Regulation of T-cell activation and migration by the kinase TBK1 during neuroinflammation

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1 Received Oct Accepted 9 Dec Published Jan DOI:.8/ncomms77 OPEN Regulation of T-cell activation and migration by the kinase TBK during neuroinflammation Jiayi Yu,, Xiaofei Zhou,, Mikyoung Chang, Mako Nakaya,, Jae-Hoon Chang,, Yichuan Xiao, J. William Lindsey, Stephanie Dorta-Estremera,, Wei Cao,, Anna Zal, Tomasz Zal,, & Shao-Cong Sun,, Development of an immune or autoimmune response involves T-cell activation in lymphoid organs and subsequent migration to peripheral tissues. Here we show that T-cell-specific ablation of the kinase TBK promotes T-cell activation but causes retention of effector T cells in the draining lymph node in a neuroinflammatory autoimmunity model, experimental autoimmune encephalomyelitis (EAE). At older ages, the T-cell-conditional TBK-knockout mice also spontaneously accumulate T cells with activated phenotype. TBK controls the activation of and its downstream kinase mtorc by a mechanism involving TBK-stimulated ubiquitination and degradation. The deregulated -mtorc signalling in turn contributes to enhanced T-cell activation and impaired effector T-cell egress from draining lymph nodes. Treatment of mice with a small-molecule inhibitor of TBK inhibits EAE induction. These results suggest a role for TBK in regulating T-cell migration and establish TBK as a regulator of the -mtorc signalling axis. Department of Immunology, The University of Texas MD Anderson Cancer Center, 7 Fannin Street, Box 9, Houston, Texas 77, USA. Division of Pharmacology, School of Medicine, University of Fukui, Fukui 9-87, Japan. College of Pharmacy, Yeungnam University, Gyeongsan 7-79, Republic of Korea. Department of Neurology, University of Texas Health Science Center at Houston, Houston, Texas 77, USA. The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, Texas 77, USA. Center for Inflammation and Cancer, The University of Texas MD Anderson Cancer Center, 7 Fannin Street, Box 9, Houston, Texas 77, USA. These authors contributed equally to this work. Correspondence and requests for materials should be addressed to S.-C.S. ( ssun@mdanderson.org). NATURE COMMUNICATIO :7 DOI:.8/ncomms77 & Macmillan Publishers Limited. All rights reserved.

2 NATURE COMMUNICATIO DOI:.8/ncomms77 Autoimmunity occurs as a result of T-cell activation by antigens derived from self-tissues. Following priming by the self-antigens in peripheral lymphoid organs, autoimmune effector T cells migrate to target organs to mediate inflammation and tissue damage. The central nervous system (C) is an organ of a number of autoimmune and inflammatory disorders, including multiple sclerosis (MS), a disease characterized by chronic inflammation, demyelination and neuronal damage. An animal model, experimental autoimmune encephalomyelitis (EAE), has proved to be powerful for investigating the pathogenesis of MS. It is generally believed that in MS and EAE, autoimmune T cells are primed by myelin-specific antigens and then migrate across the blood brain barrier to enter the C, where they become reactivated and mediate inflammation and neuronal damage,. The T-cell priming and differentiation are governed by signal transduction mediated by the TCR and a costimulatory molecule, CD8, as well as cytokine signals. However, the signalling mechanism that regulates T-cell migration from the lymphoid organs to the tissues of autoimmunity, such as C, is still poorly defined. TBK, as well as its homologous kinase IKKe, are known as mediators of type I interferon (IFN) induction in antiviral innate immunity 7. TBK and IKKe share structural homology with IKKa and IKKb, typical IKK components mediating activation of the transcription factor NF-kB,. However, unlike the typical IKKs, TBK and IKKe are dispensable for NF-kB activation but are required for activation of IFN-responsive factor, a transcription factor mediating type I IFN gene expression.to date, the roles of the atypical IKKs in other biological processes are poorly defined. In particular, the study of the in vivo function of TBK has been hampered by the embryonic lethality of the conventional TBK-knockout (KO) mice. In the present study, we employed a conditional Tbk-KO approach and demonstrated an unexpected role for TBK in the regulation of T-cell function and autoimmunity. T-cell-specific ablation of TBK perturbed T-cell homeostasis, characterized by an increased frequency of T cells with an activated phenotype, and rendered naïve T cells more sensitive to activation by agonistic antibodies for TCR and CD8. Surprisingly, the T-cellconditional Tbk-KO (hereafter called Tbk-) mice were refractory to the induction of EAE due to impaired migration of autoimmune T cells from the draining lymph nodes to the C. Our data suggest that TBK mediates egress of effector T cells from draining lymph nodes in a mechanism that involves negative regulation of the kinases and mtorc. Results TBK is a kinase that responds to T-cell activation signals. Our initial analysis of the BioGPS database revealed that in addition to macrophages, lymphocytes had an abundant expression of TBK (data not shown). To assess the function of TBK in T cells, we examined its ability to respond to signals stimulated by the TCR and CD8 agonistic antibodies (anti-cd and anti-cd8) or mitogens (PMA and ionomycin) that activate protein kinase C and calcium pathways downstream of the TCR and CD8. Despite being known as an innate immune mediator, TBK, as well as its homologue IKKe, were strongly activated by the T-cellactivation signals, as shown by both phospho-specific immunoblot (IB) and in vitro kinase assays (Fig. a,b). Activation of the typical IKK complex by T-cell-activation signals requires a scaffold protein, CARMA (refs,7). Interestingly, CARMA was also required for the activation of TBK and IKKe (Fig. b). Furthermore, activation of IKKe was completely dependent on IKK, since it was blocked in T cells lacking the IKK regulatory subunit NEMO or the IKK catalytic subunit IKKb (Fig. b). On the other hand, the activation of TBK was only partially inhibited in the NEMO- and IKKb-deficient T cells (Fig. b). Similar results were obtained using Jurkat T cells lacking CARMA (JPM.) (ref. 7) or NEMO (JM..; ref. 8; Fig. c). Thus, both TBK and IKKe are activated by T-cell-activation signals, although the underlying mechanism appeared to be different for these kinases. TBK regulates T-cell activation. To study the role of TBK in regulating the T-cell function, we generated Tbk- mice by crossing the Tbk-floxed mice with CD-Cre mice. As expected, TBK was ablated in T cells but not in B cells (Supplementary Fig. a). The T-cell-specific TBK ablation did not appreciably alter the pattern of thymocyte development (Supplementary Fig. b,c). The young ( weeks old) Tbk- and wild-type () control mice also had comparable numbers of CD þ and CD8 þ T cells in different lymphoid organs (Supplementary α-cd + α-cd8 PMA + lono PMA + Jurkat JPM. J.SVT JM (min) lono (min) P-TBK TBK KA TBK IKKε KA IKK KA P-GST-IRF P-GST-IRF P-GST-IκBα PMA + lono (min) TBK KA IKKε KA IKK KA Carma- KO NEMMO IKKβ- P-GST-IRF P-GST-IRF P-GST-IκBα Figure Activation of TBK and IKKe by T-cell activation signals. (a) IB analysis of phosphorylated (P-) TBK (Ser7) and total TBK in CD þ T cells (-week-old mice), stimulated with anti-cd plus anti-cd8 using a crosslinking method or with the mitogens PMA plus ionomycin. (b) CD þ T cells from the indicated KO or T-cell-conditional KO () and internal control mice ( weeks) were treated with PMA plus ionomycin. TBK, IKKe and the canonical IKK complex were isolated by IP from the cell lysates and subjected to in vitro kinase assays (KA) using GST-IRF (for TBK and IKKe) or GST-IkBa (for IKK) recombinant proteins as substrates. (c) TBK, IKKe and IKK kinase assays were performed using Jurkat and derivative cells as described in b. Data are representative of three or more independent experiments. NATURE COMMUNICATIO :7 DOI:.8/ncomms77 & Macmillan Publishers Limited. All rights reserved.

