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Endoplasmic Reticulum Stress Response Promotes Cytotoxic Phenotype of CD8 βα + Intraepithelial Lymphocytes in a Mouse Model for Crohn's Disease-like Ileitis This information is current as of September 28, 2021. Jung-Su Chang, Soeren Ocvirk, Emanuel Berger, Sigrid Kisling, Uli Binder, Arne Skerra, Amy S. Lee and Dirk Haller J Immunol 2012; 189:1510-1520; Prepublished online 2 July 2012; Downloaded from doi: 10.4049/jimmunol.1200166 http://www.jimmunol.org/content/189/3/1510 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2012/07/02/jimmunol.120016 Material 6.DC1 References This article cites 57 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/189/3/1510.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Endoplasmic Reticulum Stress Response Promotes Cytotoxic Phenotype of CD8ab+ Intraepithelial Lymphocytes in a Mouse Model for Crohn’s Disease-like Ileitis

Jung-Su Chang,*,† Soeren Ocvirk,* Emanuel Berger,* Sigrid Kisling,* Uli Binder,‡ Arne Skerra,‡ Amy S. Lee,x and Dirk Haller*

Endoplasmic reticulum (ER) unfolded protein responses (UPR) are implicated in the pathogenesis of inflammatory bowel disease. Cytotoxic CD8ab+ intraepithelial lymphocytes (IEL) contribute to the development of Crohn’s disease-like ileitis in TNFDARE/+ mice. In this study, we characterized the role of ER-UPR mechanisms in contributing to the disease-associated phenotype of cytotoxic IEL under conditions of chronic inflammation. Inflamed TNFDARE/+ mice exhibited increased expression of Grp78, + + ATF6, ATF4, and spliced XBP1 in CD8ab IEL but not in CD8aa IEL or in lamina propria lymphocytes. Chromatin immu- Downloaded from noprecipitation analysis in CD8ab+ T cells showed selective recruitment of ER-UPR transducers to the granzyme B gene promoter. Heterozygous Grp782/+ mice exhibited an attenuated granzyme B-dependent cytotoxicity of CD8ab+ T cells against intestinal epithelial cells, suggesting a critical activity of this ER-associated chaperone in maintaining a cytotoxic T cell phenotype. Granzyme B-deficient CD8ab+ T cells showed a defect in IL-2–mediated proliferation in Grp782/+ mice. Adoptively transferred Grp782/+ CD8ab+ T cells had a decreased frequency of accumulation in the intestine of RAG22/2 recipient mice. The tissue DARE/+ 2/+ DARE/+ + pathology in TNF 3 Grp78 mice was similar to TNF mice, even though the cytotoxic effector functions of CD8ab http://www.jimmunol.org/ T cells were significantly reduced. In conclusion, ER stress-associated UPR mechanisms promote the development and maintenance of the pathogenic cytotoxic CD8ab+ IEL phenotype in the mouse model of Crohn’s disease-like ileitis. The Journal of Immunology, 2012, 189: 1510–1520.

nflammatory bowel disease (IBD) is a chronically relapsing turn, commensal bacteria cross the epithelial cell layer (20), inflammatory pathology largely restricted to the small in- leading to an accelerated activation of effector T cells (21) and I testine and colon (1). Because of a very heterogeneous increased recruitment of inflammatory cells into the intestinal pathogenesis, factors triggering IBD include host genetic predis- mucosa (22). position (2, 3), environmental factors (4–6), commensal bacteria Analysis of human biopsy samples from Crohn’s disease patients by guest on September 28, 2021 as well as infectious triggers (7, 8), and a deregulated intestinal identified CD8+ T cells as the predominant T cell phenotype in the T cell homeostasis (9). The balance of regulatory and effector epithelium (23). Colitogenic CD4+ T cells were predominantly T cells is disturbed in Crohn’s disease, with more naive T cells identified in the lamina propria lymphocyte (LPL) compartment of differentiating into cytotoxic effector T cells (10–15). This process the inflamed intestinal mucosa (23). Interestingly, adoptive transfer likely results in an excessive cytotoxic response (15, 16), in- of naive CD4+ T cells into an immunodeficient recipient host increased permeability of the epithelial barrier (17, 18), continuous duced colonic inflammation (24, 25), which was prevented by the epithelial erosion, and reduced production of defensins (19). In cotransfer of CD8aa+ TCRab+ intraepithelial lymphocytes (IEL) (26) or CD4+ CD8aa+ IEL (25) but not CD8ab+ TCRab+ IEL (26). Emerging evidence suggests that an unresolved unfolded protein *Chair for Biofunctionality, Research Centre for Nutrition and Food Science, Centre for Diet and Disease, Technical University of Munich, 85350 Freising- response (UPR) of the endoplasmic reticulum (ER) contributes to Weihenstephan, Germany; †School of Nutrition and Health Science, Taipei Medical intestinal inflammation and the pathogenesis of IBD (1, 27–30). University, Taipei 110, Taiwan, Republic of China; ‡Chair for Biological Chemistry, Technical University of Munich, 85350 Freising-Weihenstephan, Germany; and Dissociation of ER-resident chaperone glucose-regulated protein xDepartment of Biochemistry and Molecular Biology, Keck School of Medicine, 78 (Grp78) from the membrane-anchored receptors, such as IRE1, University of Southern California, Los Angeles, CA 90033 PERK, and ATF6, initiates UPR signal transduction (1, 31, 32), Received for publication January 13, 2012. Accepted for publication May 29, 2012. leading to a selective activation or downregulation of gene ex- This work was supported by Deutsche Forschungsgemeinschaft Grant HA 3148/2-1 pression by downstream transcription factors ATF4, XBP1, and and by the Munich Center of Integrated Protein Science. ATF6. Being the master regulator of ER stress signaling, the in- Address correspondence and reprint requests to Prof. Dirk Haller, Chair for Biofunc- duction of Grp78 expression is required to alleviate ER stress (33, tionality, Research Centre for Nutrition and Food Science, Centre for Diet and Dis- ease, Technical University of Munich, Gregor-Mendel-Strasse 2, 85350 Freising- 34). Grp78 expression levels are increased by ER stress stimuli Weihenstephan, Germany. E-mail address: [email protected] (28, 35–37), and cleavage of Grp78 protein by subtilase cytotoxin The online version of this article contains supplemental material. leads to an inappropriate ER stress response, triggering cell death Abbreviations used in this article: ARE, AU-rich element; ARE mice, TNFDARE/+ (38). ER stress-associated UPR signals are increased in the in- mice; ChIP, chromatin immunoprecipitation; ER, endoplasmic reticulum; Grp78, testinal epithelium during chronic inflammation (28) and sensitize glucose-regulated protein 78; IBD, inflammatory bowel disease; IEC, intestinal epithelial cell; IEL, intraepithelial lymphocyte; LPL, lamina propria lymphocyte; MLN, secretory epithelial cells to cell death (30). A deletion of one or mesenteric lymph node; qPCR, quantitative RT-PCR; sCD8ab+ T cell, splenic two alleles of XBP1, a downstream transcription factor of the + CD8ab T cell; siRNA, small interfering RNA; UPR, unfolded protein response; IRE1 pathway, resulted in spontaneous inflammation of the small Wt, wild type. intestine, T cell infiltration, and Paneth cell dysfunction (29), as Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 well as altered composition of microbiota (39). Although accu- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1200166 The Journal of Immunology 1511 mulating evidence suggests that unresolved ER stress contributes Isolation of primary T cells from small intestine and spleen to the mucosal pathogenesis in IBD, little is known about the Spleen and small intestine were removed and kept on ice. The small in- impact of unresolved ER stress on T cell homeostasis under testine was cut open longitudinally, and feces were washed off. IEL were chronic inflammatory conditions. released by incubation of the tissue pieces with digestion buffer (5 mM Several lines of evidence suggest that ER stress-associated UPR EDTA, 1 mM DTT, 10% FBS, RPMI 1640) two times for 20 min. LPL were signals are involved in the maintenance of lymphocyte homeo- digested twice in the presence of LPL digestion buffer (100 U/mg collagenase II and collagenase IV, 10% FBS, RPMI 1640) at 37˚C and 200 rpm. stasis and viability. First, Grp78 was shown to be noncovalently Pooled IEL from each group were placed on ice for 10 min to separate the bound to the Ig H chain (40) or the nonmatured TCRa-chain tissue debris from cells. Supernatant was carefully removed and centri- within the ER (41). TCRa-deficient mice lack functional CD4 fuged at 350 3 g, 4˚C, for 5 min. Pellets were resuspended in 2 ml FACS and CD8 T cells (42), implying a critical activity of TCRa buffer (5% FBS, 2 mM EDTA, PBS) and applied onto a nylon wool fiber column (Polysciences, Eppelheim, Germany) to remove adherent cells and maturation on conventional T cell function. Moreover, it was + tissue debris. Pooled IEL and LPL (n = 6 mice/group) were sorted into demonstrated that XBP1 facilitated the transition of CD8ab the single phenotype, with a purity of 95–98%, by FACSAria (BD Bio- effector T cells into terminally differentiated CD8ab+ Tcells sciences, Franklin Lakes, NJ). CD8ab+ T cells were isolated from pooled (43, 44). Franco et al. (45) reported that ER stress is also in- spleen by indirect positive selection through a magnetic column, resulting volved in the induction and plastic differentiation of regulatory in purities between 90 and 98% (Miltenyi Biotec, Bergisch Gladbach, Germany). peripheral T cells. Recent studies indicated that Grp78 expression in conventional CD4+ or CD8ab+ T cells is induced by CD3:TCR Flow cytometry activation or mitogenic stimulation (46, 47). This was shown to Splenic T cells, IEL, and LPL (105 cells) were used for immunopheno- be mediated by protein kinase C, because blocking of protein typing by FACS analysis. Abs used for the six-color surface staining were Downloaded from kinase C resulted in diminished ER stress response and reduced CD3-allophycocyanin-Cy7 (BD Pharmingen, Franklin Lakes, NJ), CD8a- IL-2 synthesis in T cells (47). A knock down of Grp78 protein allophycocyanin (Miltenyi Biotec), CD8b-FITC (BD Pharmingen), CD4- PE-Cy7 (BD Pharmingen), TCRab-PE (Serotec, Du¨sseldorf, Germany), induced apoptosis in a murine T cell line (46). Consistently, and CD44-PerCP-Cy7 (BD Pharmingen). Intracellular production of IL-2, Grp78 is imputed to have an antiapoptotic effect due to inhi- IFN-g, Bcl-2, and granzyme B (eBioscience, San Diego, CA) was deter- bition of the apoptosis-triggering pathways of UPR (48), and mined after incubation of T cells with 1 mg/ml brefeldin A for the last 3–4

