KDEL-retained antigen in B lymphocytes induces a proinflammatory response: a possible role for endoplasmic reticulum stress in adaptive T cell immunity.
ABSTRACT Generally, APCs activate CD4 T cells against peptides derived from exogenous Ag in the context of MHC II molecules. In this study, using transgenic B lymphocytes as model APCs, we demonstrate CD4 T cell priming in vivo against peptides derived from endogenously synthesized Ag targeted either to the cytosol or to the endoplasmic reticulum (ER). Surprisingly, priming by Ag containing the KDEL-retention motif yielded higher levels of two important proinflammatory cytokines, IFN-gamma and TNF-alpha, in responding CD4 T cells. Importantly, we found that KDEL-mediated retention of Ag up-regulates ER-stress responsive genes in primary B lymphocytes. We also found that thapsigargin treatment of A20 lymphoma cells up-regulates transcription of ER stress and proinflammatory genes along with IL-23p19. Induction of ER stress by thapsigargin also up-regulated IL-23p19 in primary B lymphocytes, macrophages, and bone marrow-derived dendritic cells. We conclude that perturbation of the secretory pathway and/or ER stress play an important role in modulating the gene program in professional APCs and in shaping CD4 T cell responses in vivo. These findings are relevant to a better understanding of the immune response after infection by viral and bacterial pathogens and the pathogenesis of certain autoimmune diseases.
- SourceAvailable from: Pablo Villoslada[Show abstract] [Hide abstract]
ABSTRACT: Celecoxib is a selective cyclooxygenase-2 (COX2) inhibitor. We have previously shown that celecoxib inhibits experimental autoimmune encephalomyelitis (EAE) in COX-2-deficient mice, suggestive for a mode of action involving COX2-independent pathways. In the present study, we tested the effect of a trifluoromethyl analogue of celecoxib (TFM-C) with 205-fold lower COX-2 inhibitory activity in two models of neuroinflammation, i.e. cerebellar organotypic cultures challenged with LPS and the EAE mouse model for multiple sclerosis. TFM-C inhibited secretion of IL-1β, IL-12 and IL-17, enhanced that of TNF-α and RANTES, reduced neuronal axonal damage and protected from oxidative stress in the organotypic model. TFM-C blocked TNF-α release in microglial cells through a process involving intracellular retention, but induced TNF-α secretion in primary astrocyte cultures. Finally, we demonstrate that TFM-C and celecoxib ameliorated EAE with equal potency. This coincided with reduced secretion of IL-17 and IFN-γ by MOG-reactive T-cells and of IL-23 and inflammatory cytokines by bone marrow-derived dendritic cells. Our study reveals that non-coxib analogues of celecoxib may have translational value in the treatment of neuro-inflammatory conditions.PLoS ONE 01/2013; 8(12):e83119. · 3.53 Impact Factor
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ABSTRACT: PURPOSE: To elucidate the mechanism of rutin in blocking macrophage-mediated inflammation and high fat diet-induced obesity and fatty liver. METHODS: Both in vitro and in vivo approaches were taken in evaluating the effects of rutin on palmitic acids-triggered inflammation in cultured macrophages, and on weight gain and development of fatty liver of mice fed a high fat diet. RESULTS: Palmitic acids increase mRNA levels of pro-inflammatory cytokines, and elevate the production of TNFα in cultured macrophages. Pre-exposure of rutin to cells greatly suppressed these elevations. The suppressed inflammation by rutin was correlated with a decrease in transcription of genes responsible for ER stress and production of reactive oxygen species. In vivo, rutin protects mice from high fat diet-induced obesity, fatty liver and insulin resistance. The protective effects were associated with lack of hypertrophy and crown-like structures in the white adipose tissue, decreased mRNA levels of marker genes for macrophages including F4/80, Cd11c and Cd68, and repressed transcription of genes involved in chronic inflammation such as Mcp1 and Tnfα in white adipose tissue. In addition, rutin increases the expression of genes responsible for energy expenditure in brown adipose tissue including Pgc1α and Dio2. Furthermore, rutin suppresses transcription of Srebp1c and Cd36 in the liver, leading to a blockade of fatty liver development. CONCLUSION: These results suggest that supplementation of rutin is a promising strategy for blocking macrophage-mediated inflammation and inflammation-induced obesity and its associated complications.Pharmaceutical Research 06/2013; · 4.74 Impact Factor
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ABSTRACT: Significance: The endoplasmic reticulum (ER) is a specialized organelle for the folding and trafficking for proteins, which is highly sensitive to changes in intracellular homeostasis and extracellular stimuli. Alterations in the protein-folding environment cause accumulation of misfolded proteins in the ER that profoundly affect a variety of cellular signaling processes including reduction-oxidation (redox) homeostasis, energy production, inflammation, differentiation, and apoptosis. The unfolded protein response (UPR) is a collection of adaptive signaling pathways that evolved to resolve protein misfolding and restore an efficient protein-folding environment. Recent Advances: Production of reactive oxygen species (ROS) has been linked to ER stress and the UPR. ROS play a critical role in many cellular processes and can be produced in the cytosol and several organelles including the ER and mitochondria. Studies suggest that altered redox homeostasis in the ER is sufficient to cause ER stress, which could in turn induces the production of ROS in the ER and mitochondria. Critical Issues: Although ER stress and oxidative stress coexist in many pathologic states, whether and how these stresses interact is unknown. It is also unclear how changes in the protein-folding environment in the ER cause oxidative stress. In addition, how ROS production and protein misfolding commit the cell to an apoptotic death and contribute to various degenerative diseases is unknown. Future Directions: A greater fundamental understanding of the mechanisms that preserve protein folding homeostasis and redox status will provide new information toward the development of novel therapeutics for many human diseases.Antioxidants & Redox Signaling 04/2014; · 8.20 Impact Factor
KDEL-Retained Antigen in B Lymphocytes Induces a
Proinflammatory Response: A Possible Role for Endoplasmic
Reticulum Stress in Adaptive T Cell Immunity1,2
Matthew C. Wheeler,*†Marta Rizzi,* Roman Sasik,*‡Gonzalo Almanza,* Gary Hardiman,*‡
and Maurizio Zanetti3*†
Generally, APCs activate CD4 T cells against peptides derived from exogenous Ag in the context of MHC II molecules. In this
study, using transgenic B lymphocytes as model APCs, we demonstrate CD4 T cell priming in vivo against peptides derived from
endogenously synthesized Ag targeted either to the cytosol or to the endoplasmic reticulum (ER). Surprisingly, priming by Ag
containing the KDEL-retention motif yielded higher levels of two important proinflammatory cytokines, IFN-? and TNF-?, in
responding CD4 T cells. Importantly, we found that KDEL-mediated retention of Ag up-regulates ER-stress responsive genes in
primary B lymphocytes. We also found that thapsigargin treatment of A20 lymphoma cells up-regulates transcription of ER stress
and proinflammatory genes along with IL-23p19. Induction of ER stress by thapsigargin also up-regulated IL-23p19 in primary
B lymphocytes, macrophages, and bone marrow-derived dendritic cells. We conclude that perturbation of the secretory pathway
and/or ER stress play an important role in modulating the gene program in professional APCs and in shaping CD4 T cell responses
in vivo. These findings are relevant to a better understanding of the immune response after infection by viral and bacterial
pathogens and the pathogenesis of certain autoimmune diseases. The Journal of Immunology, 2008, 181: 256–264.
