© 2014 Nature America, Inc. All rights reserved. to to V.H. ( Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA (N.M.). München, Munich, Germany. Biochemistry, Hannover Medical School, Hannover, Germany. Martinsried, Germany. Germany. München, Neuherberg, Germany. Australia. Research Unit Cellular Signal Integration, Helmholtz Zentrum München, Neuherberg, Germany. Germany. 1 and (CD152)) CTLA-4 receptor Ox40 IL-12p40), encodes (which of expression half-life their decrease and ‘ called OX40; and here) receptor the encodes (which mRNA ICOS), costimulator inducible the encodes (which mRNA and Roquin-1 are roquin-2 RNA-binding proteins that recognize molecules costimulatory as well as chemokines, cytokines, of ways path signaling the in receptors or ligands include this of examples Prominent genes. selected from mRNA of stability or translation of efficiency the severe changing by responses immune switch can develop regulation mice Such diseases autoinflammatory or (Mcpip1). autoimmune regnase-1 encodes which in mutations with trol over immune responses. This regulation becomes evident in mice Gene regulation on the level post-transcriptional exerts essential con repressors and differential derepression of targets to enhance T MALT1. Thus, this pathway acts as a ‘rheostat’ by translating TCR signal strength via graded inactivation of post-transcriptional Upon recognition of antigen by the T cell antigen receptor (TCR), roquin and regnase-1 proteins were cleaved by the paracaspase c-Rel, IRF4, I together with the regnase-1 endoribonuclease to repress target mRNA encoding the T accumulated as cells of the T encoding the RNA-binding protein roquin-1. Here we found that T cells lacking roquin caused pathology in the lung and Humoral autoimmunity paralleled by the accumulation of follicular helper T cells (T Daniel Krappmann Hans-Joachim Anders Lohs Claudia Katharina U Vogel Katharina M Jeltsch to promote T MALT1 releases their repressed cooperatively targets Cleavage of roquin and regnase-1 by the paracaspase nature immunology nature Received 10 August; accepted 9 September; published online 5 October 2014; Institute Institute for Immunology, München, Munich, Germany.Ludwig-Maximilians-Universität mRNA, [email protected] 10 3 6 Institute Institute for Virology, Technische Zentrum Universität München/Helmholtz München, Munich, Germany. Medizinische Medizinische Klinik und Poliklinik IV, München, Munich, Germany.Ludwig-Maximilians-Universität Institute Institute for Molecular and Clinical Immunology, Magdeburg, Magdeburg, Germany.Otto-von-Guericke-Universität Tnf mRNA (which encodes tumor-necrosis factor (TNF)) (TNF)) factor tumor-necrosis encodes (which mRNA Il6 Ctla4 k BNS BNS and I 2 mRNA (which encodes interleukin 6 (IL-6)), (IL-6)), 6 interleukin encodes (which mRNA , , Maciej Lech 12 Rc3h1 mRNA (which encodes the immunomodulatory immunomodulatory the encodes mRNA (which Department Department of Basic Medical Science, School of Medicine, University of City,Missouri-Kansas Kansas City, Missouri, USA. 2 , 15

4 1 15 , , Jürgen Ruland , which encodes roquin-1, or in in or roquin-1, encodes which , , aDV , Nina Rehage 2 Present Present addresses: Laboratory of Lymphocyte Signalling and Development, Babraham Institute, Cambridge, UK (K.U.V.), and Johns , k 6 16 6– 8 , , Ingo Schmitz Il2 B Institute Institute for Surgical Pathology, University Hospital, Zürich, Switzerland. A 9 , , Desheng Hu  . Regnase-1 negatively regulates the the regulates negatively Regnase-1 . NCE ONLINE PUBLIC ONLINE NCE (which encodes IL-2), IL-2), encodes (which H . . This cooperation required binding of RNA by roquin and the nuclease activity of regnase-1. 17 17 subset of helper T cells in the lungs. Roquin inhibited T H 6 , , Jenny E Stehklein Rel 17 17 differentiation mRNA (which encodes the the encodes (which mRNA ). 1 , 2 1– 14 , Sebastian C Warth 4 9 . Post-transcriptional Post-transcriptional . , , Axel Kallies 2 , , 10 16 , , Marc Schmidt-Supprian , , Sven Brenner A 14 TION Institut Institut für Klinische Chemie und Klinikum Pathobiochemie, rechts der Isar, Technische Universität Ox40 Icos

2 Zc3h12a , , Arie Geerlof mRNA, mRNA, mRNA’ Tnfrsf4 5 Il12b , , Mathias Heikenwalder Icos 5 16 doi:10.1038/ni.300 - - . , H 2

These These authors contributed equally to this work. should Correspondence be addressed 2 17 17 differentiation. , Stephanie L Edelmann , 16 and costimulatory receptors and costimulatory is protein the cells, T In cleaved by levels. the paracaspase MALT1 after stimulation several through the TCR on regulated is regnase-1 In contrast, unknown. remained has regulated or be can constitutive decay mRNA roquin-mediated and recognition of this CDE the in structure stem-loop a consensus binds of roquin-1 element (CDE) in the decay constitutive a to roquin-2 paralog its and roquin-1 of binding of half-life the regulate that factors for cis tures Zc3h12a 3 the vitro in of pre-microRNA loop terminal mRNA or the target cleaves that ase as well as its own mRNA ( NF- factor transcription , , Jessica Zöller elements recognized by regnase-1 has not been defined. A search A search defined. been not has by regnase-1 recognized elements 2 Institute Institute of Molecular Immunology, Helmholtz Zentrum München, Munich, 7 1 ′ , Elisabeth Kremmer , Elisabeth untranslated regions (UTRs) of of (UTRs) regions untranslated , 10 1 , , 1 mRNA that have the potential to form stem-loop struc stem-loop form to potential the have that mRNA 1 5 1 2 The The Walter and Eliza Hall Institute of Medical Research, Parkville, 11 . However, a specific consensus sequence or structure for for structure or sequence consensus specific a However, . . The regnase-1-mediated repression maps to regions in in regions to maps repression regnase-1-mediated The . , , Mingui Fu 8 FH 3 9 Helmholtz Helmholtz Centre for Infection Research, Braunschweig, , , Gitta A Heinz H cells) is linked to mutation of the gene Tnf 17 cell–promoting 17 factors cell–promoting IL-6, ICOS, 7 3 Institute Institute of Structural Biology, Helmholtz Zentrum cis & Vigo Heissmeyer κ 3 H element is required and sufficient to induce 4 Zc3h12a B subunit c-Rel; called ‘ called c-Rel; subunit B 1 Institute Institute of Toxicology and Pharmacology, ′ 17 17 cell and differentiation acted 12 UTR , 2 1 , Renee Gloury 1 , , Helmut Holtmann . . MALT1 is of part the ‘CBM’ complex, 2 7 7 , , Achim Weber . Whether the activity of roquin is is roquin of activity the Whether . . . The amino-terminal ROQ domain ) 11 1 , 10 Max Max Planck Institute of Biochemistry, , 1 2 Tnf 1 Il6 , , Daniel Nagel . Regnase-1 . is Regnase-1 an endonucle mRNA, mRNA, mRNA has identified the the identified has mRNA 5 1 , Nina Martin , 2 cRel ticles e l c i rt A 8 cRel , 13

13 mRNA’ here), Institute Institute of mRNA and and mRNA , 4

,

7

, 13 2 , 15 , 1 4  - - , ,

© 2014 Nature America, Inc. All rights reserved. with with an average survival of 130 d ( viability, reduced a observed we time, over mice these in pathology lung of worsening the with Consistent shown). not (data collagen of ( walls artery the of thickening detected we ( cells In addition, insult. goblet airway to progressive the in response presumably mucin-producing of numbers increased pathology of the lungs in fl of lungs the in cells of proliferation increased revealed Ki67 Fig. 1a in ( lung II tissue were overrepresented class (MHC) complex histocompatibility major expressing (APCs) and CD3 of numbers CD45 with and ( inflammation with concomitant space alveolar the in tion reduc progressive a Wefound pathology. lung prominent revealed of expressed from the T cell–specific of tion dele through achieved respectively), and roquin-2, roquin-1 encode of ablation combined cell–specific, T with Functional redundancy of roquin paralogs has been identified in mice T cells in roquin lacking mice in pathology Lung RESULTS T of control the in regulation gene convergence of post-transcriptional roquin- and regnase-1-mediated the demonstrate we far.Here so established been not has gene regulation post-transcriptional of pathways these between connection a roquin encoding genes the in tions microarchitecture, resemble phenotypes of mouse models with muta development of autoantibodies, postnatal death and disrupted splenic B lymphocytes, derepression of the expression of ICOS and OX40, the and most functions, prominently the activation of T lymphocytes and phenotypes autoinflammatory and T cell–intrinsic functions of regnase-1 are neededIl6 to (ref. prevent autoimmune IL-12p40 and subsequent work IL-6 has shown that combined deficiency in encoding mRNAs regnase-1 target of deregulation via responses immune innate of control inflammation and systemic of severe production autoantibodies immunoglobulins, of concentrations increased cells, T effector of accumulation death, development the attenuate EAE of to shown been has paracaspase inhibition this of pharmacological MALT1, of activity enzymatic the for (EAE) encephalomyelitis mune Malt1 T the into differentiation for critical CD4 and cells dendritic in MALT1 of presence NF- alternative signaling and canonical modulate thereby may and RelB NF- the and CYLD and A20 deubiquitinases the Bcl-10, receptor antigen the NF- for canonical required is complex CBM as well as function activity protease has scaffold it and MALT1, and Bcl-10 Carma1, adaptors the of consists which s e l c i rt A  revealed that roquin function in T cells was essential for the prevention Rc3h2

Regnase-1-deficient mice develop severe anemia, postnatal postnatal anemia, severe develop mice Regnase-1-deficient or Rc3h1 Supplementary Fig. 1a Supplementary Supplementary Fig. 1a Fig. Supplementary -deficient mice are -deficient induced autoimresistant to experimentally Zc3h12a , fl/fl 2 d lox 4 ). ). Staining with antibody specific for the proliferation marker . fl/fl 15 Cd4 P-flanked alleles ( alleles P-flanked , + 16 Rc3h2 hematopoietic cells ( cells hematopoietic , 18 -Cre mice ( 1 and and . Initially, those phenotypes were related to impaired impaired to related were phenotypes those Initially, . , 1 9 as well as bysignaling the kinase Jnk + fl/fl cls n h tsu wr icesd ( increased were tissue the in cells T Il12b Cd4 1 7 , its catalytic activity cleaves targets such as as such targets cleaves activity catalytic its , Rc3h1 does not ‘rescue’ the phenotypes not ‘rescue’ the does Fig. 1b -Cre mice between 7 and 30 weeks of age age of weeks 30 and 7 between mice -Cre ). ). The lungs of mice these were infiltrated Rc3h1 , c fl/fl ). Moreover, antigen-presenting cells cells antigen-presenting Moreover, ). 21– , c Rc3h2 Supplementary Supplementary Fig. 1f and Cd4 Supplementary Fig. 1b Fig. Supplementary 2 H fl/fl 3 1 17 differentiation. 17 H 1– . Consistent with a requirement requirement a with Consistent . 1 . Notably, various phenotypes phenotypes various Notably, . promoter ( 17 subset of helper T cells, and cells, T helper of subset 17 Rc3h2 Supplementary Supplementary Fig. 1a 3 Fig. Fig. 1b fl/fl , 8 , 11 Cd4 , 2 Rc3h1 fl/fl κ 5 Fig. 1 Fig. . Despite such overlap, overlap, such Despite . B signaling induced by induced B signaling -Cre mice also included , c ) by Cre recombinase recombinase Cre by ) 15 and + T cells seems to be be to seems cells T Cd4 , 1 and and 6 f . While . the While intact ) and deposition deposition and ) Supplementary Supplementary -Cre) 2 Rc3h2 0 1 . . Notably, the Zc3h12a ). ). These data ). However, However, ). κ i. 1d Fig. 1 8 1 ), and the the and ), B subunit subunit B . . Analysis i. 1b Fig. . Instead, . Instead, Fig. 1a Fig. Rc3h1 (which (which , e ). The and , κ e , fl/ , ), ), B b c - - - -

Rc3h2 from of lungs the and from especially lymph nodes isolated of CD4 a IL-17A-producing much frequency revealed higher (ELISA), assay immunosorbent enzyme-linked ( mice wild-type in than mice sanroque interferon- ( mice mutant the in higher type’ mice here), and the concentration of TNF and IL-10 was slightly from serum in than from serum in IL-6 and roquin- IL-17A of by production cells T cytokine effector deficient altered the addressed next We T of Control T from different cells T in in in observed Rc3h1 microarchitecture splenic perturbed the by explained be might autoantigens typical to The reactivity humoral of lack apparent autoantibodies. develop to potential the had they that proved lyd tog eciiy o acetc nies ( antigens pancreatic to reactivity strong played several from serum Notably, anti-DNA antibodies or rheumatoid factor ( Rc3h2 ( disease autoimmune lupus-like develop also and (CD95) Fas tor serum in or 199), position from at substitu methionine (with the for roquin-1 arginine of of mutant tion point a express which sanroque mice, from serum in autoreactivity humoral detected we trast, in ( immunoblotting mice by determined as immunodeficient serum, from their proteins tissue to autoreactivity Rc3h2 antigens ( Rc3h1 autoimmunity lupus-like of prevents uncontrolled autoantibody production and the development (T cells T helper follicular of accumulation and Published work has established that roquin controls the differentiation autoimmunity humoral to related not is pathology Lung viability. decreased of spontaneous lung inflammation and pathology that correlated with of differentiation into Foxp3 wild-typemice ( IL-17A-producing cells from ulated the cells We cultured naive CD4 proteins showed a pronounced bias toward T (Gr-1 neutrophils Cd4 The greater abundance of T ( CCR6 receptor chemokine the expressed ( increased frequency of cells expressing the transcription factor ROR of lungs the from 2a Fig. show increased production of the T of lungs the from cells T Supplementary Fig. 2b Fig. Supplementary i. 1 Fig. In agreement with our Rc3h1 -Cre mice was paralleled by a greater frequency and number of of number and frequency greater a by paralleled was mice -Cre MRL fl/fl fl/fl fl/fl fl/fl fl/fl g ). The predominant production of IL-17A by CD4 by IL-17A of production predominant The ). Rc3h2 ). Distinct from from Distinct ). Cd4 Cd4 fl/fl Rc3h2 Cd4 Fig. Fig. 1 aDV lpr/lpr γ Rc3h2 (IFN- H -Cre mice, most did not show immunoglobulin G (IgG) most -Cre did mice, not show immunoglobulin -Cre than in those from wild-type mice ( mice from -Cre than in wild-type those Ce ie i nt xii at-ula antibodies anti-nuclear exhibit not did mice -Cre 17 differentiation by roquin proteins roquin by differentiation 17 fl/fl in vitro A 2 fl/fl g 7 mice, which overexpress the ligand for the recep the for ligand the overexpress which mice, Fig.2 NCE ONLINE PUBLIC ONLINE NCE ). ). Despite the of accumulation T , was not higher in in higher not was , Cd4 Rc3h1 + Cd4 fl/fl γ F4/80 ), which is known to be elevated in serum from from serum in elevated be to known is which ), -Cre mice, we did not detect autoantibodies to lung Cd4 Rc3h1 , which resulted in a twofold-higher frequency of d + -Cre mice -Cre ).Consistent with that, the reciprocal program T cells under T in vivo fl/fl − Fig. 2 Fig. -Cre mice indicated a function for roquin roquin for function a indicated mice -Cre ). Most of the IL-17A-producing cells also also cells IL-17A-producing the of Most ). ) ( ) FH in vivo in MRL Rc3h2 Rc3h1 H + +/+ 3 Rc3h1 Fig. 2 Fig. cell–mediated B cell help. cell B cell–mediated regulatory T cells (T 17 17 cells in the lungs of , Fig. 2 Fig. 8 results, CD4 . Rc3h2 Analyzing humoral autoimmunity in in autoimmunity humoral Analyzing Rc3h1 a lpr/lpr ). In line with our data obtained by obtained data our with line In ). 8 fl/fl . We found higher concentrations concentrations higher found We . fl/fl . Nevertheless, the lung pathology pathology lung the Nevertheless, . fl/fl c H a and data not shown). not data and Cd4 Rc3h1 ). Notably, the concentration of of concentration the Notably, ). A Rc3h1 Rc3h2 2 2 cytokine IL-4 ( Rc3h2 and sanroque mice, mice, sanroque and +/+ fl/fl TION H 17 conditions and then restim Cd4 -Cre mice correlated with an with correlated mice -Cre Rc3h2 H + fl/fl Supplementary Fig. 1g fl/fl fl/fl fl/fl