3 NATURE COMMUNICATIO DOI:.8/ncomms77 ARTICLE Fig. d), suggesting normal survival and homing of naïve T cells in Tbk- mice at young ages. Interestingly, however, at an older age ( months old), the Tbk- mice had splenomegaly and higher splenocyte numbers (Fig. a) as well as increased numbers of CD þ and CD8 þ T cells in different lymphoid organs and the peripheral blood (Supplementary Fig. e). To examine the effect of TBK deficiency on T-cell activation, we used naïve CD þ T cells derived from the -week-old mice. When stimulated in vitro, the and TBK-deficient naïve CD þ T cells displayed similar level of apoptosis (Supplementary Fig. f). Interestingly, however, the TBK-deficient naïve CD þ T cells displayed a higher ability to proliferate in response to lower doses of anti-cd (Fig. b). Moreover, the stimulated TBK-deficient T cells produced remarkably higher levels of IFN-g, as detected by both enzyme-linked immunosorbent assay (ELISA) and flow cytometry assays (Fig. b,c). On the other hand, the TBK deficiency did not substantially promote the expression of two other cytokines, interleukin (IL-) and granulocyte macrophage colony-stimulating factor (GM-CSF; Fig. b,c). Flow cytometry analyses revealed that the Tbk- mice had a higher frequency of activated or memory-like T cells, characterized by the CDhiCDLlo surface markers, and concomitant reduction in the percentage of naïve (CDloCDLhi) T cells (Fig. d). This phenotype was not detected in mice lacking the TBK homologue, IKKe (Fig. e), suggesting a unique function of TBK. Using a Cre-ER-inducible KO approach, we found that TBK ablation in adult mice (Supplementary Fig. a) also caused an increase in the frequency of activated T cells in the spleen (Supplementary Fig. b) without affecting the thymocyte development (Supplementary Fig. c,d). Thus, TBK appeared to have a non-redundant role in regulating the steady-state activation and homeostasis of T cells. Perturbed T-cell homeostasis can be due to a cell-intrinsic mechanism or impaired Treg function. We addressed the mechanism of TBK function by mixed bone marrow adoptive transfer. Under such conditions, the and Tbk- T cells were developed and maintained in the same environment and in the presence of Treg cells, thus allowing the assessment of cell-intrinsic functions of TBK. We transferred bone marrow from mice expressing CD. congenital marker (B.SJL) along with either or Tbk- bone marrows (CD. þ ) into lethally irradiated Rag-KO mice. As expected, the CD. þ and CD. þ T cells in the mixed bone marrow chimeric mice had similar frequency of memory and naïve CD þ T cells (Fig. f, left panels). In contrast, the Tbk- (CD. þ ) CD þ T cells had a profoundly higher Splenocytes (X 7 ) 8 Tbk- α-cd (μg ml ). α-cd8 (μg ml ) CD CD Tbk CDL.7 CDL Proliferation (CPM x) 7. IKKε-KO CD naïve (%) CD naïve (%) IL- (ng ml ) IKKε- KO CD memory (%) CD memory (%) IFN-γ (ng ml ) IKKε- KO GM-CSF (ng ml ) CD. 9.7 CD. + + CD. + 8 CDL GM-CSF.... CD. + + CD. +.. CD. + CD. + CD naïve (%) CD memory (%) 8 CD. + + CD. + Figure TBK deficiency promotes T-cell activation and perturbs T-cell homeostasis. (a) A representative spleen picture and mean±s.d. values of spleen cell numbers of -month-old and Tbk- mice (each circle represents a mouse, n ¼ 8). (b) Proliferation and ELISA (IL-, IFN-g and GM-CSF) analyses of CD þ T cells purified from the spleen of young ( weeks, n ¼ ) and mice and stimulated for 8 h with the indicated concentrations of plate-bound anti-cd and anti-cd8. (c) Flow cytometric analyses of splenic CD þ T cells stimulated for h with PMA and ionomycin in the presence of monensin. Frequency of IFN-g-producing and GM-CSF-producing T cells are presented as a representative fluorescence-activated cell sorting plot (left) and mean±s.d. values (right) of multiple mice ( weeks, ¼, ¼ ). (d,e) Flow cytometric analyses of the frequency of naïve (CD lo CDL hi ) and memory (CD hi CDL lo ) CD þ Tcells in and Tbk- (d, n ¼ ) or and IKKe-KO (e, n ¼ ) mice ( weeks old). Numbers in quadrant indicate the percentage of cells among total CD þ T cells. Data are representative plot (left) and mean±s.d. values (right). (f,g) Flow cytometric analysis of naïve (CD lo CDL hi ) and memory (CD hi CDL lo ) CD þ T cells of lethally irradiated Rag-KO mice reconstituted with a mixture (: ratio) of bone marrow cells derived from B.SJL (CD. þ ) and or (CD. þ ) mice (n ¼ ). Data are representative plot (f) and mean±s.d. values (g). Data represent mean±s.d. from three or more independent experiments. Po.; Po.; Po.; Po.;, non-significant as determined by two-tailed unpaired Student s t-test, comparing the indicated groups. IFNγ IFNγ + cells (%) CD. + () GM-CSF + cells (%) CD. + () NATURE COMMUNICATIO :7 DOI:.8/ncomms77 & Macmillan Publishers Limited. All rights reserved.