prime a T cell immune response. Traditionally, peptides that bind
MHC class II are generated through a process that involves Ag
up-take, and processing in degradative organelles, such as MIIC,
that are rich in MHC class II (1). Previous studies have reached the
conclusion that a majority of peptides associated with MHC class
II molecules are derived from exogenous Ag internalized through
scavenger receptors, or from membrane-bound proteins (2, 3).
Notwithstanding this general idea, in vitro studies have shown that
immunogenic peptides can also be generated from both endoplas-
mic reticulum (ER)4- and cytosol-localized Ag (4–11). In agree-
ment with this is the fact that 30% of MHC class II molecules are
occupied by peptides from non-membrane associated intracellular
Ags (12). However, little is known on the influence presentation of
intracellular Ag may have on CD4 T cell priming in vivo, which
is a key event in the initiation of adaptive immune responses.
ajor histocompatibility complex class II molecules ex-
pressed by APCs display immunogenic peptides to
CD4 T cells, and, along with proper costimulation,
T cell priming depends on adequate cell surface display of pep-
tide-MHC complex (signal 1) by professional APCs (dendritic
cells, macrophages, and B lymphocytes), and it requires adequate
costimulation (signal 2) to orchestrate a fully functional immune
response. Lack of costimulation through CD80, CD86, CD40, or
OX40L have been shown to impact many aspects of T cell func-
tion such as priming, initial expansion, survival, and memory (13).
OX40L has been shown to play a role in the initial CD4 T expan-
sion, subsequent memory induction (14), and its expression by B
lymphocytes is necessary for the induction of Th2 immunity (15).
The ER stress/unfolded protein response (UPR) pathway is a
cellular response to an overloaded secretory pathway that is inte-
gral to normal cellular homeostasis. Cells that accumulate mis-
folded or mutant protein in the ER initiate a transcriptional gene
program to restore normal cellular folding and secretion equilib-
rium (16, 17). In general, the UPR pathway results in the up-
regulation of genes responsible for ER biogenesis, protein folding,
cell survival, and ultimately apoptosis. In B lymphocytes, it is an
important cell differentiation checkpoint as the UPR-inducible
transcription factor XBP-1 is required for plasma cell differentia-
tion (18). However, the effects of the UPR pathway on the Ag
presenting function of B cells, clonal T cell expansion, and the
activation of adaptive immune responses is unknown.
In this study, we sought to determine whether a different intra-
cellular localization of endogenously synthesized Ag has any effect
at the level of the APC and of the CD4 T cell response in vivo. To
this end, we used a model system, developed in this laboratory,
whereby adaptive T cell responses can be induced using B lym-
phocytes programmed as APCs by spontaneous transgenesis of
plasmid (p)DNA (19, 20). In this model of immunization, pDNA
codes for the H chain of a secretory Ig (IgH) molecule. Ag spec-
ificity is imparted by DNA sequences coding for heterologous T
cell epitopes engineered in the complementarity-determining re-
gions of a rearranged variable (V) domain (21). Previously, we
showed that a single i.v. injection of transgenic B lymphocytes as
*Laboratory of Immunology, Department of Medicine and Moores Cancer Center,
†Biomedical Sciences Program, and‡Biomedical Genomics Microarray, La Jolla, CA
Received for publication April 11, 2007. Accepted for publication April 19, 2008.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by National Institutes of Health Grant R01CA 92119,
University of California, La Jolla, CA 92093.
2The sequences presented in this article have been submitted to EBI Array Express
Database (accession number pending).
3Address correspondence and reprint requests to Dr. Maurizio Zanetti, Moores Can-
cer Center, University of California, San Diego, 3855 Health Sciences Drive, La Jolla,
CA 92093-0815. E-mail address: email@example.com
4Abbreviations used in this paper: ER, endoplasmic reticulum; UPR, unfolded pro-
tein response; p, plasmid; qRT-PCR, quantitative RT-PCR.
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
The Journal of Immunology
APCs primes both CD4 and CD8 T cell responses in the spleen and
lymph nodes (20), and that the generation of CD4 responses re-
quires MHC class II expression on the injected transgenic APCs.
Thus, since pDNAs (transgenes) can be easily engineered with
different targeting motifs, and the APCs can be programmed ac-
cordingly, this system constitutes an ideal model system to study
how endogenously synthesized Ag and ER stress affect the APC
and in turn the CD4 T cell response in vivo.
Materials and Methods
Mice and cell lines
C57BL/6 Mice were purchased from Harlan Sprague Dawley. MHC class
II-deficient mice on a C57BL/6 background were purchased from Taconic.
