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of June 18, 2017.
Endoplasmic Reticulum Stress Response
Promotes Cytotoxic Phenotype of CD8 αβ+
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 and Dirk
Haller
Supplementary
Material
http://www.jimmunol.org/content/suppl/2012/07/02/jimmunol.120016
6.DC1
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Copyright © 2012 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol published online 2 July 2012
http://www.jimmunol.org/content/early/2012/06/29/jimmun
ol.1200166
Published July 2, 2012, doi:10.4049/jimmunol.1200166
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*
I
nflammatory bowel disease (IBD) is a chronically relapsing
inflammatory pathology largely restricted to the small intestine and colon (1). Because of a very heterogeneous
pathogenesis, factors triggering IBD include host genetic predisposition (2, 3), environmental factors (4–6), commensal bacteria
as well as infectious triggers (7, 8), and a deregulated intestinal
T cell homeostasis (9). The balance of regulatory and effector
T cells is disturbed in Crohn’s disease, with more naive T cells
differentiating into cytotoxic effector T cells (10–15). This process
likely results in an excessive cytotoxic response (15, 16), increased permeability of the epithelial barrier (17, 18), continuous
epithelial erosion, and reduced production of defensins (19). In
*Chair for Biofunctionality, Research Centre for Nutrition and Food Science,
Centre for Diet and Disease, Technical University of Munich, 85350 FreisingWeihenstephan, Germany; †School of Nutrition and Health Science, Taipei Medical
University, Taipei 110, Taiwan, Republic of China; ‡Chair for Biological Chemistry,
Technical University of Munich, 85350 Freising-Weihenstephan, Germany; and
x
Department of Biochemistry and Molecular Biology, Keck School of Medicine,
University of Southern California, Los Angeles, CA 90033
Received for publication January 13, 2012. Accepted for publication May 29, 2012.
This work was supported by Deutsche Forschungsgemeinschaft Grant HA 3148/2-1
and by the Munich Center of Integrated Protein Science.
Address correspondence and reprint requests to Prof. Dirk Haller, Chair for Biofunctionality, Research Centre for Nutrition and Food Science, Centre for Diet and Disease, Technical University of Munich, Gregor-Mendel-Strasse 2, 85350 FreisingWeihenstephan, Germany. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: ARE, AU-rich element; ARE mice, TNFDARE/+
mice; ChIP, chromatin immunoprecipitation; ER, endoplasmic reticulum; Grp78,
glucose-regulated protein 78; IBD, inflammatory bowel disease; IEC, intestinal epithelial cell; IEL, intraepithelial lymphocyte; LPL, lamina propria lymphocyte; MLN,
mesenteric lymph node; qPCR, quantitative RT-PCR; sCD8ab+ T cell, splenic
CD8ab+ T cell; siRNA, small interfering RNA; UPR, unfolded protein response;
Wt, wild type.
Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1200166
turn, commensal bacteria cross the epithelial cell layer (20),
leading to an accelerated activation of effector T cells (21) and
increased recruitment of inflammatory cells into the intestinal
mucosa (22).
Analysis of human biopsy samples from Crohn’s disease patients
identified CD8+ T cells as the predominant T cell phenotype in the
epithelium (23). Colitogenic CD4+ T cells were predominantly
identified in the lamina propria lymphocyte (LPL) compartment of
the inflamed intestinal mucosa (23). Interestingly, adoptive transfer
of naive CD4+ T cells into an immunodeficient recipient host induced colonic inflammation (24, 25), which was prevented by the
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
response (UPR) of the endoplasmic reticulum (ER) contributes to
intestinal inflammation and the pathogenesis of IBD (1, 27–30).
Dissociation of ER-resident chaperone glucose-regulated protein
78 (Grp78) from the membrane-anchored receptors, such as IRE1,
PERK, and ATF6, initiates UPR signal transduction (1, 31, 32),
leading to a selective activation or downregulation of gene expression by downstream transcription factors ATF4, XBP1, and
ATF6. Being the master regulator of ER stress signaling, the induction of Grp78 expression is required to alleviate ER stress (33,
34). Grp78 expression levels are increased by ER stress stimuli
(28, 35–37), and cleavage of Grp78 protein by subtilase cytotoxin
leads to an inappropriate ER stress response, triggering cell death
(38). ER stress-associated UPR signals are increased in the intestinal epithelium during chronic inflammation (28) and sensitize
secretory epithelial cells to cell death (30). A deletion of one or
two alleles of XBP1, a downstream transcription factor of the
IRE1 pathway, resulted in spontaneous inflammation of the small
intestine, T cell infiltration, and Paneth cell dysfunction (29), as
well as altered composition of microbiota (39). Although accu-
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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 immunoprecipitation 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
pathology in TNFDARE/+ 3 Grp782/+ mice was similar to TNFDARE/+ mice, even though the cytotoxic effector functions of CD8ab+
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: 000–000.