4 NATURE COMMUNICATIO DOI:.8/ncomms77 frequency of memory population compared with the (CD. þ ) CD þ T cells within the same chimeric mice (Fig. f, right panels, and Fig. g). These data suggest that TBK has a cell-intrinsic role in the regulation of steady-state activation and homeostasis of T cells. TBK is essential for EAE induction. Since the reduced threshold of T-cell activation often promotes autoimmunity, we examined the role of TBK in regulating a T-cell-dependent autoimmunity, EAE. The pathogenesis of EAE involves the inflammatory action of CD þ T helper 7 (Th7) and Th cells, which are characterized by the production of the signature cytokines IL-7 and IFN-g, respectively. Immunization of mice with a myelin-specific autoantigen, myelin oligodendrocyte glycoprotein (MOG) peptide MOG, led to the induction of overt EAE clinical scores (Fig. a). Surprisingly, the Tbk- mice were not hypersensitive, but rather remarkably resistant, to EAE induction (Fig. a). Histological analyses revealed drastic reduction in C-infiltrating immune cells and CD þ T cells in the Tbk- mice (Fig. b, left and middle). In addition, consistent with the clinical scores, the spinal cord of the, but not Tbk-, mice had severe demyelination (Fig. b, right). Parallel flow cytometry analyses confirmed the drastic reduction in the number of CD þ and CD8 þ T cells in the C of EAEinduced Tbk- mice (Fig. c). The EAE-refractory phenotype of the Tbk- mice was unlikely due to a defect in inflammatory T-cell production, since the TBK-deficient CD þ T cells generated higher levels of Th and Th7 cells than CD þ T cells in an in vitro differentiation assay (Supplementary Fig. ). Consistently, despite the drastically reduced T-cell numbers in the C of Tbk- mice, the percentage of the effector T cells expressing IFN-g (Th), IL-7A (Th7) or GM-CSF within the CD þ T-cell population in the C, spleen or draining lymph node of Tbk- mice was either higher than or similar to that of the control mice (Fig. d f). Notably, the absolute number of the inflammatory effector CD þ T cells was drastically increased in the draining lymph nodes of the Tbk- mice due to the accumulation of total CD þ T cells (Fig. g). These results suggest that although TBK deficiency promotes T-cell activation and differentiation, it attenuates EAE induction probably by blocking the migration of the effector T cells from the draining lymph nodes to the C. To further investigate the EAE-regulating function of TBK, we examined the role of TBK in regulating migration of autoimmune T cells to the C. Since TBK deficiency did not decrease the cellularity or subset composition of T cells in the lymph nodes (Supplementary Fig. d,e), it is likely that TBK is Mean clinical score Day after immunization H & E α-cd IHC LFB Tbk- CD + T cells (X ) 8 C CD8 + T cells (X ) IFNγ + cells (%) IL-7A + cells (%) C 8 GM-CSF + cells (%) Spleen 8 IFNγ + cells (%) IL-7A + cells (%) GM-CSF + cells (%) dln dln IFNγ + cells (%) 8 IL-7A + cells (%) GM-CSF + cells (%)..... CD + T cells (X ) IFN-γ + cells (X ) IL-7A + cells (X ) 8 GM-CSF + cells (X ) Figure TBK is crucial for EAE induction. (a) EAE disease score of (n ¼ ) and Tbk- (n ¼ ) mice. (b) Haematoxylin and eosin (H&E), CD IHC, and Luxol Fast Blue (LFB) staining of spinal cord sections from MOG -immunized and EAE mice (8 day after immunization). Scale bars, mm. Data are representative of three independent experiments. (c) Flow cytometry determining the mean±s.d. values of CD þ T-cell number and CD8 þ T-cell number recovered from the C of and Tbk- mice 8 days after EAE induction. (d f) Flow cytometric analyses of CD þ T cells from C, spleen and lymph nodes of the EAE-induced mice described in a (8 days after EAE induction). The T cells were stimulated for h with PMA and ionomycin in the presence of monensin, followed by flow cytometry analysis of the IFN-g, IL-7A and GM-CSF-producing cells. (g) Mean±s.d. values of absolute numbers of IFN-g, IL-7A- and GM-CSF-producing CD þ Tcells recovered from the draining lymph node (dln) of or Tbk- mice described in a. Data represent mean±s.d. from three or more independent experiments. Po.; Po.; Po.;, non-significant as determined by two-way analysis of variance with Bonferroni s post test for EAE clinical scores analysis and two-tailed unpaired Student s t-test for other analysis, comparing the indicated groups. NATURE COMMUNICATIO :7 DOI:.8/ncomms77 & Macmillan Publishers Limited. All rights reserved.

5 NATURE COMMUNICATIO DOI:.8/ncomms77 ARTICLE dispensable for T-cell homing to the lymph nodes. Indeed, adoptively transferred Tbk- CD þ T cells efficiently homed to the spleen and lymph nodes in the T-cell-deficient Tcrb/d-KO mice (Fig. a). In fact, T-cell cellularity in Tbk- mice was moderately increased in the lymphoid organs and decreased in the peripheral blood, compared with mice (Fig. a). Thus, although the Tbk- mice displayed reduced frequency of CDL hi T cells (Fig. d), they did not have a defect in T-cell homing to the lymph nodes as shown for the CDLdeficient mice 9,. T-cell migration into the C is promoted by the inflammatory C microenvironment. To test whether the defect of the Tbk- T cells in C infiltration was due to the cell-intrinsic mechanisms, we employed a mixed bone marrow adoptive transfer approach. We adoptively transferred a mixture of green fluorescent protein (GFP þ ) bone marrows and GFP Tbk- bone marrows into lethally irradiated Rag-KO mice and induced EAE in the recipient mice. On the basis of GFP expression, we analysed the frequency of and Tbk- T cells in the C and draining lymph nodes of the recipient mice during the early effector phase (day ) of EAE induction. Within the same recipient mice, the T cells (GFP þ ) efficiently migrated to the C, whereas the majority of the Tbk- T cells (GFP ) were retained in the draining lymph nodes (Fig. b). Since the and TBK-deficient T cells were activated under the same conditions, these results suggest that the defect of the TBK-deficient T cells in infiltrating into C is not due to attenuated C inflammation but rather due to a cellintrinsic deficiency. TBK deficiency promotes T-cell retention in lymph nodes. Antigen-activated T cells are initially retained in draining lymph nodes to undergo differentiation and subsequently exit the lymph nodes and migrate to the peripheral site of infection or inflammation. Since the TBK-deficient T cells were accumulated in the draining lymph nodes during EAE induction (Fig. b), we examined whether the loss of TBK affected T-cell egress from T cells (%) 8 Spleen T cells (%) plns Blood C dlns T cells (%) T cells (%) T cells (%) GFP + GFP GFP + GFP () () () () 8 Spleen dlns Blood 8 8 Red:, Green: : ratio.... T-zone LV Mean clinical score Day after adoptively transfer CD + T cells (X ) Spleen C C Blood Spleen Lung Med LNs CD + T cells (X ) Figure TBK deficiency promotes T-cell retention in draining lymph nodes. (a) and Tbk- naïve CD þ cells were labelled with CMRA and CFSE, respectively, and adoptively transferred into Tcrb/d-KO mice in a mixture (at : ratio). Cell distribution in different organs was analysed h later by flow cytometry (n ¼ ). (b) Lethally irradiated Rag-KO mice were reconstituted with a mixture (: ratio) of bone marrow cells isolated from Tbk- and GFP-expressing mice (n ¼ ). The chimeric mice were immunized with MOG weeks after reconstitution. C and draining LN (dln) CD þ T cells were analysed based on GFP expression gated on CD þ cell on day after EAE induction. (c e) and Tbk- naïve CD þ cell were labelled with CMRA or CFSE respectively and adoptively transferred into mice at : ratio, followed by MOG immunization. Five days later, recipient mice were subjected to flow cytometry analysis of cell distribution in different organs (c) or confocal imaging of the differentially labelled T cells in a draining LN, showing the entire T-cell zone (left) and the peripheral region (right) (d, red, CMRA-labelled Tcells; green, CFSE-labelled Tbk- Tcells; blue, LYVE- þ lymphatic vessels). : T-cell ratio in the T-cell zone (set at an index of :) and lymphatic vessel are presented as mean±s.d. values of based on three experiments with a total of T cells analysed (e). (f,g) Clinical scores (f) and flow cytometry analysis of CD þ T cells (g, day 8) of Tcrb/d-KO mice undergoing passive EAE induction after being adoptively transferred with or Tbk- MOG-primed CD þ T cells. (h) Flow cytometry analysis of CD þ T cells in the indicated organs and tissues of mice adoptively transferred (for days) with a mixture (: ratio) of MOG-primed (CMRA labelled) and Tbk- (CFSE-labelled) CD þ T cells (: ratio). Data represent mean±s.d. from two or more independent experiments. Po.; Po.; Po.; Po.;, non-significant as determined by two-way analysis of variance with Bonferroni s post test for EAE clinical scores analysis and two-tailed unpaired Student s t-test for other analysis, comparing the indicated groups. NATURE COMMUNICATIO :7 DOI:.8/ncomms77 & Macmillan Publishers Limited. All rights reserved.