All mice were housed at the University of California San Diego Animal
Facility and handled in accordance with University of California-San Di-
ego Animal Subjects Program Guidelines. J558L cells were originally ob-
tained from Dr. S. Morrison (University of California, Los Angeles, CA).
Plasmids encoding the secretory IgH genes were made as previously de-
scribed (22) with the following modifications: for cytosolic targeting, the
leader sequence was removed using a Quick Change XL site directed mu-
tagenesis kit from Stratagene. For ER targeting, a ClaI and a NotI site was
added 3? of the ?1 coding region using a Quick Change XL site directed
mutagenesis kit from Stratagene. A pair of complementary oligonucleo-
tides coding for SEKDEL with a 3-alanine spacer were inserted between
these restriction sites. The heterologous peptide E? (ASFEAQGALANIA
VDKA) was inserted into the variable regions as previously described (23).
Plasmid pEGFP was purchased from Clontech Laboratories. pMACS Kk
plasmid was purchased from Miltenyi Biotech. All plasmid sequences were
confirmed by sequencing at the University of California San Diego Cancer
Center Core Sequencing Facility. Plasmids were prepared using Wizard
Plus Maxi-prep kit from Promega.
Peptides, CpG, and Abs
E? peptide 56–73 (ASFEAQGALANIAVDKA) was synthesized at the
Ohio State University Peptide Synthesis Facility. Tunicamycin and thap-
sigargin were purchased from Calbiochem. CpG ODN 1826 was kindly
provided by Dr. P. Lenert (University of Iowa, Iowa City, IA). Goat anti-
calnexin Ab was purchased from Santa Cruz Biotechnology. Donkey anti-
goat-Cy3 and donkey anti-human ?1 FITC Abs were purchased from Jack-
son ImmunoResearch Laboratory. Goat anti-human IgG Abs labeled with
HRP were purchased from Sigma-Aldrich. PE-conjugated Abs anti-mouse
I-Ab, CD19, CD86, CD40, OX40L, CD4, CD8; FITC-conjugated IFN-?;
and a biotinylated anti-mouse Kkwere purchased from BD Pharmingen.
Allophycocyanin-conjugated CD4 was purchased from eBioscience.
Spontaneous lymphocyte transgenesis and in vivo immunization
Spontaneous lymphocyte transgenesis was performed as previously de-
scribed (19) with the following modifications. Briefly, spleen cells were
harvested, washed with PBS and incubated with 25 mg of pDNA for 1 h
at 37 °C together with the PMACS Kkplasmid (Miltenyi Biotec) coding for
a truncated mouse H-2 Kkmolecule as selectable cell surface marker. After
overnight incubation, transgenic cells were magnetically sorted and ana-
lyzed by flow cytometry for EGFP expression on a FACSCalibur (BD
Generation of stable transfectoma
J558L cells (10 ? 106) were transfected by electroporation with 50 ?g of
linearized pDNA in ice-cold PBS without Ca2?and Mg2?and shocked
twice at 300 mV. Cells were incubated on ice for 15 min and then allowed
to recover in RPMI 1640 containing10% FBS for 48 h. Cells were then
grown under G418 selection 1 mg/ml for 14 days. Supernatants and lysates
of clones were then screened by capture ELISA for production of IgH
polypeptides (24). Detection of intracellular immunoreactive transgenic
IgH products was also performed by ELISA on cell lysates obtained by
freeze-thaw. Cell lysates were tested at 1/10 dilution.
Cell separation and isolation
CD4?and CD8?T cells were isolated from the spleens of immunized mice
by negative selection using CD4 and CD8 isolation kits from Stem Cell
Technologies. B lymphocytes were isolated from spleens of naive C57/Bl6
mice by negative selection using a B cell isolation kit from Stem Cell
Technologies. Cell populations were greater than 90% pure as determined
by flow cytometry with appropriate Abs. Kk-positive cells were isolated
using a biotinylated anti-KkAb, and a biotin selection kit from Stem Cell
Technologies. Dendritic cells were generated from the bone marrow of
C57BL/6 mice by culture in the presence of GM-CSF (40 ng/ml) and IL-4
(4 ng/ml) for 7 days. Macrophages were generated from bone marrow cells
incubated with 30% L929-conditioned medium for 7 days. Adherent cells
were removed by scraping.
J558L transfectoma were fixed and permeabilized using the CytoFix/
CytoPerm kit from BD Pharmingen. Cells were then incubated with goat
anti-calnexin primary Ab for 1 h at room temperature. Cells were then
incubated with donkey anti-goat-Cy3 and donkey anti-human-FITC Abs.
Cells were washed and spun onto slides using a cytospin centrifuge.
Stained cells were imaged at the University of California San Diego Cancer
Center Digital Imaging Shared Resource using a Photometrics CCD
mounted on a Nikon TE-220 inverted epifluorescence microscope at ?100
magnification. Images were deconvoluted using Soft Worx (Applied
T cell proliferation
A total of 4 ? 105splenocytes were incubated, in triplicate, in a round-
bottom 96-well plate with either medium or 50 ?g/ml E? peptide. At 72 h,
1?Cu of [3H]-labeled thymidine was added to each well, and cells were
harvested onto filter mats and read on a Wallac ? counter 16 h later. Results
obtained by stimulation with peptide are expressed as cpm from which cpm
of cultures in medium only were subtracted. Tests were performed in
Supernatants of 48-h triplicate cultures were pooled and assayed with a
mouse Th1/Th2 cytometric bead array (BD Pharmingen). Data were ac-
quired using a BD FACSCalibur, and analyzed by BD CBA software (BD
Biosciences). Statistical analysis was performed using Prism software from
GraphPad, and significance was determined using a two-tailed unpaired
Total RNA was extracted from cells using an RNeasy kit from Qiagen.