2
ER STRESS IN T CELL HOMEOSTASIS AND PATHOGENESIS OF IBD
Materials and Methods
Mice
ARE mice on a C57BL/6 background and wild-type (Wt) littermates were
a generous gift from Dr. G. Kollias (Institute for Immunology, Biomedical
Sciences Research Center “Al. Fleming”, Varkiza, Greece). The generation
of Grp78 heterozygous (Grp782/+) mice and Wt littermates was described
previously (50). Grp782/+ mice on a C57BL/6 or JV129 background
and Wt littermates were kindly provided by A.S.L. (50). TNFDARE/+ 3
Grp782/+ double transgenic mice were generated by breeding TNFDARE/+
mice into Grp782/+ on a C57BL/6 background. RAG22/2 mice on
a JV129 background (51), which lack functional lymphocytes owing to
their inability to initiate V(D)J rearrangement, were purchased from
Taconic. All mice were raised in a conventional manner in the animal
facility at the Technical University of Munich-Weihenstephan as approved
by the institution in charge (approval no. 55.2-1-S4-2531-74-06 and 322347/4+63, tested by meeting the requirements of the Federation of European Laboratory Animal Science Associations showing positive results
for Helicobacter typhlonius, Norovirus, and Trichomonas spp.). Mice were
killed by cervical dislocation at the age of 8, 18, or 24 wk. Tissue sections
of distal ileum were fixed in 10% neutral-buffered formalin. Histopathological changes were scored in paraffin-embedded ileal sections in a blinded fashion, as previously described (52).
Isolation of primary T cells from small intestine and spleen
Spleen and small intestine were removed and kept on ice. The small intestine was cut open longitudinally, and feces were washed off. IEL were
released by incubation of the tissue pieces with digestion buffer (5 mM
EDTA, 1 mM DTT, 10% FBS, RPMI 1640) two times for 20 min. LPL were
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.
Pooled IEL from each group were placed on ice for 10 min to separate the
tissue debris from cells. Supernatant was carefully removed and centrifuged at 350 3 g, 4˚C, for 5 min. Pellets were resuspended in 2 ml FACS
buffer (5% FBS, 2 mM EDTA, PBS) and applied onto a nylon wool fiber
column (Polysciences, Eppelheim, Germany) to remove adherent cells and
tissue debris. Pooled IEL and LPL (n = 6 mice/group) were sorted into
the single phenotype, with a purity of 95–98%, by FACSAria (BD Biosciences, Franklin Lakes, NJ). CD8ab+ T cells were isolated from pooled
spleen by indirect positive selection through a magnetic column, resulting
in purities between 90 and 98% (Miltenyi Biotec, Bergisch Gladbach,
Germany).
Flow cytometry
Splenic T cells, IEL, and LPL (105 cells) were used for immunophenotyping by FACS analysis. Abs used for the six-color surface staining were
CD3-allophycocyanin-Cy7 (BD Pharmingen, Franklin Lakes, NJ), CD8aallophycocyanin (Miltenyi Biotec), CD8b-FITC (BD Pharmingen), CD4PE-Cy7 (BD Pharmingen), TCRab-PE (Serotec, Düsseldorf, Germany),
and CD44-PerCP-Cy7 (BD Pharmingen). Intracellular production of IL-2,
IFN-g, Bcl-2, and granzyme B (eBioscience, San Diego, CA) was determined after incubation of T cells with 1 mg/ml brefeldin A for the last 3–4
h of in vitro culture. At 48 h postactivation, intracellular proteins were
detected by intracellular staining in combination with surface expression of
CD3+, together with CD8b2 or CD8b+, according to the BD Cytofix/
Cytoperm and GolgiPlug protocol (all Abs were from BD Pharmingen,
with the exception of granzyme B). To test for viability, T cells were incubated with propidium iodide (50 mg/ml) for 30 min and subsequently
acquired by flow cytometry. A total of 10,000 cells was acquired from the
LSR II (BD Biosciences) and analyzed using BD FACSDiva software.
Chromatin immunoprecipitation analysis
Unstimulated splenic CD8ab+ T cells (sCD8ab+ T cells; 3 3 106 cells),
isolated and pooled from mice (n . 3), were used for chromatin immunoprecipitation (ChIP) analysis, according to the ChIP-IT Express kit
instructions (Active Motif, La Hulpe, Belgium). Briefly, isolated CD8ab+
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
polyclonal anti-CD3/CD28 MicroBeads and with or without 100 U/ml rIL2 protein (R&D Systems, Minneapolis, MN). DNA-bound protein was
cross-linked with 1% formaldehyde for 10 min at room temperature. Enzymatic DNA shearing was performed in a 37˚C water bath for 15 min.
Sheared chromatin (300–100 bp) was immunoprecipitated with specific
Abs against ATF4, ATF6a, and XBP1 (all from Santa Cruz Biotechnology,
Santa Cruz, CA) and Abs against acetyl histone 3 (lysine 9), YY1, and
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
quantitative RT-PCR (qPCR) was DNA from total nuclear extract without
immunoprecipitation. qPCR was performed with total DNA (input control;
1 ml) and immunoprecipitated DNA (1 ml). The putative promoter regions
were evaluated using the Gene2Promoter program from Genomatix (http://
www.genomatix.de), and primers were designed using the Primer3 Web
site (http://frodo.wi.mit.edu/). Details of primer sequences and amplicon
length are listed in Supplemental Table I.
RNA isolation, reverse transcription, and qPCR
Total RNA was extracted using an RNeasy Mini Kit (QIAGEN, Hilden,
Germany). RNA yield and quality were assessed by absorbance using a
Nanodrop ND-1000 spectrophotometer (LabTech International, Brampton,
ON, Canada). A total of 200–500 ng RNA was used for reverse transcription using the SuperScript III First-Strand system (Invitrogen, Darmstadt, Germany). Primers and probes were designed using the universal
probe library (Roche, Mannheim, Germany) or the Primer3 Web site (http://
frodo.wi.mit.edu/). Details on primer concentration and sequences are
shown in Supplemental Table I. Amplicon sizes ranged between 90 and
230 bp to ensure high amplification efficiency. GAPDH was selected as the
reference gene in this study because of its stability in response to antiCD3/CD28 and ionomycin/PMA stimulation. RNA-expression profiles of
both target and reference genes were performed using the LightCycler
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mulating evidence suggests that unresolved ER stress contributes
to the mucosal pathogenesis in IBD, little is known about the
impact of unresolved ER stress on T cell homeostasis under
chronic inflammatory conditions.