6 NATURE COMMUNICATIO DOI:.8/ncomms77 the lymph nodes, by employing a mixed T-cell transfer approach. We labelled the and Tbk- naïve CD þ T cells with CellTracker Orange CMRA dye and carboxyfluorescein succinimidyl ester (CFSE) fluorescence dyes, respectively, and transferred a mixture of the labelled cells (in : ratio) into mice. We then analysed the distribution of the and Tbk- T cells during an early phase of MOG immunization (day ). We detected the accumulation of Tbk- T cells in the draining lymph nodes with concomitantly reduced frequency in the peripheral blood (Fig. c). This result indicated enhanced lymph node retention of the TBK-deficient T cells following antigen stimulation. In parallel, we also performed confocal imaging to analyse the T cells in both the T-cell zone and the lymphatic vessel region. Consistent with the flow cytometry analysis result (Fig. c), the CFSE-labelled Tbk- T cells were clearly more abundant than the CMRA-labelled T cells in the T-cell zone of the draining lymph node (Fig. d, left). Interestingly, however, a significantly lower : T-cell ratio was found in the lymphatic vessel area compared with that in T-cell zones (Fig. d, right; Fig. e), consistent with a reduced egress of T cells through the cortical sinuses (Fig. d,e). These results indicate that TBK may regulate the exit of effector T cells from the draining lymph nodes. Since the TBK deficiency only partially affected the lymph node exit of T cells, we wondered whether the Tbk- mice were competent in mounting immune responses against infections. In an influenza viral infection model, we found that the lung of the virus-infected Tbk- mice only had moderately reduced frequency of CD þ and CD8 þ T cells and increased frequency of T cells in the draining mediastinal lymph nodes (Supplementary Fig. a). Both the body weight loss (Supplementary Fig. b) and the cytokine concentration in bronchoalveolar lavage were comparable between the and Tbk- mice (Supplementary Fig. c). These results suggest competent immune responses to influenza viral infection in the Tbk- mice. To understand why the TBK deficiency particularly influenced the pathogenesis of EAE, we performed passive EAE studies. We isolated T cells from MOG-immunized or Tbk- mice and restimulated them in vitro using the MOG peptide. After expansion, we adoptively transferred the autoimmune and Tbk- effector T cells intravenously (i.v.) into Tcrb/d-KO mice. Interestingly, under these conditions that would bypass the step of T-cell exit from the draining lymph node, the TBK-deficient T cells were still significantly defective in inducing EAE (Fig. f), coupled with reduced CD T-cell number in the C and increased CD T-cell number in the spleen on day 8 following the T-cell transfer (Fig. g). Gene expression analysis revealed that the Tbk- T cells had a moderate reduction in the expression of Ninj gene and competent expression of several other cell adhesion molecules and chemokine receptors implicated in T-cell migration into the C (Supplementary Fig. ). To further assess the mechanism by which TBK mediates C infiltration of T cells, we compared the migration of the and TBK- deficient T cells by labelling the in vitro-expanded MOG-primed and Tbk- T cells with different fluorescence dyes (CMRA and CFSE, respectively) and transferring them in a mixture (: ratio) into mice. We then analysed the frequency of the and Tbk- T cells (in the same recipients) in the blood, lung and lymphoid organs during the early phase (day ) of T-cell transfer. This approach further confirmed the defect of the Tbk- effector T cells in migrating to the C (Fig. h). Interestingly, the Tbk- effector T cells were accumulated in the mediastinal lymph nodes (Fig. h). Of note, autoimmune T cells are known to migrate to the C via the lung and the lung-draining mediastinal lymph nodes. These results suggest that the impaired C infiltration by TBK-deficient autoimmune T cells may involve increased intranodal retention in different steps, although the involvement of TBK in regulating some other aspects of T-cell migration is also possible. TBK controls activation of and mtorc. To understand the signalling mechanism by which TBK regulates T-cell homeostasis and EAE pathogenesis, we examined the effect of the T-cell-specific TBK ablation on the activation of signalling molecules. A striking phenotype of the Tbk- T cells was elevated homeostatic activation of (Fig. a). The phosphorylation of Foxo, a known target of, was also higher in the TBK-deficient T cells (Fig. a). Another major downstream target of is the metabolic kinase mtorc (ref. ). Consistently, the TBK-deficient T cells had elevated phosphorylation of the mtorc substrate protein S kinase (SK) as well as the SK substrate S (Fig. a). In contrast, the loss of TBK did not alter the phosphorylation of the MAP kinases p8 and ERK (Supplementary Fig. a). The crucial role of TBK in controlling the homeostatic activation of and mtorc was unique, since it was not seen with the IKKe-KO T cells (Supplementary Fig. b). Furthermore, the deregulated signalling events in the Tbk- T cells were not due to their activation state, since similar results were obtained with both naïve and memory CD þ T cells (Supplementary Fig. c). The loss of TBK also promoted the phosphorylation of and S stimulated by anti-cd plus anti-cd8 (Fig. b). Moreover, we detected hyperphosphorylation of as well as S and SK in Tbk- T cells isolated during the effector phase of EAE induction (Fig. c). Using the Cre-ER-inducible KO system, we found that TBK ablation in adult mice also caused aberrant phosphorylation of and S in T cells (Supplementary Fig. d), further emphasizing a pivotal role for TBK in controlling the activation of and its downstream targets, Foxo and mtorc. TBK induces phosphorylation-dependent degradation. Although activation has been extensively studied, how this master kinase is negatively controlled is unclear. We found that the hyperactivation of in TBK-deficient T cells was associated with a profound increase in the level of total protein (Fig. a and Supplementary Fig. b d). This phenotype was not due to enhanced mrna expression (Supplementary Fig. e), indicating a post-translational mechanism of regulation. We thus examined the possible involvement of the ubiquitin proteasome pathway. Because of the low number of naïve T cells in the Tbk- mice, we used thymocytes for ubiquitination assays. As seen in peripheral T cells, the TBK- deficient thymocytes also had elevated level of (Fig. d). Interestingly, was undergoing lysine 8-linked polyubiquitination in the thymocytes, which was drastically reduced in the TBK-deficient thymocytes (Fig. d). This finding suggests that TBK controls the homeostatic level of by promoting ubiquitin-dependent degradation, thus explaining the accumulation of in the TBK-deficient T cells. Since the homeostasis of T cells involves constant stimulation of the TCR by self-ligands 7, we asked whether the TCR signal could stimulate degradation and whether this signalling event was regulated by TBK. To avoid the homeostatic effect of TBK deficiency, we employed CD þ T cells. Indeed, the stimulation of the CD þ T cells with anti-cd and anti-cd8 led to the gradual loss of (Fig. e). Moreover, a small-molecule inhibitor of TBK, MRT77 (ref. 8), efficiently inhibited the degradation (Fig. e). We next employed a NATURE COMMUNICATIO :7 DOI:.8/ncomms77 & Macmillan Publishers Limited. All rights reserved.