cDNA was synthesized using a poly(T) primer from 50 ng of total RNA
using Sensiscript Reverse Transcriptase (Qiagen). Primers used for PCR
analysis had the following sequence: human ?1; tcaaggactacttccccgaacc
and tactccttgccattcagccagccagtcc, MyD116; gagaagagggagtggctgagc and
agcattccgacaagggtgacc, Chop; ccctgcctttcaccttgg and ccgctcgttctcctgctc,
Grp78; ctgggtacaatttgatctgacgg and gcacctggtggctttcccagcattc, and ?-actin
tgggccgccctaggcacc and cggttggccttagggttcag. PCR was conducted for 30
cycles using platinum Pfx polymerase from Invitrogen. The nested PCR for
mIL-23p19 was performed using the following primers: IL23 Forward (5?-
GTGCCTAGGAGTAGCAGTCCGA-3?) and IL23 Reverse (5?-TGGCT
GGAGGAGTTGGCTGA-3?) for 30 cycles, and then IL23 Forward N (5?-
TGCATGCTAGCCTGGAACGC-3?) and IL23 Reverse N (5?-GGG
AGTAGAGCAGGCTCCCC-3?) also for 30 cycles.
Quantitative RT-PCR (qRT-PCR)
cDNA was generated from 200 ng of total RNA using Omniscript Reverse
Transcriptase (Qiagen). qRT-PCR was conducted using the TaqMan
method on an ABI Prism 7700, normalized to ?-actin, and analyzed by
comparative Ctanalysis. Sequences for probes and primers were as
follows: mIL-23a-p19 (NM_031252.1)-171Forward TGGCTGTGC
TCC; 200TaqMan probe TGG GCT CAG TGC CAG CAG CTC TC;
m? Actin-(GI49865)-379 Forward TTCAACACCCCAGCCATGTA;
447Reverse TGTGGTACGACCAGAGGCATAC; 402TaqMan probe
AGCCATCCAGGCTGTGCTGTCCC. Results were expressed as fold
change compared with unstimulated cells.
Biotinylated cRNA was prepared using the Illumina RNA Amplification
kit, Catalog no. 1L1791 (Ambion) according to the manufacturer’s direc-
tions starting with ?250 ng total RNA. The labeling approach uses a mod-
ified Eberwine protocol (25) by which messenger RNA is converted to
cDNA, followed by an amplification/labeling step mediated by T7 DNA
polymerase. The cDNA and cRNA filter cartridges (Ambion) were used
according to the manufacturer’s instructions for room temperature and
257 The Journal of Immunology
in vitro transcript cleanup, respectively. For microarray analysis, the Illu-
mina Mouse 6 Sentrix Expression BeadChip was used (Illumina). Hybrid-
ization of labeled cRNA to the BeadChip, washing, and scanning were
performed according to the Illumina BeadStation 500? manual. Essen-
tially the amplified, biotin-labeled human cRNA samples were resuspended
in a solution of Hyb E1 buffer (Illumina) and 25% (v/v) formamide at a
final concentration of 25 ng/?l. A total of 1.5 ?g of each cRNA was
hybridized. Hybridization was allowed to proceed at 55°C for 18 h, after
which the bead array matrix was washed for 10 min with 1? high tem-
perature buffer (Illumina), followed by a subsequent 10-min wash in Wash
E1BC buffer. The arrays were then washed with 100% ethanol for 10 min
to strip off any remaining adhesive on the chip. A 2-min E1BC wash was
performed to remove residual ethanol. The arrays were blocked for 5 min
with 1% (w/v) casein-PBS (Pierce). The array signal was developed via
10-min incubation with streptavidin-Cy3 at a final concentration of 1
?g/ml solution of (GE Healthcare) in 1% casein-PBS blocking solution.
The Mouse 6 Sentrix Expression BeadChip was washed a final time in
Wash E1BC buffer for 5 min and subsequently dried via centrifugation for
4 min at a setting of 275 ? g. The arrays were scanned on the Illumina
BeadArray reader, a confocal-type imaging system with 532 (cye3) nm
laser illumination. Image analysis and data extraction was conducted as
previously reported (26). The array images were registered using an algo-
rithm described previously (27). Essentially the bead signals were com-
puted with weighted averages of pixel intensities, and local background is
subtracted. Sequence-type signal was calculated by averaging correspond-
ing bead signals with outliers removed (using median absolute deviation).
Preliminary data analysis and QC was conducted using the BeadStudio
In vitro analysis of B cells harboring differentially targeted
To determine whether the ability of endogenously synthesized Ag
to prime a CD4 T cell response varies depending on the intracel-
lular localization of Ag, we compared secretory IgH, IgH retained
in the ER via a KDEL ER-retention motif, and IgH targeted to the
cytosol via deletion of the N-terminal leader sequence. The ex-
pressed IgH products are referred to as ?1VHdenoting the wild-
type secretory IgH, ?1VHKDEL denoting ER targeting, and ?1
?L11VHdenoting cytosolic targeting. Stable transfectants of
J558L cells, a murine plasmacytoma that possesses a ?1L chain
but expresses no endogenous H chain, were used to ensure that the
engineered ?1VHKDEL and ?1?L11VHIgH molecules were tar-
geted to the intended intracellular compartment, and not secreted.
Upon transfection with a wild-type IgH transgene, J558L cells
produce complete H2L2(IgG) molecules, which can be detected by
capture ELISA. We found no transgenic IgG in ?1VHKDEL or
?1?L11VHtransfectoma supernatants (Fig. 1A), despite the fact
that both ?1VHKDEL and ?1?L11VHtransfectoma expressed the
transgene by RT-PCR (Fig. 1B). By ELISA transgenic products
were detected in soluble intracellular fractions (data not shown).