Several lines of evidence suggest that ER stress-associated UPR
signals are involved in the maintenance of lymphocyte homeostasis and viability. First, Grp78 was shown to be noncovalently
bound to the Ig H chain (40) or the nonmatured TCRa-chain
within the ER (41). TCRa-deficient mice lack functional CD4
and CD8 T cells (42), implying a critical activity of TCRa
maturation on conventional T cell function. Moreover, it was
demonstrated that XBP1 facilitated the transition of CD8ab+
effector T cells into terminally differentiated CD8ab+ T cells
(43, 44). Franco et al. (45) reported that ER stress is also involved in the induction and plastic differentiation of regulatory
peripheral T cells. Recent studies indicated that Grp78 expression
in conventional CD4+ or CD8ab+ T cells is induced by CD3:TCR
activation or mitogenic stimulation (46, 47). This was shown to
be mediated by protein kinase C, because blocking of protein
kinase C resulted in diminished ER stress response and reduced
IL-2 synthesis in T cells (47). A knock down of Grp78 protein
induced apoptosis in a murine T cell line (46). Consistently,
Grp78 is imputed to have an antiapoptotic effect due to inhibition of the apoptosis-triggering pathways of UPR (48), and
it is constitutively expressed, with enhanced expression due to
stressful conditions like glucose deprivation, calcium depletion
in the ER, oxidative stress, and hypoxia (31, 48, 49). In summary, these data suggest a critical role for Grp78 and ER stressassociated UPR signals in the homeostasis and viability of
T cells.
To investigate the effects of ER stress-associated mechanisms
on T cell homeostasis under chronic intestinal inflammation, we
performed experiments in a mouse model that is characterized
by the genetic ablation of AU-rich elements (ARE) from the
TNF mRNA, leading to overproduction of TNF (15, 16). These
TNFDARE/+ mice (ARE mice) show an aberrant T cell homeostasis and spontaneously develop Crohn’s disease-like transmural tissue pathology in the distal ileum. Most interestingly,
the mucosal pathology of this model is attributed to the presence of pathogenic cytotoxic CD8ab+ IEL that preferentially
accumulate in the epithelium (15). In this study, we investigated the effects of ER stress-associated signals on the homeostasis of intestinal CD8ab+ T cells under chronic intestinal
inflammation.
The Journal of Immunology
(Roche) at 10 ml/PCR reaction (400 nM primers and/or 200 nM probe
concentration, 1 ml cDNA, QuantiTect Probe RT-PCR Master Mix buffer).
SYBR Green or QuantiTect Probe Master Mix (Roche) was used for target
gene quantification. The relative induction of mRNA expression was calculated using the following equation ECp (control mice 2 ARE mice) and normalized for the expression of GAPDH.
Western blot analysis
Freshly isolated, unstimulated cells were lysed in lysis buffer, and protein
concentration was quantitated by Bradford (Carl Roth, Karlsruhe, Germany). Protein lysates were boiled in 13 SDS buffer at 95˚C for 10 min.
Equal amounts of proteins were resolved on a 10% SDS-polyacrylamide
gel and transferred by electroblotting to a nitrocellulose membrane.
Membranes were probed with Abs, as indicated, and the specific signals
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
to detect immunoreactive Grp78 and b-actin, respectively. Anti-rabbit
secondary Abs were used to detect the primary Abs.
Cell culture
Cell-proliferation assay
CD8ab+ T cell and MLN cell proliferation was measured by a colorimetric immunoassay based on the measurement of BrdU incorporation
during DNA synthesis (Roche); 10 mM/ml BrdU was added to the
CD8ab+ T or mesenteric lymph node (MLN) cells (0.5 3 106 cells/well) at
48 h postactivation and left overnight. On day 3 postactivation, cells were
fixed in FixDenat to facilitate DNA denaturation. Peroxidase-labeled anti–
BrdU Abs that recognize BrdU-labeled DNA generated a photometrically
detectable signal that was measured at 450 nm in an ELISA plate reader.
In vitro stimulation experiments were performed in four to six wells for
each stimulus per time point.
Grp78 knock down using EGF-SubA fusion protein or small
interfering RNA
A total of 1 3 106 sCD8ab+ T cells was cultured with 100 pM EGF-SubA
(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
transfected with 400 nM Grp78 and a start negative control small interfering RNA (siRNA) duplex (QIAGEN) by nucleofection using a mouse
Nucleofactor Kit (Lonza, Visp, Switzerland). The transfection efficiency
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
harvested 24 h poststimulation.
T cell-mediated cytotoxicity
T cell-mediated cytotoxicity against the murine small intestinal epithelial
cell (IEC) line, Mode-K, was performed by CytoTox 96 Non-Radioactive
Cytotoxicity Assay (Promega, Mannheim, Germany) or a DNAfragmentation 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
of 2.5:1, with or without TCR cross-linking. Ex vivo cytotoxicity was
measured based on the released lactate dehydrogenase in the cell culture
supernatant upon cell lysis. Alternatively, long-term CD8ab+ T cellmediated cytotoxicity against confluent Mode-K was assessed after
4 d of coculture by a DNA-fragmentation assay. In this system, 7.5 3 103
Mode-K cells were seeded in 500 ml Mode-K cell culture medium containing 10 mM/ml BrdU in a 24-well plate, reaching 60–70% confluence
overnight. BrdU was allowed to incorporate into proliferating Mode-K
cells overnight prior to the addition of primary T cells or IEL. Unless
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
4 d in the presence or absence of anti-CD3/CD28 MicroBeads. T cellmediated killing of Mode-K cells was measured by DNA-fragmentation
assay, and granzyme B concentration in the cell culture supernatant was
quantified using a granzyme B-specific ELISA kit (R&D Systems). Ex
vivo and in vitro stimulation experiments were performed in four to six
wells for each stimulus per time point.