7 NATURE COMMUNICATIO DOI:.8/ncomms77 ARTICLE P- P-Foxo Foxo P-S S P-SK SK α-cd + α-cd min P- P-S S EAE P- P-S S P-SK SK TBK Ub- α-cd + α-cd8 (h) DMSO MRT77 HA- T9A T8A S78A FLAG-TBK HA- FLAG- TBK GFP FLAG- TBK FLAG- TBK DMSO K8A HA- MG K8A T9A T8A S78A HA- GFP DMSO Rapamycin P-S S CD Control.9 9. Rapamycin CDL Akt +/ Akt +/ P- Mean clinical score Akt +/ Akt +/ Day after immunization CD + T cells (X ) dln C Akt +/ Akt +/ Akt +/ Akt +/ Relative mrna (fold).... CCR7 CDL Naïve KLF SPR Relative mrna (fold).... CCR7 Memory CDL KLF SPR SPR + cell % KLF mrha (fold) DMSO i LY9 Figure TBK stimulates degradation and negatively regulates the activation of and mtorc pathways. (a) IB analysis of phosphorylated (P-) (Ser7), Foxo (Thr), S (Ser/), SK (Thr89), as well as their total protein controls in CD þ Tcells from and Tbk- mice ( weeks old). (b) IB analysis of the indicated phosphorylated (P-) and total proteins in CD þ T cells activated by anti-cd plus anti-cd8 using a crosslinking method. (c) IB analysis of the indicated phosphorylated (P-) and total proteins in CD þ T cells isolated from day 8 EAE-induced or Tbk- mice. (d) was isolated by IP from the lysates of thymocytes derived from or Tbk- mice ( weeks old), and the ubiquitinated (Ub-) was detected by IB using an anti-ubiquitin antibody detecting K8-linked polyubiquitin chains. (e) IB analysis using CD þ Tcells, pretreated for h with a TBK inhibitor, MRT77, of solvent control dimethylsulphoxide (DMSO) and subsequently stimulated for the indicated times with anti-cd plus anti-cd8 in the presence of a protein synthesis inhibitor, cycloheximide. (f) IB analysis of endogenous and and exogenous FLAG-TBK in whole-cell lysates of HEK9T cells, transfected as indicated and subsequently incubated for h with DMSO or MG ( mm). An arrowhead indicates the non-specific band. (g) IB analysis of the indicated exogenous proteins in the whole-cell lysates of HEK9 cells transfected with HA- or the indicated point mutants either in the presence ( þ ) or absence ( ) of FLAG-tagged TBK. An arrowhead indicates the non-specific band. (h) IB analysis of whole-cell lysates of CD þ T cells transduced with pmigr retroviruses encoding or mutant forms of as well as the GFP marker. (i) IB (left) and flow cytometry (right) analyses of splenic CD þ T cells from or Tbk- mice treated injected (i.p) with rapamycin for days (n ¼ ). (j) IB analysis of CD þ cells from spleen of the indicated mouse strains. (k,l) EAE disease score (k) and CD þ T-cell number of draining LN and C (l, day7) (l) of the indicated mouse strains, immunized for EAE induction (n ¼ ). (m) Quantitative PCR (qpcr) analysis of the indicated genes in freshly isolated naïve or memory CD þ T cells from and Tbk- mice (n ¼ ). (n) SPR flow cytometry analysis in memory CD þ cell of inguinal LN from or Tbk- mice. (o) qpcr analysis of KLF in CD þ T cells treated for h with either DMSO control or inhibitors for (i) or PI kinase (LY9). Data represent mean±s.d. from two or more independent experiments. Po.; Po.;, non-significant as determined by two-way analysis of variance with Bonferroni s post test for EAE clinical scores analysis and two-tailed unpaired Student s t-test for other analysis, comparing the indicated groups. transient transfection model to see whether overexpressed TBK was able to induce degradation. Indeed, the expression of TBK, but not its catalytically inactive mutant (K8A), caused drastic loss of endogenous, which was blocked by a proteasome inhibitor, MG (Fig. f). TBK also induced the proteolytic degradation of exogenously expressed but did not affect the co-transfected control protein GFP (Supplementary Fig. f). A previous study suggests that the overexpressed TBK phosphorylates at threonine-9 and serine-78, but the functional significance has not been determined 9. Our finding that TBK stimulated degradation led us to examine whether these phosphorylation sites regulate TBK stability. Mutation of threonine-9, or the activation-loop phosphorylation site threonine-8, of only weakly inhibited TBK-stimulated degradation (Fig. g). Interestingly, the mutation of serine-78 of completely prevented its degradation by TBK (Fig. g). When stably expressed in T cells, the mutant harbouring the serine-78 mutation also displayed markedly higher level of expression (Fig. h). These results indicate that the TBK-induced phosphorylation at serine-78 plays an important role in regulating the fate of. NATURE COMMUNICATIO :7 DOI:.8/ncomms & Macmillan Publishers Limited. All rights reserved.

