Paracaspase MALT1 Deficiency Protects Mice from Autoimmune-Mediated Demyelination

This information is current as Conor Mc Guire, Peter Wieghofer, Lynn Elton, David of October 1, 2021. Muylaert, Marco Prinz, Rudi Beyaert and Geert van Loo J Immunol 2013; 190:2896-2903; Prepublished online 11 February 2013; doi: 10.4049/jimmunol.1201351 http://www.jimmunol.org/content/190/6/2896 Downloaded from

Supplementary http://www.jimmunol.org/content/suppl/2013/02/11/jimmunol.120135 Material 1.DC1 http://www.jimmunol.org/ References This article cites 34 articles, 10 of which you can access for free at: http://www.jimmunol.org/content/190/6/2896.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Paracaspase MALT1 Deficiency Protects Mice from Autoimmune-Mediated Demyelination

Conor Mc Guire,*,† Peter Wieghofer,‡ Lynn Elton,*,† David Muylaert,*,† Marco Prinz,‡,x Rudi Beyaert,*,† and Geert van Loo*,†

The paracaspase MALT 1 is a major player in lymphocyte activation and proliferation. MALT1 mediates Ag-induced signaling to the transcription factor NF-kB by functioning both as a scaffold and cysteine . We studied the role of MALT1 in the development of experimental autoimmune encephalomyelitis (EAE), an model of multiple sclerosis. MALT1-knockout mice did not develop any clinical symptoms of EAE. In addition, lymphocyte and macrophage infiltration into the spinal cord was absent in MALT1-knockout mice, as were demyelination and proinflammatory gene expression. Adoptive transfer experiments showed that MALT1 deficiency in splenocytes is sufficient for EAE resistance. Moreover, autoreactive T cell activation was severely impaired in MALT1-deficient T cells, suggesting the inability of MALT1-deficient effector T cells to induce demyelinating Downloaded from inflammation in the CNS. Finally, the MALT1 substrates A20 and CYLD were completely processed in wild-type T cells during EAE, which was partially impaired in MALT1-deficient T cells, suggesting a contribution of MALT1 proteolytic activity in T cell activation and EAE development. Together, our data indicate that MALT1 may be an interesting therapeutic target in the treatment of multiple sclerosis. The Journal of Immunology, 2013, 190: 2896–2903.

although the trigger for this autoimmune disease has not been

xperimental autoimmune encephalomyelitis (EAE) is the http://www.jimmunol.org/ main animal model for multiple sclerosis (MS), the most elucidated, considerable efforts have been made in clarifying the E common chronic inflammatory demyelinating disease of molecular mechanisms behind autoreactive T cell activation. the CNS (1, 2). Both MS and EAE are characterized by an au- The transcription factor NF-kB plays a central role in the ac- toreactive immune response that targets the CNS, causing typical tivation and proliferation of T cells. Upon stimulation of the TCR, pathological hallmarks, such as demyelination, oligodendrocyte protein kinase C u–mediated phosphorylation of the CARD-con- cell death, and neurodegeneration. Encephalitogenic Th1/Th17– taining protein CARMA1 (also known as CARD11) results in the polarized T cells play a central role in this immune response; recruitment of B cell lymphoma-10 (BCL10) and MALT1 (3). This CARMA1/BCL10/MALT1 (CBM) complex subsequently re-