T cells lacking both roquin 17 differentiation ( Supplementary Fig. 2c Fig. Supplementary -Cre mice (called ‘wild- (called mice -Cre nature immunology nature Cd4 Rc3h2 Rc3h2 Cd4 fl/fl FH FH reg -Cre mice than from ex vivo ex Cd4 -Cre mice did not not did mice -Cre cells) and thereby thereby and cells) Rc3h1 cells cells) was moder Fig. 1 Fig. fl/fl fl/fl i. 1 Fig. Supplementary Supplementary Ce ie dis mice -Cre Fig. Fig. 2 Cd4 Cd4 8 stimulation stimulation in fl/fl g h -Cre mice mice -Cre -Cre mice mice -Cre ). In con In ). Rc3h1 b Rc3h1 Rc3h1 Rc3h2 ), which which ), + Fig. 2 + ). ). CD4 T cells cells T T cells , 3 fl/fl fl/fl h fl/fl fl/fl d , 2 γ 8 ). ). ). ). + 6 ------t ,

© 2014 Nature America, Inc. All rights reserved. from the small and large intestine showed a greater frequency of of frequency greater a IL-17A showed intestine large and small the from ( arthritis of signs no showed and normal was joints their of phology ( ( skin the in ( mice skin, of of joints sections and histology analyzed intestine also we arthritis, and colitis T of IFN- trend toward enhanced T mice stimulated under either T Cre mice ( ately impaired in naive CD4 ( ( tissue pancreas ( 1 mm. bars, scale 510 263 measurement: diameter indicate arrowheads solution; staining tissue connective Gieson elastica–van with stained mice, Cre and wild-type 23-week-old to 18- from arteries large and small 10 bar, scale ×40; magnification, Original cells. positive indicate arrowheads acid–Schiff; and wild-type ( and wild-type from tissue lung in cells ( cells. positive indicate Arrowheads rows). bottom 10 or middle) and left 100 bars, scale (right); ×80 or rows) fourth and third second, of middle and (left ×40 middle), and left (top ×20 magnification, Original anti-Ki67. or (MHCII) II class MHC to antibody anti-CD3, with or (H&E) eosin and hematoxylin Rc3h1 and wild-type 23-week-old to 18- from sections 10 or 100 bars, scale (bottom); ×40 or middle) and (top ×20 magnification, Original eosin. and hematoxylin with and (WT) wild-type from sections tissue lung of ( mice. roquin-mutant of lungs 1 Figure nature immunology nature ( counterparts wild-type their in than mice Cre mice ( mice two-tailed (unpaired, ( mean the indicate lines horizontal small mouse; individual an represents 18–23 weeks a n d c Supplementary Fig. 2f Supplementary Supplementary Fig. 2h Fig. Supplementary 7–11 weeks H ) Quantification of CD3 of ) Quantification = 4), = 4), 23-week-old to 18- of bronchioles in cells goblet of ) Microscopy ie te rmnn ivleet f T of involvement prominent the Given 2 cultures ( µ Rc3h1 m. ( m. µ µ m (bottom left) and 1,143 1,143 and left) m (bottom a fl/fl m (bottom). ( m (bottom). upeetr Fg 2d Fig. Supplementary – γ + Rc3h1

f -producing cells, and fewer IL-4-producing cells in restimulated e and IL-17A and ) or two experiments ( experiments two ) or Rc3h2 Inflammatory phenotypes in the the in phenotypes Inflammatory ) Quantification of goblet cells positively stained as in in as stained positively cells goblet of ) Quantification Fig. 2 fl/fl Rc3h2 Supplementary Fig. 2d Fig. Supplementary Rc3h1 Fig. 2f fl/fl fl/fl h e Rc3h2 ). Naive CD4 ) from immunodeficient mice, probed with serum from the following 10- to 23-week-old mice: mice: 23-week-old to 10- following the from serum with probed mice, immunodeficient ) from Cd4 g WT fl/fl , b fl/fl h t , ) Microscopy of alveoli alveoli of ) Microscopy -test). Data are representative of three independent experiments with three wild-type mice and four four and mice wild-type three with experiments independent three of representative are Data -test). + g ) Representative immunoblot analysis of extracts from lung ( lung from extracts of analysis immunoblot ) Representative µ -Cre mice, stained with with stained mice, -Cre Cd4 Rc3h2 IFN- ). m (top right, and middle and and middle and right, m (top fl/fl +

, µ , MHC class II–positive (MHCII II–positive class , MHC H g ). However, flow cytometry of CD4 of cytometry However, flow ). Cd4 -Cre mice, stained stained mice, -Cre Rc3h1 m (top and middle) middle) and m (top 1 differentiation, as assessed by the frequency and data not shown). In the addition, mor aDV + γ T cells isolated from + fl/fl a g -Cre ( -Cre CD4 H ) Microscopy ) Microscopy + , A Cd4 h T cells from 1 conditions or T µ Rc3h1 NCE ONLINE PUBLIC ONLINE NCE ). fl/fl – m (bottom right). Original magnification, ×60; ×60; magnification, Original right). m (bottom i n ). We did not find abnormalities abnormalities find not did We ). Cd4 -Cre mice, stained with Alcian blue and periodic periodic and blue Alcian with stained mice, -Cre

+ Rc3h2 = 16) and sanroque ( sanroque and = 16) T cells in in cells T fl/fl Rc3h1 -Cre Rc3h2 , e

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b 0 conditions showed a -Cre and wild-type wild-type and -Cre Anti-MHCII 18–23 weeks fl/fl

Anti-Ki67 Anti-CD3 Ab-PAS fl/fl A Rc3h2 fl/fl Supplementary Supplementary H&E + TION fl Cd4 ) and Ki67 ) and /fl Rc3h2 Rc3h1 Rc3h2 e -Cre mice. mice. -Cre fl/fl

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s.e.m.). * s.e.m.). = 16). Left margins, molecular size in kilodaltons (kDa). Each symbol ( symbol Each (kDa). kilodaltons in size molecular margins, Left = 16). roquin-1 and roquin-2 that migrated in two discernable bands just just bands discernable two in migrated that roquin-2 and roquin-1 full-length of disappearance the caused ionomycin plus PMA ester CD4 wild-type of Stimulation regulated. was dif abundance their cell whether T determine to sought controlled we ferentiation, roquin-2 and roquin-1 that shown Having MALT1by cleavage proteins roquin of Regulation gastritis. and inflammation T T of control the in teins identified a previously unrecognized cell-intrinsic role for roquin pro ( cells epithelial gastric in mainly observed we signal for phosphorylated intracellular signaling molecule STAT3 that nuclear a strong found we by IL-6, induced potentially signaling, tor ( of stomach the in present was cells 2i Fig. Supplementary Fig. 2i Fig. Supplementary

fl/fl H Rc3h1 17 cells in the lung and gut, as well as in the development of lung lung of development the in as well as gut, and lung the in cells 17 *

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). Furthermore, pronounced infiltration of CD3 of infiltration pronounced Furthermore, ). fl/fl P

- = 0.0372, ** = 0.0372,

Rc3h2

Rc3h1 Cd4 Rc3h1 WT Cd4-Cre

fl/fl - Cr fl/fl g (kDa Cd4 fl/fl e 100 135 Rc3h2 25 35 48 63 75 Rc3h2 ) -Cre

MRL fl/fl fl/fl P lpr/lpr = 0.015, *** = 0.015, Rc3h1 H , h Lung

17 differentiation and the accumulation of accumulation the and differentiation 17 +/+ ). As an indication of local cytokine recep cytokine of local ). As an indication c Rc3h1 Rc3h2 CD3+ cells per mm2 of 3 artery Big fl/fl +/+ artery Small lung tissue area (×10 )

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lpr/lpr Cd4 0 1 2 3 4

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fl/fl of lung tissue area 0. 0. +/+ 0. 0. Rc3h1 1 2 0 3 4 Cd4 Rc3h1 Rc3h1 WT , Rc3h2 Cd4 i P + fl/fl ). These data data These ). = 0.0036 = 0.0036 Cd4 and CD45 and

fl/fl +/+ - Cr fl/fl Cd4

-Cre mice mice -Cre Rc3h2 Cd4 fl/fl e - Rc3h1 Rc3h2 Cre Rc3h2 fl/fl -Cre (WT) ** -Cre -Cre san/san Cd4 c fl/fl fl/fl , -Cre e )  + - - -

© 2014 Nature America, Inc. All rights reserved. osat ewe 3 mn n 10 i o siuain ( stimulation of min 180 and min remained 30 and min between 30 constant and min 10 between intensity in increased only detected weakly in of extracts unstimulated cells, but their bands equally paralogs recognizes roquin both that antibody by monoclonal a kDa with 50–60 analysis immunoblot approximately of proteins migrating faster several ( treatment of min 60 after extracts cell in detectable was proteins roquin-2 or roquin-1 full-length no almost the amount of roquin-1 full-length and roquin-2 was diminished, and immunoblot by ( analysis respectively, (kDa), kilodaltons 135 at and below ( ( experiments of two representative are Data ( mean the indicate lines horizontal small mouse; individual 100 bars, scale ×20; ( ( cells IFN- and IL-17A for stained and T ( ( quadrants to 18-week-old 12- IFN- and of IL-17A cytometry Flow ( mean the indicate lines horizontal small mouse; an individual represents symbol Each excluded. been have points no data and dilutions at several pretested was serum Rc3h1 2 Figure s e l c i rt A  CD4 In g n d reg

) independent experiments, one experiment with two technical replicates ( replicates technical two with experiment one experiments, ) independent – = 3 per genotype), stained with hematoxylin and eosin, anti-CD3, anti-CD45 and antibody to phosphorylated STAT3 to phosphorylated (p-STAT3).antibody and magnification, anti-CD45 Original anti-CD3, eosin, and hematoxylin with stained = 3 genotype), per g cell ( cell ) ) d b a c In In vitro Lymph g Lung T + ). SSC, side scatter. ( side ). SSC, node /

H IL-17A (pg/ml) Lung +

+ 200 400 600 800 17 e Rc3h2 Enhanced T Enhanced T cells with single deficiency in roquin-1 or roquin-2, the the roquin-2, or roquin-1 in deficiency single with cells T ), ), T Fig. 3 Fig. 0 b ) indicate percent cells in each throughout; numbers adjacent to outlined areas ( areas to outlined adjacent numbers throughout; in each cells percent ) indicate differentiation of differentiation IL-17A

H IL-17A Gr-1 10 10 10 10 10 10 10 10 10 10 10 10 1 1 ( 0 0 4 3 5 2 2 3 4 5 2 3 4 5 + Rc3h1 Rc3h1 Rc3h1 / a IFN- IFN- F4/80 Cd4- + Cd4- f 1 0 3.3 01 Cd4- 25.2 0.4 01 , top), T , top), ). Specifically, as early as 30 min after stimulation stimulation after min 30 as early as Specifically, ). Cd4 ** ± 0 0 Rc3h1 0 2 2 2 s.e.m.). NS, not significant; * significant; not NS, s.e.m.). γ γ +/ +/ H Cre (WT) 10 10 Cre (WT) +/ µ Cre (WT) 10 1.9 + + 17 differentiation and gastritis in mice that lack roquin in T cells. ( in T cells. roquin lack that in mice gastritis and 17 differentiation -Cre or + m. m. ( 3 3 Rc3h2 Rc3h2 3 Rc3h2 10 10 10 4 4 0.4 5.4 1.2 4 0.1 H 0.1 +/+ 10 10 10 i 0 0 0 ( ) Quantification of CD3 ) Quantification 5 5 h +/ +/ 5 +/ Rc3h2 + + ) Microscopy of stomach sections from 26- to 30-week-old to 30-week-old 26- from sections of stomach ) Microscopy Rc3h1 + f Rc3h1 Rc3h1 Rc3h1 Rc3h1 , bottom) or T , bottom) 8 γ 15.4 52.9 6.7 ( ( IL-6 (pg/ml) 200 250 100 150 Cd4- Cd4- Fig. 3 Fig. d Cd4- γ 50 in CD4 fl/fl ), ), IFN- +/+ 0 fl/fl fl/fl fl 7.2 / Rc3h2 +/+ Rc3h2 Cre Rc3h2 fl Cre Cd4 Cre Rc3h2 Rc3h2 4.5 0.8 0.6 a 0.2 0.1 0.9 ). These smaller proteins were proteins smaller These ). γ -Cre and and -Cre fl/fl fl/fl + fl/fl ( a T cells from the lymph nodes and lungs ( lungs and nodes lymph the from T cells * f H ), seven ( ), seven ) or IL-4 ) ( or IL-4 fl 2 2 ( Fig. 3 Fig. / +/+ fl Cd4 g g e Cd4 f T T T T ) conditions, then stained for the transcription factor Foxp3 ( Foxp3 factor transcription the for stained then ) conditions, + P H H H reg Rc3h1 , , CD45 2 0 1 = 0.001, ** = 0.001, -Cre mice ( a -Cre and and -Cre ). We also detected detected We ). also b IL-4 Foxp3 g IFN-γ ) or three ( ) or three 10 10 10 10 10 10 10 10 10 10 10 10 ). Numbers adjacent to outlined areas indicate percent Foxp3 percent indicate areas to outlined adjacent ). Numbers Rc3h1 0 0 2 3 4 5 2 3 4 5 0 2 3 4 5 Rc3h1 Rc3h1 Cd4- fl/fl SSC

