Wnt/Β-Catenin Signaling Induces Integrin Α4β1 in T Cells And

Wnt/β-Catenin Signaling Induces Integrin α4 β1 in T Cells and Promotes a Progressive Neuroinflammatory Disease in Mice

This information is current as Daniele Sorcini, Stefano Bruscoli, Tiziana Frammartino, of September 27, 2021. Monica Cimino, Emanuela Mazzon, Maria Galuppo, Placido Bramanti, Mumna Al-Banchaabouchi, Dominika Farley, Olga Ermakova, Olga Britanova, Mark Izraelson, Dmitry Chudakov, Michele Biagioli, Paolo Sportoletti, Sara Flamini, Marcello Raspa, Ferdinando Scavizzi, Claus Nerlov, Graziella Migliorati, Carlo Riccardi and Oxana Downloaded from Bereshchenko J Immunol 2017; 199:3031-3041; Prepublished online 22 September 2017; doi: 10.4049/jimmunol.1700247 http://www.jimmunol.org/content/199/9/3031 http://www.jimmunol.org/

Supplementary http://www.jimmunol.org/content/suppl/2017/09/22/jimmunol.170024 Material 7.DCSupplemental References This article cites 51 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/199/9/3031.full#ref-list-1 by guest on September 27, 2021

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Wnt/b-Catenin Signaling Induces Integrin a4b1 in T Cells and Promotes a Progressive Neuroinﬂammatory Disease in Mice

Daniele Sorcini,*,1 Stefano Bruscoli,*,1 Tiziana Frammartino,* Monica Cimino,* Emanuela Mazzon,† Maria Galuppo,† Placido Bramanti,† Mumna Al-Banchaabouchi,‡ Dominika Farley,‡ Olga Ermakova,‡,x Olga Britanova,{,‖,# Mark Izraelson,{,‖,# Dmitry Chudakov,{,‖,# Michele Biagioli,* Paolo Sportoletti,* Sara Flamini,* Marcello Raspa,x Ferdinando Scavizzi,x Claus Nerlov,‡ Graziella Migliorati,* Carlo Riccardi,*,1 and Oxana Bereshchenko*,‡,1 Downloaded from The mechanisms leading to autoimmune and inflammatory diseases in the CNS have not been elucidated. The environmental triggers of the aberrant presence of CD4+ T cells in the CNS are not known. In this article, we report that abnormal b-catenin expression in T cells drives a fatal neuroinflammatory disease in mice that is characterized by CNS infiltration of T cells, glial activation, and progressive loss of motor function. We show that enhanced b-catenin expression in T cells leads to aberrant and Th1-biased T cell activation, enhanced expression of integrin a4b1, and infiltration of activated T cells into the spinal cord, without affecting regulatory T cell function. Importantly, expression of b-catenin in mature naive T cells was sufficient to drive http://www.jimmunol.org/ integrin a4b1 expression and CNS migration, whereas pharmacologic inhibition of integrin a4b1 reduced the abnormal T cell presence in the CNS of b-catenin–expressing mice. Together, these results implicate deregulation of the Wnt/b-catenin pathway in CNS inflammation and suggest novel therapeutic strategies for neuroinflammatory disorders. The Journal of Immunology, 2017, 199: 3031–3041.