*Department for Molecular Biomedical Research, Unit of Molecular Signal Trans- cruits TRAF2 and TRAF6, allowing further downstream signaling by guest on October 1, 2021 duction in Inflammation, VIB, B-9052 Ghent, Belgium; †Department of Biomedical and leading to nuclear translocation and activation of NF-kB. Gene- Molecular Biology, Ghent University, B-9052 Ghent, Belgium; ‡Department of Neu- ropathology, University of Freiburg, D-79106 Freiburg, Germany; and xBIOSS Cen- targeting strategies showed that MALT1 is indispensable for NF-kB tre for Biological Signaling Studies, University of Freiburg, D-79106 Freiburg, activation downstream of TCR stimulation, resulting in an absence Germany of T cell activation and proliferation in MALT1-deficient T cells (4, 1R.B. and G.v.L. share senior authorship. 5). Besides acting as a scaffold mediating TCR signaling, MALT1 Received for publication May 14, 2012. Accepted for publication January 10, 2013. has proteolytic activity. Indeed, recent findings demonstrated that This work was supported by research grants from the Interuniversity Attraction Poles A20 (6), BCL10 (7), RelB (8), and cylindromatosis (CYLD) (9) are Program (IAP6/18 and IAP7), the Fonds voor Wetenschappelijk Onderzoek-Vlaan- substrates of MALT1. Although the adaptor function of MALT1 is deren, the Belgian Foundation against Cancer, the Strategic Basis Research Program of the Instituut voor Innovatie door Wetenschap en Technologie, the Hercules Foun- indispensible for T cell activation, its proteolytic activity is con- dation, and the Concerted Research Actions and Ghent Researchers On Unfolded sidered critical for a full-blown NF-kB response, shaping the extent in Inflammatory Disease Multidisciplinary Research Partnership of Ghent University. C.M. and D.M. were supported as a Ph.D. and postdoctoral fellow, re- of T cell activation (3). spectively, by the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen, and L.E. Because of its essential role in T and B cell activation, MALT1 is was supported as a Ph.D. fellow by the Instituut voor Innovatie door Wetenschap considered an important therapeutic target in autoimmunity. How- en Technologie. G.v.L. was supported by a Fonds voor Wetenschappelijk Onderzoek- Vlaanderen Odysseus grant and by grants from the Geneeskundige Stichting Konin- ever, its role in the development of autoimmune disease has not gin Elisabeth and the Charcot Foundation. been reported. In this study, we sought to address the role of MALT1 C.M. performed the experiments, analyzed data, and prepared the figures; P.W., L.E., in the generation of autoreactive T cells in the context of EAE. For D.M., and M.P. contributed to experimental work and/or provided assistance; and 2/2 R.B. and G.v.L. supervised the overall research. C.M., R.B., and G.v.L. wrote the this, we induced EAE in mice deficient in MALT1 (MALT1 )and +/+ manuscript. in wild-type and heterozygous littermate control mice (MALT1 2/+ 2/2 Address correspondence and reprint requests to Dr. Geert van Loo and Dr. Rudi and MALT1 ). MALT1 mice were completely protected Beyaert, Department for Molecular Biomedical Research, VIB and Ghent University, from EAE, which was reflected in the absence of immune cell Technologiepark 927, B-9052 Ghent, Belgium. E-mail addresses: geert.vanloo@ dmbr.vib-ugent.be (G.v.L) and [email protected] (R.B.) infiltration, demyelination, and axonal damage in the spinal cord. 2/2 The online version of this article contains supplemental material. Furthermore, splenocytes from MALT1 mice failed to produce Abbreviations used in this article: APP, amyloid precursor protein; CBM, CARMA1/ an autoreactive T cell response and failed to induce autoimmune B cell lymphoma-10/MALT1; CYLD, cylindromatosis; EAE, experimental autoim- inflammation upon transfer in wild-type mice. Finally, cleavage of mune encephalomyelitis; MOG, myelin oligodendrocyte glycoprotein; MS, multiple the MALT1 substrates A20 and CYLD were shown in wild-type sclerosis; Treg, regulatory T cell. T cells from mice with EAE. Collectively, these data demonstrate Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 a crucial role for MALT1 in T cell activation and in the early www.jimmunol.org/cgi/doi/10.4049/jimmunol.1201351 The Journal of Immunology 2897 priming phase of EAE, suggesting that targeting MALT1 might be T cell recall assay an important therapeutic strategy to treat MS. T cell recall responses were assessed in splenocytes isolated from mice 10 d after immunization with MOG35–55 peptide. After erythrocyte lysis Materials and Methods using ACK lysis buffer, splenocytes were cultured in flat-bottom 96-well 5 plates at a density of 7 3 10 /well in DMEM supplemented with 5% FCS, L-glutamine, nonessential amino acids, and antibiotics. Cells were incu- 2/2 MALT1 mice (5), backcrossed to C57BL/6 mice, were kindly provided bated for 48 h in the presence of 1, 10, or 30 mg/ml MOG35–55 peptide. by Dr. Tak W. Mak (Ontario Cancer Institute, Toronto, ON, Canada). After 48 h, supernatant was collected, and concentrations of IL-2 and MALT12/+ mice were intercrossed to generate MALT1+/+, MALT12/+, and IFN-g were measured by ELISA (eBioscience). IL-10 was quantified using MALT12/2 offspring. Mice were housed in individually ventilated cages a Bio-Plex Pro kit on the Bio-Plex 200 system (both from Bio-Rad), in either specific pathogen–free or conventional animal facilities. All an- according to the manufacturer’s instructions. For analysis of cell prolif- imal experiments were performed according to institutional, national, and eration, cultures were pulsed with 0.5 mCi [3H]thymidine/well (1 mCi/ml European animal regulations. Animal protocols were approved by the [3H]TdR; GE Healthcare) during the last 18 h. Cells were harvested onto ethics committee of Ghent University. glass fiber filter membranes using a 96-well plate cell harvester (IH110-96; Inotech), and thymidine incorporation was measured by scintillation Induction and assessment of EAE counting (MicroBeta Plus 1450 reader; PerkinElmer).