Of note, the detection of immunoreactive transgenic products in
cell lysates was higher for the ?1VHKDEL transfectoma than for
the ?1VHtransfectoma (A450 duplicate values 1.55/1.67 vs 0.59/
0.62, respectively). Although the nature of the immunoreactive
products in the ?1VHKDEL transfectoma was not investigated, it
likely includes a mixture of properly folded, unfolded, and de-
graded transgenic IgH polypeptides. The higher content of trans-
genic products in the ?1VHKDEL transfectoma lysate suggests
that KDEL promotes intracellular retention, and that ?1VHKDEL
transfectoma are consequently subject to a heavier cargo of trans-
genic IgH polypeptides than transfectoma harboring the secretory
IgH transgene. Additional experiments will need to establish that
even though levels of transcription of the different transgenes are
similar (Fig. 1B), the ?1VHKDEL transfectoma do not undergo
Proper intracellular compartment localization was further as-
sessed by deconvolution microscopy. To this end, stably trans-
fected J558L cells were stained for both the ER marker calnexin
and the human ?1 portion of the IgH transgene (Fig. 1C). Both the
?1VHand the ?1VHKDEL proteins showed a predominantly ve-
sicular staining that colocalized with calnexin. The ?L11?1VH
protein showed no colocalization with calnexin and produced a
predominantly puntiform staining shown previously to correspond
to cytosolic localization of an IgH polypeptide in cell lines ex-
pressing high levels of this protein (28).
proteins. J558L cells were stably transfected with plasmids expressing the
IgH transgene targeted to the secretory pathway (?1VH), the ER (?1VH-
KDEL), or the cytosol (?1?L11VH), respectively. A, B cell transfectoma
harboring the transgene targeted to ER or the cytosol do not secrete trans-
genic IgH molecules as determined by a sandwich ELISA for the human
IgG constant region of the transgenic product. Tests were performed on
supernatants of stable tansfectants. B, Transgene expression in B cell trans-
fectoma. RT-PCR for the IgH transgene was performed on total RNA
extracted from transfected J558L cells, ?-actin serves as a control for RNA
quality. C, Intracellular localization of the transgenic product in B cell
transfectoma. J558L cells were analyzed by deconvolution microscopy as
described in Materials and Methods. Staining for human ?1 is shown in
green (left panels) and for the ER marker calnexin in red (middle panels).
The overlay (right panels) shows areas of colocalization in yellow.
Intracellular targeting and trafficking of engineered IgH
258ER STRESS AND PROINFLAMMATION
Analysis of the CD4 T cell response in vivo
To assess the in vivo APC function of B lymphocytes expressing
endogenously synthesized Ag localized to different intracellular
compartments, we engineered IgH transgenes to express in the
complementarity-determining region 3 the amino acid sequence
ASFEAQGALANIAVDKA, an immunodominant determinant
from the ?-chain of the I-Ebmolecule that is presented by I-Ab. In
C57BL/6 mice that do not express the I-Eballele, the E? peptide
is non-self and readily induces a T cell response (29). C57BL/6
mice immunized by i.v. injection of syngeneic B lymphocytes
transgenic for E? mounted a strong and specific proliferative re-
sponse irrespective of the intracellular compartment targeted (Fig.
2a). In the experiment, 7/11 mice injected with ?1E?3transgenic
B lymphocytes, 11/11 mice injected with ?1E?3KDEL transgenic
B lymphocytes, and 12/12 mice injected with ?1?L11E?3 trans-
genic cells responded to immunization. Interestingly, no signifi-
cant difference in the magnitude of the response was observed
among mice of the three groups. To verify that responding cells
were in fact CD4 T cells, splenic CD4 and CD8 T cells were
isolated by negative selection and restimulated with LPS blasts
from naive C57BL/6 mice pulsed with the E? peptide. Only CD4
T cells proliferated in vitro upon restimulation with the E? peptide
(Fig. 2b), indicating that E?-specific CD4 T cells had been primed.
T cell priming also required MHC class II expression on the in-
jected B lymphocytes (Fig. 2c), despite normal levels of transfec-
tion (data not shown). Thus, B lymphocytes presenting endog-
enously synthesized Ag can prime CD4 T cells irrespective of its
When we profiled the cytokines secreted by responding T cells
using a cytometric bead array, we found that there was no signif-
icant effect on IL-2 or IL-4 secretion (Fig. 2, f and g). However, T
cells primed by B lymphocytes transgenic for ?1E?3KDEL pro-
duced significantly more IFN-? and TNF-? (Fig. 2, d and e) than
T cells primed by transgenic B lymphocytes presenting cytosolic
with Ag presenting B lymphocytes transgenic for ?1E?3(inverted triangles), ?1E?3KDEL (circles) or ?1?L11E?3(triangles) or ?1 NV2NA3(diamonds).
Splenocytes were harvested on day 14 and proliferation after restimulation with E? peptide was measured by [3H]thymidine incorporation. Each data point
represents the mean of triplicate wells. Only values from responding mice are shown. b, Naive C57BL/6 mice were immunized with B lymphocytes
transgenic for ?1E?3(left), ?1E?3KDEL (middle), or ?1?L11E?3(right), respectively. On day 14, CD4 and CD8 T cells were isolated by negative
selection. Cells were then restimulated with LPS-treated B lymphocytes with or without Ea peptide, and then assayed for proliferation as indicated above.
Results refer to two mice/group. c, B lymphocytes from either wild-type C57BL/6 mice (filled symbols) or MHC class II ?/? mice (open symbols) were
rendered transgenic with either ?1E?3KDEL (squares) or ?1?L11E?3(circles), and used to immunize naive C57BL/6 mice. Splenocytes were harvested
14-days postimmunization and proliferative responses were analyzed as described in a. d–g, Mice were immunized with Ag presenting B lymphocytes
transgenic for ?1E?3KDEL (black bars, n ? 6), ?1E?3(gray bars, n ? 7), or ?1?L11E?3(open bars, n ? 8). Splenocytes were harvested at 14 days and
stimulated with the E? peptide for 48 h. Levels of cytokines in culture supernatants were measured by the cytometric bead array (BD Biosciences) for IFN-?