n increased presence of activated T cells in organs is T (Treg) cell subsets define the duration of immune responses and associated with the development of autoimmune and the outcome of many autoimmune and inflammatory diseases (1, inflammatory diseases (1–4). Balanced activation and 3, 5). Although there is strong evidence for the involvement of A by guest on September 27, 2021 differentiation of T lymphocytes into specific Th and regulatory CD4+ T cells in the pathogenesis of several neuroinflammatory diseases, their causative role in disease initiation has not been demonstrated. The environmental triggers and molecular deter- *Department of Medicine, University of Perugia, Perugia 06132, Italy; minants of T cell–mediated diseases of the CNS have not been † Istituto di Ricovero e Cura a Carattere Scientifico, Centro Neurolesi “Bonino fully characterized and require further exploration. Pulejo,” Messina 98124, Italy; ‡Mouse Biology Unit, European Molecular Biology Laboratory, Monterotondo 00015, Italy; xConsiglio Nazionale delle Ricerche, Istituto The deregulation of the Wnt/b-catenin pathway was recently di Biologia Cellulare e Neurobiologia, Monterotondo 00015, Italy; {Shemyakin- detected in several neurodegenerative diseases, including Alz- Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science, 117997 Moscow, Russia; ‖Central European Institute of Technology, Masaryk Uni- heimer’s disease (6), Parkinson’s disease (7), and multiple scle- versity, Brno 625 00, Czech Republic; and #Pirogov Russian National Research rosis (8–10). Elevated expression of Wnt3a and b-catenin was Medical University, 117997 Moscow, Russia detected in spinal cords during experimental autoimmune en- 1D.S., S.B., C.R., and O. Bereshchenko contributed equally to this work. cephalomyelitis (EAE) (11). Activation of the Wnt signaling ORCIDs: 0000-0002-5073-717X (E.M.); 0000-0001-9173-324X (D.F.); 0000-0001-8365- pathway was detected in T cells in relapsing uveitis in rats (12), 4904 (O.E.); 0000-0003-0430-790X (D.C.); 0000-0002-2995-6896 (M.B.); 0000-0002- 5630-9862 (P.S.); 0000-0002-6994-8428 (S.F.); 0000-0002-3437-2430 (M.R.); 0000- and it underlies the pathogenesis of neuropathic pain in rodents 0001-9257-3997 (C.R.); 0000-0002-2772-6042 (O. Bereshchenko). (13). The expression of Wnt signaling components in immune-like Received for publication February 21, 2017. Accepted for publication August 22, cells of the CNS, including macrophages/microglia and astrocytes, 2017. as well as in oligodendrocytes, suggests that they contribute to This work was partly supported by the Italian Ministry of Education and Research inflammation-driven brain repair or damage (14, 15). However, (Progetti di Ricerca di Rilevante Interesse Nazionale, Bando 2015 2015ZT9HXY to C.R. and 20153NBRS3 to S.B., and Programma Futuro in Ricerca 2013 RBFR13BN6Y whether specific activation of the Wnt/b-catenin pathway in to O. Bereshchenko) and by Russian Science Foundation Project 16-15-00149. T cells triggers the pathogenesis of autoimmune and inflammatory Address correspondence and reprint requests to Dr. Oxana Bereshchenko, University diseases in the CNS has not been investigated. of Perugia, Department of Medicine, Section of Pharmacology, Severi Square, 1, S. b-Catenin is a key component of the Wnt signaling pathway, Andrea delle Fratte, 06132 Perugia, Italy. E-mail address: oxana.bereshchenko@ unipg.it and its levels are tightly controlled via Gsk3b-mediated phos- The online version of this article contains supplemental material. phorylation and ubiquitin-mediated degradation (16, 17). Phos- phorylated b-catenin, but not the nonphosphorylated form, is Abbreviations used in this article: BM, bone marrow; ChIP, chromatin immunoprecipitation; DN, double-negative; DP, double-positive; EAE, experimental autoim- rapidly degraded by the ubiquitin–proteasome pathway. Activation mune encephalomyelitis; qPCR, quantitative real-time PCR; SP, single-positive; of the Wnt pathway, as well as the cancer-associated point mu- Tconv, conventional T; Treg, regulatory T; WT, wild-type. tations in the Ctnnb1 gene, prevents phosphorylation-dependent Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 degradation of b-catenin and triggers the accumulation of b-catenin www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700247 3032 Wnt/b-CATENIN IN T CELLS AND NEUROINFLAMMATION in the nucleus, where, in complex with its DNA-binding and forelimbs and combined forelimb and hindlimb grip strength in rodents, chromatin-modifying partners, it activates the transcription of including mice (36). In this study, the forelimb and hindlimb strength of the target genes (17, 18). In addition to its documented role in T cell mice was measured by gently lowering the mice over the top of the grid of the apparatus so that, in the first instance, only their forelimbs touched the development (19–25), a role for Wnt/b-catenin in T effector and grid, and in the second instance, their front and hind paws could grip the regulatory cell function is emerging. It was shown to inhibit grid. The torso was kept parallel to the grid, and the mouse was gently T cell activation (26), promote Th2 and Th17 differentiation (27, pulled back steadily until the grip was released down the complete length 28), and either promote (29, 30) or inhibit (28, 31) the function of the grid. The maximal grip strength value of the mice was displayed on the screen. The average of three trials in each condition was taken into of Treg cells. However, the causative role of Wnt/b-catenin in consideration. initiating autoimmune and inflammatory diseases of the CNS has not been demonstrated. Footprint test In this article, we demonstrate that stable b-catenin over- We assessed motor dysfunction using the footprint test, as described pre- expression in mice results in aberrant T cell activation, Th1 viously (37). Mice were encouraged to walk down a narrow corridor (40 skewing, expression of integrin a4b1, and infiltration of Th1 cells cm long) on a floor covered with absorbent white paper. Mice were mo- into the CNS, leading to the development of progressive lethal tivated to walk the course by placing a small box with an opening at the other end of the corridor. The hind paws of the mice were colored with neurologic disease associated with T cell infiltration in the CNS nontoxic ink. and neuroinflammation. Our results implicate T cell–specific deregulation of the Wnt/b-catenin pathway in the initiation of CNS Disease scoring inflammation. To score the clinical signs of disease, we used a previously described