Mouse myelin oligodendrocyte glycoprotein (MOG)35–55 peptide T cell isolation and Western blot (MEVGWYRSPFSRVVHLYRNGK) was synthesized by Sigma Genosys. Ten- to 15-wk-old male mice were immunized s.c. with an emulsion of 200 CD4+ T cells were isolated from splenocytes using a MACS CD4+ T cell mg MOG35–55 peptide in 200 ml sterile PBS and an equal volume of CFA isolation kit II (Miltenyi Biotec), according to the manufacturer’s (Sigma-Aldrich) supplemented with 10 mg/ml Mycobacterium tuberculo- instructions. The purity of CD4+ T cells was analyzed on a FACS LSRII sis H37RA (Becton Dickinson Bioscience). Mice also received 50 ng apparatus using CD3-PE (clone 145-2C11; BD Pharmingen) and CD4- pertussis toxin (Sigma-Aldrich) in 200 ml sterile PBS at the time of im- FITC (clone GK1.5; BD Pharmingen). For Western blots, CD4+ T cells Downloaded from munization and 48 h later. For passive induction of EAE, spleens from were lysed in 50 mM HEPES (pH 7.6), 250 mM NaCl, 5 mM EDTA, and immunized mice were isolated 10 d postimmunization. Splenocytes were 0.5% (v/v) Nonidet P-40, including phosphatase and protease inhibitors. cultured for 48 h in RPMI 1640 supplemented with 10% FBS, sodium The proteins were separated by SDS-PAGE, followed by semidry immu- pyruvate, L-glutamine, nonessential amino acids, antibiotics, 30 mM noblotting and detection via ECL (PerkinElmer) for analysis. The Abs used MOG35–55 peptide, and 10 ng/ml recombinant mouse IL-23 (eBioscience). were anti-A20 (clone A12, sc-166692; Santa Cruz Biotechnology), anti- After 48 h, splenocytes were washed and resuspended in PBS. A total of CYLD (clone E10; Santa Cruz Biotechnology), and anti-actin (clone MP 7 2 3 10 live splenocytes was injected i.v. into recipient mice, which were 6472J; MP Biomedicals). http://www.jimmunol.org/ sublethally irradiated (400 rad) 1 d prior to splenocyte injection. Clinical signs of disease were scored, as described previously (10), on a scale of 0 to Flow cytometry 6, with 0.5 points for immediate clinical findings as follows: 0, normal; 1, Splenocytes and thymocytes were isolated in PBS containing 0.5% BSA weakness of tail; 2, complete loss of tail tonicity; 3, partial hind limb pa- (Sigma-Aldrich). Cells were stained with Aqua Live/Dead (Life Tech- ralysis; 4, complete hind limb paralysis; 5, forelimb paralysis or moribund; nologies), anti-CD16/CD32 (clone 2.4G2; Fc Block; Becton Dickinson and 6, death. To eliminate any diagnostic bias, mice were scored blindly. Biosciences), anti–CD3-eFluor 450 (clone 145-2C11; eBioscience), anti– Histological analysis CD4-FITC (clone GK1.5; Pharmingen), anti–CD8-PE-Cy7 (clone 53-6.7; eBioscience), anti–CD25-allophycocyanin-Cy7 (clone PC61; BD Bio- Mice were transcardially perfused with PBS containing 5 IU/ml heparin sciences), anti–CD44-allophycocyanin (clone IM7; eBioscience), anti–

(De Pannemaeker, Ghent, Belgium), followed by perfusion with 4% para- CD25-PerCP-Cy5.5 (clone PC61; BD Biosciences), and anti–CTLA4- by guest on October 1, 2021 formaldehyde. Spinal cords were dissected, dehydrated, and embedded in paraffin blocks. Sections of 2 mm were stained with H&E, Luxol fast blue (Solvent Blue 38, practical grade; Sigma Genosys) for assessment of de- myelination, and Abs against CD3 (clone CD3-12; Serotec), Mac-3 (clone CD107b, M3/84; Becton Dickinson Biosciences), B220 (clone RA3-6B2; Becton Dickinson Biosciences), or amyloid precursor protein (APP; clone 22C11; Millipore). Sections were rehydrated and incubated in 10 mM citrate buffer for 5 min at 94˚C. Nonspecific binding was blocked by in- cubating sections in 0.1 M PBS containing 10% FCS and 1% Triton X-100 for 30 min. Primary Abs were incubated overnight at 4˚C. Histological quantification was described previously (11). Quantitative real-time PCR Total RNA was isolated using TRIzol reagent (Invitrogen) and an Aurum Total RNA Isolation Mini Kit (Bio-Rad), according to manufacturer’s instructions. Synthesis of cDNA was performed using an iScript cDNA synthesis kit (Bio-Rad), according to the manufacturer’s instructions. A total of 10 ng of cDNA was used for quantitative PCR in a total volume of 10 ml with LightCycler 480 SYBR Green I Master Mix (Roche) and spe- cific primers on a LightCycler 480 (Roche). Real-time PCR reactions were performed in triplicates. The following mouse-specific primers were used: HPRT forward, 59-AGTGTTGGATACAGGCCAGAC-39, HPRT reverse, 59-CGTGATTCAAATCCCTGAAGT-39; TNF forward, 59-ACCCTGGTA- TGAGCCCATATAC-39, TNF reverse, 59-ACACCCATTCCCTTCACAG- AG-39; IL-1b forward, 59-CACCTCACAAGCAGAGCACAAG-39, IL-1b reverse, 59-GCATTAGAAACAGTCCAGCCCATAC-39;IFN-g forward, 59-GCCAAGCGGCTGACTGA-39, IFN-g reverse, 59-TCAGTGAAGTA- AAGGTACAAGCTACAATCT-39; IL-6 forward, 59-GAGGATACCACT- CCCAACAGACC-39, IL-6 reverse, 59-AAGTGCATCATCGTTGTTCAT- ACA-39; MCP1 forward, 59-GCATCTGCCCTAAGGTCTTCA-39, MCP1 reverse, 59-TGCTTGAGGTGGTTGTGGAA-39; RANTES forward, 59-CG- FIGURE 1. MALT1 is critical for the induction of EAE. EAE was in- TCAAGGAGTATTTCTACAC-39, RANTES reverse, 59-GGTCAGAATCAA- +/+ 2/+ 2/2 GAAACCCT-39;TGF-b forward, 59-GCTGAACCAAGGAGACGGAATA-39, duced in male MALT1 (n = 4), MALT1 (n = 9), and MALT1 (n = TGF-b reverse 59-GAGTTTGTTATCTTTGCTGTCACAAGA-39;and 5) littermates. (A) Clinical symptoms. (B) Loss in body weight. Results are IP-10 forward, 59-GTCACATCAGCTGCTACTC-39,IP-10reverse,59- displayed as mean 6 SEM and are representative of three independent GTGGTTAAGTTCGTGCTTAC-39. experiments. *p , 0.05. 2898 MALT1 IN AUTOIMMUNE ENCEPHALOMYELITIS