SSC 0 + 0 TNF (pg/ml)

Cd4- FS

SSC 0 Cd4- ± 15 50K 50K 10

and phosphorylated STAT3–positive as in in mice cells phosphorylated and 50 C 0 5 Rc3h2 s.e.m.). * s.e.m.). 100K 100KK 100K +/ Cre (WT) Rc3h1 +/ + Cre (WT) +/ n 150K Cre (WT) 150K Rc3h2

150 + + = 4 (wild-type) or 5 (mutant) (IL-17A and IL-6), or 84.1 Rc3h2 P Rc3h2 i. 3 Fig. 18.6 20.8 200K K 2.9 200K c 200K = 0.0007, *** = 0.0007, ) independent experiments with one mouse per genotype, three ( three genotype, per mouse one with experiments ) independent fl/fl 250K 250 250K **** +/ fl/fl +/

+ K +/ Cd4 P + + Rc3h1 Rc3h2 a Rc3h1 = 0.0049, ** = 0.0049, Rc3h1 ). ). -Cre mice, stimulated stimulated mice, -Cre

Cd4- Cd4- h Cd4- fl/fl ) or QQ experiments ( ) or QQ experiments fl/fl fl/fl to the coreceptor CD28 (anti-CD28) also induced the faster migrating body to the invariant signaling protein CD3 (anti-CD3) plus antibody with Consistent that, with combined anti stimulation costimulation. and TCR the by activation strong very mimic ionomycin and PMA ( observed changes the induce that determined stimulation of cells with PMA alone was to sufficient ( ionomycin and PMA with stimulation upon versions truncated of appearance the showed also and cells unstimulated in CD4 human only were present proteins roquin naive full-length the that showed T cells of extracts of analysis immunoblot ( Similarly, appeared roquin-2 or a roquin-1 of and form stimulation migrating faster upon disappeared similarly protein full-length 72.1 Rc3h2 fl/fl Rc3h2 8.5 Cre Cre Rc3h2 Cre 34.6 9.1 Cd4 P

= 0.0003 and **** and = 0.0003 IL-10 (pg/ml) fl/fl 100 150 fl/fl fl/fl 50 -Cre CD4 -Cre b 0 P ) and of Gr-1 and F4/80 on cells from the lungs ( lungs the from ) of on Gr-1 cells and F4/80 and = 0.0023 and *** and = 0.0023 Rc3h1 a aDV ) ELISA of cytokines in serum from 12- to 18-week-old to 18-week-old 12- from in serum of cytokines ) ELISA i c *** ) indicate percent Gr-1 percent ) indicate + 2 h + A

+/+ CD3 cells per mm of CD62L NCE ONLINE PUBLIC ONLINE NCE in in vitro gastric mucosal area (×102) Rc3h2 i 10 15 ). 0 5 Anti-p-STAT3 Anti-CD45 + P Anti-CD3 with PMA and ionomycin. Numbers in Numbers ionomycin. and PMA with CD44 < 0.0001 (unpaired, two-tailed two-tailed (unpaired, < 0.0001 +/+ * P Cd4 H&E = 0.0005 (unpaired, two-tailed two-tailed (unpaired, = 0.0005 IFN-γ (pg/ml) − Rc3h1 Rc3h1 10 15 20 25 T cells cultured under T under cultured T cells e 0 5 -Cre and and -Cre Rc3h1 ) or stimulated with PMA plus ionomycin ionomycin plus PMA with ) or stimulated Fig. 3 Fig. + 2 Cd4- fl/fl +/ n CD45 cells per mm of h + = 10 (TNF), 11 (IL-10) or 4 (IFN-

3 Rc3h2 Rc3h2 . Each symbol represents an represents symbol . Each +/ hi gastric mucosal area ( 10 ) Cre (WT)

+ × + A F4/80 d 2 4 6 cells ( cells 0 NS Rc3h2 TION ). The pharmacological agents agents pharmacological The ). Rc3h1 fl/f +/ + l Cd4- ** Cd4- +/ −

+ e cells (neutrophils). (neutrophils). cells * Rc3h1 ), ), IFN- fl/fl nature immunology nature Cre Cre (WT) Rc3h2 Cd4- d + 2 fl/fl – γ

p-STAT3 cells per mm of Rc3h2 Cd4- Rc3h1 Cd4- Rc3h1 Cre f + 3 ) ) or two H cells ( cells

c gastric mucosal area (×10 ) 17 17 ( fl/fl 2 4 6 8 0 ) ) of t -test). ( -test). Cre Cre (WT) fl/f fl/f +/ Cd4 Fig. 3 Fig. l + d l

t Rc3h2 Rc3h2 -test). -test). ), ), f ** ) ) or IL-4

-Cre mice mice -Cre Fig. 3 Fig.

b

fl/f

+/+ , c c l ). We ).

) γ b )); +

). ). + -

© 2014 Nature America, Inc. All rights reserved. pharmacological inhibitors of this paracaspase this of inhibitors two pharmacological thioridazine, and mepazine as well as z-VRPR-fmk, inhibitor peptide the including of MALT1, inhibitors with of cells incubation by pre inhibited or completely partially was that of roquin cleavage ( by ome or caspases cleavage sistent with protein resynthesis and incomplete proteolysis. the reappearance of the full-length protein at later times ( observed we agonists, TCR weaker potentially with stimulation term roquin protein by 8 h or 16 h of stimulation ( acids 61–80) with APCs and cognate antigen induced faster migrating amino glycoprotein virus choriomeningitis lymphocytic of peptide a CD4 or anti-CD3 alone was less effective ( ( protein roquin of forms ( ionomycin. with min 10–60 for or PMA with min 10–120 for stimulated (25 MG132 in as min 60 for stimulated or untreated left and blots) (above mice ( throughout. products degradation indicate asterisks with arrowheads and roquin-2, and roquin-1 full-length indicate (top) arrowheads + iono); (PMA ionomycin and PMA with lanes) (above CD4 wild-type in throughout) control (loading tubulin and roquin of ( receptors. costimulatory and TCR the of 3 Figure nature immunology nature representative of two ( two of representative (5 peptide cognate 1 h with for loaded +) (APC APCs wild-type irradiated h with 16 8 h or for cultured or −) (APC unstimulated ( anti-CD3 h with 1–4 for tution of Arg 560 or Arg 488 had no effect ( effect no had 488 Arg or 560 Arg of tution substi while Arg579, of substitution by eliminated was site cleavage MALT1 cleaved roquin-1 ‘preferentially’ after Arg510. The alternative ( product migrating faster the with together migrated (roquin-1(1–510)) 1–510 acids amino of ing product migrating cleavage ( appeared migrating faster slower the the of more yield instead, to fragment; failed protein roquin-1(R510G) of ( fragments two showed truncated roquin-1 type presumably by favoring the formation of dimers activity, paracaspase its of activate to MALT1 able is Overexpression (roquin-1(R510G)). 510 position at arginine the for glycine of tion substitu with roquin-1 mutant or roquin-1 wild-type with together in MALT1 human overexpressed Weretrovirally roquin-1. in residues arginine didate ( ionomycin plus full-length PMA with of tion stimula upon amounts products cleavage roquin of decreased presence the or either roquin exhibit not did cells T roquin, of cleavage the in MALT1 for role a critical ( effect no had (Ac-DEVD-CHO) caspases effector of inhibitor an and MG132 inhibitor proteasome the trast, b a Roquin-1 Roquin-2 Time (min) ) Immunoblot analysis of CD4 of analysis ) Immunoblot Tubulin We then assessed degradation of roquin proteins by the proteas the by proteins roquin of degradation assessed We then Since MALT1 is an arginine-specific protease, we assessed can assessed we protease, arginine-specific an is MALT1 Since + + T cells left untreated (0) or stimulated for 10–180 min min 10–180 for stimulated or (0) untreated left T cells T cells (which have transgenic expression of a TCR specific for expressionhave of specific TCR a transgenic (which cells T * *

Full-length roquin proteins are degraded after triggering triggering after degraded are proteins roquin Full-length 1 0 Fig. 4 Fig. µ M) and then left untreated or stimulated for 30 or 60 min as in in as min 60 or 30 for stimulated or untreated left then and M) 0 PMA +iono c 30 ). Consistent with that, truncated roquin-1 consist roquin-1 truncated that, with ). Consistent 60 Malt1 a – 120 c α ,

f -CD3) or anti-CD28 ( anti-CD28 or -CD3) ) or three ( three ) or Fig. 3 Fig. 180 aDV −/− mouse embryonic fibroblasts (MEFs) (MEFs) fibroblasts embryonic mouse + Fig. 4 Fig. (kDa) A T cells isolated from mutant mutant from isolated T cells 100 135 63 75 Fig. 4 Fig. e NCE ONLINE PUBLIC ONLINE NCE ), while stimulation with anti-CD28 anti-CD28 with stimulation while ), Fig. 4 Fig. d a b Roquin-1 Roquin-2 , Fig. 3 Time (min) ). PMA plus ionomycin induced induced ). plus ionomycin PMA e Tubulin c ) independent experiments. ) independent a ). These findings proved that that proved findings These ). ) Immunoblot analysis analysis ) Immunoblot b * * ). e ). Stimulation of SMARTA Fig. 3 Fig. 4 Fig. α Fig. 4 Fig. 6 0 -CD28) alone or together. ( together. or alone -CD28) Rc3h1 2 Cd4 9 0 0 f 2 . Expression of wild- Fig. 4 Fig. +/+ ). During such long- a 8 -Cre (WT) c ( ). Consistent with with Consistent ). Rc3h2 ). Expression of a of Expression ). A Fig. 4 Fig. Malt1

TION 60 Rc3h1 +/+ c

). Expression ). Expression fl/fl Fig. 3

6 0

Cd4 a

-deficient -deficient ). In con In ). Rc3h2 -Cre

0 0

f fl/fl ), con a

. . ( Cd4 Rc3h1 60 c -Cre Cd4 ) Immunoblot analysis of naive human CD4 human naive of analysis ) Immunoblot e Roquin-1 Roquin-2 e ------fl/f Tubulin ) Immunoblot analysis of wild-type CD4 wild-type of analysis ) Immunoblot l

(kDa -Cre Time (h)

f Rc3h2 100 135 63 75 ) Immunoblot analysis of roquin in SMARTA (SMtg) CD4 SMARTAin (SMtg) roquin of analysis ) Immunoblot a cleavage site at Arg509 is present in mouse roquin-2 and is is and roquin-2 mouse in present LIPRGTD- is the ( at (LISRTDS) only in its Arg509 human ortholog Arg509 conserved humans, at and site mice cleavage in conserved are sites and alternative cleavage at Arg579 in MVPRGSQ. While both roquin-1 tial’ cleavage of roquin-1 by MALT1 at Arg510 in the motif LIPRGTD ( cleavage carboxy-terminal endogenous product induced an by the stimulation detected of T cells with roquin-1 PMA plus ionomycin of required for post-transcriptional repression firmed the published observation that carboxy-terminal sequences are of roquin-1(1–510) did not affect ICOS expression ( mRNA is a known target of roquin in CD4 wild-type roquin-1 in downregulating surfacethan effective moreexpression roquin-1(R510G,R579G)wasmutant the of sion of ICOS, whose IRES sequences ( with equal amounts of green fluorescent protein (GFP)β coexpressed from sackie adenovirus receptor (CAR) driven by the CAG promotertransfer of genes(chicken by adenovirus due to transgenic expression of the cox Rc3h1 roquin-1deficientblyroquin-2.isolatedandinfromwe Forcells this ( z-VRPR-fmk inhibitor peptide specific the by blocked ( was it since cells specific, was cleavage enzymatic kidney embryonic human 293T in expressed of roquin-1(1–510) with fragment together migrated that kDa cleavage 55 approximately main a of appearance the to led and protein that the Immunoblot roquin-1. with recombinant analysis showed full-length of protein glutathione fusion active catalytically a incubating by MALT1 by roquin-1 of cleavage direct reconstitute to (PM) protein MALT1 inactive catalytically a used we when produced not ( roquin-1(1–579) or roquin-1(1–510) with together migrated that products cleavage ate to failed gener likewise compound mutant roquin-1(R510G,R579G) f . . ( ) l/f Supplementary Fig. 3b Supplementary * * *

-actin promoter-actin cytomegaloviruswith enhancers) l Immunoblot analysis with antibody to a carboxy-terminal epitope to a antibody carboxy-terminal with analysis Immunoblot We then reconstituted roquin-1 expression in naive CD4 d ) Immunoblot analysis of wild-type CD4 wild-type of analysis ) Immunoblot 1 0 c fl/fl Roquin-1 Roquin-2 Time (min Tubulin α in vitro Rc3h2 -CD2 4 3 * * * 8 ) fl/fl 3 1 cleavage reaction decreased the cleavage amountdecreased reaction of full-length 3 0 α Supplementary Fig. 3a -CD3 CAG-CAR + iono PM 6 0 1 4 A Fig. 4 Fig. Malt1 0 α α -CD3 -CD2 ). These data ). were ‘preferenwith These consistent ( 4 3 kDa 63 75 100 135 stop-fl −/− d ) 8 + ). Roquin cleavage products were also also were products cleavage Roquin ). d Roquin-1 Roquin-2 MEFs ( MEFs (kDa Time (min Tubulin 63 75 100 135 Cd4 Fig. 4 Fig. + T cells left untreated or stimulated stimulated or untreated left T cells ) + T cells pretreated for 30 min with with min 30 for pretreated T cells S -Cre mice, which are susceptible to * * -transferase (GST) and MALT1 (GST) -transferase f Roquin-1 Roquin-2 Tubulin e ) ), we found that ectopic expres Fig. 4 Fig. ). 1 0 + + T cells left untreated or untreated left T cells SMtg T cells ( APC 6 * , 8 0 . d 30 µ PM ). We then confirmed confirmed Wethen ). g/ml). Data are are Data g/ml). + + –

60 A Fig. 4 Fig. 4 + 8 + + – ticles e l c i rt A 120 . Comparing. cells T cells left left T cells 8 h + 10 i. 4 Fig. f f ), which con + ). Expression Table i T cells dou ono 30 – 16 60 h + + e ). The The ). 1 ). ). All (kDa (kDa 63 75 100 100 135 135 63 75 ) )  ------