(d), TNF-? (e), IL-2 (f), and IL-4 (g). Results represent average amounts ? SEM after peptide stimulation. Significance (p ? 0.05) was determined using
an unpaired two-tailed t test and is indicated with a ?. Values (pg/ml) obtained from splenocytes cultured with medium alone are as follows: ?1E?3KDEL;
IFN-? (11.4 ? 16.2), TNF-? (8.8 ? 5.3), IL-2 (21. ? 13.9), IL-4 (5.4 ? 1.9), ?1E?3; IFN-? (12.1 ? 16.6), TNF-? (11.0 ? 10.2), IL-2 (16.7 ? 15.1),
IL-4 (3.94 ? 1.7), ?1?L11E?3; IFN-? (5.9 ? 7.7), TNF-? (8.3 ? 4.6), IL-2 (21.3 ? 10.8), IL-4 (7.1 ? 2.1).
Endogenously synthesized Ag retained in the ER or in the cytosol primes a CD4 T cell response. a, Naive C57BL/6 mice were immunized
259 The Journal of Immunology
or secreted Ag. Interestingly, ER-retained Ag showed an increase
in these two cytokines compared with secretory Ag, which also
traffics through the ER. Since this was unexpected, new experi-
ments were planned to understand the reasons for this difference.
To rule out the possibility that the above effects could be due to
intrinsic properties of the plasmids used (e.g., immunostimulatory
DNA motifs), we isolated B lymphocytes from naive mice by neg-
ative selection, incubated them with 25 ?g/ml of either the
?1E?3KDEL or ?1E?3plasmid, and recorded proliferation at 48 h.
Since B cell proliferation is a classic response to B cell mitogens
like CpG (30) or endotoxin, this would rule out a contribution by
these factors to the previous experiments. We found that when
compared with LPS or CpG ODN, pDNA induced a similar mag-
nitude of proliferation as control ODN (Fig. 3), confirming that
pDNA is not mitogenic for B lymphocytes (31, 32). In contrast, B
lymphocytes incubated with ?1E?3KDEL or ?1E?3pDNA did
up-regulate the costimulatory molecule CD86 (data not shown),
another marker of B cell activation. Together these experiments
show that the two plasmids did not differ in their ability to stim-
ulate B lymphocytes during the initial stages of activation.
Modulation of costimulatory molecules in transgenic APCs
To elucidate factors that could influence the Ag presenting func-
tion of B lymphocytes, we engineered plasmids containing the
selectable surface marker H2 Kkto aid the identification and iso-
lation after injection in vivo. C57BL/6 mice were injected i.v. with
1 ? 106B lymphocytes transgenic for ?1E?3KDEL-Kkor ?1E?3-
Kk. Control mice were injected with B lymphocytes transgenic for
a plasmid that expressed only Kk(pMACS Kk). Twenty four hours
after transgenesis, we saw comparable up-regulation of I-Abor of
the costimulatory molecules CD86, CD40, and OX40L in trans-
genic B lymphocytes irrespective of the plasmid used for their
transfection (Fig. 4A, a–d). Both ?1E?3KDEL-Kkand ?1E?3-Kk
transgenic lymphocytes showed up-regulation of I-Ab, CD86, and
CD40 when compared with non-transfected B lymphocytes iso-
lated from the spleens of naive mice, indicating that some activa-
tion had already occurred before injection. However, after 3 days,
that is at the time when T cells reach the height of clonal expansion
after encounter with Ag (33), Kk-positive B lymphocytes re-iso-
lated from the spleens of mice injected with B lymphocytes trans-
genic for either ?1E?3KDEL-Kkor ?1E?3-Kkshowed marked up-
regulation of I-Ab, CD86, and CD40 when compared with mice
injected with B lymphocytes transgenic for pMACS Kkonly
(Fig. 4A, e–g). Oddly, we also noticed a small decrease in OX40L
expression by ?1E?3KDEL-Kkcells (Fig. 4Bh), but further inves-
tigation using an in vitro priming system revealed that this did not
correlate with enhanced IFN-? production (data not shown). Of
note, B lymphocytes transgenic for ?1E?3KDEL-Kkharvested on
ferent immune responses in vivo. Splenic B cells were isolated by negative
selection and incubated with non-CpG ODN (black bar), 25 ?g/ml
E?3?1(gray bar), 25 ?g/ml E?3?1KDEL (open bar), 2.5 ?g/ml LPS
(checkered bar), or 0.1 ?M CpG ODN 1826 (cross-hatched bar) for 24 h.
Proliferation was determined by [3H]thymidine incorporation. Values rep-
resent means ? SD of triplicate wells.
Differences in pDNA composition do not account for dif-
cytes that express ER retained Ag
show diminished OX40L expression
in vivo and up-regulate transcription
of ER stress responsive genes. A, B
lymphocytes were subjected to spon-
(black line) or ?1E?3
KDEL-Kk(gray line). Kk-positive
lymphocytes were isolated 24 h later,
and analyzed for expression of I-Ab
(a and e), CD86 (b and f), CD40 (c
and g), or OX40L (d and h) at days 0
(top row) and 3 (bottom row) after i.v.
injection and compared with naive B
lymphocytes (gray fill at day 0) or
pMACS-Kktransfected B lympho-
cytes (gray fill at day 3). B, B lym-
phocytes were rendered transgenic for
either ?1E?3-Kkor ?1E?3KDEL-Kk
and Kk-positive cells were isolated at
24 and 48 h. Total RNA was ex-
tracted and RT-PCR was performed
for ER stress induced genes Myd116,
Chop, and Grp78. RNA from splenic
B lymphocytes isolated by negative
selection, and treated with 5 ?g/ml
tunicamycin for 24 h served as a pos-
Primary B lympho-
260 ER STRESS AND PROINFLAMMATION
day 3 (in vivo) showed similar recovery relative to the other
groups since the yield of live Kk?transgenic lymphocytes was 69
(?1E?3-Kk), 59 (?1E?3KDEL-Kk), and 60% (pMACS Kk) of the
injected 1 ? 106lymphocytes, respectively.