it is constitutively expressed, with enhanced expression due to h of in vitro culture. At 48 h postactivation, intracellular proteins were http://www.jimmunol.org/ detected by intracellular staining in combination with surface expression of stressful conditions like glucose deprivation, calcium depletion 2 CD3+, together with CD8b or CD8b+, according to the BD Cytofix/ in the ER, oxidative stress, and hypoxia (31, 48, 49). In sum- Cytoperm and GolgiPlug protocol (all Abs were from BD Pharmingen, mary, these data suggest a critical role for Grp78 and ER stress- with the exception of granzyme B). To test for viability, T cells were in- associated UPR signals in the homeostasis and viability of cubated with propidium iodide (50 mg/ml) for 30 min and subsequently T cells. acquired by flow cytometry. A total of 10,000 cells was acquired from the LSR II (BD Biosciences) and analyzed using BD FACSDiva software. To investigate the effects of ER stress-associated mechanisms on T cell homeostasis under chronic intestinal inflammation, we Chromatin immunoprecipitation analysis performed experiments in a mouse model that is characterized Unstimulated splenic CD8ab+ T cells (sCD8ab+ T cells; 3 3 106 cells), by the genetic ablation of AU-rich elements (ARE) from the isolated and pooled from mice (n . 3), were used for chromatin immu- by guest on September 28, 2021 TNF mRNA, leading to overproduction of TNF (15, 16). These noprecipitation (ChIP) analysis, according to the ChIP-IT Express kit TNFDARE/+ mice (ARE mice) show an aberrant T cell homeo- instructions (Active Motif, La Hulpe, Belgium). Briefly, isolated CD8ab+ stasis and spontaneously develop Crohn’s disease-like trans- T cells were directly fixed with 1% formaldehyde for 10 min at room temperature or incubated at 37˚C and 5% CO2 for 48 h in the presence of mural tissue pathology in the distal ileum. Most interestingly, polyclonal anti-CD3/CD28 MicroBeads and with or without 100 U/ml rIL- the mucosal pathology of this model is attributed to the pres- 2 protein (R&D Systems, Minneapolis, MN). DNA-bound protein was ence of pathogenic cytotoxic CD8ab+ IEL that preferentially cross-linked with 1% formaldehyde for 10 min at room temperature. En- accumulate in the epithelium (15). In this study, we investi- zymatic DNA shearing was performed in a 37˚C water bath for 15 min. Sheared chromatin (300–100 bp) was immunoprecipitated with specific gated the effects of ER stress-associated signals on the ho- + Abs against ATF4, ATF6a, and XBP1 (all from Santa Cruz Biotechnology, meostasis of intestinal CD8ab T cells under chronic intestinal Santa Cruz, CA) and Abs against acetyl histone 3 (lysine 9), YY1, and inflammation. phosphorylated c-Jun (all from Cell Signaling Technology, Boston, MA) at 4˚C overnight. Isolated DNA was purified using a PCR purification kit (STRATEC Biomedical, Birkenfeld, Germany). The input control for the Materials and Methods quantitative RT-PCR (qPCR) was DNA from total nuclear extract without Mice immunoprecipitation. qPCR was performed with total DNA (input control; 1 ml) and immunoprecipitated DNA (1 ml). The putative promoter regions ARE mice on a C57BL/6 background and wild-type (Wt) littermates were a generous gift from Dr. G. Kollias (Institute for Immunology, Biomedical were evaluated using the Gene2Promoter program from Genomatix (http:// Sciences Research Center “Al. Fleming”, Varkiza, Greece). The generation www.genomatix.de), and primers were designed using the Primer3 Web of Grp78 heterozygous (Grp782/+) mice and Wt littermates was described site (http://frodo.wi.mit.edu/). Details of primer sequences and amplicon 2/+ length are listed in Supplemental Table I. previously (50). Grp78 mice on a C57BL/6 or JV129 background DARE/+ and Wt littermates were kindly provided by A.S.L. (50). TNF 3 RNA isolation, reverse transcription, and qPCR Grp782/+ double transgenic mice were generated by breeding TNFDARE/+ mice into Grp782/+ on a C57BL/6 background. RAG22/2 mice on Total RNA was extracted using an RNeasy Mini Kit (QIAGEN, Hilden, a JV129 background (51), which lack functional lymphocytes owing to Germany). RNA yield and quality were assessed by absorbance using a their inability to initiate V(D)J rearrangement, were purchased from Nanodrop ND-1000 spectrophotometer (LabTech International, Brampton, Taconic. All mice were raised in a conventional manner in the animal ON, Canada). A total of 200–500 ng RNA was used for reverse transcrip- facility at the Technical University of Munich-Weihenstephan as approved tion using the SuperScript III First-Strand system (Invitrogen, Darm- by the institution in charge (approval no. 55.2-1-S4-2531-74-06 and 32- stadt, Germany). Primers and probes were designed using the universal 2347/4+63, tested by meeting the requirements of the Federation of Eu- probe library (Roche, Mannheim, Germany) or the Primer3 Web site (http:// ropean Laboratory Animal Science Associations showing positive results frodo.wi.mit.edu/). Details on primer concentration and sequences are for Helicobacter typhlonius, Norovirus, and Trichomonas spp.). Mice were shown in Supplemental Table I. Amplicon sizes ranged between 90 and killed by cervical dislocation at the age of 8, 18, or 24 wk. Tissue sections 230 bp to ensure high amplification efficiency. GAPDH was selected as the of distal ileum were fixed in 10% neutral-buffered formalin. Histopatho- reference gene in this study because of its stability in response to anti- logical changes were scored in paraffin-embedded ileal sections in a blin- CD3/CD28 and ionomycin/PMA stimulation. RNA-expression profiles of ded fashion, as previously described (52). both target and reference genes were performed using the LightCycler 1512 ER STRESS IN T CELL HOMEOSTASIS AND PATHOGENESIS OF IBD