© 2014 Nature America, Inc. All rights reserved. Zc3h12a including regnase-1, of targets known the of regulation negative the same regnase-1 set of target mRNAs. and We determined that roquin roquin also exerted of regulate roquin-1 and regnase-1 whether functions investigate to us prompted the of overlap extensive The regnase-1 and roquin of cooperation Post-transcriptional proteins. roquin of control of its own expression to regulate is which that known In regnase-1, suggested they addition, signaling. TCR of downstream MALT1 by cleaved was roquin regnase-1, like ‘acute’ of deletion with cells in state at steady present was protein more regnase-1 tially substan upon however, note, Of regnase-1 ionomycin. plus PMA or with stimulation CYLD of cleavage MALT1-mediated prevent of deletion ‘acute’ Such roquin. of dance Rc3h1 Rc3h2 deleted ‘acutely’ we MALT1, of targets other of cleavage ( CYLD and NIK kinase the Bcl-10, A20, to homology lesser and RelB and in regnase-1 sites target to homology extensive showed sites cleavage roquin GFP (pre-gated ICOS for stained and PMA T 3 d under for IRES-GFP,cultured then coexpressing adenovirus from expressed roquin-1(1–510) or 1(R510G,R579G) of cytometry ( z-VRPR-fmk. of (−) absence or (+) GST-MALT1 presence the in recombinant and roquin-1 full-length (rec) recombinant with reactions cleavage ( lanes). (above roquin-1 mutant or wild-type expressing retrovirus with infected ( lanes). (above mutants roquin-1 various or roquin-1 wild-type expressing retrovirus with infected or (−) uninfected MALT1,left with then reconstituted ( ionomycin. and PMA with min 60 or 15 for stimulated or unstimulated left T cells ( throughout. controls loading as served Tubulin GAPDH or ionomycin. and PMA with min 60 for (+) stimulated or (−) unstimulated left then lanes), (above thioridazine or mepazine z-VRPR-fmk, Ac-DEVD-CHO, 4 Figure s e l c i rt A  of form doxycycline-inducible a with MEFs regnase-1-deficient or ionomycin. Data are representative of three ( three of representative are Data ionomycin. Rc3h2 d Roquin-1 Roquin-2 d Roquin-1 Roquin-2 a PMA +iono

GAPDH ) Immunoblot analysis of roquin and MALT1 and in roquin of analysis ) Immunoblot MALT1 Tubulin Roquin-1 To determine whether the absence of roquin proteins altered the the altered proteins roquin of absence the whether Todetermine MAL fl/fl in T in fl/ * * * * T1

fl CD4 , , The paracaspase MALT1 cleaves roquin. ( roquin. MALT1 cleaves paracaspase The Rc3h2 cRel

+ –

H MG132 Rc3h1 17 cells ( cells 17 + WT and and

T cells cultured under T under cultured T cells WT +

fl/fl Ac-DEVD-CHO

Table R510G Rc3h1 fl/fl mice with Cre effectively diminished the abun the diminished effectively Cre with mice + Ctla4 z-VRPR-fmk

Rc3h2 1–510 + Fig. 4 Fig. 1–579 Mepazine 1 and (ref. (ref. ).

R510G,R579G – +

fl/fl Thioridazine PM (kDa) g

Rc3h2 WT CAG-CAR + ). Transduction of CD4 of Transduction ). 1 DMSO 1 100 63 75 100 135 ). Notably, reconstitution of roquin- roquin- of Notably, reconstitution ). Zc3h12a . Together these data showed that . showed data Together these Rec roquin-1 GST-MALT1 z-VRPR-fmk Roquin-1 (kDa) H stop-fl 17 conditions and infected with retrovirus expressing Cre–IRES–Thy-1.1, then stimulated with PMA and and PMA with stimulated then Cre–IRES–Thy-1.1, expressing retrovirus with infected and conditions 17 63 75 100 135 e + cells). ( cells). a mRNA, was also under the under mRNA, was also , * Cd4 Rc3h1 c , f PMA +iono(min) ) or two ( two ) or Malt1 -Cre CD4 -Cre b + – – + + a g ) Immunoblot analysis of roquin in wild-type CD4 wild-type in roquin of analysis ) Immunoblot ) Immunoblot analysis of total and cleaved roquin, regnase-1 and CYLD in wild-type or or wild-type in CYLD and regnase-1 roquin, cleaved and total of analysis ) Immunoblot and and + + – Roquin-1 Roquin-2 GAPDH MALT1 −/− + MEFs reconstituted with wild-type MALT1 or catalytically inactive MALT1 (PM), then MALT1then (PM), inactive MALT1 catalytically or wild-type with reconstituted MEFs b NS Rc3h2 + + , * d T cells from from cells T Roquin-1 (1–510) T cells infected with empty virus (EV) or reconstituted with wild-type roquin-1, roquin- roquin-1, wild-type with reconstituted or (EV) virus empty with infected T cells , e (kDa) Rc3h1 , g 55 1 0 ) independent experiments. ) independent did not not did Malt1 6 5 and and b Il6 ) Immunoblot analysis of roquin and MALT1 and in roquin of analysis ) Immunoblot +/ –

Cells (%) 0 - - 100 , 20 40 60 80 f 0 0

ICOS residues notincluded here. acid positionofthearginine residueafterwhichMALT1 cleaves;ellipses(…)indicate regnase-1, A20,Bcl-10,RelB,NIKandCYLD; subscriptednumbersindicatetheamino site inroquin-2orhomologouscleavagesites inthepreviouslyidentifiedMALT1 targets Alignment oftheidentifiedMALT1 cleavagesitesinroquin-1withaconserved Human CYLD Mouse CYLD Human NIK Human RelB Mouse RelB Human Bcl-10 Mouse Bcl-10 Human A20 Human regnase-1 Mouse regnase-1 Human roquin-1 Mouse roquin-1 Human roquin-2 Mouse roquin-2 MALT1 targets Table 1 abundance of the transcription factor IRF4 (ref. CD4 of into Cre transduction by retroviral genes of roquin-encoding tion ( IL-6 of production TNF-induced and IL-17- Rc3h1 Malt1 0 15 e 10 ) Immunoblot analysis of roquin and the products of its its of products the and roquin of analysis ) Immunoblot 2

GFP –/ + c – 60 T cells from from cells T ) Immunoblot analysis of roquin and MALT1 and in roquin of analysis ) Immunoblot or 10

+ 3

cells Alignments Alignments of MALT1 target sites Zc3h12a (kDa) 10 aDV 100 100 135 63 75 4 10

1–510 R510G,R579G WT EV A 5 NCE ONLINE PUBLIC ONLINE NCE , respectively , ( respectively Roquin-1 Roquin-2 c Rc3h1 Tubulin MALT1 Roquin-1 + MALT1 T cells preincubated with MG132, MG132, with preincubated T cells g * * fl/fl Regnase-1 Roquin-1 Roquin-2

Rc3h2 – Time (min) GAPDH CYLD WT

Supplementary Fig. 4a Supplementary WT * * * fl/fl

A R488G FMSR FMSR CLSR LVSR LVPR LRSR LRSR GASR LVPR LVPR LIPR LIPR LISR LIPR Malt1 TION mice effectively increased the the increased effectively mice Homology ofcleavagesites H 1 0

17 conditions, stimulated with with stimulated conditions, 17 R510G 510 510 509 509 85 64 111 111 325 228 228 439 324 324 WT

+/− GAA GPA GTD…MVPR GTD…MVPR TDS GTD Cre–IRES–Thy-1.1 GGG GGS 5 GAH TVS ALS GEA

GVG GVG 1–510 nature immunology nature and and 60 8 Fig. 5 Fig.

) ) and increased c-Rel – Malt1 Malt1 Rc3h1 WT 0

a WT in vitro in ). ‘Acute’ ). dele 579 579 1 −/− −/−

fl/f , 5 b

Rc3h1 l GSQ GSQ Rc3h2 MEFs MEFs ), ), repressed CD4 R560G 60

f R579G ) Flow ) Flow fl/fl + fl/fl

(kDa) (kDa)

100 63 75 100 135 63 75 100 63 75 63 75 100 135 -

© 2014 Nature America, Inc. All rights reserved. inducible ( additional target of regnase-1. We used MEFs deficient in either roquin post-transcriptional targets of roquin-1 and identified wild-type in low very but ( MEFs MEFs, regnase-1-deficient and MEFs post-translational unknown an to I due modification. immunoblots in kDa 70 NF- fore of target a roquin predicted Nfkbid ( CD4 naive from T cells culturing after CTLA-4 intracellular increased found also we OX40, and ICOS of expression surface elevated the Beyond ( cells wild-type in that with compared expression protein ( four condition), each of triplicates ( two technical of of representative are Data tamoxifen. with treatment RV) ICOS (+ without or tam) + RV ICOS (+ with Cre-ERT2, responsive tamoxifen- of expression stable with MEFs in as ( regnase-1. of re-expression for cassette doxycycline-inducible stable in as ( dox). + RV ICOS (+ doxycycline with treated or RV) ICOS (+ uninduced left then plots) 3 with 3 natural its with or (CDS) without mRNA reporter containing retrovirus with transduced or (UT) untransduced left either roquin-1 of re-expression for cassette doxycycline-inducible a Rc3h1 ( MEFs. regnase-1-deficient or wild-type deficient, I and regnase-1 roquin, ( conditions. T under h 48 for cultured and mice and wild-type from CD4 naive of CTLA-4 intracellular and OX40 ( retrovirus. Cre–IRES–Thy-1.1 with ( or Rc3h1 of extracts in c-Rel and roquin of ( Tukey’s test). with variance of analysis (one-way * detectable. not ND, graph). (below TNF and IL-17A doxycycline, of combinations various with h 24 for stimulated then respectively, of re-expression doxycycline-inducible allowing vector with ( ( roquin-deficient of supernatants ( targets. shared 5 Figure nature immunology nature contrast, ICOS expression was substantially decreased by the induction sion of ICOS generated from constructs without a 3 expression of roquin-1 or regnase-1 did not change the surface expres 3 various with or without human a containing retrovirus with cells these we infected induction, Before c Rc3h1 Zc3h12a Rc3h1 Fig. 5 i. 5 Fig. ) Flow cytometry of surface ICOS and and ICOS surface of cytometry Flow ) Rc3h1 We next identified identified Wenext + κ T cells sorted by flow cytometry cytometry flow by sorted cells T e e B inhibitor I inhibitor B ′ of of of −/− +/+ UTRs of various mouse genes (above (above genes mouse various of UTRs e mRNA, which contains two CDEs in its 3 its in CDEs two contains which mRNA, fl/fl −/− c e Fig. 5 Fig. ICOS ) or regnase-1 (

) Flow cytometry of ICOS in in ICOS of cytometry Flow ) ). We then investigated whether regnase-1 also regulated regulated also regnase-1 whether investigated then We ). ′ Rc3h2 Rc3h2 fl/fl Regnase-1 and roquin-1 regulate regulate roquin-1 and Regnase-1 UTR ( UTR Rc3h1 Zc3h12a −/− Rc3h2 Rc3h2 Rc3h1 Rc3h2 ) MEFs stably transduced transduced stably MEFs ) d P Rc3h1 ) Immunoblot analysis of of analysis Immunoblot ) reporter gene expressed from its coding sequence (CDS) d = 0.034 and ** and 0.034 = b ). ) Immunoblot analysis analysis Immunoblot ) −/− +/+ ICOS a Rc3h1 fl/fl κ −/− fl/fl or ) ELISA of IL-6 in in IL-6 of ELISA ) BNS expression was high in both roquin-deficient roquin-deficient both in high was expression BNS MEFs containing containing MEFs CAG-CAR fl/fl −/− Rc3h2 ) or regnase-1-deficient regnase-1-deficient or ) Rc3h1 ) T cells transduced transduced cells T ) κ fl/fl Zc3h12a 3 CAG-CAR BNS, which produces specific signals at 35 and and 35 at signals specific produces which BNS, MEFs reconstituted with a with reconstituted MEFs or or Rc3h2 ′ κ cRel UTR) or as fusion fusion as or UTR) Fig. 5 BNS in roquin- in BNS

fl/fl fl/fl Zc3h12a aDV stop-fl ′ , , g CAG-CAR Rc3h2 UTRs of selected target genes. Inducing Inducing genes. target selected of UTRs , , respectively ( ) Flow cytometry cytometry Flow ) Ctla4, Il6 Ctla4, fl/fl f P stop-fl ) that we reconstituted with doxycycline- < 0.0001 0.0001 < A (WT) (WT)

Cd4

NCE ONLINE PUBLIC ONLINE NCE 7

H . . , fl/fl

Nfkbid 17 17

-Cre mice under T under mice -Cre f ICOS stop-fl

Cd4

) Flow cytometry cytometry Flow )

and and

-Cre -Cre

mRNA encodes the nuclear mRNA encodes

Supplementary Fig. Supplementary 4a Irf4

e

) or three independent experiments ( experiments independent three or ) mRNAs as additional additional as mRNAs

IL-17A +TNF ′ ′ e c a f UTR and is there is and UTR UTR ( Cells (%) Cells (%) Cells (%)

A 100 100 100 Irf4 20 40 60 80 TION 20 40 60 80 20 40 60 80 0 H 0 0 10 10 ICOS ICOS IL-6 (ng/ml) 17 conditions conditions 17 ICOS 0 10 15 20 1 Dox 1 mRNA as an 10 0 5 10 10 Fig. 5e 2

2 2 10 CDS CDS 10 10 ND – – Fig. 5 Fig. 3 3 3 10 10 10 Zc3h12a Rc3h1 + 4 – 4 4 10 , 10 10 f ND 5 ** ). In – 5 + 5 – 100 b c 20 40 60 80 –/– 0 ). ). ). ). + + + ICOS - - ICOS –/– Rc3h2

OX40 1 0

ND – – – 0 g 2

vector encoding regnase-1 and the alloantigen Thy-1.1 connected connected Thy-1.1 alloantigen the and regnase-1 encoding vector a with cells retroviral of those superinfection After protein mCherry. fluorescent red the and P2A peptide cleavage the roquin-1, of sion expres the for vector doxycycline-inducible a and ICOS expresses stably that clone MEF wild-type a Weused together. effectors both of in context its the own 3 reporter the coexpressing by mRNAs target of regulation the in nase-1 regnase-1. and by roquin 3 identified of regulation experiments the in differences loss-of-function or gain- these Together activation of tamoxifen-induced a Cre-ERT2 fusion protein ( in a MEF cell line after deletion of the ( cells, wild-type we assessed the effect of depleting the cells of roquin to fused the 3 Irf4 cRel, 3 the with regnase-1 and roquin-1 by repression 3 own its the when regnase-1 or roquin-1 of much greater than with achieved the 3 This derepression via the regulatory elements of 3 3

Cells (%) 10 Supplementary Fig. 4d Supplementary + –/– ′ ′ UTR UTR 100 3 e eosrtd ucinl oprto o rqi- ad reg and roquin-1 of cooperation functional demonstrated We 20 40 60 80 10 ND 0 * was moderately inhibited ( inhibited moderately was + – 4 while the ICOS reporter in the context of the 3 the of context the in reporter ICOS the while 10 10 ICOS ICOS 1 + + 5 100 10 20 40 60 80 b cRel cRel 0 2 ′ – 10