Table I. Clinical features of MOG-induced EAE in MALT1+/+, MALT12/+, and MALT12/2 littermate mice

Day of Disease Onset Maximal Clinical Score Genotype Incidence (%) (mean 6 SEM) (mean 6 SEM) Minimal % Body Weight MALT1+/+ 4/4 (100) 15.3 6 0.6 4.1 6 0.1 79.6 6 3.4 MALT12/+ 7/9 (78) 15.0 6 0.5 3.7 6 0.5 79.3 6 2.8 MALT12/2 0/5 (0) – 1.0 6 0.1 97.1 6 0.4 Disease incidence, day of onset, maximal clinical score, and minimal percentage body weight. Results are displayed as mean 6 SEM. –, No disease onset. allophycocyanin (clone UC10-4B9; eBioscience) for 30 min at 4˚C. Prior spinal cord sections isolated at day 20 postimmunization from to staining for Foxp3 with anti–Foxp3-PE (clone FJK-16s), cells were fixed MALT+/+, MALT2/+, and MALT12/2 littermate mice. In line with and permeabilized using the Anti-Mouse/Rat Foxp3 Staining Set PE (both the findings that MALT12/2 mice are protected from EAE, histo- from eBioscience), according to the manufacturer’s instructions. Mea- 2/2 surements were performed on a BD LSR II cytometer (BD Biosciences), pathological hallmarks of EAE were virtually absent in MALT1 and data were analyzed using FACSDiva Software (BD Biosciences). mice compared with control littermates. Large areas of demyelin- +/+ Statistical analysis ation of the white matter were present in the spinal cord of MALT and MALT12/+ mice 20 d after immunization (Fig. 2A, Table II). Results are expressed as mean 6 SEM. Statistical analysis between mul- This was accompanied by axonal degeneration as visualized by tiple groups was assessed using one-way ANOVA, followed by the Bon- + 2/2

APP aggregates (Fig. 2B, Table II). In contrast, MALT mice Downloaded from ferroni posthoc correction. Statistical analysis between two groups was assessed using a two-tailed Student t test. showed no signs of pathology in the spinal cord after immuniza- tion (Fig. 2A, 2B, Table II). Furthermore, although inflammatory Results infiltration of CD3+ T cells, Mac-3+ macrophages, and B220+ B MALT1 ablation completely protects against EAE cells was present to a similar degree in spinal cords of MALT+/+ and MALT2/+ mice at 20 d post-EAE induction, these lympho- To determine the importance of MALT1 in the pathogenesis of 2/2 2/2 mononuclear infiltrates were completely absent in MALT mice EAE, we immunized mice lacking MALT1 (MALT1 ) and both http://www.jimmunol.org/ heterozygous (MALT12/+) and wild-type (MALT1+/+) control (Fig. 2C, Table II). Next, we determined the expression of proin- flammatory cytokines and chemokines in spinal cord tissue of littermates with MOG35–55 peptide and followed disease progres- +/+ 2/+ 2/2 sion by assessing both clinical disease symptoms as well as body MALT , MALT , and MALT1 mice by quantitative real- weight. As expected, all MALT+/+ mice developed EAE and fol- time PCR on tissues isolated at day 16 postimmunization. Al- lowed a typical disease course, starting ∼15 d after immunization though all inflammatory cytokines and chemokines tested were clearly upregulated in MALT1+/+ and MALT12/+ mice during and reaching a mean maximal clinical score of 4.1 (Fig. 1A, Table 2/2 I). MALT2/+ mice developed similar clinical symptoms, with a EAE, they remained undetectable in MALT mice (Fig. 3). mean maximal clinical score of 3.7 (Fig. 1A, Table I). No differ- These results demonstrate that the absence of MALT1 protects ences in incidence or disease severity were found between wild- mice from EAE by inhibiting inflammatory cell infiltration into by guest on October 1, 2021 type and heterozygous mice (Table I). In contrast, MALT12/2 mice the spinal cord, as well as by preventing the expression of key were completely protected and did not develop any clinical sign of mediators involved in CNS inflammation. EAE over a period of 20 d postimmunization (Fig. 1A, Table I). In +/+ 2/+ Peripheral autoreactive T cell activation is impaired in addition, although MALT and MALT mice showed a decrease MALT12/2 mice in body weight after immunization reflecting disease progression, 2/2 MALT12/2 mice did not lose any weight (Fig. 1B, Table I). T cell activation was shown to be impaired in MALT1 mice 2/2 (4, 5). To test whether this is also the case for the autoreactive Absence of spinal inflammatory infiltration in MALT1 mice T cell compartment during EAE, we analyzed the generation of 2/2 To further characterize the lack of clinical manifestations of EAE MOG35–55-autoreactive T cells in MALT1 mice. Splenocytes in MALT12/2 mice, histopathological analysis was performed on from MALT1+/+, MALT1+/2 and MALT12/2 were isolated 10 d