(Roche) at 10 ml/PCR reaction (400 nM primers and/or 200 nM probe supernatant upon cell lysis. Alternatively, long-term CD8ab+ T cell- concentration, 1 ml cDNA, QuantiTect Probe RT-PCR Master Mix buffer). mediated cytotoxicity against confluent Mode-K was assessed after SYBR Green or QuantiTect Probe Master Mix (Roche) was used for target 4 d of coculture by a DNA-fragmentation assay. In this system, 7.5 3 103 gene quantification. The relative induction of mRNA expression was cal- Mode-K cells were seeded in 500 ml Mode-K cell culture medium con- culated using the following equation ECp (control mice 2 ARE mice) and nor- taining 10 mM/ml BrdU in a 24-well plate, reaching 60–70% confluence malized for the expression of GAPDH. overnight. BrdU was allowed to incorporate into proliferating Mode-K cells overnight prior to the addition of primary T cells or IEL. Unless Western blot analysis otherwise stated, BrdU-labeled Mode-K cells were coincubated with primary sCD8ab+ T cells (50,000 cells/well) or IEL (10,000 cells/well) for Freshly isolated, unstimulated cells were lysed in lysis buffer, and protein 4 d in the presence or absence of anti-CD3/CD28 MicroBeads. T cell- concentration was quantitated by Bradford (Carl Roth, Karlsruhe, Ger- mediated killing of Mode-K cells was measured by DNA-fragmentation many). Protein lysates were boiled in 13 SDS buffer at 95˚C for 10 min. assay, and granzyme B concentration in the cell culture supernatant was Equal amounts of proteins were resolved on a 10% SDS-polyacrylamide quantified using a granzyme B-specific ELISA kit (R&D Systems). Ex gel and transferred by electroblotting to a nitrocellulose membrane. vivo and in vitro stimulation experiments were performed in four to six Membranes were probed with Abs, as indicated, and the specific signals wells for each stimulus per time point. were detected using an ECL detection system (GE Healthcare, Barrington, IL). Anti-mouse Grp78 (Sigma-Aldrich, Munich, Germany) and b-actin (MP Biomedicals, Santa Ana, CA) Abs were used at a dilution of 1:3000 Adoptive transfer to detect immunoreactive Grp78 and b-actin, respectively. Anti-rabbit A total of 1.5 3 106 sCD8ab+ T cells isolated from donor mice (Grp782/+ secondary Abs were used to detect the primary Abs. or Wt littermates on a JV129 background) was resuspended in 300 ml PBS and transferred to 8-wk-old RAG22/2 recipient mice on a JV129 back- Cell culture ground by i.p. injection. RAG22/2 mice were killed by cervical dislocation