UTR ( UTR 3 d 1 0 CTLA-4 3 ′ UTR ,

10 Roquin-1 Roquin-2 b 0 3 3 f 2 , ′ ′ Tubulin 4 UTR UTR ′ g 10 10 UTR of c-Rel ). 3 5 10 Fig. 5e Fig. * 4 Cre–IRES–Thy-1.1 10 Irf4 5 Ctla4 Ctla4 3 Cd4 Rc3h2 Rc3h1 WT ′ UTR Nfkbiz WT , -Cre f 3 3 , ). We observed similar post-transcriptional post-transcriptional similar We). observed e ′ ′ UTR UTR Rc3h1 fl/fl fl/fl ). Both ). of Both were reporters those derepressed Rc3h2 fl/fl Nfkbiz or

63 75 63 75 100 135 (kDa) fl/fl Nfkbid Fig. 5e Fig. Il6 Il6 3

′ ′ ′ UTR 3 3 UTR with individual effectors or effectors individual with UTR UTRs of target mRNAs shared shared mRNAs target of UTRs ICOS ′ ′ UTR UTR Rc3h1 ′ UTR of Regnase-1 d was already fully repressed in repressed fully was already , Roquin-1 Roquin-2 Nfkbid f reporter gene was fused to to fused was gene reporter ). Since the the Since ). Tubulin I I κ κ fl/fl BN BN Irf4 Irf4 3 S S * and ′ UTR a ICOS Nfkbiz 3 3 ; mean and s.d. s.d. and mean ; ′ ′ UTR UTR ′ Rc3h2 ticles e l c i rt A

UTR of of UTR Rc3h1

or Rc3h2 + tam(Creactivation) + ICOSRV + ICOSRV UT and

′ –/– ICOS UTR of of UTR UT + dox(regnase-1) + ICOSRV + ICOSRV –/–

Irf4 WT + dox(roquin-1) + ICOSRV + ICOSRV UT fl/fl Nfkbid

( Zc3h12 alleles by reporter reporter Ctla4 Fig. Fig. 5 Fig. Fig. 5 48 63 63 63 75 100 135 (kDa) –/– a Il6 ICOS was or or or or g g 1 ). ). ). ).  3 - -

© 2014 Nature America, Inc. All rights reserved. ( activity deubiquitinase its for not but activity nuclease its for ment a require or revealed mutant forms of regnase-1 wild-type with cells the presence of roquin ( 4f Fig. deubiquitinase function (C157A) or both (D141N) been shown to interfere with its nuclease function (D225A,D226A) or with roquin, we introduced point substitutions in that regnase-1 have ( reporter the of expression on effect small a only or no had cells roquin-deficient in regnase-1 or cells of overexpression pression of in regnase-1-deficient roquin-1 ( expression reporter downregulate to able equally was cells or regnase-1-deficient cells of roquin-deficient regnase-1–IRES–Thy-1.1 retroviral vector. Notably, the reconstitution 3 the roquin duced trans we first experiments, In these regulation. post-transcriptional in regnase-1 and roquin of interdependence demonstrated We then of in regulators both effects the repression of cooperative tially 1.1 (Thy- more roquin-1 and was regnase-1 coexpressed that cells downregulation in pronounced its however, roquin-1; or regnase-1 of expression by individual similarly of was decreased ICOS expression surface The roquin-1. express to doxycycline with them induced or untreated cells the left we (IRES), site entry ribosome internal an by in (as ROQ–regnase-1 or roquin-1 regnase-1, expressing ( (bottom). regnase-1 of sequence coding the to linker GSGGRIP a flexible with fused roquin-1 of domain ROQ the containing 131–360) acids (amino a region of protein artificial an of and (middle) regnase-1 in domains finger zinc CCCH and nuclease the of (top), roquin-1 of domain coiled-coil and (PRR) region proline-rich finger, zinc CCCH ROQ, finger, ( regnase-1 mutant or wild-type expressing retrovirus IRES–Thy-1.1 with cells of ( RV). regnase-1 (+ regnase-1 or RV) roquin-1 (+ roquin-1 expressing retrovirus IRES–Thy-1.1 with 3 the with fused mRNA reporter ICOS the encoding ( (throughout). cells untransduced line, dashed (roquin-1)); RV + dox regnase-1 (+ both or (roquin-1)) dox (+ roquin-1 doxycycline-induced or the 6 Figure s e l c i rt A  controlled b d a i. 6 Fig. Zc3h12a ′

UTR ) Flow cytometry of of cytometry ) Flow To define the catalytic activity of regnase-1 required for cooperation Rc3h2 Rc3h1 + ICOS ICOS

mCherry Cells (%) 100 20 40 60 80 0 ). All mutant and wild-type regnase-1 proteins depended on on depended proteins regnase-1 wild-type and mutant All ). –/ –/ –/ c 10 1

+ regnase-1RV + dox(roquin-1) + dox(roquin-1) + regnase-1RV – – – reporter with its 3 its with reporter . nlzn dwrglto o epeso o te CDE- the of expression of downregulation Analyzing ). 3 ICOS Roquin and regnase-1 cooperate in post-transcriptional gene regulation. ( regulation. gene post-transcriptional in cooperate regnase-1 and Roquin 1 reporter fused to 3 the fused reporter , then transduced them with a roquin-1–IRES–Thy-1.1 or or roquin-1–IRES–Thy-1.1 a with them transduced then , 10

ICOS Cells (%) 2 100 10 20 40 60 80 0 3 + - 10 ) ) ( or regnase-1-deficient MEFs with a vector encoding encoding vector a with MEFs regnase-1-deficient or 10 ICOS 4 1 reporter by wild-type and mutant by forms of reporter wild-type roquin-1 10 Fig. Fig. 6 10 5 2 WT 10 Rc3h1 3 10 4 a 10 Fig. 6 ). These findings indicated additive or additive indicated ). poten findings These b 5 ′ UTR alone (shaded curve) or together with retrovirus expressing regnase-1–IRES–Thy-1.1 (+ regnase-1 RV) regnase-1 (+ regnase-1–IRES–Thy-1.1 expressing retrovirus with together or curve) (shaded alone UTR Zc3h12a −/− Rc3h2 Rc3h1 Fig. 6 Fig. K239A,R260A Rc3h2 c ). ). Reconstitution of regnase-1-deficient –/ –/ –/ ′ -terminal 260 nucleotides of the nucleotides 260 -terminal – – – b ). −/− Cells (%) 100 and and 20 40 60 80 0 Fig. 6 Fig. 10 ICOS 1 + roquin-1RV K239A,R260A K220A, 10 Zc3h12a 2 10 b 3 10 ). In contrast, overex contrast, In ). ′ 4 -terminal 260 nucleotides of the the of nucleotides 260 -terminal 10 1 , 5 3 −/− 0 ( e MEFs transduced with retrovirus encoding the the encoding retrovirus with transduced MEFs + regnase-1RV Supplementary ). Data are representative of three or more experiments. more or three of representative are Data ). e 1 RING 131 f ) Flow cytometry as in in as cytometry ) Flow ROQ ROQ ICOS Tnf GSGG 360 - - - - .

1 CCCH c Zc3h12a ( increased of I and expression protein regnase-1 in diminution efficient found we Zc3h12a CAR DO11.10 from those T cells (CAR domain cytosolic a lacking receptor coxsackie-adenovirus human of and (DO11.10) TCR OVA-specific an of expression transgenic with mice from cells T the promoted regnase-1 of amount cellular in T regnase-1 and roquin by regulation gene of importance the assessed Wethen I respectively. regnase-1, and roquin-1 through of defined activities nuclease and RNA-binding was of the interaction functional cooperation the that showed findings these ulated reporter expression in roquin-deficient cells ( of with roquin, as independently cooperation it functioned downreg but cells regnase-1-deficient in regnase-1 wild-type as active as was ( reporter regnase-1 diminished full-length encoding frame reading open the to roquin-1 of structures domain CDE to ( repression the roquin-1 with of interfered binding (K220A,K239A,R260A) completely more or sion ( wild-type roquin-1 in roquin-deficient cells inhibited reporter expres ( cells regnase-1-deficient in (ref. Rc3h2 Rc3h1 k upeetr Fg 5a Fig. Supplementary Nuclease Nuclease c BNS and I and BNS ) or roquin-1 ( roquin-1 ) or a PR –/ –/ –/ 1 Fig. 6 ) Flow cytometry of ICOS in a wild-type MEF clone stably expressing expressing stably clone MEF a wild-type in ICOS of cytometry ) Flow – – – 3 Tnf C R ( ) -specific short hairpin RNA -specific (shRNA) and GFP,coexpressing

CCCH CCCH Cells (%) b 100 3 but with superinfection of cells with IRES–Thy-1.1 retrovirus retrovirus IRES–Thy-1.1 with cells of superinfection with but 20 40 60 80 Supplementary Fig. 4g Fig. Supplementary aDV 0 d Fig. 6 Fig. ′ 10 UTR. Reporter alone (shaded curves) or then superinfected superinfected then or curves) (shaded alone Reporter UTR. Fig. 6 Fig. ). ). In contrast, substitutions that partially (K239A,R260A) ICOS 1 k 10 H A κ 2 B 17 differentiation. First, we the showed that reducing 17 differentiation. oiled-coil d NCE ONLINE PUBLIC ONLINE NCE 10 B WT  e ) (above plots). ( plots). ) (above 597 3 ζ control T control and and d 10 c , which is encoded by the regnase-1 target target regnase-1 by the encoded is , which Regnase- ROQ–regnase- ). We then fused a region containing the ROQ ROQ the containing region a fused then We ). , 4 d 10 1,130 ) Flow cytometry as in in as cytometry ) Flow 5 Supplementary Fig. 4h Fig. Supplementary ∆ Roquin- ICOS -I) ( -I) , 1 b ). To assess the effect of regulation of of regulation of effect the assess To ). D141N H reporter transduced with retrovirus retrovirus with transduced reporter 1 Fig. 7 Fig. 1 17 differentiation 17 i. 6 Fig. e ∆ f ) Domain organization of the RING RING the of organization ) Domain Zc3h12a -I mice with adenovirus encoding encoding adenovirus with -I mice Rc3h2 Rc3h1 ), we again found no regulation regulation no found again we ), a A ). Upon infection of CD4 naive ). Upon infection d TION ). As expected, expression of of expression expected, As ). D225A,D226A –/ –/ –/ – – – b Cells (%) 100 but with superinfection superinfection with but 20 40 60 80 nature immunology nature 0 10 H ICOS ). This fusion protein protein fusion This ). 1 17 differentiation of of differentiation 17 10 2 10 Fig. Fig. 6 3 10 + roquin-1RV + regnase-1RV + ROQ–regnase-1RV

C157A 4 10 5 f

). ). Together Nfkbiz + - -

© 2014 Nature America, Inc. All rights reserved. as in in as ( (gMFI). intensity fluorescence geometric µ in as analysis ( OVA of peptide. lanes) (above concentrations various with loaded +) (APC APCs wild-type irradiated with or −) (APC h alone 18 for ( (+). ionomycin and PMA with min 60 for stimulation or (−) stimulation no by followed (Mep), mepazine or (DMSO) sulfoxide demethyl with min 180 for ( ionomycin. and CD4 I as well as regnase-1, and roquin-2 ( targets. 8 Figure expressed we when normalized partially T increased results, those with Consistent of IFN- regulation I of effect ( I of down for production to shown IL-17 be required has been which directed shRNA of use the against through proteins these of abundance I ( four from are from T cells naive in plots) (above roquin-1 mutant and wild-type of expression adenoviral ectopic fl IFN- and flow cytometry from a GFP T 3 d under for cultured then shRNAs), different indicate sh2 and (sh1 Nfkbiz for specific shRNA and GFP encoding adenovirus with transduced T cells CAR DO11.10 in ( T inhibits regnase-1 and differentiation I 7 Figure nature immunology nature ( s.d. and mean show bars (error experiments independent a a κ Fig. 7 κ Regnase-1 Rc3h2 , but with cells cultured under T under cultured cells with , but g/ml. ( g/ml. a ) Intracellular IL-17A IL-17A ) Intracellular B

Roquin-1 Roquin-2 + B

Time (min) IL-17A cells (fold) ζ Tubuli ζ + + I I 0.5 1.0 1.5 2.0 promote T promote κ κ cells); results are presented relative to those of control empty virus empty control of those to relative presented are results cells); T cells left unstimulated or stimulated for 30–180 min with PMA PMA with min 30–180 for stimulated or unstimulated left T cells c or I or IRF4 BN BN I κ , , , generating , generating a B e fl/fl Nfkbid S S Nfkbiz n ), which identified a previously unrecognized T * * * ζ

γ a ) Flow cytometry of IRF4 in CD69 in IRF4 of cytometry ) Flow

TCR signal strength determines the derepression of roquin roquin of derepression the determines strength signal TCR I H assessed as in in as assessed ) Immunoblot analysis of total and cleaved roquin-1, roquin-1, cleaved and total of analysis ) Immunoblot κ Zc3h12a Nfkbid Nfkbiz CAG-CAR κ 17 differentiation. differentiation. 17 κ

κ sh1 BNS and and BNS BNS on T on BNS BNS resulted in markedly decreased IL-17A production production IL-17A decreased markedly in resulted BNS 0 BNS. The effect was specific, because we did not find find not did we because specific, was effect The BNS. sh2 c a or or and

, but with APCs loaded with wild-type, R9 or F9 OVA peptide (high, low and very low affinity, respectively) at a concentration of 10 of a concentration at respectively) affinity, low very and low (high, OVA F9 peptide or R9 wild-type, with loaded APCs with , but sh1 H ) or two ( two ) or 30 17 17 b

∆ sh2 Zc3h12a ) Immunoblot analysis as in in as analysis ) Immunoblot -I CD4 -I sh1 60 in vitro in PMA +iono Rc3h1 stop-fl Nfkbid γ

-producing cells under T under cells -producing Ctrl 90 H

b Cd4 + 17 differentiation, we decreased the cellular cellular the decreased we differentiation, 17 b 120 a ) independent experiments ( experiments ) independent

T fl/fl , but with naive CD4 naive with , but

+ (

H IFN- cells (fold) aDV γ 180 Supplementary Supplementary Fig. 5c Rc3h2 -Cre mice and transduced with shRNA specific for for specific shRNA with transduced and mice -Cre 0.5 1.0 1.5 2.0 17 cultures for 3 d. ( 3 d. for cultures 17

A (kDa) H e NCE ONLINE PUBLIC ONLINE NCE 75 63 75 63 75 100 135 35 48 63 48 1-polarizing conditions. ( conditions. 1-polarizing Zc3h12a Nfkbid Nfkbiz fl/fl