grading system for clinical assessment of EAE (38). Briefly, each mouse Downloaded from Materials and Methods was monitored and assigned a score ranging from 0 to 5 defined by the following clinical criteria: 0, normal mouse, no overt signs of disease; 1, Mice limp tail or hind limb weakness (clasping) but not both; 2, limp tail and Mice expressing bcatS33Y in T cells from two R26 alleles were generated hind limb weakness (clasping); 3, partial hind limb paralysis; 4, complete as described previously (32, 33). CD4-Cre–transgenic and Rag12/2 mice hind limb paralysis; and 5, moribund state, death by disease or sacrifice for were purchased from The Jackson Laboratory. Foxp3YFP-Cre mice were humane reasons. provided by A. Rudensky (Memorial Sloan-Kettering Cancer Center, New Competitive bone marrow chimeras http://www.jimmunol.org/ York, NY) (34). All procedures were approved by the Ethical Committees of the European Molecular Biology Laboratory and the University of Generation of radiation chimeras was performed as described (39). In brief, Perugia. lethally irradiated (two doses of 600 rad with a 3-h pause; RAD GIL EN 60601-1 GILARDONI) CD45 1/2+ syngeneic (“recipient”) mice were in- Abs and flow cytometry jected with a 1:1 mixture of bone marrow (BM) cells isolated from wild- + + Staining with surface or intracellular Abs and Annexin V were done as type (WT) or CD4bcat/+ (CD45.2 , “donor”) and WT (CD45.1 , “helper”) 3 6 described previously (1, 35). The following Abs (purchased from eBio- mice. BM cells (2 10 per mouse) were injected into the tail vein. In- science or Tonbo Bioscience) were used: anti-CD4 (GK1.5), anti-CD8 jected hosts were treated with Baytril (enrofloxacin; Bayer) in drinking (2.43), anti-CD44 (IM7), anti-CD25 (53-7.3), anti-Foxp3 (FJK-16s), water for the entire observation period (5 wk). anti–IFN-g (XMG 1.2), anti–IL-17 (eBio17B7), anti–IL-4 (BVD6-24G2), by guest on September 27, 2021 anti-CD62L (MEL-14), anti-CD45 (30F-11), anti–CTLA-4 (UC10-4F10- CNS mononuclear cell isolation 11), anti-GITR (DTA-1), anti-CD127 (A7R34), anti-CD45 (clone 30F-11), Mice were sacrificed with CO2, and spinal cords were collected by flushing anti-CD45.1 (A20), anti-CD45.2 (104), anti-CD11b (M1/70), anti-B220 of the spinal column with sterile balanced-salt saline solution (PBS). We (RA3-6B2), anti-CD25 (PC61.5), anti-CD69 (H1.2F3), anti-CD103 (2E7), dissociated spinal cords into single cells, and filtered them through a 70-mm anti-CD122 (5H4), anti-CD127 (clone A7R34), anti–CTLA-4 (clone UC10- strainer (BD Biosciences) in RPMI 1640 containing 10% heat-inactivated 4F10-11), anti-GITR (clone DTA-1), anti–Ki-67 (clone B56), anti–integrin FBS and 10 mM HEPES. Cell suspensions were centrifuged on a 30/70% a4 (R1-2), anti–integrin b1 (EBIOHMB1-1), and anti–integrin a4b7 isotonic Percoll gradient (Sigma) for 20 min without braking. Leukocytes (DATK32). Staining for Foxp3 was performed using the Foxp3/ were isolated from the gradient interface and resuspended in PBS with 2% Transcription Factor Staining Buffer Set (eBioscience/Tonbo Bioscience). FBS. Analysis and cell sorting were done using a three-laser standard con- figuration FACSAria II (BD Biosciences) and an Attune NxT Acoustic Immunohistochemistry Focusing Cytometer (equipped with red, blue and violet lasers; Invi- trogen). Analysis of flow cytometry data was performed with FlowJo Paraffin-embedded tissues were cut into 7-mm-thick sections and treated software (Tree Star). as described previously (40). All images were obtained using light mi- croscopy (Leica DM2000 combined with a Leica ICC50 HD camera) and Behavioral tests studied via an imaging computer program (Leica Application Suite V4.1). We analyzed the behavior of male and female mice between 12 and 18 mo of age. Animals were housed with three to five mice per cage. Food and water Quantitative real-time PCR were provided ad libitum. The light in the animal room was controlled on a 12-h light/12-h dark cycle, and the ambient temperature was 22 6 1˚C. On RNA of cells was isolated using an RNeasy Plus Micro Kit (QIAGEN), and experiment days, mice were transferred to the test room to habituate for cDNA synthesis was performed with a High-Capacity cDNA Reverse Tran- $30 min. scription Kit (Applied Biosystems), as described (41). All real-time PCR Mice were analyzed using the SHIRPA primary screen test series de- reactions were performed using the 7300 Real-Time PCR System (Applied veloped for rapid phenotype testing in mutation screens. Motor defects Biosystems). Amplifications were done using TaqMan Gene Expression Master were then tested to assess motor function and strength in CD4bcat/+ mice Mix and qPCR TaqMan probes (Applied Biosystems): Tnfa (Mm00443258_m1), and littermate controls. To check for simple cognitive changes in those Il1b (Mm00434228_m1), Itga4 (Mm 01277951_M1), and Itgb1 (Mm01253230). mice, an additional Y-maze test was performed. Western blotting Limb clasping Western blotting studies were performed as described previously (42) using Limb clasping was scored quantitatively based on the direct observation of anti–b-catenin (D10AB; Cell Signaling Technology), b-actin (AC-40; its severity. Each mouse was lifted by its tail and scored for extending its Sigma), and Laminin B1 (Ab16048; Abcam) Abs. hind paws wide open or closing/joining them toward its abdomen. Chromatin immunoprecipitation assay Grip strength Chromatin immunoprecipitation (ChIP) assays were performed as described Grip strength performance is an indicator of neuromuscular function. It is previously (1). In brief, 1 3 106 purified CD4+CD252 T cells were used measured using an automated apparatus that assesses muscle strength of for immunoprecipitation for each Ab. Cells were fixed in 1% parafor- The Journal of Immunology 3033 maldehyde and sonicated on ice. Precleared lysates were incubated overnight exhibited a block at the DP stage (Supplemental Fig. 1B) reported at 4˚C with polyclonal anti–b-catenin (clone D10A8; Cell Signaling Tech- in CD4bcat/bcat mice and an alternative model of b-catenin nology), anti–acetyl histone H3 (Millipore), or control rabbit IgG (Cell Sig- stabilization in T cells (33, 44). CD4bcat/+ mice showed accu- naling Technology). Primers were designed to amplify the putative Lef1/Tcf binding site in the promoter of the Itgb1 gene (position 21102/21085), the mulation of cells at the double-negative (DN) stage (Supplemental Inta4 gene (position 21003/2986), the Tbx21 gene (position 223/26), the Fig. 1B, 1C) similar to that observed in the published model of Rorc gene, and the Ifng gene identified using Matinspector software. Quan- biallelic b-catenin overexpression in T cells (figure 1D in Ref. 33). titative real-time PCR (qPCR) analysis was performed using Power SYBR This was likely a result of increased DN cell proliferation in Green PCR Master Mix (Applied Biosystems). The quantitative real-time PCR primers are available upon request. The relative presence of specific CD4bcat/+ mice compared with littermate control mice that did indicated factors at specific DNA regions is calculated using the percentage of not overexpress b-catenin (hereafter called WT mice), as revealed input method and relative fold induction compared with IgG levels. by Ki-67 staining (Supplemental Fig. 1D). In addition, b-catenin– Nucleofection experiments overexpressing cells exhibited decreased apoptosis at the DN1–3 stages, as revealed by Annexin V staining (Supplemental Fig. 1E); Transfection of native T cells was performed using an Amaxa Mouse T Cell however, apoptosis increased at the DN4 stage (Supplemental Fig. Nucleofector Kit (Lonza), according to the manufacturer’s instructions. In + 2 1E), suggesting selective death at the b-selection checkpoint in brief, CD4 C25 T cells were purified from the spleens of WT mice (8-wk- old C57BL/6 males) by magnetic sorting. Multiple aliquots of 2 3 106 cells CD4bcat/+ mice. Consistently, DP T cells, as well as the subse- were electroporated using a 4D-Nucleofector X Unit (Lonza) with 4 mgof quently formed single-positive (SP) T cells, significantly decreased pcDNA3–bcatS33Y or pcDNA3 control vector resuspended in fully supple- in CD4bcat/+ animals compared with WT mice (Supplemental Fig. mented T cell culture medium (Lonza). A total of 0.6 3 106 T cells was 2 2 1F–H). Importantly, the DP/SP ratio was preserved in CD4bcat/+ transferred into Rag1 / mice, which were sacrificed 15 d later. pcDNA3– bcatS33Y was a gift from E. Fearon (plasmid number 19286; Addgene) (43). mice (Supplemental Fig. 1I), and none of the CD4bcat/+ mice Downloaded from showed the leukemia predisposition observed in two alternative a b Treatment with integrin 4 1 inhibitor in vivo mouse models of b-catenin stabilization (data not shown), suggesting Bio5192 sterile powder (Tocris) was reconstituted at 20 mg/ml in DMSO that the monoallelic dose of stable b-catenin overexpression does and then used as a 200 mg/ml solution in PBS. Animals received 1.5 mg/kg not stall the DP to SP transition, allowing us to analyze the impact of Bio5192 by i.v. injection every 2 d for a total of 1 wk. b-catenin overexpression on mature T cells.