Unless otherwise stated, the density of CD8ab+ T cells used in cell culture 6 wk after adoptive transfer. Downloaded from was as follows: 2 3 105 CD8ab+ T cells for gene-expression profiling, 5–10 3 105 cells for BrdU-incorporated cell-proliferation analysis, and Data analysis 10,000–50,000 CD8ab+ T cells for DNA-fragmentation assay. CD8ab+ The nonparametric Mann–Whitney-U test was used to analyze differences T cells were cultured in T cell culture medium (RPMI 1640, 10% FBS, between two genotypes. The paired Student t test was used to analyze the 2mML-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin; all from differences in response to treatment within the same group. Time-course Life Technologies, Darmstadt, Germany) at 37˚C, 5% CO in the pres- 2 experiments were analyzed by one-way ANOVAwith Bonferroni posttests ence or absence of ionomycin/PMA (1 mg/ml ionomycin and 50 ng/ml and correction or by one-way ANOVA and the Tukey t test for qPCR time- http://www.jimmunol.org/ PMA; Sigma-Aldrich) and anti-CD3/CD28 Ab-coated MicroBeads (2:1 course experiments. Analysis was conducted using Prism 4.03 software MicroBeads/T cell ratio; Miltenyi Biotec). In some experiments, CD8ab+ (GraphPad). Differences were considered significant if p values were , T cells were treated or not with stimuli, including 10 ng/ml TNF (R&D 0.05, and data are presented either as mean 6 SEM or median (25th–75th Systems), 3 mg/ml anti-TNF mAb (TN3-19.12 clone; Abcam, Cambridge, percentiles). Unless otherwise noted, all data are representative of a mini- U.K.), or 100 U/ml murine rIL-2 (R&D Systems), for various lengths of mum of three independent experiments. time. Mode-K cells were kept in a 37˚C incubator in a T75 cell culture flask containing Mode-K cell culture medium (DMEM culture medium, 10% FCS, 2 mM L-glutamine, antibiotic/antifungal agent [100 U/ml pen- Results icillin, 100 mg/ml streptomycin, 0.25 mg/ml amphotericin B]). Mode-K cells were trypsinized, split, and seeded in a 24-well plate at a density that Induction of ER stress-associated UPR in disease-relevant 3 + reached 60–70% confluence after 24 h of incubation (7.5 3 10 cells in CD8ab IEL under conditions of Crohn’s disease-like ileitis by guest on September 28, 2021 500 ml Mode-K cell culture medium). All in vitro experiments were per- To investigate the induction of ER stress-associated UPR in the formed using a minimum of three wells/treatment. small intestine under chronic inflammatory conditions, we used Cell-proliferation assay inflamed ARE mice and Wt littermate control mice at the age of CD8ab+ T cell and MLN cell proliferation was measured by a colori- 8 and 18 wk. H&E staining of representative paraffin-embedded metric immunoassay based on the measurement of BrdU incorporation distal ileal segments showed increased total histopathological during DNA synthesis (Roche); 10 mM/ml BrdU was added to the scores, which were determined by assessing infiltration of immune + 6 CD8ab T or mesenteric lymph node (MLN) cells (0.5 3 10 cells/well) at cells, crypt hyperplasia, and villus atrophy, in ARE mice at 8 and 48 h postactivation and left overnight. On day 3 postactivation, cells were 18 wk of age compared with Wt mice (Fig. 1A, 1B). The devel- fixed in FixDenat to facilitate DNA denaturation. Peroxidase-labeled anti– BrdU Abs that recognize BrdU-labeled DNA generated a photometrically opment of ileal inflammation in ARE mice was associated with detectable signal that was measured at 450 nm in an ELISA plate reader. phenotypic changes in the IEL and LPL fractions (Table I), In vitro stimulation experiments were performed in four to six wells for resulting in a significantly increased ratio of cytotoxic CD8ab+/ each stimulus per time point. CD8aa+ IEL in 18-wk-old ARE mice (median: 0.57; 25th–75th Grp78 knock down using EGF-SubA fusion protein or small percentiles: 0.53–0.75) compared with Wt mice (median: 0.37; interfering RNA 25th–75th percentiles: 0.34–0.39, p , 0.01). We next sought to characterize ER stress-associated UPR gene 3 6 ab+ A total of 1 10 sCD8 T cells was cultured with 100 pM EGF-SubA expression in isolated IEL and LPL from the small intestine of ARE (SibTech, Brookfield, CT). Cells were harvested at 24 h posttreatment. A total of 5 3 106 sCD8ab+ T cells was isolated from Wt mice and mice compared with Wt mice at the age of 18 wk. Interestingly, we transfected with 400 nM Grp78 and a start negative control small inter- detected increased mRNA expression of the ER stress chaperone fering RNA (siRNA) duplex (QIAGEN) by nucleofection using a mouse Grp78 specifically in the CD8ab+ IEL, but not in the CD8aa+ Nucleofactor Kit (Lonza, Visp, Switzerland). The transfection efficiency ∼ IEL, from ARE mice (Fig. 1C). Increased Grp78 transcript levels was estimated at day 1 or 2 posttransfection and was 15–20%. After 24 h, + transfected cells were activated using ionomycin/PMA, and cells were in ARE CD8ab IEL were associated with upregulated UPR harvested 24 h poststimulation. signal transducers ATF4, ATF6, CHOP, and sXBP1. In contrast to IEL subpopulations, LPL exhibited only minor changes with T cell-mediated cytotoxicity regard to the expression levels of Grp78 and UPR signal trans- T cell-mediated cytotoxicity against the murine small intestinal epithelial ducers (Fig. 1C). Focusing on the IEL compartment, we next in- cell (IEC) line, Mode-K, was performed by CytoTox 96 Non-Radioactive vestigated the gene expression of IFN-g, granzyme B, and Bcl-2 Cytotoxicity Assay (Promega, Mannheim,Germany)oraDNA- of isolated ARE CD8ab+ IEL.AsshowninFig.1D,ARE fragmentation assay (Roche). For the analysis of ex vivo cytotoxicity, + CD8ab+ T cells were cultured for 4 h with Mode-K cells at an E:T ratio CD8ab IEL exhibited markedly increased transcript levels for of 2.5:1, with or without TCR cross-linking. Ex vivo cytotoxicity was IFN-g, granzyme B, and Bcl-2 under conditions of experimental measured based on the released lactate dehydrogenase in the cell culture ileitis. The differences observed in the expression profiles of ER The Journal of Immunology 1513

tion with anti-CD3/CD28–coated MicroBeads or ionomycin/PMA (Fig. 2B), suggesting that TCR activation induces UPR signaling. Interestingly, TCR activation of ARE CD8ab+ T cells resulted in signiﬁcantly greater increases in Grp78 gene expression compared with CD8ab+ T cells from Wt mice. This was conﬁrmed by ChIP analysis, which was performed to determine the binding of downstream UPR signal transducers to the native Grp78 promoter. In contrast to Wt mice, sCD8ab+ T cells from ARE mice showed selective recruitment of ATF6, XBP1, and the DNA-binding transcription factor YY1 to the Grp78 promoter (Fig. 2C). UPR transducers are selectively recruited to the gene promoter of granzyme B To determine to what extent enhanced expression levels of UPR signal transducers and Grp78 in ARE CD8ab+ T cells affect T cell effector functions, we performed a promoter analysis to identify potential bindings sites of UPR signal transducers, such as XBP1, ATF4, and ATF6. The promoter regions of granzyme B and Bcl-2

were selected for ChIP analysis. Indeed, XBP1, ATF4, and ATF6a Downloaded from selectively bound to the gene promoter of granzyme B in splenic ARE T cells but not in Wt CD8ab+ T cells (Fig. 2D). This was mediated by the recruitment of nuclear CREB and EBOX to the proximal promoter region (data not shown) and suggests that active granzyme B gene transcription is under the control of ER

stress-associated mechanisms. In addition to granzyme B, the http://www.jimmunol.org/ analysis of Bcl-2 promoter revealed partial binding of UPR signal transducers under conditions of chronic ileitis in ARE CD8ab+ T cells (Fig. 2D). Furthermore, ARE CD8ab+ T cells from spleen showed remarkably increased expression of intracellular proin- ﬂammatory (granzyme B) and antiapoptotic molecules (Bcl-2)