FIGURE 2. Absence of CNS demyelination, inflammatory cell infiltration, and axonal damage in MALT1-deficient mice. Immunohistochemistry on spinal cord sections from MALT1+/+ (n = 4), MALT12/+ (n = 6), and MALT12/2 (n = 5) mice. Assessment of demyelination on sections from the lumbar spinal cord by Luxol fast blue (blue) staining (A) and axonal damage by APP (brown) immunohistochemistry (B). (C) Staining for infiltrating T cells (CD3, brown), macrophages (Mac-3, brown), and B cells (B220, brown) by immunohistochemistry. (A) Scale bars, 500 mm(top panels) and 200mm(lower panels). (B and C) S bars, 200 mm. (middle panels). Results are representative of two independent experiments. The Journal of Immunology 2899

Table II. Quantification of spinal cord cell infiltrates, demyelination, and axonal damage from histological sections shown in Fig. 2

Genotype Demyelination (%) APP+/mm2 CD3+/mm2 B220+/mm2 MAC3+/mm2 MALT1+/+ 36.3 6 3.9 17.9 6 4.1 102.3 6 24.4 19.0 6 5.8 7780.6 6 116.6 MALT12/+ 29.3 6 3.5 23.5 6 8.0 82.8 6 21.2 18.3 6 3.2 7750.1 6 140.8 MALT12/2 0.0 6 0.0 0.3 6 0.2 1.4 6 0.6 0.2 6 0.1 20.4 6 1.0 Percentage of demyelination and numbers of APP+ depositions, infiltrating T cells (CD3), B cells (B220), and macrophages (MAC3) in MALT1+/+ (n = 4), MALT12/+ (n = 9), and MALT12/2 (n = 5) littermate mice 25 d after EAE induction. Data are mean 6 SEM. For all parameters between MALT1+/+ and MALT12/+ versus MALT12/2, p , 0.05. after immunization, after which their in vitro response to sec- deletion might influence regulatory T cells (Tregs). Indeed, we 2/2 ondary exposure to MOG35–55 peptide was analyzed. Autoreactive found that naive MALT1 mice show a severe reduction in Treg T cell responses to secondary MOG35–55 stimulation were com- population (Supplemental Fig. 1A, 1B). Moreover, the percentage parable between MALT1+/+ and MALT12/+ mice, as assessed by of activated Tregs is also decreased in these mice, as shown by proliferation and IL-2 and IFN-g production (Fig. 4A–C). In CTLA4 expression (Supplemental Fig. 1A, 1B). Similar obser- 2/2 contrast, however, T cells from MALT1 mice were impaired in vations were made in splenocytes isolated from MOG35–55-stim- generating an immune response to secondary MOG35–55 stimula- ulated mice that were restimulated with MOG35–55 in vitro tion (Fig. 4A–C). These results show that peripheral T cell acti- (Supplemental Fig. 1C, 1D). 2/2 Downloaded from vation is severely impaired in MALT1 mice and suggest that 2/2 2/2 MOG-specific MALT1 splenocytes fail to induce EAE in the protective phenotype of MALT1 mice in EAE results from +/+ this impaired autoreactive immune response. MALT mice Interestingly, MALT12/2 splenocytes also produced less IL-10 An impaired peripheral immune response can result in protection after in vitro stimulation with MOG35–55 (Fig. 4D). These findings from EAE because of the inability of autoreactive T cells to cause suggest that, in addition to affecting effector T cells, MALT1 pathological damage in the spinal cord. To test this hypothesis, http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 3. Impaired proinflammatory gene expression in CNS of MALT1-deficient mice. Quantitative measurements of the indicated cytokine and chemokine mRNA expression in spinal cord from MALT1+/+ (n = 2), MALT1+/2 (n = 4), and MALT12/2 (n = 5) littermate mice 16 d after immunization. Results are displayed as mean 6 SEM and are representative of two independent experiments. *p , 0.05, **p , 0.01. 2900 MALT1 IN AUTOIMMUNE ENCEPHALOMYELITIS

FIGURE 4. Peripheral T cell acti- vation is impaired in MALT-deficient mice. Splenocytes from MOG pep- tide–immunized control (MALT1+/+ and MALT12/+)andMALT12/2 mice were cultured and stimulated with the indicated concentrations of MOG peptide. (A and B) Culture superna- tants were collected 48 h after MOG peptide stimulation and assayed for IL-2 and IFN-g by ELISA. Results are shown as means 6 SEM and are representative of two independent experiments. (C) T cell proliferation was assessed by [3H]thymidine in- corporation. Results are expressed as cpm of triplicate cultures and are representative of two independent experiments. (D) Culture superna- tant was collected from splenocytes Downloaded from 48 h after MOG peptide stimulation andassayedforIL-10byBio-Plex. Results are shown as means 6 SEM. **p , 0.01, ***p , 0.001.