κ IL-17A sh1 10 10 10 10 B

CAG-CAR sh2 0 2 3 4 5 ζ Cd4- CAG-CAR Rc3h1 IFN- , IRF4 and I and , IRF4 sh1 13.5 0 Regnase-1 b

10 sh2 f Roquin-1 Roquin-2 + ) Flow cytometry of cells cultured as in in as cultured cells of cytometry ) Flow Cre (WT) PMA +iono γ GAPD 2

cells among OT-II among CD4 cells sh1 +/ I I κ κ 10 + Ctrl IRF4 Nfkbiz- BNS BNS I Ctrl Rc3h2 a κ 3 + stop-fl stop-fl B , but with preincubation preincubation with , but H T cells purified by flow cytometry from from cytometry flow by purified T cells H 10 * * * ζ H 17 differentiation was was differentiation 17 0.04 4 c 1 conditions ( 1 conditions h 0.1 +/ 10 ) Intracellular IL-17A in cells as in in as cells in IL-17A ) Intracellular

Cd4 IL-17A κ + 10 10 10 10 – 5 BNS, in wild-type wild-type in BNS, , 0 or or a 2 3 4 5 d , IFN- -Cre mice and transduced with shRNA specific for ). ). Similar to I b 28.2 A 54.0 0 + + ; average and s.d.) or are representative of two ( two of representative are or s.d.) and ; average 10 TION DMSO Nfkbid- H e γ 2 , 17-promoting c f CAG-CA 10 Ctrl Ctrl , ) or variance ( variance ) or Mep d 3 Rc3h1

(kDa) ) Intracellular IL-17A and IFN- and IL-17A ) Intracellular

10 3 35 48 63 48 75 63 75 63 75 100 135 1 Fig. 7 Fig. 4 , , knock 0.2 0.2 specific specific 0.1 10 + R 0 T cells cultured for 24 h in T h in 24 for cultured T cells fl/fl 5

stop-f κ Rc3h2 OVA(323–339) B H 58.6 c e 37. b

l

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Zc3h12a 17-polarizing conditions and stimulated with PMA plus ionomycin (pregated on (pregated ionomycin plus PMA with stimulated and conditions 17-polarizing – ζ ). ). IRF4 Nfkbiz - 0 3 transduced cells, set as 1 (dotted line). ( line). 1 (dotted as set cells, transduced ,

3 g

(gMFI × 10 ) fl/fl , Cr h 10 15 ) of three ( three ) of e c insensitive roquin-1(R510G,R579G) mutant showed enhanced enhanced showed mutant roquin-1(R510G,R579G) insensitive adenovirus roquin-1-expressing their T normalized with cells T Reconstitution functions. roquin-deficient of nonredundant had also regnase-1 and ( cells IL-17A-producing of abundance in a greater resulted T cells in roquin-deficient regnase-1 through part I of in expression the least of at upregulation mediated was cells T roquin-deficient in observed differentiation increased the that showed findings Those CD4 shRNA and in roquin-2-deficient roquin-1- 0 5 Roquin-1 Roquin-2 sh1 GAPDH ) Immunoblot analysis of roquin and IRF4 in OT-IIin IRF4 CD4 and roquin of analysis ) Immunoblot Zc3h12a sh1 1. 1. IRF4 0.1 0. (

d µ .1 OT-I 1 4 2 g/ml) AP , analyzing IRF4 expression as in in as expression IRF4 , analyzing 1 * * C 10 OVA(323–339) I f 38. d IL-17A Rc3h1 . Data are representative of two ( two of representative are . Data 10 10 10 10 e Nfkbiz . . ( + + – – – 0 0 , 0 2 3 4 5 f f ) or two ( two ) or IFN- ) Intracellular IL-17A and IFN- and IL-17A ) Intracellular 45. 0 f + – 10 γ sh2 +/+ IRF4 0 6 assessed as in in as assessed γ 2

3 0. + 10 (gMFI 10 ) 18 h

× 1 1

Ctrl Rc3h2 H ( 3 µ 10 15 0 Rc3h1 H 17 conditions with APCs as in in as APCs with conditions 17 0 5 g/ml + + 1 10 g 17 17 differentiation (

, 4 d h ) WT 0. + + 10 ) technical replicates). ) technical 0 .1 IL-17A +/+ 1 10 10 10 10 5 Nfkbiz c fl/fl R9 0 ) or three ( three ) or 2 3 4 5 CAG-CAR (kDa) IFN- 40. Rc3h2 100 135 0 63 75 22. 10 Fig. 7 Fig. 9 a 1 γ 2 ( , but with naive CD4 naive with , but c 10 WT g Ctrl ) or

+ 3 fl/fl IL-17A cells b d 10 OVA(323–339) stop-fl ) Intracellular IFN- ) Intracellular e d κ e (%) CAG-CAR 4 Nfkbid 0. 0.2 ), which indicated that roquin roquin that indicated which ), – . . ( Roquin-1 Roquin-2 0. 0.2 10 20 30 40 B 10 f GAPDH 2 0 8 ) independent experiments. ) independent ζ 5 g Fig. Fig. 7 Cd4 a IRF4 ) Intracellular IL-17A in cells cells in IL-17A ) Intracellular and I and , γ OT-I b 0. APC 36. assessed as in in as assessed 10. R510G,R579G ( ), three ( three ), * * 1 1 -Cre (WT) or or (WT) -Cre Nfkbid d 1 0 7 I 0 0 ). ( f stop-fl ). ). Notably, the MALT1- OVA(323–339) 0 κ + + – – – e c BNS. Knockdown of of Knockdown BNS. ) Intracellular IL-17A ; results presented as presented ; results sh1 + d Cd4 + + – T ( cells c ) Immunoblot ) Immunoblot T cells purified by purified T cells ticles e l c i rt A 0. 0.

, h + .2 .2 d 6 WT γ 7 T cells cultured cultured T cells + + Data mice. -Cre assessed as in as assessed , 18 h

g IL-17A cells Rc3h1 ,

h (%) 15. R9 45. a ( + + µ ) or four ( four ) or , but with with , but 10 20 30 40

g/ml Nfkbid 0 4 0 0 3 Fig. Fig. 7c F9 1–510 + + ) WT fl/ sh2 R9 (kDa) 0.04 100 135 48 63 75 0. , e d .2 .1 3 , f ). ). 

)

© 2014 Nature America, Inc. All rights reserved. changes in the lungs in both mouse models include thickening of the thickening include models in mouse changes in lungs the both expres the I of of sion induction prevented also but regnase-1 of and cleavage roquin the inhibited only not mepazine inhibitor paracaspase ( min 30 after plete closely followed the cleavage of roquin and regnase-1, which was com by mRNAs encoded proteins that are of and targets regnase-1 roquin ( min 180 and 60 between I IRF4 of and expression increasing continuously and min 120 and 30 of I expression high induced ionomycin and PMA with T cells downstream of MALT1. Stimulation of CD4 wild-type and IRF4 were placed under critical post-transcriptional regulation in ( effect no had expression of the MALT1-induced cleavage product roquin-1(1–510) while cells, IL-17A-producing of frequency the reducing in activity s e l c i rt A 1 airway epithelium and increased mucus production and are associated of IRF4 in activated T cells (CD4 cells T activated in IRF4 of TCR OT-II the for affinity less with F9) and (R9 OVA mutant peptides with stimulation upon occurred ( IRF4 of and expression fragments of cleavage roquin to led accumulation the 323–339) acids OVA(amino cognate of peptide amounts with increasing TCR) (OVA)-specific ovalbumin an of expression transgenic have (which cells T OT-II and of ‘decisions’. Stimulation targets differentiation of derepression regulators, post-transcriptional of age mRNAs. target of translation the control also might regnase-1 and roquin stability, of mRNAto that regulation the in addition suggested findings These ug ad s soitd ih h atato o neutrophils of attraction the with associated is and lungs T of accumulation induces similarly which cells, T peripheral STAT3in protein ‘hyperactive’ a of expression ectopic of helper T cell in cytokines, IL-17A. of particular This is observation reminiscent overexpression and pathology lung and inflammation in to regulate ICOS expression or T roquin-1; however, ectopic expression of the cleavage product is unable dominant-negative function for the amino-terminal cleavage product of mRNAs from repression. We cannot exclude the possibilityvated roquin-1 and roquin-2 ofand released roquina and regnase-1potential target inactivate regnase-1(ref. to shown been has MALT1 paracaspase the by Cleavage of roquin was required and sufficient for cooperative target repression. contributed through its nuclease activity, while the RNA-bindingUTRs of overlappingactivity target3 post- of mRNAs.coregulation demonstrating in Our by resultsregulation regnase-1 gene indicated transcriptional and that roquin regnase-1 of cooperation identified have We DISCUSSION of T promote that targets derepression post-transcriptional the controls that regnase-1 and roquin of tivation inac proteolytic graded MALT1, of activity graded into ‘translated’ is strength TCR signal in which a model Togethersupport data these strength. signal TCR with correlated positively were differentiation ( cells of IL-17A-producing frequency lower much a yielded (R9) OVA mutant peptide the with concentrations of OVAwild-type ( peptide 17A-producing cells when OT-II cells were stimulated with increasing TCR affinity ( ( avidity TCR with correlated positively that expression 0 We then demonstrated that target mRNA encoding I encoding mRNA target that demonstrated Wethen We then investigated how TCR signal strength led to the cleav the to led strength signal TCR how investigated then We The combined T cell–specific loss of roquin-1 and roquin-2 resulted

κ Fig. 8 Fig. B ζ , I , Fig. 8 κ Fig. 7 Fig. 1 c BNS and IRF4 after 60 min of stimulation ( stimulation of min 60 after IRF4 and BNS 1 ). In contrast, diminished or no cleavage of roquin roquin of cleavage no or diminished contrast, In ). ). Our results Our showed). cleavedthat itandalso inacti f Fig. 8 Fig. ). Notably, we found an increased frequency of IL- f ). a ). Pretreatment of cells with the MALT1 the with cells of Pretreatment ). Fig. 8 Fig. H h 17 cell differentiation + ). Thus, roquin cleavage and T cleavage ). Thus, roquin CD69 3 2 Fig. 8 Fig. ( Fig. 8 Fig. + ) confirmed ‘graded’ IRF4 IRF4 ‘graded’ confirmed ) Fig. Fig. 8 H a 17 differentiation. 17 ). This upregulation of of upregulation This ). d ). Single-cell analysis analysis Single-cell ). g ), while ), stimulation while H 17 cells in the the in cells 17 6 , κ 8 Fig. 8 Fig. κ . B B ζ ζ between between Fig. 8 Fig. , I , + 3 T cells 3 e κ κ . The The . ) and and ) BNS BNS BNS BNS H b 17 17 ). ). - - - - - ′

ifrne i te xrsin f hs fcos a aat coopera control the in differences allow adapt situationsand specific to activity may tive factors these of expression the in Differences transcriptional, post-transcriptional and post-translational levels on regulated be can expression whose regnase-1, with together acted T pathway of metabolic aerobic glycolysis Hif1a tion could involve the graded transactivation of its target genes, such as T regulate can effectively abundance IRF4 which CD8 for shown stimulation been antigenic has strong by differentiation cell T effector and expression IRF4 high of induction the for concept ‘rheostat’ a Such EAE the differentiation of T IRF4 and T and IRF4 established that MALT1-mediated cleavage of roquin, derepression of Foxp3 of development the in T of precursors transcription of modulator a as so far,c-Rel with but reported to together shown it act been has been cell differentiation. Involvement of I c-Rel, as well as the transcriptional modulators I and regnase-1 of mRNAs encoding the transcription factors IRF4 and TCR signaling strength. We found that post-transcriptional regulation loss of roquin function may have duebeen to shared requirements for mice regnase-1-deficient in in cells that develops for phenotype as has reported the gastritis been bodies, T of lacked differentiation and that organ, this barrier stomach antigens could also be targets of autoanti priming to mice for relate again in conditions might special phenotype phenotype this Although cells. gastritis T in a roquin observed also differentiation. Wedrives that state hyperactive the into locked being before activation cell T initial the on depend still STAT3molecules T antigens of may and airborne priming differentiation enable effective model. One explanation for the selective pathology in the lungs is that approaches have will genetic to a establish of importance similar IL-17 Future in this cells. T in proteins roquin lack that mice in feasible are not experiments analogous at times, much later viability reduced IL- antibodies with 17-neutralizing ‘rescue’ by cells T in STAT3 hyperactive expressing mice in shown been has viability and decreased opment of lung pathology concentrations IL-17A serum T enhanced reveal viability decreased with down down results indicated that I ROR factors T to promote is and sufficient is its overexpression required expression shownbeen to be important for the proliferation of T targets includes ICOS, OX40 and CTLA-4,of layer among second which A ICOSfibroblasts. and has alsocells epithelial in production IL-6 cells T drive will TNF and IL-6. Loss of roquin thus leads to overproduction of IL-6 that upstream was the repression of mRNAs encoding cytokines, including T affect also which of T promote that molecules encoding by roquin and regnase-1 converged on the regulation of target mRNAs H H H Our data showed that constitutively expressed roquin-1 and roquin-2 We have also identified post-transcriptional regulation by roquin roquin by regulation post-transcriptional identified Wealso have The selective differentiation toward selective T The 17 cells. This is consistent with the observation that the ‘hyperactive’ 17 cell differentiation, and I 17 cell differentiation cell 17 3 37– 4 , which encodes the transcription factor HIF-1 . Increased concentrations of TNF and IL-17A induce further further induce IL-17A and TNF of concentrations Increased . 3 9 . . Finally, IRF4 is required for T aDV H H α 17 differentiation correlated with TCR signal strength. strength. signal TCR with correlated differentiation 17 17 cell differentiation and impair the induction of T of induction the impair and differentiation cell 17 and ROR A reg NCE ONLINE PUBLIC ONLINE NCE el i te thymus the in cells H 17 cell differentiation, and the mice have higher higher have mice the and differentiation, cell 17 H H 17 cells, and c-Rel-deficient mice are resistant to γ 1 cell– and T and cell– 1 t t to IL-17 expression enhance 43 3 3 , Nie CD4 Naive . 3 + 4 3 4 4 cells T κ . Due to the onset of lung pathology and . to of Due onset the lung pathology . . BNS BNS was as important as I 3 κ 3 B . A critical role . for A in IL-17 the critical devel ζ acts together with the transcription κ 41 BNS in T A FH TION H , 4 H 7 el ifrnito, many differentiation, cell 17 2 4 cell–related ‘decisions’. cell–related Most 3 + FH . One possible scenario by by scenario possible One . 1 17 17 differentiation 6 T cells from both models models both from cells T and is likewise required for required and is likewise . c-Rel itself is critical for for critical is itself c-Rel . , T , nature immunology nature H H 17 cell function has not 1 andT 1 κ BNS BNS and I H 17 cell differentia 17 cell α that controls the H 17 17 cells 3 H 1 κ 17 cells upon cells 17 . . Our knock B 4 ζ 0 κ . . We have for T B 3 ζ 5 . . I 1 . , 10 κ H , B 17 17 reg 1 1 ζ − - - - - - .