+ (Fig. 2E). This suggests a greater cytotoxic capacity for ARE FIGURE 1. Induction of ER stress response in disease-relevant CD8ab + IEL from Crohn’s disease-like TNFDARE/+ mice. (A) Histopathological mice-derived CD8ab T cells and was confirmed in an ex vivo + analysis revealed an inflamed distal ileum in ARE mice compared with 8- cytotoxicity assay: ARE CD8ab T cells, activated with anti- and 18-wk-old Wt mice (n = 6/group). (B) Representative H&E-stained CD3/CD28–coated MicroBeads, showed a significantly increased by guest on September 28, 2021 ileal tissue segments from Wt and ARE mice (original magnification cytotoxicity against an IEC line (Mode-K cells) compared with 3100). Quantitative gene expression analysis of Grp78 and UPR trans- Wt CD8ab+ T cells (Fig. 3A). ducers (C) and T cell effector molecules (IFN-g, granzyme B, Bcl-2) (D)in To investigate the impact of Grp78 on downstream UPR-signal + + freshly isolated and unstimulated pooled CD8aa IEL, CD8ab IEL, transducers, we performed a gene-expression analysis of sCD8ab+ + + CD4 LPL, and CD8 LPL from 18-wk-old ARE mice. Gene expression T cells from Wt mice treated with siRNA to transiently knock data were normalized to the expression level of GAPDH and are presented down Grp78. As a result of the siRNA treatment, the expression as fold difference between ARE and Wt mice. (E) Flow cytometric analysis + + + + levels of Grp78, as well as ATF4, sXBP1, and ATF6, were sig- of CD44 CD8aa and CD44 CD8ab IEL isolated from the intestinal + epithelium of 18-wk-old Wt and ARE mice (n = 6/group). Dot plot data are nificantly reduced in Wt CD8ab cells after 24 h (Supplemental + a representative graph from individual mice and are presented as per- Fig. 1A–D). By stimulating Wt CD8ab T cells with ionomycin/ centage under the gated lymphocyte region. Significant differences are PMA for 24 h post-siRNA treatment, we were able to show that shown with *p , 0.05, **p , 0.01, ***p , 0.001. the transient knock down of Grp78 abolished the expression of Grp78 (Supplemental Fig. 1E), as well as that of granzyme B and Bcl-2 (Supplemental Fig. 1F, 1G), suggesting that downstream stress-associated genes were not related to the level of CD44+ UPR signaling and cytotoxic effector functions are mediated by expression, because CD8ab+ IEL from Wt and ARE mice had Grp78. similar levels of CD44+ IEL (Fig. 1E). The cytotoxic phenotype of ARE CD8ab+ T cells is not directly TCR activation triggers UPR signaling dependent on TNF Considering the enhanced expression levels of UPR signal trans- To determine the impact of TNF overproduction on the pathogenic ducers and the ER stress chaperone Grp78 in CD8ab+ IEL from cytotoxic CD8ab+ T cell phenotype, we performed an ex vivo cy- ARE mice, we next investigated the mechanisms of ER stress- totoxicity assay with added anti-TNF Abs. ARE sCD8ab+ Tcells associated UPR induction and functional consequences in ARE show significantly increased cytotoxicity against Mode-K cells, CD8ab+ T cells derived from spleen. Similar to CD8ab+ IEL independent of intrinsic TNF overproduction (Fig. 3A). Consis- from ARE mice, ARE-derived sCD8ab+ T cells showed an in- tently, the addition of exogenous TNF, in combination with anti- creased baseline level of Grp78 gene expression compared with CD3/CD28 MicroBeads, enhanced the cytotoxicity of Wt sCD8ab+ Wt T cells (Fig. 2A). Because the transcriptional activation of T cells, but anti-TNF Abs did not attenuate the expression level of Grp78 is widely regarded as an important event in the onset of granzyme B in ARE sCD8ab+ Tcellsafter18hofincubation(Fig. UPR, we tested whether TCR cross-linking activates the ER stress 3B). Moreover, the addition of exogenous TNF to Wt sCD8ab+ response in vitro. ARE and Wt mice-derived sCD8ab+ T cells T cells and of anti-TNF Abs to ARE sCD8ab+ Tcellsshowed showed significantly higher expression of Grp78 after stimula- minimum effects on Grp78 expression (Fig. 3C) after 18 h of in- 1514 ER STRESS IN T CELL HOMEOSTASIS AND PATHOGENESIS OF IBD

Table I. Immunophenotypic changes of IEL and LPL in the small intestine of Crohn’s disease-like TNFDARE/+ mice

Immune Compartment Phenotype Wk Wt Micea TNFDARE+/2 Micea p Valueb IEL CD3+CD8aa+ 8 9.9 (9.4–10.2) 3.6 (3.5–3.6) ,0.05 18 24.6 (19.3–31.4) 15.6 (12.8–20.2) ,0.01 IEL CD3+CD8ab+ 8 3.4 (3.0–3.8) 0.85 (0.7–1.0) .0.05 18 8.7 (7.3–11.7) 9.8 (8.3–14.3) .0.05 IEL CD3+CD4+ 8 1.8 (1.65–1.85) 1.1 (0.8–1.2) .0.05 18 6.75 (4.1–10.7) 3.7 (3.1–5.4) .0.05 IELc CD8aa+CD44+ 18 53.8 (38.3–57.4) 15.8 (13.6–20.3) ,0.01 IELc CD8ab+CD44+ 18 27.9 (22.1–33.3) 56.8 (55.5–64.7) ,0.01 Splenocytesc CD8ab+CD44+ 18 25.6 (24.4–29.2) 55.2 (47.3–70) ,0.01 Splenocytes CD3+CD4+ 18 15.1 (14–20.1) 14 (12.8–20.5) .0.05 LPL CD3+CD4+ 8 3.3 (2.8–4.4) 2.6 (2.5–2.7) .0.05 18 4.45 (2.9–6.3) 10.5 (9.8–10.9) ,0.01 Cells were stained for CD3ε+, CD8b+, CD8a+, CD4+, and CD44+, followed by flow cytometric analysis. Expression of CD44+ on CD8+ T cell subsets was quantified within the gated CD3+CD8+ population of 18-wk-old mice. aMedian (25th–75th percentiles); n = 6/group/time point. bNonparametric Mann–Whitney U test. cGated under CD3+CD8+. Downloaded from cubation, suggesting that the pathogenic cytotoxic phenotype of that the presence of Grp78 is required for the granzyme B- ARE CD8ab+ T cells is not directly maintained by TNF. mediated cytotoxicity of CD8ab+ T cells. In contrast, Wt sCD8ab+ T cells showed no significant changes in granzyme B ab+ Grp78 is required for granzyme B-dependent CD8 T cell secretion after EGF-SubA–mediated Grp78 knock down (Fig. 4B). cytotoxicity

The transient knock down of Grp78 by EGF-SubA induced similar http://www.jimmunol.org/ Increased levels of Grp78 and UPR signal transducers are asso- rates of apoptosis in ARE CD8ab+ T cells compared with un- ciated with higher levels of intracellular granzyme B and increased treated ARE CD8ab+ T cells at 24 h post-knock down (Fig. 4C, cytotoxicity against Mode-K cells. Thus, we next investigated the Table II). Interestingly, sCD8ab+ T cells from Wt mice were more effect of Grp78 on cytotoxic effector functions. First, a transient susceptible to apoptosis than were T cells from ARE mice after knock down of intracellular Grp78 protein by endotoxin EGF- Grp78 knock down (Fig. 4C). SubA in ARE CD8ab+ T cells from spleen (Fig. 4A) resulted in Next, we performed a set of experiments with isolated CD8ab+ the depletion of basal granzyme B secretion (Fig. 4B), suggesting T cells from a Grp78-deﬁcient mouse model (Grp782/+). Al- by guest on September 28, 2021

FIGURE 2. Regulation of Grp78 gene expression in isolated sCD8ab+ T cells and impact on transcription of T cell effector molecules. (A) Grp78 gene expression was quantified in sorted splenic CD44+CD8ab+ T cells from Wt and ARE mice. Gene expression data were normalized to the expression level of GAPDH and are presented as fold difference between ARE and Wt mice. (B) sCD8ab+ T cells, isolated and pooled from Wt and ARE mice (n . 3), were stimulated with anti-CD3/CD28–coated polyclonal MicroBeads and mitogens (50 ng/ml PMA and 1 mg/ml ionomycin) for 24 and 48 h. (C) Promoter binding activity was analyzed in pooled (n . 3), freshly isolated unstimulated sCD8ab+ T cells ex vivo. ChIP analysis was performed with unstimulated sCD8ab+ T cells (30 3 106 cells) isolated from ARE and Wt mice. (D) ChIP analysis was performed with freshly isolated unstimulated sCD8ab+ T cells (30 3 106 cells) pooled from Wt and ARE mice (n . 3). (E) Intracellular expression of granzyme B (Gzm B) and Bcl-2 proteins in CD3+CD8ab+ T cells, isolated from spleen and pooled from Wt and ARE mice, was quantified at 48 h postactivation by FACS. Dot plot data are presented as percentage under the gated region. Significant differences are shown with *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 1515 Downloaded from