Statistical analysis Mice with monoallelic b-catenin overexpression did not de- http://www.jimmunol.org/ velop leukemia, but they showed a strikingly different phenotype. Statistical analysis was performed with Prism 6.0 (GraphPad). The two- Starting from 3–4 mo of age, CD4bcat/+ mice exhibited decreased tailed unpaired Student t test or nonparametric Mann–Whitney U test was used for statistical comparisons. For multiple comparisons, two-way muscle strength (Fig. 1B), loss of tail flaccidity, altered walking ANOVAwas performed. Differences in mice survival were evaluated using pattern (data not shown), and hind limb clasping (Fig. 1A), which Kaplan–Meier analysis. progressed to complete limb paralysis over time (Fig. 1A). Accelerated lethality observed in CD4bcat/+ mice (Fig. 1D) was Results almost invariably preceded by severe motor function impairment b-Catenin overexpression in T cells leads to a progressive and significant body weight loss (Fig. 1C). These data demonstrate lethal neurologic disorder in mice that T cell–specific overexpression of b-catenin results in pro- by guest on September 27, 2021 Because contrasting results have been reported for b-catenin’s role in gressive fatal disease with motor function impairment. T cell function, we reanalyzed the effect of Wnt/b-catenin signaling b in T cells using a previously described mouse model that expresses a -Catenin overexpression leads to partial T cell activation degradation-resistant mutant form of human b-catenin in which the without affecting Treg development and function serine residue at position 33 was substituted for tyrosine (bcatS33Y) CD4bcat/+ mice revealed spontaneous T cell activation. Expression (18, 33, 43). We used mice that expressed bcatS33Y in T cells from of the CD62L “naive” T cell marker was strongly decreased con- one of the two ubiquitously active Rosa26 alleles specifically in T cells comitantly with an increased expression of the CD44 activation/ (named CD4bcat/1 mice), which show, on average, an ∼2–3-fold memory marker on CD4+ and CD8+ cells in the thymus (data not difference in b-catenin protein expression compared with biallelic shown) of CD4bcat/+ mice. Splenic (Fig. 2A, 2B) and peripheral expression (CD4bcat/bcat mice) (33) (Supplemental Fig. 1A). lymph node (data not shown) CD4+ and CD8+ cells in CD4bcat/+ Mice with biallelic b-catenin overexpression develop lethal leu- mice exhibited further loss of CD62L and enhancement of CD44 kemia due to a block at the CD4+CD8+ double-positive (DP) stage expression, which correlated with a mild increase in CD69 expres- of T cell development (33). Instead, none of the CD4bcat/+ mice sion (Supplemental Fig. 2A), and increased T cell proliferation, as