© 2014 Nature America, Inc. All rights reserved. 9. 8. 7. 6. 5. 4. 3. 2. 1. reprints/index.htm R The authors declare no competing interests.financial S.B. and V.H. wrote the paper. V.H. conceived of the project and the designed experiments; and K.M.J., D.H., M.H. provided reagents, advice, and design and of supervision experiments; experiments; A.G., E.K., A.W., H.-J.A., I.S., M.S.-S., M.F., H.H., D.K., J.R. and S.C.W.,N.R., S.L.E., N.M., R.G., C.L., M.L., J.E.S. and A.K. contributing to specific K.M.J., D.H. and S.B. did most of the experiments, with J.Z., G.A.H., D.N., K.U.V., (K.U.V.). Societies Biochemical Ingelheim(M.H.), Boehringer Fonds (G.A.H.) and The Federation of European M.H. and HO1116/5-2 to H.H.) the (M.H.), PCCC the Hofschneider Foundation to D.K.; SFB 1054 TP-A02 to M.S.-S; GRK1202 to H.-J.A. and M.L.; SFB TR36 to (SFB 1054 TP-A03 and Z02 to V.H.; SFB 1054 TP-B01 to J.R.; SFB 1054 TP-A04 reading of the manuscript. Supported by the Deutsche Forschungsgemeinschaft Germany) for the cloned 3 We thank H.-M. Jäck and J. Wittmann (Friedrich-Alexander-Universität Erlangen, onli Note: Any Supplementary Information and Source Data files are available in the the in versi available are references associated any and Methods M in peripheral tolerance and the development of autoimmune disease. ferentiation pathway and highlights a role for the paracaspase MALT1 T the in regnase-1 and roquin of aretargets that genes of of the available antigens. Such regulation enables rheostat-like control activated in a graded manner that is tightly linked to avidity and affinity T fatecell ‘decisions’. data Our have provided that MALT1evidence is regulation of other known and unknown MALT1 targets contributes to extends to redundant functions of other regnase paralogs and how the also roquin of cooperation the however,whether unknown, remains It regnase-4. and regnase-2 regnase-1, roquin-2, roquin-1, in present were Notably,sites MALT1homologouscleavageshown). not highly controlled by roquin-1 as well as roquin-2 (refs. function 7,8 and data Nfkbiz of repression post-transcriptional that showed 3 the presentin CDEs of regulation potential the and roquin by as well of control the includes tion autoregulanegative Such cells. which T of desensitization the prevent may autoregulation, negative extensive exhibits also system The roquin. or regnase-1 to sensitivity ‘preferential’ with genes target of nature immunology nature C AU A ′ eprints and permissions information is available online at at online available is information permissions and eprints ckn O

UTRs of mRNAs encoding roquin-1 and roquin-2 (ref. ethods T MPE ne version of the pape Yu, D. K.U. Vogel, K. Leppek, E. Glasmacher, immunity. in networks regulatory Post-transcriptional P. Anderson, & P. Ivanov, Z. Zhou, Vinuesa, C.G. Miao, R. K. Matsushita, messenger RNA. messenger differentiation. cell T helper follicular control Immunity and mRNAs costimulator Ox40 motifs. recognition stem-loop of Immunol. Nat. repression. post-transcriptional microRNA-independent induce to decay mRNA of Rev. Immunol. mechanisms. autoimmune autoimmunity. and cells T helper follicular disease. decay. mRNA regulating by responses H on of the pape the of on o O mRNAs and T and mRNAs T wledgmen R R C et al. ING FINANCIAL IN FINANCIAL ING Immunol. Cell Biol. Cell Immunol. et al.

O t al. et 38 Roquin represses autoimmunity by limiting inducible T-cell co-stimulator N et al. et t al. et , 655–668 (2013). 655–668 , T Targeted disruption of MCPIP1/Zc3h12a results in fatal inflammatory et al.

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t al. et et al. et t al. et The NF- The et al. et The Th17 immune response is controlled by the Rel-ROR the by controlled is response immune Th17 The Pharmacologic inhibition of MALT1 protease by phenothiazines as phenothiazines by MALT1protease of inhibition Pharmacologic 10 T cell-derived IL-17 mediates epithelial changes in the airway and airway the in changes epithelial mediates IL-17 cell-derived T The I HIF1 The development of inflammatory T inflammatory of development The 2 ngtvl rglts cl rcpo sgaig o NF- to signaling receptor cell T regulates negatively A20 t al. et MCPIP1 ribonuclease antagonizes dicer and terminates microRNA Pharmacological inhibition of MALT1 protease activity protects mice , 4–21 (2012). 4–21 , at-nue cevg o rgae1 n CD4 in regnase-1 of cleavage Malt1-induced nefrngma xes ed t ptoei accumulation pathogenic to leads excess Interferon-gamma Nat. Immunol. Nat. infection. h NF- The oto o T of Control os f oun nue ery et ad mue deregulation immune and death early induces Roquin of Loss , 167–175 (2009). 167–175 , Paracaspase MALT1Paracaspase autoimmune- from mice protects deficiency T cell antigenreceptorcellstimulation T induces MALT1 paracaspase- Nature h poeltc ciiy f h prcsae AT i ky in key is MALT1 paracaspase the of activity proteolytic The Structural basis for RNA recognition in roquin-mediated post- roquin-mediated in recognition RNA for basis Structural c-Rel promotes type 1 and type 17 immune responses during responses immune 17 type and 1 type promotes c-Rel I The costimulatory molecule ICOS regulates the expression of expression the regulates ICOS molecule costimulatory The Selective C-Rel activation via Malt1 controls anti-fungal T anti-fungal controls Malt1 via activation C-Rel Selective Nat. Immunol. Nat. at-eedn Rl cevg pooe cnncl NF- canonical promotes cleavage RelB Malt1-dependent J. Exp. Med. Exp. J. EMBO J. EMBO I κ κ B α κ B kinase complex regulates the stability of cytokine-encoding κ J. Exp. Med. Exp. J. tutrl eemnns f AT poes activity. protease MALT1 of determinants Structural Immunity κ ζ B signaling. B dpnet lcltc aha ocetae a metabolic a orchestrates pathway glycolytic -dependent (S poen eits euaoy cl development cell T regulatory mediates protein B(NS) J. Clin. Invest. Clin. J. euae T regulates B transcription factor c-Rel is required for Th17 effector Th17 for required is c-Rel factor transcription B

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© 2014 Nature America, Inc. All rights reserved. directed against I against directed shRNA Adenoviral mCherry. encoding gene the of frame reading open the and P2A encoding sequence a to codon stop without roquin-1 fused was encoding regnase-1 or gene the of frame reading open the vectors, expression regnase-1-P2A-mCherry and roquin-1–P2A–mCherry For Metabion. from generated by mutagenesis site-directed II; (QuikChange Agilent) with primers were thereof mutants Any vector. expression MSCV–IRES–Thy-1.1 the into CDSs their of each integrationg by generated were hMALT1 or Cre nase-1, reg roquin-1, for constructs Expression Technologies). (Life recombination Gateway by vector retroviral KMV-IRES-GFP the into transferred then was the CDE260 element) were ligated downstream. The reporter–3 acetate was from Chembridge. DNaseI was from Roche. Ac-DEVD-CHO and DNaseI was Ac-DEVD-CHO from was Roche. from acetate Chembridge. leupeptin, collagenase IV and brefeldin A were from Sigma-Aldrich. Mepazine cates amino acids that differ) were from Biotrend. Thioridazine hydrochloride, (ISQAVHAA (ISQAVHAA (wild-type 323–339) acids (amino peptide OVA and 61–80) acids (amino Lymphocytic peptide glycoprotein Signaling. virus Cell choriomeningitis from was (D3A7) STAT3 phosphorylated to Antibody Bioscience. BD from was (30-F11) Anti-CD45 from was Biologicals. Novus (EPR1394Y) II Class MHC to Antibody NeoMarkers. from were (SP7)) and (SP6) anti-Ki67 (anti-CD3 for immunohistochemistry Antibodies 12-myristate-13-actetate (PMA), ionomycin and MG132 were anti-I from Novartis. Calbiochem. Rabbit from was (ProleukinS) IL-2 human Recombinant Systems. R&D from was (JES6-5H4) mouse IL-4, Miltenyi Recombinant IL-6 Biotec. and human TGF- Anti-mIL-2 eBioscience. from streptavidin- were as well as phycoerythrin, (GK1.5), anti-CD4 (FJK16s), anti-Foxp3 (11B11), anti-IFN- (BM8), (RB6-8C5), anti-F4/80 anti-Gr-1 ISA-3), (clone anti-hICOS (HIS51), Thy-1.1 rat and Anti-I from BD Pharmingen. anti-ROR and IL-12, mouse recombinant (140706), CCR6 anti- (Ox7), Anti-Thy-1.1 Systems. R&D from was (604421) anti-regnase-1 Monoclonal Signaling. Cell from was (P173) Anti-IRF4 Biotech. Cruz Santa from were (B-5-1-2) anti-tubulin and (E10) anti-CYLD (B12), anti-MALT1 (B-6), Anti-c-Rel Laboratories. was from Bethyl (A300-514A) anti-roquin-1y with no signal in regnase-1-deficient cells ( MEFs protein in wild-type regnase-1 of by endogenous recognition evaluated ated by injection of rats with recombinant full-length regnase-1 protein and was house (V.H. and E.K.) in produced were and described been have (2.4G2) anti-CD16/32 (Xmg1.2) (145-2C11), anti-CD28 (37N), anti-IL-12 (C17.8), anti-IL-4 (11B11), anti-IFN- anti-CD3 and 3F12) clone (anti-roquin, roquin-2 and to roquin-1 antibodies reagents. and peptides antibodies, of Origin ACACAGATATTA-3 GAGATCAAGAGCAACA-3 5 sh1, or Nfkbid 3 the of sequences and Technologies) human the vectors. knockdown and overexpression of Generation and München University 55.2-1-54-2532-24-13). Az (RvOb, guidelines Ludwig- federal and state institutional, Technical München, München, Zentrum Helmholtz the Maximilians-University with barrier pathogen-free accordance a in in housed facility were animals All background. genetic Cd4- SMARTA mice consortium. Archive mice Mutant Mouse (SMARTATg( European the laboratory. Jackson from from were mice from Taconic Farms, and NOD.Cg- Mice. METHODS ONLINE nature immunology nature Biochemicals. Alexis or Sciences Life Enzo from were zVRPR-fmk Nfkbiz 4 Cre mice Cre 8 6 5B/ ad BALB/cJ- and C57BL/6 (K. Heger and M. Schmidt-Supprian, personal communication), communication), personal Schmidt-Supprian, M. and Heger (K. ′ , , (1-559), (1-559), -CAGCTGGATATTCGTGAACAT-3 Malt1 h, 5 sh2, ICOS R κ TcrLCMV 4 −/− AEINEAGR) and F9 (ISQAVHAA 7 N hs en described been has BNS and compound mutant mice were maintained on a C57BL/6 C57BL/6 a on maintained were mice mutant compound and Nfkbiz mice CDS was cloned into the pCR8/GW/TOPO vector (Life (Life vector pCR8/GW/TOPO the into cloned was CDS κ ′ -GCGTCAATGTACCAGTATTCG-3 B ζ 8 ′ ( ) was from Sirion Biotech. Sirion from was ) . . Monoclonal antibody to regnase-1 (15D11) was gener (1-1333), (1-1333), 14 Nfkbiz )Aox) mice , , Rc3h1 γ κ ′ ) or regnase-1 ) ( or regnase-1 (XMG1.2), anti-IL-17A (eBio17B7), anti-IL-4 anti-IL-4 (eBio17B7), anti-IL-17A (XMG1.2), sh1, 5 sh1, B Tg(DO11.10) ζ fl/fl (LK2NAP), monoclonal antibody to mouse antibody monoclonal (LK2NAP), Il6 Prkdc 4 mice 5 (1-403), (1-403), , , and OT-II (OT-IITg( ′ ′ -CAGCACCTCATTGTGCAAGAT-3 UTRs of of UTRs scid 2 3 Rc3h1 Supplementary Fig. 4h 5 6 , ,

At-AD (C) phorbol- (6C5), Anti-GAPDH . Il2rg Rc3h2 10Dlo Zc3h12a Irf4 ′ , or or , Hybridomas and monoclonal monoclonal and Hybridomas san tm1Wjl cRel F (1-3260) and and (1-3260) AEINEAGR); bolding indi fl/fl mice (EM:02168) were were (EM:02168) mice Tg(CAR ; Nfkbid (1-537), (1-537), mice /SzJ as well as MRL sh1, 5 sh1, As a reporter-gene, As a reporter-gene, ′ H , I ), γ h, 5 sh2, TcraTcrb 8 AEINEAGR), R9 R9 AEINEAGR), t (Q31-378) were were (Q31-378) t ′ , , -AGCGAGGCC ∆ CAG-CAR Ctla4 ′ κ UTR sequence -1) N ( BNS Tnf ). Polyclonal β mice were were mice were from ′ (490-749; (490-749; (1-1090), (1-1090), -GCCAG )425Cbn) )425Cbn) Nfkbid stop-fl lpr/lpr ′ γ - - - ,