8 NATURE COMMUNICATIO DOI:.8/ncomms77 Functional significance of -mtorc deregulation. Our finding that TBK controls the activation of and mtorc provides molecular insight into the impaired T-cell homeostasis in Tbk- mice, since the -mtorc signalling axis has a crucial role in the regulation of T-cell homeostasis. Indeed, we found that treatment of Tbk- mice with an mtorc inhibitor, rapamycin, blocked the homeostatic activation of mtorc and corrected the T-cell homeostasis phenotype (Fig. i). It is known that activated T cells initially downregulate homing receptors, particularly sphingosine--phosphate receptor (SPR), and then regain the expression of SPR for migrating out of the lymph nodes. This dynamic change is coupled with the initial activation of and the subsequent decline of the activity. Since Tbk- T cells had aberrant accumulation and activation, we tested whether the ablation of could correct the EAE phenotype of the Tbk- mice. We crossed the Tbk- mice with Akt-KO mice to generate Akt þ / Tbk- mice. Interestingly, ablation of one allele of Akt, which reduced the level of and P- in Tbk- T cells (Fig. j), largely restored the sensitivity of the Tbk- mice to EAE induction (Fig. k) as well as the C migration of the Tbk- T cells (Fig. l). A hallmark of the C-migrating autoimmune T cells is the high level expression of the transcription factor KLF and its target gene SPR, the latter of which has become an attractive target for MS therapy,,. In naïve CD þ T cells, the TBK deficiency only moderately reduced the expression of KLF and SPR as well as two other T-cell homing genes, CCR7 and CDL (Fig. m). Interestingly, however, the TBK deficiency greatly inhibited the expression of KLF and SPR, although only moderately reduced expression of CCR7 and CDL, in the memory population of CD þ T cells (Fig. m). Flow cytometry analyses also revealed reduced level of surface expression of SPR in Tbk- CD þ T cells (Fig. n). The expression of KLF is known to be mediated by Foxo, a transcription factor that is negatively regulated by. Interestingly, incubation of Tbk- CD þ T cells with small-molecule inhibitors for (i) and its upstream regulator PI kinase (LY9) greatly promoted the expression of KLF (Fig. o). On the other hand, these inhibitors only slightly increased the expression of KLF in the T cells (Fig. o), consistent with the low basal activity in T cells (Fig. a). Thus, the TBK deficiency attenuated the expression of KLF and SPR, which in turn appeared to be due to the deregulated activation. Collectively, these data indicate that the aberrant activation in TBK-deficient T cells contributes to their attenuated C migration and induction of EAE. To examine the role of TBK in regulating -mtorc signalling and gene expression in human T cells, we silenced the expression of TBK using short hairpin RNAs (shrnas) expressed in a lentiviral vector. Interestingly, shrna-mediated TBK silencing promoted the phosphorylation of as well as the -mtorc downstream proteins Foxo and S (Supplementary Fig. 7a). Consistently, the TBK knockdown also reduced the expression of SPR and KLF as well as CDL (Supplementary Fig. 7b). Using a well-defined in vitro T-cell migration model, we found that TBK knockdown in human CD þ T cells significantly inhibited their ability to transmigrate through a human brain microvascular endothelial cell (EC) monolayer (Supplementary Fig. 7c). Similar results were obtained when the T cells were treated with a TBK inhibitor, amlexanox (Supplementary Fig. 7d). In parallel with the functional studies, we analysed the potential alterations of TBK expression in MS patients. Our data revealed that the expression of TBK is significantly increased in the peripheral blood mononuclear cell (PBMC) of MS patients compared with the healthy donors (Supplementary Fig. 7e). Consistently, TBK expression was also shown to be elevated (.- and.79-folds) in two MS PBMC microarray databases,. These results suggest that TBK controls the mtorc signalling axis in both murine and human T cells. A TBK inhibitor ameliorates EAE pathogenesis. The data described above not only revealed a previously unknown signalling mechanism regulating T-cell function and C inflammation but also implicated TBK as an attractive therapeutic target for the treatment of MS. To further assess the therapeutic value of TBK, we tested the effect of TBK pharmacological inhibition on the induction of EAE. In this regard, a recent study identified the Food and Drug Administration-approved therapeutic compound amlexanox as a selective inhibitor of TBK (ref. ). We asked whether amlexanox could ameliorate EAE and, if so, whether it acted through modulating T-cell migration into the C. We treated the mice daily with amlexanox (via intraperitoneal (i.p.) injection) during the induction of EAE. The amlexanox treatment drastically delayed the onset, and reduced the severity, of the EAE disease (Fig. a), which was associated with a profound reduction in the number of CD þ and CD8 þ T cells in the C of the EAE-induced mice (Fig. b). As seen with the TBK gene ablation, the amlexanox treatment did not inhibit T-cell expansion but rather caused the sequestration of T cells in the draining lymph nodes and spleen (Fig. c). Moreover, the T cells isolated from the amlexanox-treated mice displayed aberrant activation of and its target protein Foxo as well as the mtocr pathway component S (Fig. d). To further assess the therapeutic potential of amlexanox, we employed a remitting relapsing EAE model, which involved the immunization of SJL mice with a peptide (9 ) derived from the proteolipid protein (PLP). As expected, the immunized SJL mice developed EAE, recovered from the disease after about days and then relapsed (Fig. e). On day after immunization, we treated the mice (via i.p. injection) with vehicle control or the TBK inhibitor amlexanox ( mg per kg body weight) for days (n ¼ ). Importantly, the amlexanox treatment greatly inhibited the EAE relapse (Fig. e). Collectively, these results highlight the potential of TBK as a therapeutic target for the treatment of MS. Of course, it is currently unclear whether the effect of amlexanox was solely on T cells or also on innate immune cells, such as monocytes or macrophages. Future studies will further address this question by generating myeloid cell-conditional Tbk-KO mice. Discussion The data presented in this study establish TBK as a crucial signalling factor that regulates the homeostasis and inflammatory function of T cells. A prominent phenotype of the Tbk- mice was the increased frequency of memory-like T cells and decreased frequency of naïve T cells in the lymphoid organs. Although the TBK deficiency promotes T-cell activation and differentiation, the Tbk- mice are refractory to the induction of EAE, an animal model of the autoimmune neuroinflammatory disease MS. We obtained genetic and biochemical evidences that TBK served as a negative regulator of. T-cell-specific ablation of TBK caused heightened activation of and its downstream targets, mtorc and Foxo. The deregulated mtorc activation appeared to contribute to the perturbed homeostasis of TBK-deficient T cells. We found that the injection of Tbk- mice with an mtorc inhibitor could largely, although not completely, correct their abnormal phenotype in T-cell 8 NATURE COMMUNICATIO :7 DOI:.8/ncomms77 & Macmillan Publishers Limited. All rights reserved.