FIGURE 1. b-Catenin overexpression causes the development of a progressive motor function disorder in mice. (A) Incidence of EAE-like symptoms in 3–15-mo-old WT and CD4bcat/+ mice (mean 6 SD). p , 0.001, two-way ANOVA. (B) Grip strength in 9-mo-old WT and CD4bcat/+ mice. Signiﬁcant differences between WT and CD4bcat/+ mice were detected for both sexes (mean 6 SEM), n = 4 or 5 per group. p , 0.05, two-way ANOVA. (C) Loss of body weight in 11–14-mo-old CD4bcat/+ mice and age-matched WT mice. Data are pooled from three independent experiments. (D) Kaplan–Meier survival analysis of WT (n = 32) and CD4bcat/+ mice (n = 28). ***p , 0.001. 3034 Wnt/b-CATENIN IN T CELLS AND NEUROINFLAMMATION Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 2. b-Catenin overexpression causes partial T cell activation. Flow cytometry analysis of CD44 and CD62L expression in CD4+ (A) and CD8+ (B) SP cells isolated from the spleens of 6-wk-old WT and CD4bcat/+ mice. (C) Flow cytometry analysis of IFN-g and IL-17 expression in CD4+ cells isolated from the spleens of age-matched 9–12-mo-old WT and CD4bcat/+ mice. Graphs represent the frequency of IFN-g, IL-17, and IL-4–expressing cells among splenic CD4+ cells (right panels). Data are pooled from three (A and B)orfour(C) independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001. measured by analysis of Ki-67 expression (Supplemental Fig. 2B). from CD4bcat/+ mice was predominantly composed of CD45.2+ Of note, expression of other memory/activation/migration markers, cells (Supplemental Fig. 3E). CD4+ cells exhibited increased cycling such as CD122, CD25, GITR, CD5, and CD103, was not increased (Supplemental Fig. 3F), resulting in a 4-fold increase in the number in CD4bcat/+ mice (Supplemental Fig. 2C–G, respectively), sug- of b-catenin–expressing CD4+ cells compared with WT CD45.2+ gesting that b-catenin overexpression results in a “partial” T cell CD4+ cells (Supplemental Fig. 3G). Therefore, b-catenin exerts a activation state. With age, CD4bcat/+ mice, but not WT mice, cell autonomous function in T cell activation, leading to proliferative exhibited elevated production of proinﬂammatory cytokines, in- expansion of CD4+ cells in radiation chimeras. cluding IFN-g, IL-17, and IL-4 (Fig. 2C). b To determine whether the b-catenin–induced T cell activation was -Catenin overexpression does not impair Treg cell a cell autonomous defect and not a consequence of cytopenia ob- development and function served in CD4bcat/+ mice, we generated mixed BM chimeras by Contrasting results have been reported about the effect of b-catenin reconstituting lethally irradiated WT mice (CD45 1/2 allotype) with overexpression on Treg cell development and function. Because a1:1mixtureofWTorCD4bcat/+ BM cells (CD45.2+, donor) and T cell activation may occur as a result of Treg cell deﬁciency, we WT (CD45.1+, helper) BM cells. We observed that b-catenin–driven analyzed Treg cell development and function in our model, which T cell activation occurred in donor-derived CD45.2+CD4+ cells expressed elevated levels of b-catenin in the Treg-enriched CD4+ (Supplemental Fig. 3A) but not in helper-derived CD45.1+CD4+ CD25+ fraction as well (data not shown). The frequency of cells cells (Supplemental Fig. 3B). Although the CD45.2/CD45.1 ratio expressing Treg master regulator Foxp3 among CD4+ cells was was similar in Mac-1+ and CD8+ cells in mice reconstituted with similar in WT and CD4bcat/+ mice (Fig. 3A). However, the WT and CD4bcat/+ donor cells (Supplemental Fig. 3C, 3D), the overall number of CD4+Foxp3+ Treg cells was decreased in CD4+ compartment in mice that received donor cells originating CD4bcat/+ mice (Fig. 3B). This is likely a result of the decreased The Journal of Immunology 3035 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 3. b-Catenin overexpression does not impair Treg development and function. (A) Flow cytometry analysis of Foxp3 expression in CD4+ cells in the thymus of 6-wk-old WT and CD4bcat/+ mice. (B) Frequency (left and middle panels) and number (right panel) of CD4+Foxp3+ cells in the thymus of WT and CD4bcat/+ mice. (C) CD4+Foxp32 (Tconv)/CD4+Foxp3+ (Treg) ratio in spleens of WT and CD4bcat/+ mice. (D) Foxp3 mean fluorescence intensity (MFI) in CD4+CD25+ cells in spleens of WT and CD4bcat/+ mice. (E) Flow cytometry analysis of CD25, GITR, CTLA-4, and CD127 expression in CD4+Foxp3+ cells in spleens of WT and CD4bcat/+ mice. A representative plot for each genotype is shown. (F) Flow cytometry analysis of CD62L and CD44 expression (left panel) and relative frequency (right panel) in splenic CD4+Foxp3+ WT and CD4bcat/+ cells. (G) Change in body weight of Rag12/2 mice following adoptive transfer with WT Tconv cells, transferred alone or together with CD4+CD25+ cells (Treg cells) isolated from WT or CD4bcat/+ mice (mean 6 SEM, n = 6–7 per group). Data are pooled from two independent experiments. *p , 0.05, ***p , 0.001. number of CD4+ SP T cells that serve as a reservoir for Treg cell maintained (Fig. 3C). The stable b-catenin did not alter the ex- development and is not due to their defective generation, because pression levels of Foxp3 (Fig. 3D) or the Treg signature genes the ratio of conventional T (Tconv)/Treg cells in these mice was CD25, GITR, CTLA-4, and CD127 (Fig. 3E). It also did not lead 3036 Wnt/b-CATENIN IN T CELLS AND NEUROINFLAMMATION to aberrant expression of Th1-, Th2-, or Th17-related genes in tivated T cells that infiltrate the CNS. Indeed, the spinal cords of CD4+CD25+ cells (data not shown), despite the fact that CD4+ CD4bcat/+ mice, but not age-matched WT mice, showed signif- Foxp3+ cells largely lost CD62L expression and acquired the icant accumulation of CD4+ T cells (Fig. 5A) and, to a lesser CD44+ “effector Treg” phenotype (Fig. 3F). Importantly, b-catenin– extent, CD8+ T cells (data not shown) that showed an activated expressing and WT Treg cells suppressed colitis equally in an phenotype (Fig. 5B), as well as an increased expression of the adoptive T cell–transfer assay (Fig. 3G), suggesting that b-catenin Th1-type cytokine IFN-g but not IL-17 or IL-4 (Fig. 5C, data not does not impair the function of Treg cells. shown). Brains of CD4bcat/+ mice also revealed a moderate in- Adoptive transfer of 106 WT Treg cells did not restore normal crease in the frequency of infiltrating CD4+ and CD8+ T cells T cell activation in CD4bcat/+ mice (Supplemental Fig. 4A), further (data not shown). The total number of mononuclear cells was ele- supporting the idea that the effect of b-catenin overexpression on vated in the spinal cords of aged CD4bcat/+ mice (Fig. 5D). The T cell activation is intrinsic to Tconv cells and is not due to a Treg microglia compartment (CD11b+CD45lo/+) was augmented in the deficiency. To rule out the possibility that the neurologic phenotype spinal cords of CD4bcat/+ mice, but not in age-matched WT mice in CD4bcat/+ mice results from a loss of Treg function, we analyzed (Fig. 5E), along with increased expression of TNF-a and IL-1b T cell activation in mice with conditional overexpression of stabi- (Fig. 5F). The robust increase in GFAP positivity in CD4bcat/+ lized b-catenin in Treg cells, by crossing R26-stop-bcatS33A mice mice compared with WT mice (Fig. 5G) indicated that T cell with mice expressing Cre recombinase under the Foxp3 promoter infiltration in the spinal cords of CD4bcat/+ mice was accompa- (hereafter called Foxp3bcat/+ mice), thus achieving monoallelic nied by activation of astroglia. The infiltration of CD4+ T cells in b-catenin overexpression specifically in Treg cells (Supplemental Fig. the spinal cords of CD4bcat/+ mice was detected as early as at 3–

4B). Treg compartment–confined expression of stable b-catenin did 4 mo of age in the absence of an evident increase in lymphocytes Downloaded from not alter CD62L and CD44 expression in peripheral Tconv cells, as it in other organs (data not shown). However, increased numbers of isobservedinmicewithb-catenin overexpression in all CD4+ and CD4+ cells were detected in colons, livers, and muscles, but not in CD8+ cells (Supplemental Fig. 4C, 4D), nor did it lead to increased lungs and skin, of aged CD4bcat/+ mice (data not shown), sug- T cell CNS infiltration (Supplemental Fig. 4E) or development of the gesting that activated b-catenin–overexpressing T cells may also neurologic phenotype (data not shown) in Foxp3bcat/+ mice. To- infiltrate additional organs. Together, our data show that b-catenin