Adoptive transfer
A total of 1.5 3 106 sCD8ab+ T cells isolated from donor mice (Grp782/+
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 background by i.p. injection. RAG22/2 mice were killed by cervical dislocation
6 wk after adoptive transfer.
Data analysis
The nonparametric Mann–Whitney-U test was used to analyze differences
between two genotypes. The paired Student t test was used to analyze the
differences in response to treatment within the same group. Time-course
experiments were analyzed by one-way ANOVA with Bonferroni posttests
and correction or by one-way ANOVA and the Tukey t test for qPCR timecourse experiments. Analysis was conducted using Prism 4.03 software
(GraphPad). Differences were considered significant if p values were ,
0.05, and data are presented either as mean 6 SEM or median (25th–75th
percentiles). Unless otherwise noted, all data are representative of a minimum of three independent experiments.
Results
Induction of ER stress-associated UPR in disease-relevant
CD8ab+ IEL under conditions of Crohn’s disease-like ileitis
To investigate the induction of ER stress-associated UPR in the
small intestine under chronic inflammatory conditions, we used
inflamed ARE mice and Wt littermate control mice at the age of
8 and 18 wk. H&E staining of representative paraffin-embedded
distal ileal segments showed increased total histopathological
scores, which were determined by assessing infiltration of immune
cells, crypt hyperplasia, and villus atrophy, in ARE mice at 8 and
18 wk of age compared with Wt mice (Fig. 1A, 1B). The development of ileal inflammation in ARE mice was associated with
phenotypic changes in the IEL and LPL fractions (Table I),
resulting in a significantly increased ratio of cytotoxic CD8ab+/
CD8aa+ IEL in 18-wk-old ARE mice (median: 0.57; 25th–75th
percentiles: 0.53–0.75) compared with Wt mice (median: 0.37;
25th–75th percentiles: 0.34–0.39, p , 0.01).
We next sought to characterize ER stress-associated UPR gene
expression in isolated IEL and LPL from the small intestine of ARE
mice compared with Wt mice at the age of 18 wk. Interestingly, we
detected increased mRNA expression of the ER stress chaperone
Grp78 specifically in the CD8ab+ IEL, but not in the CD8aa+
IEL, from ARE mice (Fig. 1C). Increased Grp78 transcript levels
in ARE CD8ab+ IEL were associated with upregulated UPR
signal transducers ATF4, ATF6, CHOP, and sXBP1. In contrast
to IEL subpopulations, LPL exhibited only minor changes with
regard to the expression levels of Grp78 and UPR signal transducers (Fig. 1C). Focusing on the IEL compartment, we next investigated the gene expression of IFN-g, granzyme B, and Bcl-2
of isolated ARE CD8ab+ IEL. As shown in Fig. 1D, ARE
CD8ab+ IEL exhibited markedly increased transcript levels for
IFN-g, granzyme B, and Bcl-2 under conditions of experimental
ileitis. The differences observed in the expression profiles of ER
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Unless otherwise stated, the density of CD8ab+ T cells used in cell culture
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
10,000–50,000 CD8ab+ T cells for DNA-fragmentation assay. CD8ab+
T cells were cultured in T cell culture medium (RPMI 1640, 10% FBS,
2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin; all from
Life Technologies, Darmstadt, Germany) at 37˚C, 5% CO2 in the presence or absence of ionomycin/PMA (1 mg/ml ionomycin and 50 ng/ml
PMA; Sigma-Aldrich) and anti-CD3/CD28 Ab-coated MicroBeads (2:1
MicroBeads/T cell ratio; Miltenyi Biotec). In some experiments, CD8ab+
T cells were treated or not with stimuli, including 10 ng/ml TNF (R&D
Systems), 3 mg/ml anti-TNF mAb (TN3-19.12 clone; Abcam, Cambridge,
U.K.), or 100 U/ml murine rIL-2 (R&D Systems), for various lengths of
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 penicillin, 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
reached 60–70% confluence after 24 h of incubation (7.5 3 103 cells in
500 ml Mode-K cell culture medium). All in vitro experiments were performed using a minimum of three wells/treatment.
3
4
ER STRESS IN T CELL HOMEOSTASIS AND PATHOGENESIS OF IBD
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
stress-associated genes were not related to the level of CD44+
expression, because CD8ab+ IEL from Wt and ARE mice had
similar levels of CD44+ IEL (Fig. 1E).
TCR activation triggers UPR signaling
The cytotoxic phenotype of ARE CD8ab+ T cells is not directly
dependent on TNF
Considering the enhanced expression levels of UPR signal transducers and the ER stress chaperone Grp78 in CD8ab+ IEL from
ARE mice, we next investigated the mechanisms of ER stressassociated UPR induction and functional consequences in ARE
CD8ab+ T cells derived from spleen. Similar to CD8ab+ IEL
from ARE mice, ARE-derived sCD8ab+ T cells showed an increased baseline level of Grp78 gene expression compared with
Wt T cells (Fig. 2A). Because the transcriptional activation of
Grp78 is widely regarded as an important event in the onset of
UPR, we tested whether TCR cross-linking activates the ER stress
response in vitro. ARE and Wt mice-derived sCD8ab+ T cells
showed significantly higher expression of Grp78 after stimula-
To determine the impact of TNF overproduction on the pathogenic
cytotoxic CD8ab+ T cell phenotype, we performed an ex vivo cytotoxicity assay with added anti-TNF Abs. ARE sCD8ab+ T cells
show significantly increased cytotoxicity against Mode-K cells,
independent of intrinsic TNF overproduction (Fig. 3A). Consistently, the addition of exogenous TNF, in combination with antiCD3/CD28 MicroBeads, enhanced the cytotoxicity of Wt sCD8ab+
T cells, but anti-TNF Abs did not attenuate the expression level of
granzyme B in ARE sCD8ab+ T cells after 18 h of incubation (Fig.