FIGURE 4. Transient knock down of Grp78 diminishes expression of granzyme B. (A–C) Pooled nonstimulated sCD8ab+ T cells isolated from Wt and ARE mice (n . 3) were incubated with 100 pM bacterial endotoxin EGF-SubA for 24 h. Cell viability was analyzed by propidium iodide (PI) staining, and Grp78 level was analyzed by Western blot at 24 h post- http://www.jimmunol.org/ knock down. The single FACS plots are representative plots for the PI+ (%) CD8ab+ T cells as shown in Table II. Signiﬁcant differences are shown with *p , 0.05, **p , 0.01, ***p , 0.001.

proliferation compared with Wt CD8ab+ T cells after 3 d of stimulation with anti-CD3/CD28–coated MicroBeads or ionomycin/ PMA (Fig. 6A). MLN cells derived from Grp782/+ mice exhibited + similar defects in proliferation (Fig. 6B). Consistent with the

FIGURE 3. Cytotoxic phenotype of ARE CD8ab T cells is indepen- by guest on September 28, 2021 + 2/+ dent of TNF. (A) Ex vivo cytotoxicity of sCD8ab+ T cells pooled from Wt previous findings, sCD8ab T cells and MLN cells from Grp78 and ARE mice (n . 3) against murine epithelial Mode-K cells was mea- mice showed significantly reduced levels of granzyme B secretion sured at an E:T cell ratio of 2.5:1 after 4 h of TCR activation by anti-CD2/ (data not shown). CD28 MicroBeads using a nonradioactive cytotoxicity assay. To investi- Gene expression analysis revealed decreased IL-2 mRNA gate the effect of TNF on T cell cytotoxicity, anti-TNF Abs were added (3 levels in Grp782/+ sCD8ab+ T cells after 48 h of stimulation with + mg/ml) prior to coculture. (B and C) Pooled sCD8ab T cells isolated from polyclonal anti-CD3/CD28–coated MicroBeads (Fig. 7A). ChIP . Wt and ARE mice (n 3 mice) were incubated for 18 h. TNF production analysis was used to further evaluate the binding of endogenous by ARE CD8ab+ T cells was neutralized by the addition of 3 mg/ml anti- UPR transducers to the IL-2 gene promoter in sCD8ab+ T cells. TNF Ab for 3 h prior to anti-CD3/CD28 MicroBead activation. Wt + We demonstrated increased binding of ATF6a, ATF4, and p-cJun CD8ab T cells were incubated with 10 ng/ml TNF recombinant protein + and activated by polyclonal anti-CD3/CD28 MicroBeads. Gene expression to the IL-2 promoter sites in activated Wt sCD8ab T cells 2/+ data were normalized to the expression level of GAPDH and are presented compared with Grp78 T cells (Fig. 7B). Intracellular staining as fold difference between ARE and Wt mice. Significant differences are of IL-2 (Fig. 7C) confirmed markedly reduced levels of IL-2 in 2 shown with *p , 0.05, **p , 0.01, ***p , 0.001. Grp78 /+ sCD8ab+ T cells after 48 and 72 h of stimulation compared with Wt CD8ab+ T cells (Fig. 7D). Most interestingly, the addition of exogenous rIL-2 rescued the proliferative blockade 2/+ + though freshly isolated Grp78 sCD8ab T cells exhibited an ex of spleen-derived Grp782/+ CD8ab+ T cells after 24 h of stim- vivo cytotoxicity similar to Wt T cells (Supplemental Fig. 2A), ulation (Fig. 7E). The addition of rIL-2 led to significantly in- 2/+ + Grp78 CD8ab T cells, isolated from small intestine (Fig. 5A) creased granzyme B production by Grp782/+ CD8ab+ Tcells or spleen (Fig. 5B), exhibited a significantly reduced in vitro cytotoxicity against Mode-K cells compared with Wt T cells after 4 d of coculture under TCR activation. This was attributed to the Table II. Viability of sCD8ab+ T cells with transient knock down of significantly attenuated granzyme B secretion in the same assay Grp78 by EGF-SubA treatment demonstrated for intestinal (Fig. 5C) or splenic (Fig. 5D) CD8ab+ T cells. Propidium Iodide+ Cells (%)a

b Grp78 intrinsically controls CD8ab+ T cell proliferation h Group Control EGF-SubA p Value through IL-2–mediated mechanisms 24 Wt 16.35 (16.05–17.8) 27.35 (24.75–33.3) ,0.05 DARE/+ . Because reduced cytotoxicity can also result from reduced pro- 24 TNF 28.1 (27–28.2) 28.7 (28.4–31.5) 0.05 liferation, we next sought to investigate the impact of a Grp78 CD8ab+ T cells isolated from spleen of Wt and ARE mice were incubated with + 2/+ + EGF-SubA for 24 h and stained with propidium iodide to test for viability of cells. deﬁciency on CD8ab T cell proliferation. Grp78 CD8ab aMedian (25th–75th percentiles); n = 3/group. T cells isolated from spleen exhibited a signiﬁcantly reduced bNonparametric Mann–Whitney U test. 1516 ER STRESS IN T CELL HOMEOSTASIS AND PATHOGENESIS OF IBD

FIGURE 5. Grp78 is essential for granzyme B-dependent CD8ab+ T cell cytotoxicity. Mode-K cells were cocultured with 10,000 CD8ab+ IEL (A, C) or 50,000 sCD8ab+ T cells/ well (B, D), isolated and pooled from Wt and Grp782/+ mice (n . 3), for 4 d in the presence or absence of anti-CD3/CD28 polyclonal Micro- Beads. T cell-mediated cytotoxicity against IEC was quantiﬁed by DNA-fragmentation assay (A, B), and secreted granzyme B was detected in the supernatant by ELISA (C, D). Signiﬁcant differences are shown with *p , 0.05, **p , 0.01, ***p , 0.001. Downloaded from http://www.jimmunol.org/

after 24 h of stimulation with polyclonal anti-CD3/CD28–coated from Grp782/+ mice compared with Wt mice (Fig. 8A, 8B). The MicroBeads (Fig. 7F). Importantly, the absolute amount of gran- observed reduced repopulation in the intestine was not due to zyme B secreted by Grp782/+ CD8ab+ T cells stimulated with defects in b7 integrin expression on Grp782/+ donor T cells (data MicroBeads and rIL-2 was lower than for the MicroBead- not shown). As a ﬁnal step, we generated TNFDARE/+ 3 Grp782/+ stimulated Wt T cells, arguing that both proliferation and gran- (ARE 3 Grp782/+) double-transgenic mice and evaluated histo-

zyme B secretion are attenuated by reduced levels of Grp78. pathological changes in distal ileum, as well as the cytotoxic ac- by guest on September 28, 2021 tivity of CD8ab+ T cells. Interestingly, and despite the fact that Heterozygous Grp78 deficiency results in attenuated splenic ARE 3 Grp782/+ CD8ab+ T cells had a significantly repopulation of CD8ab+ T cells in vivo but does not protect reduced cytotoxic activity against intestinal epithelial Mode-K from experimental ileitis cells (Fig. 8C), the degree of ileal pathology was not different To validate the role of Grp78 deficiency in T cell distribution to the between ARE 3 Grp782/+ and ARE mice (Fig. 8D). In addition to intestine, MLN, and spleen, we next performed adoptive transfer of the total pathology scores, no differences were observed with 6 + 2/+ 1.5 3 10 CD8ab T cells isolated from spleen of Grp78 and regard to the subscores, including leukocyte infiltration, crypt loss, 2/2 Wt mice into RAG2 recipient mice. As expected, the transfer or ulcus formation (data not shown). Together with the observa- + of CD8ab T cells alone did not induce colitis in the recipient tions that splenocytes and distal ileal tissue of ARE 3 Grp782/+ 2/2 RAG2 mice (data not shown). Flow cytometric analysis of mice showed no significant difference in Grp78 protein (Fig. 8E) 2/2 donor T cell expansion in the RAG2 recipient mice showed and Grp78 and TNF mRNA expression (data not shown) compared + a lower percentage of repopulating CD8ab T cells originating with ARE mice, the similar level of ileal pathology may suggest that compensatory mechanisms in the heterozygous genotype of ARE 3 Grp782/+ mice prevent a reduction in ileal inflammation.