gether, our data suggest that forced b-catenin expression in naive causes recruitment of activated T cells primarily into the CNS that http://www.jimmunol.org/ T cells results in their spontaneous cell-intrinsic activation and loss initiate the Th1-type inflammatory cascade, involving microglia of tolerance, without an apparent loss of Treg function. and astroglia activation, resulting in neurologic abnormalities and motor impairment in mice. b-Catenin induces Th1 priming in T cells b A recent study demonstrated that forced expression of b-catenin in -Catenin overexpression leads to an increase in integrin a b mice with targeted deletion of exon 3 (bcatDex3 mice), which en- 4 1 expression codes the region responsible for b-catenin degradation, results in In multiple sclerosis, the integrin a4b1 (VLA-4) is essential for elevated expression of the Th17 master regulator Rorgtandim- T cells to gain access to the brain (45). Interestingly, the expression printing of the Th17 program in T cells, leading to colon inflam- of b-catenin was associated with a strong increase in the frequency by guest on September 27, 2021 mation (27). Interestingly, we observed elevated expression of the of integrin a4b1+ cells among CD4+CD44+ cells in peripheral blood Th1 signature cytokine IFN-g in CD4+ (Fig. 4A) and CD8+ (Fig. 4B) (Fig. 6A). The increased proportion of T cells expressing integrin SP thymocytes isolated from CD4bcat/+ mice, whereas expression a4b1inCD4bcat/+ mice suggests that b-catenin regulates the ex- of the Th17 cytokine IL-17 was unaltered (Fig. 4A, 4B), suggesting pression of Itgb1 and Itga4 genes that encode for the integrin b1 that T cell activation in the thymus was associated with differentiation and a4 subunits of integrin a4b1. We detected a high-probability along the Th1 pathway in CD4bcat/+ mice. The Th1 bias was con- b-catenin DNA binding site in the Itgb1 promoter region, and ChIP sistent with increased mRNA expression of Th1-type cytokines, such assays demonstrated direct binding (Fig. 6B) and activation (Fig. 6C) as IFN-g,IL-2,andTNF-a (Fig. 4C–E, respectively). The mecha- of the Itgb1 promoter by b-catenin. Similarly, b-catenin binding nisms underlying the observed Th1 skewing involve transcriptional (Fig. 6D) and promoter activation (Fig. 6E) were detected in the regulation of the Th1 master regulator Tbet (encoded by the Tbx21 proximity of the predicted Tcf/Lef1 binding site in the promoter gene), as evidenced by increased occurrence of acetylated histone 3 at region of the Itga4 gene but not at intronic control regions in the the Tbx21 gene in the region encompassing predicted binding sites for Itgb1 and Itga4 genes (Fig. 6B, 6D, respectively) and other pro- b-catenin DNA binding partners Tcf-1/Lef1 (position 223/26, moters tested, such as Rorc and Ifng (data not shown). Consistent identified using Matinspector software) in CD4+CD252 cells purified with increased specific b-catenin DNA binding and histone acety- from CD4bcat/+ mice but not from WT mice (Fig. 4F). Therefore, lation in the respective promoter regions, expression of Itgb1 and b-catenin likely regulates chromatin accessibility at the Tbet pro- Itga4 mRNA was increased in T cells from CD4bcat/+ mice (Fig. moter. Consistently, Tbx21 mRNA expression increased in the thy- 6F). These data demonstrate that integrin a4b1 is a novel direct target mus of 6-wk-old CD4bcat/+ mice (Fig. 4G). Consistent with of b-catenin in T cells. facilitated Th1 differentiation, CD4+CD252 cells purified from b a b CD4bcat/+ mice showed enhanced production of IFN-g over IL-17 -Catenin expression in mature T cells induces integrin 4 1 upon adoptive transfer into Rag12/2 mice compared with WT cells expression and CNS migration (Fig. 4H), resulting in an increased ratio of IFN-g/IL-17–producing To exclude that enhanced CNS migration develops as a result of CD4+ cells (Fig. 4I). These data suggest that aberrant activation of possible altered thymocyte selection in CD4bcat/+ mice, we tested T cells induced by b-catenin overexpression is associated with en- whether b-catenin overexpression by itself was sufficient to induce a hanced differentiation into a Th1 proinflammatory subset. CNS migration program in mature postselection T cells. Transduc- tion of naive WT mature T cells with a construct expressing stable b -Catenin overexpression results in T cell CNS infiltration and b-catenin (bcatS33Y), but not the empty control vector, was suffi- neuroinflammation cient to induce integrin a4b1 expression in mature T cells, as detected We hypothesized that abnormal motor function in CD4bcat/+ mice in the spleens of adoptively transferred Rag12/2 mice(Fig.7A).CNS develops as the result of an inflammatory reaction caused by ac- of these mice showed increased numbers of infiltrating mononuclear The Journal of Immunology 3037 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 4. b-Catenin induces Th1 priming in T cells. (A) Flow cytometry analysis of IFN-g and IL-17 expression (left panel) in thymi isolated from 6-wk-old WT and CD4bcat/+ mice. Frequency of IFN-g+ and IL-17+ cells among CD4+ SP thymocytes (right panel). (B) Frequency of IFN-g+ and IL-17+ cells among CD8+ SP thymocytes. qPCR analysis of Ifng (C) (mean 6 SEM, n = 6 per group), Il2 (D) (mean 6 SEM, n = 7–12 per group), and Tnfa (E) (mean 6 SEM, n =9– 12 per group) mRNA expression in the thymi of 6-wk-old WT and CD4bcat/+ mice. (F) ChIP assay of histone acetylation at the Tbx21 promoter region in WT and CD4bcat/+ splenic CD4+CD252 T cells. Cell lysates were immunoprecipitated with anti-acetyl histone 3 (AcH3) or control IgG, and the presence of specific regions in the immunoprecipitates was determined by qPCR (mean 6 SEM, n = 4 per group). (G)qPCRanalysisofTbx21 (mean 6 SEM, n = 6 per group) mRNA expression in the thymi of 6-wk-old WT and CD4bcat/+ mice. (H) Flow cytometry analysis of IFN-g and IL-17 expression in mediastinal lymph nodes isolated from Rag12/2 mice adoptively transferred with CD4+CD252 Tconv cells isolated from WT or CD4bcat/+ mice. (I)RatioofIFN-g+/IL-17+ cells among CD4+ cells analyzed in (H). Data were pooled from two (A–F)orone(H–I) independent experiment. *p , 0.05, **p , 0.01. cells (Fig. 7B) and integrin a4b1–expressing CD4+ cells (Fig. 7C, Th1-biased T cells with an elevated expression of integrin a4b1 7D). To demonstrate that integrin a4b1 induction was a critical step that enables them to infiltrate the CNS. These activated T cells in in aberrant CNS migration occurring in CD4bcat/+ mice, we used a the CNS initiate neuroinflammation, leading to the development pharmacologic approach to inhibit integrin a4b1functioninWT of a progressive neurologic disease associated with progressive and CD4bcat/+ mice. Inhibition of integrin a4b1 by the selective loss of hind limb motor function and lethality. a4b1 inhibitor (Bio5192) prevented the infiltration of activated CD4bcat/+ mice that express stable b-catenin in T cells from T cells into the spinal cord of CD4bcat/+ mice, resulting in an one of the two Rosa26 alleles do not develop T cell leukemia, overall decrease in the presence of CD4+ cells in their spinal cords unlike mice with the biallelic overexpression (33), but they display (Fig. 7E). These data demonstrate that integrin a4b1 is required for an important neurologic phenotype. Thus, the differences in b-catenin–mediated CNS infiltration. b-catenin expression levels in the same mouse model can either result in the block of T cell development at the DP stage, causing a Discussion predisposition to leukemia (33), or they can initiate the develop- In this study, we demonstrate that stabilization of b-catenin at a ment of an autoimmune/inflammatory disease in the CNS (as specific dosage in T cells results in the production of activated and in the current study). Although both models showed a survival 3038 Wnt/b-CATENIN IN T CELLS AND NEUROINFLAMMATION Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 5. b-Catenin overexpression results in Th1-type T cell CNS inﬁltration. (A) Flow cytometry analysis of CD4 and CD8 expression in the spinal cords of age-matched 9–12-mo-old WT and CD4bcat/+ mice (left panel). Frequency and number of CD4+ cells in the spinal cords of 9–12-mo-old WT and CD4bcat/+ mice (right panel). (B) Flow cytometry analysis of CD44 expression on CD4+ cells in mononuclear cells of 9–12-mo-old WT and CD4bcat/+ mice (left panel). Frequency of CD44+ cells within CD4+ cells (right panel). (C) Flow cytometry analysis of IFN-g and IL-17 expression in CD4+ cells in the spinal cords of 9–12-mo-old WT and CD4bcat/+ mice (left panel). Frequency of IFN-g+ cells within CD4+ cells (right panel). (D) Total number of mononuclear cells isolated from spinal cords of 9–12-mo-old CD4bcat/+ and WT mice (mean 6 SEM, n = 7–8). (E) Flow cytometry analysis of CD45 and CD11b expression in mononuclear cells isolated from spinal cord of diseased CD4bcat/+ and age-matched WT mice (left panel). Frequency and number of CD45lo/+CD11b+ microglia cells (mean 6 SEM, n = 5) (right panel). (F) qPCR analysis of Il1b (left panel) and Tnfa (right panel) mRNA expression in mononuclear cells isolated from the spinal cord of diseased CD4bcat/+ and age-matched WT mice relative to Actnb mRNA expression (mean 6 SEM, n =7–8).(G) GFAP expression in longitudinal sections of thoracic spinal cords of 9–12-mo-old WT and CD4bcat/+ mice. Scale bars, 100 mM. Data are pooled from two (D and E)orthree(A–C and F) independent experiments or are representative of three independent experiments (G). *p , 0.05, ***p , 0.001.