3B). Moreover, the addition of exogenous TNF to Wt sCD8ab+
T cells and of anti-TNF Abs to ARE sCD8ab+ T cells showed
minimum effects on Grp78 expression (Fig. 3C) after 18 h of in-
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FIGURE 1. Induction of ER stress response in disease-relevant CD8ab+
IEL from Crohn’s disease-like TNFDARE/+ mice. (A) Histopathological
analysis revealed an inflamed distal ileum in ARE mice compared with 8and 18-wk-old Wt mice (n = 6/group). (B) Representative H&E-stained
ileal tissue segments from Wt and ARE mice (original magnification
3100). Quantitative gene expression analysis of Grp78 and UPR transducers (C) and T cell effector molecules (IFN-g, granzyme B, Bcl-2) (D) in
freshly isolated and unstimulated pooled CD8aa+ IEL, CD8ab+ IEL,
CD4+ LPL, and CD8+ LPL from 18-wk-old ARE mice. Gene expression
data were normalized to the expression level of GAPDH and are presented
as fold difference between ARE and Wt mice. (E) Flow cytometric analysis
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
a representative graph from individual mice and are presented as percentage under the gated lymphocyte region. Significant differences are
shown with *p , 0.05, **p , 0.01, ***p , 0.001.
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
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
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 proinflammatory (granzyme B) and antiapoptotic molecules (Bcl-2)
(Fig. 2E). This suggests a greater cytotoxic capacity for ARE
mice-derived CD8ab+ T cells and was confirmed in an ex vivo
cytotoxicity assay: ARE CD8ab+ T cells, activated with antiCD3/CD28–coated MicroBeads, showed a significantly increased
cytotoxicity against an IEC line (Mode-K cells) compared with
Wt CD8ab+ T cells (Fig. 3A).
To investigate the impact of Grp78 on downstream UPR-signal
transducers, we performed a gene-expression analysis of sCD8ab+
T cells from Wt mice treated with siRNA to transiently knock
down Grp78. As a result of the siRNA treatment, the expression
levels of Grp78, as well as ATF4, sXBP1, and ATF6, were significantly reduced in Wt CD8ab+ cells after 24 h (Supplemental
Fig. 1A–D). By stimulating Wt CD8ab+ T cells with ionomycin/
PMA for 24 h post-siRNA treatment, we were able to show that
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
UPR signaling and cytotoxic effector functions are mediated by
Grp78.
The Journal of Immunology
5
Table I. Immunophenotypic changes of IEL and LPL in the small intestine of Crohn’s disease-like TNFDARE/+
mice
Phenotype
Wk
Wt Micea
TNFDARE+/2 Micea
p Valueb
IEL
CD3+CD8aa+
IEL
CD3+CD8ab+
IEL
CD3+CD4+
8
18
8
18
8
18
18
18
18
18
8
18
9.9 (9.4–10.2)
24.6 (19.3–31.4)
3.4 (3.0–3.8)
8.7 (7.3–11.7)
1.8 (1.65–1.85)
6.75 (4.1–10.7)
53.8 (38.3–57.4)
27.9 (22.1–33.3)
25.6 (24.4–29.2)
15.1 (14–20.1)
3.3 (2.8–4.4)
4.45 (2.9–6.3)
3.6 (3.5–3.6)
15.6 (12.8–20.2)
0.85 (0.7–1.0)
9.8 (8.3–14.3)
1.1 (0.8–1.2)
3.7 (3.1–5.4)
15.8 (13.6–20.3)
56.8 (55.5–64.7)
55.2 (47.3–70)
14 (12.8–20.5)
2.6 (2.5–2.7)
10.5 (9.8–10.9)
,0.05
,0.01
.0.05
.0.05
.0.05
.0.05
,0.01
,0.01
,0.01
.0.05
.0.05
,0.01
Immune Compartment
IELc
IELc
Splenocytesc
Splenocytes
LPL
CD8aa+CD44+
CD8ab+CD44+
CD8ab+CD44+
CD3+CD4+
CD3+CD4+
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.
a
Median (25th–75th percentiles); n = 6/group/time point.
b
Nonparametric Mann–Whitney U test.
c
Gated under CD3+CD8+.
Grp78 is required for granzyme B-dependent CD8ab+ T cell
cytotoxicity
Increased levels of Grp78 and UPR signal transducers are associated with higher levels of intracellular granzyme B and increased
cytotoxicity against Mode-K cells. Thus, we next investigated the
effect of Grp78 on cytotoxic effector functions. First, a transient
knock down of intracellular Grp78 protein by endotoxin EGFSubA in ARE CD8ab+ T cells from spleen (Fig. 4A) resulted in
the depletion of basal granzyme B secretion (Fig. 4B), suggesting
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.
that the presence of Grp78 is required for the granzyme Bmediated cytotoxicity of CD8ab+ T cells. In contrast, Wt
sCD8ab+ T cells showed no significant changes in granzyme B
secretion after EGF-SubA–mediated Grp78 knock down (Fig. 4B).