Discussion The development of Crohn’s disease-like ileitis in TNFDARE/+ mice has been attributed to an aberrant CD8ab+ IEL cytotoxicity (15, 16). We demonstrate in this study that ER stress-associated UPR plays an essential role in the development of this disease-relevant cytotoxic IEL phenotype. We found strikingly increased levels of Grp78 expression in CD8ab+ IEL from inflamed ARE mice, suggesting persistent ER stress conditions in this lymphocyte compartment. CD8ab+ IEL from ARE mice exhibited a selec- FIGURE 6. Grp78 controls CD8ab+ T cell proliferation. CD8ab+ A B tive binding of downstream UPR transducers to the Grp78 and T cells isolated and pooled from spleen cells ( ) or MLN cells ( )ofWt + 2/+ . granzyme B promoters. Consistently, CD8ab T cells from and Grp78 mice (n 3) were treated or not with anti-CD3/CD28 2/+ MicroBeads for 3 d (0.5 3 106 cells/well). BrdU was added to the cells at Grp78 mice exhibited decreased granzyme B-dependent cyto- 48 h postactivation and left overnight. The cell proliferation was measured toxicity against epithelial cells, reduced proliferation, and lower based on the BrdU incorporation to the newly synthesized DNA. Signifi- efficiency with regard to repopulation of the IEL compartment. cant differences are shown with *p , 0.05, **p , 0.01, ***p , 0.001. Interestingly, heterozygous Grp78 deficiency was not effective in The Journal of Immunology 1517

FIGURE 7. CD8ab+ T cell proliferation is mediated by IL-2. (A) Isolated sCD8ab+ T cells from Wt and Grp782/+ mice (n . 3) were pooled and treated or not with anti-CD3/CD28 Micro- Beads or mitogens (50 ng/ml PMA and 1 mg/ml ionomycin) for 24, 48, or 72 h. The kinetic expression of IL-2 was quantified at the level of mRNA. Gene expression data were normalized to the expression level of GAPDH and are presented as fold difference between Grp782/+ and Wt mice. (B) ChIP analysis was performed with unstimulated sCD8ab+ T cells (3 3 106 cells), isolated and pooled from Wt and Grp782/+ mice (n . 3), to determine the IL-2 promoter regulation in T cells at 48 h post-TCR activation. The different conditions are shown on representative individual blots and were repeated three to five Downloaded from times. (C and D) Representative FACS blots are shown (C), which are used for the quantification of kinetic expression of IL-2 at the level of intracellular protein (D). (E and F) In another ex- periment, exogenous IL-2 (100 U/ml) was present or absent in a cell culture of isolated sCD8ab+ T cells, pooled from Wt and Grp782/+ mice http://www.jimmunol.org/ (n . 3), and stimulated with polyclonal anti- CD3/CD28 MicroBeads for 72 h. Cell proliferation was quantified according to the overnight BrdU incorporation to the newly synthesized DNA (E), and granzyme B secretion was detected by ELISA (F). Significant differences are shown with *p , 0.05, **p , 0.01, ***p , 0.001. by guest on September 28, 2021

reducing intestinal inflammation in ARE 3 Grp782/+ mice, sug- Grp78-mediated control of CD8ab+ T cell homeostasis is im- gesting that the remaining plasticity of T cell responses in Grp782/+ pacted at the level of two major effector mechanisms. First, an mice compensates for the partial loss of Grp78. In addition, effective CD8ab+ T cell cytotoxic activity by granzyme B se- dysregulation of ER stress-associated UPR in the epithelium cretion requires a tightly controlled signal from the Grp78- primed mice to increased disease susceptibility (29), suggesting mediated signaling cascade, which, in turn, is linked to the acti- that unconditioned Grp78 deficiency may differentially affect vation of the TCR. CD8ab+ T cells generally showed increasing tissue compartments and lead to a net compensation of effects. levels of Grp78 expression after TCR stimulation with polyclonal Nevertheless, the findings of this study strongly support the hy- anti-CD3/CD28–coated MicroBeads. This implies that the acti- pothesis that ER stress-associated UPR mechanisms contribute vation of TCR induces ER stress-associated UPR, supporting re- to an aberrant cytotoxic CD8ab+ IEL phenotype in experimental cent studies (44, 46, 47). Interestingly, the cytotoxic effector ileitis. function of Grp782/+ CD8ab+ T cells is maintained at basal levels Two recent reports attempted to identify the signaling pathways compared with Wt cells; however, the impact of Grp78 deficiency triggering Grp78 expression after TCR activation (46, 47). Takano becomes significant after TCR activation and results in reduced et al. (46) reported that this process might be regulated by the granzyme B-dependent cytotoxicity. calcineurin–NFAT pathway. This hypothesis was supported by the Moreover, low levels of IL-2 were shown to promote effector finding that Ca2+ chelators abrogated TCR-mediated Grp78 ex- T cell expansion and effector memory development (53). Effector pression. In contrast, Pino et al. (47) argued that the protein kinase CD8+ T cells are short-lived and either die or convert into memory C-signaling pathway controls TCR-mediated Grp78 expression cells after Ag exposure. This cell fate decision seems to rely on and additionally affects IL-2 expression. Consistently, we dem- the endogenous production of IL-2 by terminally differentiated onstrated that IL-2 expression and T cell proliferation are con- effector CD8+ T cells. Only memory precursor cells (KLRG1low trolled by Grp78. A deficiency in Grp78 resulted in strikingly ILR7Rahigh) that are capable of producing IL-2, as well as cyto- attenuated proliferative responses, which were rescued by the toxic (granzyme B) and effector molecules (IFN-g, TNF), differ- addition of exogenous IL-2. In addition, adoptively transferred entiate into memory cells (53). Terminally differentiated effector Grp782/+ sCD8ab+ T cells exhibited a reduced ability to repo- CD8+ T cells (KLRG1highILR7Ralow) are non–IL-2–producing pulate the intestine of RAG22/2 recipient mice, independent of cells that are eventually programmed for cell death. A recent study gut-homing factors, suggesting a role for Grp78 in the prolifera- showed that IL-2–mediated IRE1-XBP1–splicing machinery tive responsiveness in vivo. contributes to the development of terminally differentiated effec- 1518 ER STRESS IN T CELL HOMEOSTASIS AND PATHOGENESIS OF IBD