advantage at the DP stage (data not shown), consistent with overexpression model are likely insufﬁcient to block T cell de- evidence for a protective role of the Wnt/b-catenin pathway in velopment at the DP stage, explaining the lack of leukemia thymocytes (24, 25, 33, 46), the lower levels of b-catenin in the predisposition in mice with hemizygous b-catenin overexpression monoallelic overexpression model compared with the biallelic and allowing us to analyze the effect of b-catenin in mature T cells. The Journal of Immunology 3039 Downloaded from http://www.jimmunol.org/

FIGURE 6. b-Catenin induces integrin a4b1 expression in CD4+ T cells via direct binding to Intb1 and Inta4 promoter regions. (A) Flow cytometry analysis of integrin a4b1+ expression in CD4+CD44+ cells in the peripheral blood of WT and CD4bcat/+ mice (far left panel). Frequency of integrin a4b1+ cells among CD4+CD44+ cells (middle left panel), CD44+ cells among CD4+ cells (middle right panel), and integrin a4b1+ cells among CD4+ cells (far right panel) cells in the peripheral blood of 6–9-mo-old WT and CD4bcat/+ mice. ChIP assay for b-catenin presence (B) and histone acetylation (C) at the Intb1 promoter region in WT and CD4bcat/+ peripheral blood CD4+ cells. ChIP assay to detect b-catenin (D) and histone acetylation (E) at the Inta4 promoter region. (F) qPCR analysis of Intb1 (left panel) and Inta4 (right panel) mRNA expression in the thymi of 6-wk-old WT and CD4bcat/+ mice (mean 6 SEM, n = 6–7 per group). Data are from four (A), two (B, C,andF), or one (D and E) experiment. *p , 0.05, ***p , 0.001. N.D., not detectable. by guest on September 27, 2021 These data highlight the previously reported importance of b-catenin reported inhibitory role of the Wnt pathway in CD8+ cell differ- expression levels for its biological activity (32, 47), which has also entiation (52). Altogether, these experimental data demonstrate been observed for other transcription factors (48, 49). that specific differences in the dose and mode of expression may Similar to what is observed in an alternative b-catenin–over- affect the functional outcome of b-catenin overexpression in expression model (28), b-catenin overexpression in T cells T cells in various models, as described for other hematopoietic changed their function. Overexpression of b-catenin in T cells lineages (32, 47). results in increased expression of the activation marker CD44 Our data suggest that the main mechanisms underlying patho- and a concomitant loss of expression of the naive T cell marker logic infiltration of activated Th1 cells into the CNS involved CD62L. CD44 was previously implicated as a target of the Wnt upregulation of the expression of CNS-homing receptor integrin pathway (50) and was induced in the biallelic b-catenin–over- a4b1. We demonstrate that b-catenin directly binds to promoter expression model (33), suggesting that its expression could be regions and activates the transcription of both components of induced by b-catenin. A mild increase in T cell proliferation and integrin a4b1. Thus, we identified a novel direct target of Wnt/ CD69 expression in the absence of deregulation of other activation b-catenin signaling in T cells with an implication for disease markers suggests that b-catenin overexpression imparts a partially development. Pharmacologic treatment with a small-molecule activated state to T cells, as observed in other genetic mouse inhibitor of integrin a4b1 restored a normal number of CD4+ models (51). The early T cell activation is associated with aberrant T cells in the CNS of CD4bcat/+ mice, demonstrating that integrin differentiation along the Th1 pathway that is associated with a4b1 induction is necessary for the aberrant CNS migration in transcriptional activation of the expression of the Th1 master CD4bcat/+ mice. It is possible that additional b-catenin–induced regulator Tbet and elevated expression of Th1-type cytokines. The gene expression changes are involved in controlling the migration Th17-differentiation program was not activated in the thymus of of activated T cells to specific tissues in CD4bcat/+ mice. CD4bcat/+ mice, differently from the alternative b-catenin–sta- The most prominent outcome of T cell activation in CD4bcat/+ bilization model in which b-catenin activated rorc expression in mice is CNS targeting of the inflammatory disease, which has not the thymus (28). been described previously. CNS infiltration occurs as the first A comparable frequency of Foxp3+ cells among CD4+ cells event in pathogenesis, in the absence of detectable T cell migra- suggests that the Treg-differentiation program is not perturbed by tion to other sites in 3–4-mo-old CD4bcat/+ mice (data not b-catenin. These results are in line with a previous report sug- shown). We detected elevated expression of TNF-a and IL-1b gesting a survival-enhancing activity of b-catenin in Treg cells inflammatory cytokines in the CNS of diseased mice. Although (29), but they contrast with other studies reporting b-catenin–mediated restricted to T cells, b-catenin stabilization leads to activation of Treg functional impairment (28, 31). b-Catenin–expressing CD8+ other immune and nonimmune cell types, underlying the initiating cells also revealed an activated phenotype, in contrast with the role of T cells in CNS disease development. Thus, the described 3040 Wnt/b-CATENIN IN T CELLS AND NEUROINFLAMMATION Downloaded from http://www.jimmunol.org/