The transient knock down of Grp78 by EGF-SubA induced similar
rates of apoptosis in ARE CD8ab+ T cells compared with untreated ARE CD8ab+ T cells at 24 h post-knock down (Fig. 4C,
Table II). Interestingly, sCD8ab+ T cells from Wt mice were more
susceptible to apoptosis than were T cells from ARE mice after
Grp78 knock down (Fig. 4C).
Next, we performed a set of experiments with isolated CD8ab+
T cells from a Grp78-deficient mouse model (Grp782/+). Al-
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cubation, suggesting that the pathogenic cytotoxic phenotype of
ARE CD8ab+ T cells is not directly maintained by TNF.
6
ER STRESS IN T CELL HOMEOSTASIS AND PATHOGENESIS OF IBD
FIGURE 3. Cytotoxic phenotype of ARE CD8ab+ T cells is independent of TNF. (A) Ex vivo cytotoxicity of sCD8ab+ T cells pooled from Wt
and ARE mice (n . 3) against murine epithelial Mode-K cells was measured at an E:T cell ratio of 2.5:1 after 4 h of TCR activation by anti-CD2/
CD28 MicroBeads using a nonradioactive cytotoxicity assay. To investigate the effect of TNF on T cell cytotoxicity, anti-TNF Abs were added (3
mg/ml) prior to coculture. (B and C) Pooled sCD8ab+ T cells isolated from
Wt and ARE mice (n . 3 mice) were incubated for 18 h. TNF production
by ARE CD8ab+ T cells was neutralized by the addition of 3 mg/ml antiTNF Ab for 3 h prior to anti-CD3/CD28 MicroBead activation. Wt
CD8ab+ T cells were incubated with 10 ng/ml TNF recombinant protein
and activated by polyclonal anti-CD3/CD28 MicroBeads. Gene expression
data were normalized to the expression level of GAPDH and are presented
as fold difference between ARE and Wt mice. Significant differences are
shown with *p , 0.05, **p , 0.01, ***p , 0.001.
though freshly isolated Grp782/+ sCD8ab+ T cells exhibited an ex
vivo cytotoxicity similar to Wt T cells (Supplemental Fig. 2A),
Grp782/+ CD8ab+ T cells, isolated from small intestine (Fig. 5A)
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
significantly attenuated granzyme B secretion in the same assay
demonstrated for intestinal (Fig. 5C) or splenic (Fig. 5D) CD8ab+
T cells.
Grp78 intrinsically controls CD8ab+ T cell proliferation
through IL-2–mediated mechanisms
Because reduced cytotoxicity can also result from reduced proliferation, we next sought to investigate the impact of a Grp78
deficiency on CD8ab+ T cell proliferation. Grp782/+ CD8ab+
T cells isolated from spleen exhibited a significantly reduced
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
previous findings, sCD8ab+ T cells and MLN cells from Grp782/+
mice showed significantly reduced levels of granzyme B secretion
(data not shown).
Gene expression analysis revealed decreased IL-2 mRNA
levels in Grp782/+ sCD8ab+ T cells after 48 h of stimulation with
polyclonal anti-CD3/CD28–coated MicroBeads (Fig. 7A). ChIP
analysis was used to further evaluate the binding of endogenous
UPR transducers to the IL-2 gene promoter in sCD8ab+ T cells.
We demonstrated increased binding of ATF6a, ATF4, and p-cJun
to the IL-2 promoter sites in activated Wt sCD8ab+ T cells
compared with Grp782/+ T cells (Fig. 7B). Intracellular staining
of IL-2 (Fig. 7C) confirmed markedly reduced levels of IL-2 in
Grp782/+ 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
of spleen-derived Grp782/+ CD8ab+ T cells after 24 h of stimulation (Fig. 7E). The addition of rIL-2 led to significantly increased granzyme B production by Grp782/+ CD8ab+ T cells
Table II. Viability of sCD8ab+ T cells with transient knock down of
Grp78 by EGF-SubA treatment
Propidium Iodide+ Cells (%)a
h
Group
Control
EGF-SubA
p Valueb
24
Wt
16.35 (16.05–17.8) 27.35 (24.75–33.3) ,0.05
28.1 (27–28.2)
28.7 (28.4–31.5) .0.05
24 TNFDARE/+
CD8ab+ T cells isolated from spleen of Wt and ARE mice were incubated with
EGF-SubA for 24 h and stained with propidium iodide to test for viability of cells.
a
Median (25th–75th percentiles); n = 3/group.
b
Nonparametric Mann–Whitney U test.
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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 postknock 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.
The Journal of Immunology
7
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 MicroBeads. 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.