FIGURE 8. Heterozygous Grp78 deficiency results in reduced repopulation of CD8ab+ T cells in an adoptive transfer model, but it does not protect from experimental ileitis. (A and B)A total of 1.5 3 106 sCD8ab+ T cells from donor mice (Grp782/+ or Wt) was transferred to 8-wk- old RAG 22/2 recipient mice by i.p. injection (n = 6/group). After 6 wk, mice were sacrificed, and a flow cytometric analysis of expanded CD8ab+ T cells in the RAG22/2 recipient mice was performed. (C) A total of 100,000 CD8ab+ T cells/ well, pooled and isolated from spleen of Wt, ARE, and ARE 3 Grp782/+ mice (n . 3), were cocultured with the murine intestinal epithelial Mode-Kcelllinefor3dinthepresenceor absence of anti-CD3/CD28–coated polyclonal

MicroBeads. T cell-mediated cytotoxicity against Downloaded from IEC was quantified by DNA-fragmentation assay. (D) Histopathological analysis of distal ileum of 18-wk-old ARE 3 Grp782/+ mice revealed no difference compared with ARE mice with regard to intestinal inflammation. (E) Western blot analysis of Grp78 showed no difference between 2/+ single ARE and ARE 3 Grp78 mice with http://www.jimmunol.org/ regard to Grp78 protein expression in splenocytes and ileal tissue. Significant differences are shown with *p , 0.05, **p , 0.01, ***p , 0.001.

tor CD8+ T cells in vivo (44). Additionally, we showed that ex- duced cytotoxicity in vitro compared with ARE CD8ab+ T cells, by guest on September 28, 2021 ogenous IL-2 rescued the cytotoxic phenotype of CD8ab+ T cells tissue pathology was not affected in ARE 3 Grp782/+ mice under Grp78 deficiency. However, and in contrast to proliferation, compared with ARE mice. Interestingly, there is no difference in the presence of exogenous IL-2 failed to induce a complete rescue Grp78 protein and mRNA expression levels detectable between at the level of granzyme B expression. Although this may argue ARE 3 Grp782/+ and ARE CD8ab+ T cells, indicating that for a contribution of both reduced proliferation and diminished Grp78 heterozygosity is compensated by the remaining T cells’ granzyme B secretion to the impaired cytotoxicity of Grp782/+ plasticity. The reason for this remains unclear, but TNF was shown CD8ab+ T cells in response to TCR activation, the granzyme B to improve the responsiveness of CD8+ T cells to IL-2–mediated secretion of Wt CD8ab+ T cells was also diminished. A study by proliferation and to enhance IL-2–induced expression of the IL-2R Kambayashi et al. (54) showed that the CD8b-chain is downreg- of CD8+ T cells (55, 56), supporting the hypothesis that T cells ulated in the presence of IL-2, even in concentrations as low as 10 from a heterozygous Grp78 deficiency may partially maintain U/ml. IL-2–stimulated CD8+ T cells have a decreased affinity for their effector functions. Indeed, we also observed a similar pro- MHC class I–peptide complexes, so the downregulation of CD8b liferation of ARE 3 Grp782/+ sCD8ab+ T cells compared with expression in IL-2–stimulated T cells may compromise the ability ARE cells (data not shown), suggesting that an enhanced prolif- of T cells to respond to their cognate Ags and, subsequently, eration of ARE 3 Grp782/+ CD8ab+ T cells due to TNF over- attenuates granzyme B secretion. production may abolish the effects of a Grp78 deficiency. In the complex scenario of intestinal immune homeostasis, the Although TNF plays a role in the induction of the aberrant cyto- importance of CD8+ IEL was recently highlighted (11, 15, 53). In toxic phenotype of ARE CD8ab+ T cells, it is not implicated in the small intestine of Wt mice, the majority of IEL harbor the IL- the short-term cytotoxicity, as reflected by the similar ex vivo 10–producing CD8aa+ T cell phenotype (.70%), whereas con- cytotoxicity of ARE CD8ab+ T cells with TNFR blocked by anti- ventional CD8ab+ IEL account for ∼20% of all IEL. IL-10 was TNF Abs. Consistent with our results, a recent study by Kollias shown to inhibit TNF-induced Grp78 expression in IEC by and colleagues (57) demonstrated that TNF overproduction spe- modulating ATF6 nuclear recruitment to the Grp78 promoter cifically by IEC in the TNFDARE/+ mouse model is sufficient to (28). Consistently, the loss of IL-10–secreting CD8aa+ IEL in induce mucosal pathology. We demonstrated that this localized TNFDARE/+ mice is associated with aberrant Grp78 expression scenario in the ileum induces a complex immunopathology, which in the disease-relevant CD8ab+ IEL exerting aberrant cytolytic is triggering an excessive cytotoxic response of CD8ab+ IEL function. This intriguing observation raises the question whether maintained by the UPR. the aberrant cytotoxicity of ARE CD8ab+ IEL due to unresolved In summary, this study identifies the ER stress-associated ER stress with high levels of Grp78 expression can be antagonized chaperone Grp78 as a critical factor that intrinsically mediates under conditions of Grp78 deficiency. Although ARE 3 Grp782/+ intestinal T cell homeostasis. In the TNFDARE/+ mouse model of CD8ab+ T cells isolated from spleen showed a significantly re- Crohn’s disease-like ileitis, unresolved ER stress, reflected by The Journal of Immunology 1519 high levels of Grp78, accounts for the development and mainte- junction permeability requires NF-kappa B activation. Am. J. Physiol. Gastro- + intest. Liver Physiol. 286: G367–G376. nance of a pathogenic cytotoxic CD8ab IEL phenotype. This 19. Simms, L. A., J. D. Doecke, M. D. Walsh, N. Huang, E. V. Fowler, and + CD8ab IEL phenotype exerts aberrant cytolytic function that G. L. Radford-Smith. 2008. Reduced alpha-defensin expression is associated further causes IEC death and, thus, contributes to the mucosal with inflammation and not NOD2 mutation status in ileal Crohn’s disease. Gut 57: 903–910. immunopathology in chronic intestinal inflammation. Although 20. Elson, C. O., Y. Cong, V. J. McCracken, R. A. Dimmitt, R. G. Lorenz, and + a Grp78 deficiency in CD8ab T cells was shown to result in C. T. Weaver. 2005. Experimental models of inflammatory bowel disease reveal attenuated granzyme B-mediated cytotoxicity and reduced T cell innate, adaptive, and regulatory mechanisms of host dialogue with the microbiota. Immunol. Rev. 206: 260–276. proliferation, it does not protect from intestinal inflammation in 21. Cruickshank, S. M., L. D. McVay, D. C. Baumgart, P. J. Felsburg, and experimental ileitis, thus not completely unraveling the role of S. R. Carding. 2004. Colonic epithelial cell mediated suppression of CD4 T cell unresolved ER stress in CD8ab+ T cells under chronic intestinal activation. Gut 53: 678–684. 22. Hatoum, O. A., J. Heidemann, and D. G. Binion. 2006. The intestinal micro- inflammation. vasculature as a therapeutic target in inflammatory bowel disease. Ann. N. Y. Acad. Sci. 1072: 78–97. 23. Caballero, T., F. Nogueras, M. T. Medina, M. D. Caracuel, C. de Sola, Acknowledgments F. J. Martıńez-Salmeroń, M. Rodrigo, and R. Garcıá del Moral. 1995. 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