FIGURE 7. b-Catenin induces integrin a4b1 expression and CNS migration in mature T cells. (A) Flow cytometry analysis of integrin a4b1 expression in spleens of Rag12/2 mice adoptively transferred with CD4+ cells nucleofected with pcDNA3.1 or pcDNA3.1bcatS33Y vectors (left panel). Frequency of integrin a4b1+ cells by guest on September 27, 2021 among CD4+ cells (right panel). (B) Total number of mononuclear cells (MNC) infiltrating the spinal cord of Rag12/2 mice 15 d after adoptive transfer with CD4+ cells nucleofected with pcDNA3.1 or pcDNA3.1bcatS33Y vectors. (C) Flow cytometry analysis of integrin a4b1+ expression in the spinal cords of Rag12/2 mice adoptively transferred with CD4+ cells nucleofected with pcDNA3.1 or pcDNA3.1bcatS33Y vectors. (D) Number of CD4+ cells (left panel) and CD4+ integrin a4b1+ cells (right panel) in the spinal cord of Rag12/2 mice adoptively transferred with CD4+ cells nucleofected with pcDNA3.1 or pcDNA3.1bcatS33Y vectors. (E) Number of CD4+ cells (left panel) and CD4+ integrin a4b1+ cells (right panel) in the spinal cord of 4-mo-old WT and CD4bcat/+ mice treated with Bio5192 or PBS (mean 6 SEM, n = 3–6 per group). Data were obtained from one (E)ortwo(A–D) independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001. mouse model represents a novel tool for studying the mechanisms Our data suggest that deregulation of the Wnt/b-catenin pathway, of aberrant T cell CNS migration and the development of neuro- specifically in T cells, may be implicated in human CNS inflam- inflammation. matory conditions, as well as other autoimmune/inflammatory dis- A previous report showed Th17-mediated colitis development in eases. These data suggest that further study of the involvement of b-catenin–overexpressing mice (28). In our model, T cells over- this pathway in human inflammatory and neurodegenerative diseases expressing b-catenin show a Th1 bias and a higher expression of is warranted. the a4b1 integrin. We found that the ratio of a4b1/a4b7 integrin– expressing T cells is much higher in CD4bcat/+ mice (data not Disclosures shown), providing a hypothesis for the preferential targeting of the The authors have no financial conflicts of interest. inflammatory disease to the CNS in our model compared with the colon in a previous study (28). However, we also detected T cell infiltration into colon, muscles, and, to a lesser extent, liver in References some of the diseased CD4bcat/+ mice (data not shown), sug- 1. Bereshchenko, O., M. Coppo, S. Bruscoli, M. Biagioli, M. Cimino, T. Frammartino, D. Sorcini, A. Venanzi, M. Di Sante, and C. Riccardi. 2014. GILZ promotes pro- gesting that b-catenin–induced integrin a4b1 may also mediate duction of peripherally induced Treg cells and mediates the crosstalk between T cell infiltration to other organs. Thus, this model may be useful glucocorticoids and TGF-b signaling. Cell Reports 7: 464–475. 2. Bettelli, E., M. Oukka, and V. K. Kuchroo. 2007. T(H)-17 cells in the circle of for studying other autoimmune/inflammatory conditions. Although immunity and autoimmunity. Nat. Immunol. 8: 345–350. we did not detect direct peripheral nerve damage in CD4bcat/+ 3. Josefowicz, S. Z., L. F. Lu, and A. Y. Rudensky. 2012. Regulatory T cells: mice, we frequently detected leukocyte infiltration in the muscle mechanisms of differentiation and function. Annu. Rev. Immunol. 30: 531–564. 4. Santucci, L., M. Agostini, S. Bruscoli, A. Mencarelli, S. Ronchetti, E. Ayroldi, tissues of diseased mice that was associated with elevated expression A. Morelli, M. Baldoni, and C. Riccardi. 2007. GITR modulates innate and of IFN-g (data not shown). Thus, we cannot exclude that systemic adaptive mucosal immunity during the development of experimental colitis in or muscle tissue inflammation contributes to the development of mice. Gut 56: 52–60. 5. Ja¨ger, A., V. Dardalhon, R. A. Sobel, E. Bettelli, and V. K. Kuchroo. 2009. Th1, the hind limb paralysis, in addition to the CNS inflammation, in Th17, and Th9 effector cells induce experimental autoimmune encephalomy- CD4bcat/+ mice. elitis with different pathological phenotypes. J. Immunol. 183: 7169–7177. The Journal of Immunology 3041

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