Heterozygous Grp78 deficiency results in attenuated
repopulation of CD8ab+ T cells in vivo but does not protect
from experimental ileitis
To validate the role of Grp78 deficiency in T cell distribution to the
intestine, MLN, and spleen, we next performed adoptive transfer of
1.5 3 106 CD8ab+ T cells isolated from spleen of Grp782/+ and
Wt mice into RAG22/2 recipient mice. As expected, the transfer
of CD8ab+ T cells alone did not induce colitis in the recipient
RAG22/2 mice (data not shown). Flow cytometric analysis of
donor T cell expansion in the RAG22/2 recipient mice showed
a lower percentage of repopulating CD8ab+ T cells originating
from Grp782/+ mice compared with Wt mice (Fig. 8A, 8B). The
observed reduced repopulation in the intestine was not due to
defects in b7 integrin expression on Grp782/+ donor T cells (data
not shown). As a final step, we generated TNFDARE/+ 3 Grp782/+
(ARE 3 Grp782/+) double-transgenic mice and evaluated histopathological changes in distal ileum, as well as the cytotoxic activity of CD8ab+ T cells. Interestingly, and despite the fact that
splenic ARE 3 Grp782/+ CD8ab+ T cells had a significantly
reduced cytotoxic activity against intestinal epithelial Mode-K
cells (Fig. 8C), the degree of ileal pathology was not different
between ARE 3 Grp782/+ and ARE mice (Fig. 8D). In addition to
the total pathology scores, no differences were observed with
regard to the subscores, including leukocyte infiltration, crypt loss,
or ulcus formation (data not shown). Together with the observations that splenocytes and distal ileal tissue of ARE 3 Grp782/+
mice showed no significant difference in Grp78 protein (Fig. 8E)
and Grp78 and TNF mRNA expression (data not shown) compared
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
FIGURE 6. Grp78 controls CD8ab+ T cell proliferation. CD8ab+
T cells isolated and pooled from spleen cells (A) or MLN cells (B) of Wt
and Grp782/+ mice (n . 3) were treated or not with anti-CD3/CD28
MicroBeads for 3 d (0.5 3 106 cells/well). BrdU was added to the cells at
48 h postactivation and left overnight. The cell proliferation was measured
based on the BrdU incorporation to the newly synthesized DNA. Significant differences are shown with *p , 0.05, **p , 0.01, ***p , 0.001.
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 selective binding of downstream UPR transducers to the Grp78 and
granzyme B promoters. Consistently, CD8ab+ T cells from
Grp782/+ mice exhibited decreased granzyme B-dependent cytotoxicity against epithelial cells, reduced proliferation, and lower
efficiency with regard to repopulation of the IEL compartment.
Interestingly, heterozygous Grp78 deficiency was not effective in
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after 24 h of stimulation with polyclonal anti-CD3/CD28–coated
MicroBeads (Fig. 7F). Importantly, the absolute amount of granzyme B secreted by Grp782/+ CD8ab+ T cells stimulated with
MicroBeads and rIL-2 was lower than for the MicroBeadstimulated Wt T cells, arguing that both proliferation and granzyme B secretion are attenuated by reduced levels of Grp78.
8
ER STRESS IN T CELL HOMEOSTASIS AND PATHOGENESIS OF IBD
reducing intestinal inflammation in ARE 3 Grp782/+ mice, suggesting that the remaining plasticity of T cell responses in Grp782/+
mice compensates for the partial loss of Grp78. In addition,
dysregulation of ER stress-associated UPR in the epithelium
primed mice to increased disease susceptibility (29), suggesting
that unconditioned Grp78 deficiency may differentially affect
tissue compartments and lead to a net compensation of effects.
Nevertheless, the findings of this study strongly support the hypothesis that ER stress-associated UPR mechanisms contribute
to an aberrant cytotoxic CD8ab+ IEL phenotype in experimental
ileitis.
Two recent reports attempted to identify the signaling pathways
triggering Grp78 expression after TCR activation (46, 47). Takano
et al. (46) reported that this process might be regulated by the
calcineurin–NFAT pathway. This hypothesis was supported by the
finding that Ca2+ chelators abrogated TCR-mediated Grp78 expression. In contrast, Pino et al. (47) argued that the protein kinase
C-signaling pathway controls TCR-mediated Grp78 expression
and additionally affects IL-2 expression. Consistently, we demonstrated that IL-2 expression and T cell proliferation are controlled by Grp78. A deficiency in Grp78 resulted in strikingly
attenuated proliferative responses, which were rescued by the
addition of exogenous IL-2. In addition, adoptively transferred
Grp782/+ sCD8ab+ T cells exhibited a reduced ability to repopulate the intestine of RAG22/2 recipient mice, independent of
gut-homing factors, suggesting a role for Grp78 in the proliferative responsiveness in vivo.
Grp78-mediated control of CD8ab+ T cell homeostasis is impacted at the level of two major effector mechanisms. First, an
effective CD8ab+ T cell cytotoxic activity by granzyme B secretion requires a tightly controlled signal from the Grp78mediated signaling cascade, which, in turn, is linked to the activation of the TCR. CD8ab+ T cells generally showed increasing
levels of Grp78 expression after TCR stimulation with polyclonal
anti-CD3/CD28–coated MicroBeads. This implies that the activation of TCR induces ER stress-associated UPR, supporting recent studies (44, 46, 47). Interestingly, the cytotoxic effector
function of Grp782/+ CD8ab+ T cells is maintained at basal levels
compared with Wt cells; however, the impact of Grp78 deficiency
becomes significant after TCR activation and results in reduced
granzyme B-dependent cytotoxicity.
Moreover, low levels of IL-2 were shown to promote effector
T cell expansion and effector memory development (53). Effector
CD8+ T cells are short-lived and either die or convert into memory
cells after Ag exposure. This cell fate decision seems to rely on
the endogenous production of IL-2 by terminally differentiated
effector CD8+ T cells. Only memory precursor cells (KLRG1low
ILR7Rahigh) that are capable of producing IL-2, as well as cytotoxic (granzyme B) and effector molecules (IFN-g, TNF), differentiate into memory cells (53). Terminally differentiated effector
CD8+ T cells (KLRG1highILR7Ralow) are non–IL-2–producing
cells that are eventually programmed for cell death. A recent study
showed that IL-2–mediated IRE1-XBP1–splicing machinery
contributes to the development of terminally differentiated effec-
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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 MicroBeads 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
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 experiment, exogenous IL-2 (100 U/ml) was present
or absent in a cell culture of isolated sCD8ab+
T cells, pooled from Wt and Grp78 2/+ mice
(n . 3), and stimulated with polyclonal antiCD3/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.