cells per bronchial epithelial cell area) with a SCN400 slide scanner analysis analysis scanner slide SCN400 a with area) cell (positive epithelial bronchial identity per cells sample to ‘blinded’ researchers by analyzed and selected stained for alcian acid blue–periodic Schiff, four different areas were randomly with a slide SCN400 scanner.Leica For of quantification goblet cells positively the total tissue area. Images were obtained by scanning of whole tissue to sections normalized were results and area) total per stained (area densitometry by or cells) total per cells (positive numerically for II MHC were class quantified stained positively areas Tissue automatically. counted were sections intestine Ki67 CD45 area. tissue to normalized were results and sections tissue embedded paraffin- whole on (Leica) software analysis image TissueIA SlidePath with Detection Kit (Ventana). Cells positive for the various markers were quantified DAB IVIEW an NEXES with a Instruments) (Ventana on robot staining immuno-histochemistry by followed buffer, Ventana in incubated was tissue and cells goblet lung for paraffin-embedded for arteries, Gieson staining pulmonary elastica–van staining Schiff acid periodic blue– alcian For scanner. slide SCN400 a Leica with acquired were DAB.for Images solution detection BOND-MAX a on refine polymer BOND with performed Biosystems) robot (Leica immunohistochemistry was staining The (Leica). diluent antibody various above) and primary (identified antibodies in secondary Bond primary or eosin and hematoxylin with stained were tissues The staining. for cut and paraffin in embedded were legs the Then motion. slow in shaker horizontal a on 7.4) EDTA(pH 12.5% in decalcification of weeks 2 by followed d, 1 for paraformaldehyde 4% in fixed were legs hind preparation, For joint sections. as 2- cut were tissues skin intestine, small stomach, lung, embedded evaluation. Histopathological and IL-2 (20 U/ml); T U/ml); (20 IL-2 and (10 T above): identified antibodies (all bodies above) in (identified culture conditions with and the following cytokines anti (1 anti-CD3 with precoated plates 96-well Novartis). ProleukinS, For naive differentiation, CD4 of blasts were by expanded the addition of recombinant human IL-2 (20 U/ml, pre-coated with goat anti-hamster IgG (0855397, MP Biochemicals). Populations(0.25 (20 dazine Ac-DEVD-CHO (50 (1 mycin and iono nM) (20 PMA with stimulated were Dynabeads via selected T cells as CD4 of naive or T by tions (Invitrogen) population were cell cytometry sorted flow by according with to Dynabeads positive selection the manufacturer’s instruc for CD4 enriched were samples If required, analysis. for further used were suspensions IV. Single-cell collagenase mg/ml 0.4 containing medium RPMI in °C 37 at h 1 for digested were tissues remaining being the PBS, in After washed DTT. without medium same the in °C 20 at min 40 for shaken were EDTA, mM then 1.35 and DTT mM 1 FCS, 1% with supplemented PBS in °C 20 at min 20 for shaken were fragments The pieces. small into cut PBS and ice-cold in washed longitudinally, opened were segments intestine the and removed, carefully were Peyer’s patches intestine, the from cells propria 70- through sieved was solution in a strainer. cell The or were sue homogenized lymph or spleens nodes intact 20 IV (Sigma), collagenase small pieces, and digested for 1 h at 37 °C in RPMI medium containing 4 mg/ml sions, lungs with were 10 ml perfused PBS through the right ventricle, cut into as described and grown prepared were lines cell Rc3h1 obtained from American Type stimulation. Culture and Collection Invitrogen, respectively. and culture isolation, Cell (Olympus). ‘cellSens’ software measured on an Olympus BX53 Microscope and a and DP72 camera together with obtained were staining Gieson elastica–van of Images (Leica). software toethanol (Sigma) and 100 U/ml penicillin-streptomycin (Invitrogen). CD4 (Invitrogen). penicillin-streptomycin U/ml 100 and (Sigma) toethanol mM 2 (Lonza), mixture vitamin mM sodium-pyruvate (Lonza), 10 mM HEPES pH 7.4, 1× eagle MEM essential medium supplemented with 10% (vol/vol) FBS, 1 × nonessential amino acids, 1 µ + g/ml); T g/ml); µ , , p-STAT3 −/− g/ml) and anti-CD28 (2.5 + CD62L Rc3h2 µ M) with or without preincubation for 3 h with MG132 (25 (25 MG132 with h 3 for preincubation without or with M) µ M). Alternatively, CD4 Alternatively, M). H 2, 2, IL-4 (20 anti-IL-12 (10 ng/ml), −/− + + CD44 or CD3 , , Rc3h1 µ M), M), z-VRPR-fmk (75 − H on a FACSAria III (BD). T cells were cultured in RPMI 17, TGF- 17, fl/fl + cells on paraffin-embedded lung, stomach, skin or skin stomach, lung, on paraffin-embedded cells µ Rc3h2 g/ml DNase g/ml lung I tis and 10% Predigested FBS. µ Paraformaldehyde (4%)-fixed and paraffin- and (4%)-fixed Paraformaldehyde µ g/ml) (both identified above) on six-well plates l m fine mesh. For the preparation of lamina lamina of preparation For the mesh. fine m fl/fl -glutamine (Invitrogen), 50 50 (Invitrogen), -glutamine β (10 (10 + Cre-ERT2 T cells were stimulated with anti-CD3 anti-CD3 with stimulated were cells T µ g/ml), IL-6 (60 ng/ml), anti-IL-12 anti-IL-12 ng/ml), (60 IL-6 g/ml), H µ HEK293T and 293A cells were were cells 293A and HEK293T µ 1, IL-12 (10 ng/ml) and anti-IL-4 anti-IL-4 and ng/ml) (10 IL-12 1, g/ml) and anti-CD28 (10 (10 anti-CD28 and g/ml) M), M), mepazine (20 , , Malt1 8 µ , 13 g/ml), anti-IFN- g/ml), , 4 8 + −/− . For single-cell suspen . For single-cell T cells were cultured in and doi: Zc3h12a 10.1038/ni.3008 µ µ M M M) M) or thiori γ β (5 -mercap µ −/− + m thick m thick µ µ T cells T cells g/ml) g/ml) g/ml) g/ml) MEF µ M), M), + + ------,

© 2014 Nature America, Inc. All rights reserved. then collected for immunoblot analysis. Lung or pancreas protein extracts extracts protein pancreas or Lung analysis. immunoblot for collected then were thawed again and (10 min, centrifuged 10,000 then frozen, shock were EDTA), without mix inhibitor protease and EDTA MgCl mM 1.5 Nonidet-P40, (vol/vol) 0.25% NaCl, mM 150 7.5, pH Cg- to standard according protocols. Lung anddetected pancreas were tissue lysates wereproteins prepared and from NOD.lane per loaded were protein total 50–70 analysis, immunoblot For assay. Protein BioRad a by measured 10,000 min, (10 centrifugation by was cleared lysate mixture, EDTAAfter and without 1 mM DTT). (Roche) NaCl, 0.25% (vol/vol) Nonidet-P40, 1.5 mM MgCl mM 150 7.5, pH Tris-HCl, mM (20 buffer lysis with ice on min 15 for bated Protein by detection immunoblot analysis. as performed was transduction cell T or described MEF and adenovirus transduction. or cell virus and production Virus product. cleavage main the to addition in bands migrating faster produces also assay cleavage to the roquin-1, Due truncated represent that DTT). of polypeptides mM copurification 10 and citrate sodium 1M CHAPS, (wt/vol) 0.1% saccha rose, (wt/vol) 10% NaCl, mM 150 7.0, pH MES, mM (50 buffer cleavage of (5 ence or the absence MALT1z-VRPR-fmk inhibitor peptide GST-MALT1 at 30 °C human with (5 full-length recombinant described as system chromatography liquid Äkta an via lysate bacterial from purified 50 with °C 18 at overnight induction after bacteria RILP Codonplus BL21 in produced was GST-MALT1 1-thioglycerol. (vol/vol) 0.01% and NaCl mM 300 8.0, pH HCl, Tris-mM 50 was buffer purification The Healthcare). StrepTrap(GE column a and Healthcare) (GE column HiTrap chelating a on steps chromatography affinity- consecutive two by purified was protein The (DE3). Rosetta2 strain the in II amino-terminal Strep-tag an carboxy-terminal a containing and tag construct six-histidine a from expressed was roquin-1 MALT1 and purification Protein CD4 OT-II or SMARTA purified 10:1. of at ratio a cells T with plated being before experiments differentiation for washed low not were or to peptide excess (high of removal for F9 ments or R9 experi stimulation h) (18 wild-type, short-term for twice washed were APCs affinity)). 323–339; acids (amino peptide 0.1–10 OVA or 61–80) acids (amino glycoprotein virus choriomeningitis for pulsed 1 and h rad) (2000 subsequently at 37 °C with 5 were prepared from and spleen lymph mice, irradiated of node cells wild-type 50 serum, pooled human (vol/vol) 10% with supplemented (Invitrogen) Aim-V the manufacturer’s (Miltenyi Biotec) instructions and were cultured in further CD4 Naive a with sorting cell activated (Pan human Biotech, Pancoll density 1,077on g/l). Human naive centrifugation CD4 density by obtained were (PBMCs) cells mononuclear blood Peripheral of cytokines. staining before intracellular (1 ionomycin and cells were washed twice with PBS and were incubated for 4 h with PMA (20 nM) cells were cultured for 3 d and T (2.5 (10 doi: µ 10.1038/ni.3008 Prkdc µ g/ml gentamycin, 2 mM L-glutamine and 10 mM HEPES, pH 7.4. APCs APCs 7.4. pH HEPES, mM 10 and L-glutamine mM 2 gentamycin, g/ml µ g/ml); and T g/ml); g/ml), anti-IL-4 (10 (10 anti-IL-4 g/ml), scid 6 2 , 8 8 . Recombinant full-length roquin-1 (5 (5 roquin-1 full-length Recombinant . ,

4 Il2rg 9 . µ M isopropyl isopropyl M tm1Wjl reg µ , , TGF- M) in the presence of brefeldin A (10 (10 A brefeldin of presence the in M) /SzJ mice. Organs were lysed on Organs were mice. /SzJ lysed ice (20 mM Tris-HCl, β (10 µ β /l, ni IFN- anti- g/ml), H g - d , 4 °C). The protein content in the lysate was was lysate the in content protein The °C). 4 , 2 cells were cultured for 5 d. For restimulation, µ -thiogalactopyranoside. The proteins were were proteins The -thiogalactopyranoside. g/ml), IL-2 (100 U/ml). T IL-2 (100 U/ml). g/ml), in vitro in + T Cell Isolation Kit II according to to according II Kit Isolation Cell T CD4 + cleavage reaction. cleavage T cells were sorted by magnetic- elcto-eiin retro Replication-deficient γ + 2 (5 (5 T cells or MEFs were incu and protease inhibitor mix g µ , , 4 °C). Supernatants were g) was incubated for 1 h 1 for incubated was g) µ g/ml) and anti-IL-2 anti-IL-2 and g/ml) µ g/ml lymphocytic lymphocytic g/ml H 1, 1, T Escherichia coli Escherichia µ µ g) g) in the pres g/ml) for 2 h 2 for g/ml) µ H Full-length Full-length M) M) in 50 17 17 and T 2 , 1 mM mM 1 , in in vitro µ µ g/ml g/ml g of of g reg µ + - - - - - l

with 2% FBS and 2% 2 FBS with mM EDTA)for at 20 min 4 and anti were stained °C with (PBS buffer for in staining 10 min at incubated 4 (CD16/32) °C Fc-block with staining. and intracellular Flow cytometry curve. standard the to according nm with a plate and were Sun-rise (TECAN) reader concentrations calculated 450 at measured was Absorbance (eBioscience). instructions manufacturer’s IFN- of IgG antibody. mouse (A90-131P, For as was used detection secondary Bethyl) samples were diluted 1:10, and horseradish peroxidase–conjugated antibody to °C 4 at 10 overnight with coated were plates ELISA factor, For rheumatoid of Labs). analysis (Bethyl ELISA by detected were DNA double-stranded for cific spe IgG of titers serum, mouse with incubation After cells. stem embryonic were plates ELISA poly- with coated maxisorp NUNC DNA, double-stranded to antibodies described as ELISA by determined were concentrations h 3 for ice on incubated for at 10 min 16,000 then and centrifuged heart, the from drawn was blood and killed were mice ELISA, and analysis immunoblot for serum mouse of preparation and autoantibodies. and of ELISA preparation Mouse cytokines serum detection. by ECL followed Signaling), Cell IgG (7076, mouse to antibody peroxidase–conjugated horseradish with incubated subsequently BSA 5% were in and 1:200 diluted serum mouse with °C 4 at overnight probed were (25 50. 49. 48. 47. 46. 45. test. Tukey’swith multiple-comparisons P Statistical analysis. (TreeStar). software FlowJo with analyzed were samples and above). factors were Cells (identified acquired on an LSR transcription Fortessa (BD) device to antibodies with stained were finally and eBioscience) from kit to the Foxp3 staining (according buffer 20 min at 4 °C in permeabilization for incubated were buffer, then fixation/permeabilization with 1 h at for 4 °C fied above). For staining of factors, unstimulated transcription cells were fixed for were cells stained then 50 min at (identi 4 to °C cytokines with antibodies PBS containing 0.5% saponin (VWR international) and 1% BSA (ROTH). The 4% paraformaldehyde (MERCK) and for were 20 permeabilized min at 4 °C in staining (antibodies identified above). Cells were fixed for 10 min at 20 °C with cell staining (Invitrogen) was performed for 30 min at 4 °C, followed by surface dead fixable Then added. was Sigma) ng/ml; A (10 brefeldin 2 h, final the for for 4 T were stimulated h cells PMA (20 nM) and with (1 ionomycin CD4 isolated staining, cytokine intracellular For above). (identified bodies -values were calculated by Student’s by calculated were -values

Lech, M. study to cells T CD4 naive Heissmeyer,of V.& transduction S.C. Warth,Adenoviral K.M. Ansel, Lee, P.P. expression TCR F.R.Defective Carbone, & W.R. Heath, J., Allison, M.J., Barnden, Virus-specific H. Hengartner, & R.M. Zinkernagel, M.F., Bachmann, A., Oxenius, effects of lupus autoantigens. lupus of effects differentiation. treg processing. survival. and function, elements. (1998). regulatory heterologous of control cDNA-based using constructed mice transgenic in cellular and humoral on infection. viral effects after responses immune mice: TCR-transgenic II-restricted MHC-class µ g per lane )were separated by SDS-PAGE (10% polyacrylamide). Blots Blots polyacrylamide). (10% SDS-PAGE by separated )were lane per g γ µ , IL-17A, IL-6, IL-10 and TNF, ELISA kits were used following the the following used were kits TNF,ELISA and IL-10 IL-6, IL-17A, , g/ml rabbit IgG (011-000-003, Jackson Immunoresearch). Serum Serum Immunoresearch). Jackson (011-000-003, IgG rabbit g/ml et al. et al. Nat. Struct. Mol. Biol. Mol. Struct. Nat. et al. et Tir8/Sigirr prevents murine lupus by suppressing the immunostimulatory A critical role for Dnmt1 and DNA methylation in T cell development, l -lysine (Trevigen) and double-stranded DNA from mouse DNAmouse from double-stranded and (Trevigen) -lysine Mouse Eri1 interacts with the ribosome and catalyzes 5.8S rRNA 5.8S catalyzes and ribosome the with interacts Eri1 Mouse GraphPad Prism 5.0d was used for statistical analyses, and J. Vis. Exp. Vis. J. Immunity J. Exp. Med. Exp. J.

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15 , 763–774 (2001). 763–774 , g , 523–530 (2008). 523–530 , to yield the supernatant. Autoantibody Autoantibody supernatant. the to yield t Eur. J. Immunol. J. Eur. -test or one-way analysis of variance variance of analysis one-way or -test

205 Single-cell suspensions were Single-cell pre muo. el Biol. Cell Immunol. , 1879–1888 (2008). 1879–1888 , α 5 - and - nature immunology nature (13 August 2013). August (13

28 β -chain genes under the under genes -chain 5 , 390–400 (1998). 390–400 , 0 . For detection of of detection For .

76 µ 34–40 , M) M) and For + - - - -