Up-regulation of ER stress genes in B lymphocytes transgenic
for the KDEL motif
We reasoned that the KDEL retention motif might lead to up-
regulation of canonical ER stress/UPR genes in transgenic B lym-
phocytes and that this may provide us with new clues to elucidate
their Ag presenting function. To this end, we first sought differ-
ences in expression of ER stress responsive genes in primary B
lymphocytes rendered transgenic with ?1E?3KDEL as compared
with B lymphocytes transgenic for the ?1E?3plasmid. Generally,
cells that undergo ER stress up-regulate the expression of a num-
ber of stress responsive genes, including Myd116, Grp78 (BiP),
and Chop (Gadd 153) (17). When we analyzed B lymphocytes
transgenic for either ?1E?3KDEL-Kkor ?1E?3-Kkat 24 h, we
found no up-regulation of the ER stress markers Myd116, Grp78,
and Chop. However, at 48 h post-transfection we saw up-regula-
tion of ER stress genes in ?1E?3KDEL-Kktransgenic B lympho-
cytes (Fig. 4B), but not in lymphocytes transgenic for ?1E?3-Kk.
Purified B lymphocytes treated with tunicamycin, a compound that
induces ER stress by preventing N-linked glycosylation (34),
served as a positive control and resulted in a robust expression of
all three ER stress responsive genes at 24 h (Fig. 4B). These results
indicate that transgenic B lymphocytes that retain KDEL-tagged
Ag accumulate enough protein to induce up-regulation of canon-
ical ER stress/UPR genes.
Genome-wide array of ER stressed B cells
To elucidate factors that influence T cell priming we resorted to
genome wide expression profiling of a B cell line treated with the
ER stress inducing compound thapsigargin. We treated A20 cells
with thapsigargin and collected RNA at 8- and 24-h time points.
Induction of ER stress responsive genes such as Chop, Myd116,
Grp78, Wfs1, and Atf4 was evident at both time points after treat-
ment (Fig. 5A), indicating that thapsigargin adequately induced an
ER stress response. Not surprisingly, we observed significant
down-regulation of the BCR complex and cell cycle pathways
(data not shown). We also saw up-regulation of IFN regulatory
factor 4 (Irf4) (Fig. 5B), a transcription factor implicated in plasma
cell development (35). These results were expected since ER stress
serves as a physiological checkpoint for plasma cell development.
Finally, we noticed a significant down-regulation of the MHC class
II Ag processing pathway (data not shown), and various accessory
molecules such as DM and the CIITA (Fig. 5B). This is consistent
with plasma cell development, although it should be noted that
filing of A20 B lymphoma cells under
conditions of ER stress. A20 B lym-
phoma cells were treated for 8 or 24 h
with 300 nM thapsigargin. Total RNA
was extracted and analyzed using Il-
lumina bead arrays. Levels of gene
expression were compared with un-
treated A20 cells. Two independent
shown. In the clustergrams, genes are
grouped into (A) ER stress responsive
genes and (B) immunologically rele-
vant genes. The extremal fold changes
are noted on the respective color
scales. C, Levels of IL-23p19 gene
expression as determined by qPCR.
IL-23p19 levels in A20 cells were de-
termined at 8 and 24 h after thapsigar-
gin treatment and compared with un-
analysis. Two independent experi-
ments are shown (experiment 1, black
bars; experiment 2, gray bars).
Gene expression pro-
261 The Journal of Immunology
while the overall transcriptional levels of MHC class II fell (data
not shown), they remained sufficiently high to be detected in vivo
(Fig. 4A), suggesting that treatment with thapsigargin may be
harsher than ER retention of Ag in promoting ER stress. Alterna-
tively, one cannot rule out the possibility that the cell surface levels
of MHC class II in transgenic B lymphocytes in vivo returns to
normal by day 3.
Next, we turned our attention to genes that are involved in the
inflammatory pathway. We observed an up-regulation of the
NF-?B family members p105, Rel-B, and c-rel, and C/EBP ? and
? (Fig. 5B). Together, this points to the activation of a proinflam-
matory transcriptional program. In fact, we saw up-regulation of
notable proinflammatory cytokines such as IL-23p19, IL-6, TNF-?,
and the T cell survival factor IL-2 (Fig. 5B). Among these, IL-
23p19 stands out due to the magnitude of the up-regulation, ?18-
fold at the 8-h time point (Fig. 5B). This up-regulation was con-
firmed by qPCR analysis (Fig. 5C). Curiously, the p40 subunit that
is shared between IL-23 and IL-12 (36) was not up-regulated and
neither was IL-12 p35. This suggests that up-regulation of the p40
subunit would require additional signals provided through T cell
interaction via CD40, which was up-regulated at 8 h (Fig. 5B).
Alternatively, such a robust up-regulation of the p19 subunit may
be sufficient to drive assembly of a functional IL-23 p19/p40 het-
erodimer even though the expression of p40 is not increased. Fi-
nally, we noted that the only TLR that was up-regulated
Transcriptional up-regulation of IL23p19 in professional APCs
To ensure that the above phenomenon was not unique to A20 cells,
experiments were performed in primary B lymphocytes, macro-
phages, and bone marrow-derived dendritic cells. Cells were
treated with thapsigargin and the RNA was collected at the time
points indicated (Fig. 6A). By qRT-PCR, IL23p19 was up-regu-
lated in all three APCs, suggesting that ER stress-induced activa-
tion of IL23p19 is a general phenomenon in these cells. Compa-
rable results were obtained by treating the cells with tunicamycin
(not shown). Finally, it was important to show a transcriptional
up-regulation of IL23p19 in B lymphocytes after transgenesis with
the ?1KDEL plasmid. As shown, 48 h after transgenesis a IL23p19
transcript was detected in B lymphocytes transgenic for
?1E?3KDEL-Kkbut not ?1E?3-Kk(Fig. 6B) suggesting that trans-
genic B lymphocytes that retain Ag intracellularly via the KDEL
motif also activate transcriptionally the IL23p19 gene. RNA ex-
tracted from bone marrow-derived dendritic cells harvested after
1 h of thapsigargin treatment was used solely as positive indicator
of IL23p19 transcription.