a series of adoptive-transfer EAE experiments was performed. cytes also fail to induce clinical manifestations of EAE in both http://www.jimmunol.org/ Splenocytes from MALT1+/+ and MALT12/2 mice were isolated MALT1+/+ and MALT2/2 mice (Fig. 5, Table III, Supplemental 2/2 10 d postimmunization and restimulated in vitro with MOG35–55 Fig. 2). Collectively, these results show that MALT1 mice do peptide and IL-23, after which donor splenocytes were injected not develop EAE because of an impaired peripheral immune re- into either MALT1+/+ or MALT12/2 recipient mice that were sponse. sublethally irradiated 1 d prior to injection to deplete hemato- poietic cells. As expected, donor MALT+/+ splenocytes induced A20 and CYLD processing is MALT1 dependent in T cells EAE in recipient MALT1+/+ mice, reflected in both clinical dis- during EAE ease progression and body weight loss (Fig. 5, Table III, Sup- Recently, it was demonstrated that MALT1 not only functions as plemental Fig. 2). Similarly, splenocytes from MALT1+/+ donor an adaptor protein, it also acts as a protease capable of cleaving by guest on October 1, 2021 mice induced EAE in acceptor MALT12/2 mice to a comparable a number of proteins, including A20 and CYLD, which both degree. In line with findings that MALT12/2 splenocytes fail to negatively regulate TCR signaling to NF-kB (6–9, 12). To in- induce an effective immune response in vitro, MALT2/2 spleno- vestigate MALT1’s proteolytic activity during the course of EAE,

FIGURE 5. MOG-specific MALT12/2 splenocytes fail to induce EAE in MALT+/+ mice. Splenocytes from MOG-immunized MALT1+/+ and MALT12/2 donor mice were injected into sublethally irradiated MALT1+/+ and MALT12/2 recipient mice. Clinical disease progression (A) and loss of body weight (B) were scored. Results are displayed as mean 6 SEM and are representative of two independent experi- ments. **p , 0.01, ***p , 0.001, MALT1+/+ → MALT1+/+ versus MALT2/2 → MALT1+/+. The Journal of Immunology 2901

Table III. Quantification of spinal cord cell infiltrates, demyelination, and axonal damage from histological sections shown in Supplemental Fig. 2