9 NATURE COMMUNICATIO DOI:.8/ncomms77 ARTICLE Mean of clinical score Vehicle Amlexanox Days after immunization Total cells (X ) CD + T cells (X ) C. CD8 + T cells (X )..... Vehicle Amlexanox T cells (X ) Vehicle Amlexanox Spleen T cells (X ) dlns Vehicle Amlexanox P- P-S S P-Foxo Foxo Mean clinical score No treatment Vehicle Amlexanox Day after immunization Figure A small-molecule inhibitor of TBK promotes -mtorc activation and inhibits EAE induction. (a d) mice were immunized for EAE induction. Starting from days before the immunization, the mice were treated daily (via i.p. injection) with vehicle control or the TBK inhibitor amlexanox ( mg per kg body weight) for days (n ¼ ). The immunized mice were monitored for EAE disease scores (a) or killed on day post immunization for flow cytometric analysis of the indicated cell populations in the C (b) and peripheral lymphoid organs (c) or for IB analysis of the indicated phosphorylated (P-) and total proteins in the splenic CD þ T cells (d). (e) EAE clinical score of SJL.J mice immunized with PLP 9 to induce the remitting relapsing EAE and then treated daily (via i.p. injection) with vehicle control or the TBK inhibitor amlexanox ( mg per kg body weight) for days starting from day (n ¼ ). Data represent mean±s.d. from two or more independent experiments. Po.; Po.; Po.; Po.;, non-significant as determined by two-way analysis of variance with Bonferroni s post test for EAE clinical scores analysis and two-tailed unpaired Student s t-test for other analysis, comparing the indicated groups. homeostasis. These data suggest that the uncontrolled mtorc signalling contributes to, but may not be solely responsible for, the perturbed T-cell homeostasis in Tbk- mice. Besides mtorc, several other factors in the related pathways, including the transcription factors Foxo and KLF, are required for maintaining T-cell homeostasis 7,8. Indeed, the TBK deficiency attenuated Foxo activation and KLF expression in T cells, indicating the involvement of these transcription factors in the perturbed T-cell homeostasis in the Tbk- mice. Migration of autoimmune T cells from lymphoid organs to the target tissues represents a crucial and complex step in the development of autoimmunity. Newly activated T cells express low levels of the factors involved in T-cell migration, particularly the transcription factor KLF and its target gene product SPR, which is correlated with the lymphoid retention of the T cells 9. Over time, the activated T cells differentiate into effector T cells and gradually regain the ability to express the migrationregulatory genes, which is important for the egress of T cells from the lymphoid organs. The role of KLF and SPR in mediating T-cell migration has been directly demonstrated in a number of studies 7,,. Our data suggest that TBK promotes the expression of KLF and SPR in T cells and, thereby, facilitates the lymph node egress of T cells. We obtained genetic and pharmacological evidence that deregulated activation in Tbk- T cells contributed to the reduced expression of KLF and SPR and attenuated migration of T cells to the C. Ablation of one allele of Akt in the Tbk- largely corrected their phenotype in T-cell migration and EAE sensitivity. The transcription factor Foxo, which is negatively regulated by, has been implicated in the induction of KLF and other migration-regulatory genes 8,,. We propose that TBK promotes T-cell migration by downregulating and, thereby, maintaining a sufficient level of active Foxo for mediating the expression of migration genes. Indeed, we found that TBK deficiency causes elevated Foxo phosphorylation in T cells. It is important to note that the TBK deficiency only partially impaired the lymph node egress of T cells. Consistently, the Tbk- mice remained competent in mounting immune responses to influenza virus infection. This finding suggests that TBK may regulate additional functions of the C autoimmune T cells other than facilitating their exit from the draining lymph nodes. It is also possible that the complex route of autoimmune T-cell migration in EAE may contribute to the remarkable resistance of the Tbk- mice to EAE induction. Following their exit from draining lymph nodes, C autoimmune T cells migrate to the C via the lung and the lung-draining mediastinal lymph nodes. Our data suggest that the Tbk- effector T cells not only have the tendency to be retained in the draining lymph nodes but also accumulate in the mediastinal lymph nodes. These results suggest that the impaired C infiltration of TBK-deficient T cells may involve different steps of lymph node retention, although it remains possible that TBK may also regulate additional mechanisms of T-cell migration to the C. TBK and its homologue IKKe are known as an innate immune kinase that mediates induction of type I IFNs. Although TBK is thought to be functionally redundant with IKKe in innate immune cells, our data suggest that these two kinases have fundamentally different functions in T cells. While NATURE COMMUNICATIO :7 DOI:.8/ncomms & Macmillan Publishers Limited. All rights reserved.

10 NATURE COMMUNICATIO DOI:.8/ncomms77 TBK regulates T-cell homeostasis and mediates T-cell migration into the C, IKKe is dispensable for these biological functions. IKKe is also dispensable for the control of -mtorc signalling axis. Nevertheless, like TBK, IKKe is activated by the T-cell activation signals. Further studies are required to examine whether IKKe regulates any aspects of T-cell functions. The functional differences between TBK and IKKe have also been shown in B cells, in which TBK, but not IKKe, regulates antibody class switching to IgA. Studies using immortalized mouse embryonic fibroblast and cancer cell lines suggest a positive role of TBK in the regulation of 9,. TBK phosphorylates at several residues, including threonine-9 and serine-78, although the functional significance has not been defined 9. Our current work provides the first evidence for the in vivo role of TBK in the regulation of. Our data clearly demonstrated a negative role for TBK in regulation. TBK does not seem to inhibit the catalytic activation, but rather controls the fate, of. In TBK-deficient T cells, the steady level of is drastically elevated, which contributes to the aberrant activity in naïve and effector T cells. We have obtained biochemical evidence that the serine- 78 phosphorylation site of is crucial for TBK-stimulated degradation. Our work not only demonstrated a novel signalling mechanism that regulates T-cell homeostasis and EAE pathogenesis but also have profound implications for MS therapy. Regarding the human relevance of our findings, we found that TBK knockdown in human PBMCs causes hyperactivation of mtorc signalling and reduced expression of the T-cell homing genes KFL and SPR. Furthermore, TBK knockdown or pharmacological inhibition attenuates the transmigration of human CD þ T cells across a human brain microvascular endothelial monolayer in