The Journal of Immunology
9
tor CD8+ T cells in vivo (44). Additionally, we showed that exogenous IL-2 rescued the cytotoxic phenotype of CD8ab+ T cells
under Grp78 deficiency. However, and in contrast to proliferation,
the presence of exogenous IL-2 failed to induce a complete rescue
at the level of granzyme B expression. Although this may argue
for a contribution of both reduced proliferation and diminished
granzyme B secretion to the impaired cytotoxicity of Grp782/+
CD8ab+ T cells in response to TCR activation, the granzyme B
secretion of Wt CD8ab+ T cells was also diminished. A study by
Kambayashi et al. (54) showed that the CD8b-chain is downregulated in the presence of IL-2, even in concentrations as low as 10
U/ml. IL-2–stimulated CD8+ T cells have a decreased affinity for
MHC class I–peptide complexes, so the downregulation of CD8b
expression in IL-2–stimulated T cells may compromise the ability
of T cells to respond to their cognate Ags and, subsequently,
attenuates granzyme B secretion.
In the complex scenario of intestinal immune homeostasis, the
importance of CD8+ IEL was recently highlighted (11, 15, 53). In
the small intestine of Wt mice, the majority of IEL harbor the IL10–producing CD8aa+ T cell phenotype (.70%), whereas conventional CD8ab+ IEL account for ∼20% of all IEL. IL-10 was
shown to inhibit TNF-induced Grp78 expression in IEC by
modulating ATF6 nuclear recruitment to the Grp78 promoter
(28). Consistently, the loss of IL-10–secreting CD8aa+ IEL in
TNFDARE/+ mice is associated with aberrant Grp78 expression
in the disease-relevant CD8ab+ IEL exerting aberrant cytolytic
function. This intriguing observation raises the question whether
the aberrant cytotoxicity of ARE CD8ab+ IEL due to unresolved
ER stress with high levels of Grp78 expression can be antagonized
under conditions of Grp78 deficiency. Although ARE 3 Grp782/+
CD8ab+ T cells isolated from spleen showed a significantly re-
duced cytotoxicity in vitro compared with ARE CD8ab+ T cells,
tissue pathology was not affected in ARE 3 Grp782/+ mice
compared with ARE mice. Interestingly, there is no difference in
Grp78 protein and mRNA expression levels detectable between
ARE 3 Grp782/+ and ARE CD8ab+ T cells, indicating that
Grp78 heterozygosity is compensated by the remaining T cells’
plasticity. The reason for this remains unclear, but TNF was shown
to improve the responsiveness of CD8+ T cells to IL-2–mediated
proliferation and to enhance IL-2–induced expression of the IL-2R
of CD8+ T cells (55, 56), supporting the hypothesis that T cells
from a heterozygous Grp78 deficiency may partially maintain
their effector functions. Indeed, we also observed a similar proliferation of ARE 3 Grp782/+ sCD8ab+ T cells compared with
ARE cells (data not shown), suggesting that an enhanced proliferation of ARE 3 Grp782/+ CD8ab+ T cells due to TNF overproduction may abolish the effects of a Grp78 deficiency.
Although TNF plays a role in the induction of the aberrant cytotoxic phenotype of ARE CD8ab+ T cells, it is not implicated in
the short-term cytotoxicity, as reflected by the similar ex vivo
cytotoxicity of ARE CD8ab+ T cells with TNFR blocked by antiTNF Abs. Consistent with our results, a recent study by Kollias
and colleagues (57) demonstrated that TNF overproduction specifically by IEC in the TNFDARE/+ mouse model is sufficient to
induce mucosal pathology. We demonstrated that this localized
scenario in the ileum induces a complex immunopathology, which
is triggering an excessive cytotoxic response of CD8ab+ IEL
maintained by the UPR.
In summary, this study identifies the ER stress-associated
chaperone Grp78 as a critical factor that intrinsically mediates
intestinal T cell homeostasis. In the TNFDARE/+ mouse model of
Crohn’s disease-like ileitis, unresolved ER stress, reflected by
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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-wkold 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-K cell line for 3 d in the presence or
absence of anti-CD3/CD28–coated polyclonal
MicroBeads. T cell-mediated cytotoxicity against
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
single ARE and ARE 3 Grp782/+ mice with
regard to Grp78 protein expression in splenocytes
and ileal tissue. Significant differences are shown
with *p , 0.05, **p , 0.01, ***p , 0.001.
10
ER STRESS IN T CELL HOMEOSTASIS AND PATHOGENESIS OF IBD
high levels of Grp78, accounts for the development and maintenance of a pathogenic cytotoxic CD8ab+ IEL phenotype. This
CD8ab+ IEL phenotype exerts aberrant cytolytic function that
further causes IEC death and, thus, contributes to the mucosal
immunopathology in chronic intestinal inflammation. Although
a Grp78 deficiency in CD8ab+ T cells was shown to result in
attenuated granzyme B-mediated cytotoxicity and reduced T cell
proliferation, it does not protect from intestinal inflammation in
experimental ileitis, thus not completely unraveling the role of
unresolved ER stress in CD8ab+ T cells under chronic intestinal
inflammation.
19.
20.
21.
22.
23.
Acknowledgments
DARE/+
We thank Dr. George Kollias for providing the TNF
mice, Silvia
Pitariu for excellent technical assistance with intestinal T cell isolation,
and Nico Gebhardt for general assistance. We are grateful to Dr. Ingrid
Schmöller for helpful advice on gene promoter analysis.
Disclosures
24.
25.
26.
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