Transfer Demyelination (%) APP+/mm2 CD3+/mm2 MAC3+/mm2 MALT1+/+ → MALT1+/+ 26.1 6 3.3 105.9 6 21.7 192.4 6 75.9 7482.3 6 87.8 MALT1+/+ → MALT12/2 45.2 6 14.3 100.2 6 33.9 385.0 6 190.8 7877.8 6 376.9 MALT12/2 → MALT1+/+ 0.0 6 0.0 4.3 6 1.5 0.5 6 0.5 00.0 6 0.0 MALT12/2 → MALT12/2 0.0 6 0.0 5.1 6 3.5 0.0 6 0.0 20.0 6 0.0 Percentage of demyelination, and numbers of APP+ depositions, infiltrating T cells (CD3), and macrophages (MAC3) in spinal cord sections from MALT1+/+ → MALT1+/+ (n = 5), MALT1+/+ → MALT12/2 (n = 3), MALT12/2 → MALT1+/+ (n = 3), and MALT12/2 → MALT12/2 (n = 3) transfers 26 d after EAE induction. Data are mean 6 SEM. For all parameters between MALT1+/+ → MALT1+/+ versus MALT12/2 → MALT1+/+, p , 0.05. we analyzed the expression and processing of A20 and CYLD in in the spleen or lymph nodes, but leads to a decrease in the fre- purified CD4+ T cells at different time points, ranging from 14 to quency of activated T cells in the periphery. Consistent with these 24 d postimmunization. Full-length A20 and CYLD are present in observations, peripheral lymphocytes from MALT1-deficient mice naive CD4+ T cells of both MALT1+/+ and MALT12/2 mice (Fig. are defective in AgR-mediated T cell activation, proliferation, and 6). Interestingly, both A20 and CYLD are no longer detectable in IL-2 production (4, 5). Therefore, MALT1 was suggested to be an MALT1+/+ CD4+ T cells from EAE-diseased mice at days 14–20 attractive drug target for the treatment of autoimmune disease. postimmunization, most likely reflecting their proteolytic pro- In this study, we investigated the sensitivity of MALT1-deficient Downloaded from cessing. Interestingly, A20 and CYLD expression reappears at 22 mice to the development of EAE as a mouse model for MS. and 24 d postimmunization, reflecting their resynthesis. We were MALT1-deficient mice are completely resistant to myelin auto- not able to clearly detect the 65- and 70-kDa fragments that were antigen-induced disease development because of severely reduced previously described to result from MALT1-mediated cleavage of peripheral autoantigen-specific T cell responses, consistent with A20 and CYLD, respectively (6, 9). However, a role for MALT1 MALT1’s essential role in TCR activation and effector functions in the processing of A20 and CYLD in MALT1+/+ CD4+ T cells in vivo. In addition to the crucial role of MALT1 in mediating an http://www.jimmunol.org/ from EAE-diseased mice is supported by our observation that effector T cell response, we demonstrate that MALT1 deficiency MALT1 deficiency at least partially prevents the disappearance of leads to a severe impairment in the generation and activation of full-length A20 and CYLD (Fig. 6). The inability to detect specific Tregs. Previous studies demonstrated that Tregs are essential in A20 and CYLD fragments that are generated by MALT1 is most the dampening of an autoreactive T cell response in the context of likely explained by their further processing by other that EAE (13). However, we believe that, in conditions of MALT1 are activated in CD4+ T cells during EAE. Altogether, these data deficiency, this Treg defect is secondary to the effector T cell de- suggest the activation of MALT1 proteolytic activity in T cells fect, resulting in complete protection of MALT1-deficient mice from EAE-diseased mice, leading to the MALT1-mediated pro- from EAE. Most likely, the protective effect of MALT1 deficiency by guest on October 1, 2021 cessing of two of its known substrates, A20 and CYLD. also reflects its role in TCR-induced NF-kB signaling, because mice lacking the NF-kB isoforms NF-kB1 (p50) or c-Rel and mice Discussion specifically lacking IkB kinase b in T cells were reported to be MALT1 is an essential signaling protein in TCR-induced NF-kB completely resistant to EAE (14–16). One mechanism by which and JNK activation. Although MALT1 is essential for thymic T cell–specific NF-kB responses promote inflammation and T cell– T cell maturation, reflected by a block in T cell development at mediated autoimmune pathology is through their control of Th17 the DN3 to DN4 transition in thymocytes from MALT2/2 mice cell differentiation, because studies showed that c-Rel regulates the (Supplemental Fig. 3) (4), deletion of MALT1 has no effect on the development and severity of EAE by influencing the balance be- total number or distribution of CD4+ and CD8+ T cell populations tween Th17/Th1 cells and Tregs (17, 18). TCR-induced signaling to NF-kB involves the formation of a CBM scaffolding complex, which subsequently recruits down- stream signaling proteins, such as TRAF6, -8, and TAK1, to collaboratively activate the IKK complex (3). Besides func- tioning as an adaptor protein in the CBM complex, MALT1 exerts proteolytic activity to further promote TCR-induced NF-kB and JNK activation (6–9). In this context, MALT1 was shown to cleave the NF-kB and JNK inhibitory proteins A20 and CYLD in AgR-stimulated T cells in vitro (6, 9), which is believed to con- tribute to a full-blown NF-kB and JNK response that is essential for T cell function. Interestingly, our data now demonstrate a role for MALT1 in the processing of A20 and CYLD in T cells from mice suffering from EAE, which, to our knowledge, is the first evidence for A20 or CYLD processing in vivo. Surprisingly, al- though one would expect that not all of the CD4+ T cells in the FIGURE 6. A20 and CYLD processing in purified T cells from mice + spleen would be activated MOG35–55-specific T cells, splenic with EAE is partially MALT1 dependent. CD4 T cells were isolated from + the spleen of MALT1+/+ and MALT12/2 naive and EAE mice every 2 d CD4 T cells from the wild-type EAE mice showed a fully cleaved from days 14 to 24 postimmunization and assayed for A20 and CYLD form of A20 and CYLD. We hypothesize that part of the A20/ expression and processing by Western blot analysis. Full-length A20 and CYLD processing comes from T cells that are indirectly activated CYLD are indicated by an arrow. Results are representative of two inde- by Ags other than MOG35–55 and possibly Ags released from pendent experiments. dying cells. However, processing of A20 and CYLD was strongly 2902 MALT1 IN AUTOIMMUNE ENCEPHALOMYELITIS impaired in CD4 T cells derived from MALT1-deficient mice, 8. Hailfinger, S., H. Nogai, C. Pelzer, M. Jaworski, K. Cabalzar, J. E. Charton, M. Guzzardi, C. De´caillet, M. Grau, B. Do¨rken, et al. 2011. Malt1-dependent indicating an important role for MALT1 in the observed A20 and RelB cleavage promotes canonical NF-kappaB activation in lymphocytes and CYLD processing. It should be mentioned that we were unable lymphoma cell lines. Proc. Natl. Acad. Sci. USA 108: 14596–14601. to detect specific A20 or CYLD fragments that were previously 9. Staal, J., Y. Driege, T. Bekaert, A. Demeyer, D. Muyllaert, P. Van Damme, K. Gevaert, and R. Beyaert. 2011. T-cell receptor-induced JNK activation described to be generated by MALT1 in TCR-stimulated cells requires proteolytic inactivation of CYLD by MALT1. EMBO J. 30: 1742–1752. in vitro (6, 9). Most likely, A20, CYLD, and the fragments gen- 10. van Loo, G., R. De Lorenzi, H. Schmidt, M. Huth, A. Mildner, M. Schmidt- erated by MALT1 are also processed by other proteases that are Supprian, H. Lassmann, M. R. Prinz, and M. Pasparakis. 2006. Inhibition of transcription factor NF-kappaB in the central nervous system ameliorates au- activated in T cells from EAE mice. In this context, increased toimmune encephalomyelitis in mice. Nat. Immunol. 7: 954–961. proteolytic activity in lymphocytes of rats with EAE was de- 11. Dann, A., H. Poeck, A. L. Croxford, S. Gaupp, K. Kierdorf, M. Knust, D. Pfeifer, C. Maihoefer, S. Endres, U. Kalinke, et al. 2012. Cytosolic RIG-I-like helicases scribed, but the identity of the protease(s) remains unclear (19). In act as negative regulators of sterile inflammation in the CNS. Nat. Neurosci. 15: this context it is worth mentioning that CYLD was demonstrated 98–106. to be processed by caspase-8 (20, 21). In addition, proteasomes 12. Du¨wel, M., V. Welteke, A. Oeckinghaus, M. Baens, B. Kloo, U. Ferch, B. G. Darnay, J. Ruland, P. Marynen, and D. Krappmann. 2009. A20 negatively were shown to contribute to A20 degradation in activated T cells regulates T cell receptor signaling to NF-kappaB by cleaving Malt1 ubiquitin (12), and a similar proteasome-mediated regulation was suggested chains. J. Immunol. 182: 7718–7728. for CYLD (22). 13. O’Connor, R. A., and S. M. Anderton. 2008. Foxp3+ regulatory T cells in the control of experimental CNS autoimmune disease. J. Neuroimmunol. 193: 1–11. While revising our manuscript, a similar study describing an 14. Greve, B., R. Weissert, N. Hamdi, E. Bettelli, R. A. Sobel, A. Coyle, essential role for MALT1 in EAE was published (23). In that study, V. K. Kuchroo, K. Rajewsky, and M. Schmidt-Supprian. 2007. I kappa B kinase 2/beta deficiency controls expansion of autoreactive T cells and suppresses ex- MALT1 deficiency was shown to abolish the expression of the perimental autoimmune encephalomyelitis. J. Immunol. 179: 179–185. Th17 effector cytokines IL-17 and GM-CSF, which are essential 15. Hilliard, B., E. B. Samoilova, T. S. Liu, A. Rostami, and Y. Chen. 1999. Ex- for a pathogenic inflammatory response (23). Moreover, the MALT1 perimental autoimmune encephalomyelitis in NF-kappa B-deficient mice: roles Downloaded from of NF-kappa B in the activation and differentiation of autoreactive T cells. J. substrate RelB was shown to be cleaved and inactivated in wild- Immunol. 163: 2937–2943. type Th17 cells but not in MALT1-deficient Th17 cells, indicating 16. Hilliard, B. A., N. Mason, L. Xu, J. Sun, S. E. Lamhamedi-Cherradi, H. C. 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32. Gilli, F., N. D. Navone, S. Perga, F. Marnetto, M. Caldano, M. Capobianco, Molecule Inhibitors Specifically Suppress ABC-DLBCL In Vitro and In Vivo. A. Pulizzi, S. Malucchi, and A. Bertolotto. 2011. Loss of braking signals during Cancer Cell 22: 812–824. inflammation: a factor affecting the development and disease course of multiple 34. Nagel, D., S. Spranger, M. Vincendeau, M. Grau, S. Raffegerst, B. Kloo, D. Hlahla, sclerosis. Arch. Neurol. 68: 879–888. M. Neuenschwander, J. Peter von Kries, K. Hadian, et al. 2012. Pharmacologic 33. Fontan, L., C. Yang, V. Kabaleeswaran, L. Volpon, M. J. Osborne, E. Beltran, Inhibition of MALT1 Protease by Phenothiazines as a Therapeutic Approach for M. Garcia, L. Cerchietti, R. Shaknovich, S. N. Yang, et al. 2012. MALT1 Small the Treatment of Aggressive ABC-DLBCL. Cancer Cell 22: 825–837. Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021 SUPPLEMENTARY FIGURE 1. MALT1 deficiency causes a decrease in regulatory T cells. (A, B) Splenocytes from naïve MALT1+/+ (n=3) and MALT-/- (n=3) mice at 10 – 15 weeks of age were analyzed by flow cytometry for FoxP3 and CTLA4 expression. (C, D)