vitro. Our finding that TBK ablation does not affect T-cell activation or immune responses against flu viral infection, but attenuates EAE induction, makes TBK an attractive therapeutic target to be exploited in the treatment of MS. We found that amalexanox, a Food and Drug Administration-approved drug that selectively inhibits TBK (ref. ), greatly attenuated the induction of EAE and inhibited the disease relapse in a remitting relapsing model of EAE. Amlexanox also inhibited human T-cell migration in vitro. Of note, the mode of action of TBK differs substantially from that of SPR. First of all, TBK appears to regulate the re-expression of SPR following its downregulation in activated T cells, but is dispensable for the overall expression of SPR. Thus, unlike the SPR ablation, the TBK deficiency does not influence the homing of naïve T cells or the thymic emigration of newly generated T cells. Furthermore, the TBK deficiency only partially inhibits the expression of SPR and the egress of effector T cells from the lymph nodes. Consistently, the loss of TBK does not appreciably compromise immune responses against influenza virus infection. These findings along with the fact that amlexanox is a relatively safe drug that has been used for many years in the clinic indicate that amlexanox may be an attractive compound to be evaluated in MS clinical trials. Methods Mice. The Tbk-flox mice were generated in B 9 mixed genetic backgrounds and further backcrossed to the B mice for four generations. To generate T-cell-conditional Tbk-KO mice, Tbk-flox mice were crossed with CD-Cre transgenic mice (B genetic background, Jackson Laboratories), producing Tbk þ / þ CD-Cre (named ) and Tbk fl/fl CD-Cre (named Tbk- mice) mice. Tbk-flox mice were also crossed with Rosa-Cre-ER mice (B genetic background, Jackson Laboratories) and the progeny Tbk þ / þ Rosa-Cre-ER and Tbk fl/fl Rosa-Cre-ER mice were injected i.p. with tamoxifen ( mg per mouse) in corn oil daily for four consecutive days to induce Cre function. The tamoxifentreated Tbk þ / þ Rosa-Cre-ER and Tbk fl/fl Rosa-Cre-ER mice were named -ER and Tbk-ER mice, respectively, and used weeks after the treatment. Carma-KO mice (B 9 genetic background) were provided by Dr Josef M. Penninger (Austrian Academy of Sciences). Mice with loxp-flanked allele encoding IKKb (IKK) or NEMO alleles were provided by Dr Manolis Pasparakis (University of Cologne) 7. These mice were crossed with CD-Cre mice to generate the T-cell-conditional IKKb KO (IKKb-) and NEMO KO (NEMO-) mice. IKKe-KO, B.SJL (CD. þ ), C7BL/, Rag-KO and TCRb/d KO mice were from Jackson Laboratory. Experiments were performed with age-matched mice. Mice were maintained in a specific pathogen-free facility of The University of Texas MD Anderson Cancer Center, and all animal experiments were done in accordance with protocols approved by the Institutional Animal Care and Use Committee of the University of Texas MD Anderson Cancer Center. Cell lines. The NEMO-deficient JM.. cell line and its NEMO-competent control J.SVT are Jurkat derivatives 8. The CARMA-deficient JPM. mutant cell line and its parental Jurkat cell line were provided by Dr Xin Lin (UT MD Anderson Cancer Center) 7. Plasmids and antibodies and reagents. The pcdna expression vector encoding haemagglutinin (HA)-tagged human was provided by Dr Keqiang Ye (Emory University). pmigr-h was generated by inserting human cdna into the EcoRI and BglII sites of pmigr vector, and the mutants (T9A, T 8A, S78A) were created by site-directed mutagenesis. Flag-tagged TBK and its catalytically inactive mutant, K8A, were provided by Dr Chen Wang (Shanghai Institutes for Biological Sciences). GST-IkBa (amino acids of IkBa) was as described 8 and GST-IRF (amino acids 8 7) was provided by Dr Rongtuan Lin (McGill University). Functional grade anti-mouse (m) CDe (-C) and anti-mcd8 (7.) antibodies and blocking antibodies for mifn-g (XMG.) and mil- (B) were from ebioscience. Antibodies for (B-, :,), phospho-erk/ (E-, :,), ERK/ (K-, :,), p8 (H-7, :,), and GFP (B-, :,) were from Santa Cruz Biotechnology. Anti-IKKi (IKKe, :,), anti- (C-, :,) and horseradish peroxidase-conjugated anti-flag (M, :,) were from Sigma-Aldrich. Antibodies for phospho-tbk Ser7 (DC, :,), phospho- Ser7 (D9E, :,), phospho-sk Thr89 (A, :,), phospho-s Ser/ (D7..E, :,), phospho-foxo Thr/FoxOa Thr (:,), phospho-p8 Thr8/Tyr8 (D7, :,), TBK, SK (9D7, :,), S (D, :,) and FoxO (C9H, :,) were from Cell Signaling Technology. Horseradish peroxidase-conjugated anti-haemagglutinin (HA-7, :,) was from Roche. Anti-K8-Ubiquitin (Apu, :,) was from Millipore. Fluorescence-labelled antibodies for murine (m) CD (LT, :), mcd8 (-.7, :), mcd (-C, :), CD (IM7, :), CDL (MEL-, :), mtcrb (H7-97, :), mcd. (A, :), mcd. (, :), mthy. (HIS, :), IL-7A (ebio7b7, :), and IFN-g (XMG., :) were purchased from ebioscience. Annexin V was from BD. The fluorescencelabelled phospho-specific antibodies were all from Cell Signaling Technology. The inhibitor / was from Calbiochem, the PI kinase inhibitor LY9 was from Selleck, the TBK inhibitor MRT77 was from Chemexpress, the TBK inhibitor amlexanox was from Cayman Chemical and the protein synthesis inhibitor cycloheximide was from Sigma-Aldrich. Flow cytometry analysis and cell sorting. Single-cell suspensions of splenocytes and lymph node cells were subjected to flow cytometry and cell sorting as previously described 9 using a FACSAria (BD Biosciences). For intracellular cytokine staining (ICS) assays, T cells isolated from the spleen, draining lymph nodes or C (brain and spinal cord) of immunized mice or from in vitro cultures were stimulated for h with PMA ( ng ml ) and Ionomycin ( ng ml )inthe presence of monensin ( mgml ). The stimulated cells were fixed in % paraformaldehyde and permeablized in.% saponin and then subjected to cytokine staining flow cytometry analyses. In vivo inhibition of mtorc. Mice were injected daily (i.p) with the mtorc inhibitor rapamycin ( mg per kg body weight) or solvent control (% ethanol in PBS) for days and then killed for experiments. Influenza viral infection. Influenza virus A/PR8 (HN) was provided by Dr Brian E. Gilbert (Baylor College of Medicine). For influenza infection, mice were first anaesthetized and then inoculated intranasally with ml of influenza virus A/PR8 diluted in DMEM medium. Control mice were inoculated with ml DMEM as control. Body weight was monitored every other day for 8 days. On day 8, bronchoalveolar lavage fluid was collected in anaesthetized mice. Then, ml cold PBS was instilled into the lungs through the trachea and aspirated back. After collecting bronchoalveolar lavage fluid, mice were killed for cell distribution analysis. Induction and evaluation of EAE. The encephalitogenic peptide of MOG (residues, Met-Glu-Val-Gly-Trp-Tyr-Arg-Ser-Pro-Phe-Ser-Arg-Val-Val-His- Leu-Tyr-Arg-Asn-Gly-Lys) and the encephalitogenic peptide of myelin PLP NATURE COMMUNICATIO :7 DOI:.8/ncomms77 & Macmillan Publishers Limited. All rights reserved.