to be death receptor- − − the tFADD / T cells corroborates this role for FADD in early and autophagy-independent, thus pointing to the existence of an cell cycle transitions. Activated FADD-deficient T-cell cultures alternate RIP1-dependent necroptotic pathway downstream of have a larger percentage of cells that have failed to enter the cell TCR signaling. This necroptotic signal may normally act at the end cycle and undergo only one cell division. For comparison, T cells of the immune response to aid in T-cell contraction, but loss of from Caspase-8 knockout mice also have proliferative defects FADD leads to uncontrolled, early induction of necrotic death. that can be rescued with Necrostatin-1 (8, 22), but they do not Wild-type T cells, like FADD-deficient T cells, are positively possess the cell cycle phenotype of the FADD-deficient T cells; affected by Necrostatin-1 treatment. Necrostatin-1 has also been Caspase-8 knockout T cells divide at the same rate as control T shown to increase the life span of activated primary macrophages cells (22). This suggests a cell cycle-related role for FADD that is (26). Thus, necroptosis may be a general mechanism for medi- independent of Caspase-8. Interestingly, the defect of FADD- ating immune system homeostasis and may play a role in abating deficient cell cycle entry can be overcome by itself to a certain the late phase of the T-cell response. In this regard, it is possible

13038 | www.pnas.org/cgi/doi/10.1073/pnas.1005997107 Osborn et al. Downloaded by guest on September 27, 2021 that an undetectable increase in the steady-state level of nec- T-Cell Analysis. Purified mature T cells were activated with plate-bound anti- − − roptosis in tFADD / mice may enhance proinflammatory sig- CD3 and anti-CD28. Necrostatin-1 (30 μM) or 3-MA were added at the start of naling and contribute to the increased red blood cell and B-cell the culture and supplemented when cultures were expanded. 3-MA was also populations we observe in these mice. added at 48 h postactivation, instead of at culture initiation. Cells were Recently, a genome-wide siRNA screen to identify regulators of counted in triplicate per condition each day of culture by trypan blue and necroptosis found many key immune system proteins to be in- analyzed by flow cytometry. For more detailed information regarding T-cell volved, further suggesting that necroptosis is indeed a relevant culture conditions, see Materials and Methods in SI Text. cell-death mechanism within the immune system (26). Although − − our tFADD / data show how uncontrolled necroptosis is specif- T. gondii Infections. Parasites of the Prugnaud strain of T. gondii were grown ically detrimental to T-cell expansion and function, especially in in fibroblasts and harvested by standard protocol (43). Mice were infected the case of T. gondii infection, we can speculate the need for intraperitoneally with 400 Pru parasites. necroptosis of other cell types during times of infection in which caspases have been specifically targeted and inactivated. It is also Cytokine Bead Array Assay. Blood was collected from mice at the indicated likely that in certain pathological contexts, necroptosis is preferred days postinfection via tail-vein bleed. The cytokine bead array assay was ’ over apoptosis, as necrotic death increases inflammation and im- performed according the manufacturer s protocol and samples were read on fl mune cell infiltrates to the affected area. However, our FADD an LSR II ow cytometer (BD Biosciences). The data are representative of two conditional knockout model clearly demonstrates the importance independent experiments, with each experiment having 300 replicates of of keeping necroptosis in check within certain cell types, especially each cytokine reading. T cells, during specific phases of the immune response in which cell See SI Text for other procedures and additional details on materials death would be counter to a productive immune response. and methods. Materials and Methods ACKNOWLEDGMENTS. We thank Vicky Liu and Yuefang Sun for help with mouse genotyping, and Namsil An and Paul Herzmark (Center for Host- −/− Generation of tFADD Mice. LoxP sites were placed around FADD exon 1 (Fig. Pathogen Studies core facilities, University of California, Berkeley), for S1A). Mice generated were crossed to CD4-Cre (42) to generate CD4-Cre/ technical help. This work is supported by grants from the National Institutes fl fl − − FADD / (tFADD / ) mice. of Health (to E.A.R. and A.W.).

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