+/+ -/- Splenocytes were isolated from MOG35-55 immunized MALT1 and MALT mice and restimulated in vitro with MOG35-55 for 48 hours. Flow cytometry analysis was performed for

FoxP3 and CD25 expression. Results are representative for 2 independent experiments * p <

0.05 ; ** p < 0.01 ; *** p < 0.001

SUPPLEMENTARY FIGURE 2. MOG-specific MALT1-/- splenocytes fail to induce EAE in MALT+/+ mice. Splenocytes from MOG-immunized MALT1+/+ and MALT1-/- donor mice were injected into sublethally irradiated MALT1+/+ and MALT1-/- recipient mice.

Immunohistochemistry on spinal cord sections for assessment of demyelination by LFB (blue) staining, axonal damage by APP (brown) immunohistochemistry, and infiltrating T cells

(CD3, brown) and macrophages (Mac-3, brown). Scale bar, 200 m. Results are representative for 2 independent experiments.

SUPPLEMENTARY FIGURE 3. MALT1 deficiency and thymic T cell development.

Thymocytes from MALT1+/+ and MALT1-/- mice at 6 (MALT1+/+ n=3; MALT1-/- n=5) and 26 weeks (MALT1+/+ n=4 ; MALT1-/- n=6) of age were isolated and analyzed by flow cytometry.

(A) Expression of CD4 and CD8 in CD3-gated cells. (B) Percentage of DN (CD3+CD4-CD8-

) cells. At both ages, the CD3+CD4-CD8- DN population was increased in MALT1 deficient mice when compared to control littermates. (C) Expression of CD44 and CD25 in DN cells.

(D) Percentage of CD25+CD44- DN3 and CD25-CD44- DN4 cells, showing a decreased DN3 and increased DN4 population in MALT1 deficient thymus, indicating a block in T cell development at the DN3 to DN4 transition ** p < 0.01 ; *** p < 0.001.

A B MALT1+/+ MALT1-/-

C D

Supplementary Figure 1 MALT1-/- → MALT1-/- MALT1-/- → MALT1+/+ MALT1+/+ → MALT1-/- MALT1+/+ → MALT1+/+

LFB

APP

CD3

Mac-3

Supplementary Figure 2 A C MALT1+/+ MALT1-/- MALT1+/+ MALT1-/-

B D

Supplementary Figure 3