In this paper, we show that endogenously synthesized Ag pro-
cessed and presented by B lymphocytes can prime naive mice in
vivo to generate CD4 T cell responses irrespective of whether Ag
is targeted to the secretory pathway, the cytosolic compartment, or
is retained in the ER. Surprisingly, by appending a model Ag with
the KDEL retention motif in the APC, we induced a qualitatively
different in vivo CD4 T cell response marked by a heightened
production of the proinflammatory cytokines IFN-? and TNF-?. In
vitro genome-wide array analysis showed that ER stress of the
APC-induced by thapsigargin treatment activates the transcription
of the proinflammatory cytokines IL-23, IL-6, and TNF-?. To-
gether these results suggest that ER stress can affect Ag presenta-
tion at different levels.
Our data suggest a relation between intracellular retention of Ag
via the KDEL motif and up-regulation of ER stress genes. This is
in line with a previous report showing that the accumulation of
misfolded protein subsequent to impairment of retrieval by the
KDEL receptor induces ER stress and modulation of MAPK sig-
naling (37). However, short-lived up-regulation of the UPR has
been documented in B cells after cross-linking the BCR with anti-
Ig Ab in the absence of, or before, increased expression and ac-
cumulation of misfolded immunoglobulins in the ER (38–40). Al-
though this may apply to physiological regulation of B cell
biology, it may not directly relate to our studies where B lympho-
cytes are activated by spontaneous transgenesis (19, 41), a process
that, to the best of our knowledge, is independent of cross-linking
of the BCR. Thus, a tentative interpretation of the studies pre-
sented herein is that up-regulation of ER stress genes is linked with
mary APCs. A, Primary B lymphocytes (a), bone marrow derived dendritic
cells (b), and macrophages (c) were treated with 300 nM thapsigargin and
IL-23p19 levels were determined by qPCR analysis at the indicated time
B,B lymphocytes transfected
?1E?3KDEL-Kk(lane 2), or untransfected (lane 3) were harvested and
purified by Kkselection as in Fig. 4 after 48 h of culture in vitro. Total
RNA was extracted, normalized, and IL-23p19 transcripts were detected by
nested RT-PCR. RNA from dendritic cells after 1 h treatment with thap-
sigargin (lane 4) served as positive indicator.
Up-regulation of IL-23p19 in response to ER stress in pri-
262 ER STRESS AND PROINFLAMMATION
the accumulation of KDEL-tagged H chain protein in transgenic B
Previous studies in vitro indicated that ER stress activates
NF-?B transcription resulting in the production of the proinflam-
matory cytokine COX-2 (42), and also increases T cell adhesion to
endothelial cells (43), suggesting an indirect role on T cell function
during adaptive immunity. Interestingly, mice that lack the ER
stress sentinel IRE-1? are more susceptible to ER stress, are prone
to colitis (44), and show expression of the inflammatory marker
ICAM-1 in colonic sections, indicating that chronic ER stress can
lead to inflammation and autoimmunity. However, a link between
ER stress, APC function, and adaptive T cell immunity has, thus
far, been relatively unexplored. The experiments presented herein
begin to elucidate this possible link and shed light on how ER
stress in the APC is anticipatory of a proinflammatory T cell re-
sponse. Several arguments favor this tentative link. First, pertur-
bation of the secretory pathway function in primary B lymphocytes
modifies gene expression favoring the transcriptional activation of
proinflammatory cytokines such as IL-23p19, IL-6, TNF-?, and
IL-2. Second, intracellular retention of Ag in primary B lympho-
cytes influences the cell surface expression of at least one costimu-
latory molecule, OX40L, in vivo. Although we were unable to
formally prove that ER stress directly inhibited OX40L expression
(data not shown), it remains that perturbation of the secretory path-
way may influence the expression of a costimulatory molecule
found to be involved in the generation of memory T cell responses
(15, 45, 46). Since we recently argued for an inverse correlation
between inflammation at the time of priming and the generation of
memory T cell responses (47) the present findings suggest that ER
stress may produce T cell responses with diminished capacity to
evolve into memory responses. Third, since as demonstrated here,
all three types of professional APCs, B lymphocytes, dendritic
cells, and macrophages are capable of transcriptional up-regulation
of IL23p19 in response to ER stress (Fig. 6A), a direct immuno-
logical consequence of ER stress on the APC is priming of T cell
responses, which then produce larger amounts of IFN-? and
TNF-?. Taken together, these results suggest a scenario where in
the presence of, or in reaction to, ER stress, the APC activates
genes involved in a proinflammatory response. In turn, this im-
prints a proinflammatory characteristic on the T cells that are
primed in vivo. This view is also consistent with an emerging
central role of ER stress in regulation of the immune microenvi-
ronment by cytokines since TNF-? can sustain a UPR response
(48) and the anti-inflammatory cytokine IL-10 can inhibit the in-
flammation-induced ER stress response as demonstrated in intes-
tinal epithelia cells (49). Interestingly, the up-regulation of TLR2
in response to ER stress documented here is consistent with the
observed transcriptional up-regulation of IL-23p19 and other
proinflammatory cytokines, and absence of counter-regulation by
In conclusion, our results indicate that events that alter traffick-
ing through the ER causing retention of Ag and/or ER stress are
likely to lead to a generalized proinflammatory response. As such,
the molecular and genome-wide studies presented herein may have
important implications to better understand the adaptive immune
response against ER stress-inducing viruses, such as hepatitis C
virus (51), and against cancer cells in which ER stress has been
documented (52). Because a proinflammatory response dominated
by IL-23 has been recently linked with the pathogenesis of certain
autoimmune diseases (53) and IL-23 seems to be required for in-
flammatory responses against bacterial and mycobacterial infec-
tion (54, 55), our findings are relevant to better understand the
nexus between infection and immune pathology. Finally, the
proinflammatory effects of ER stress in the APC may be relevant
to vaccine design against pathogens and self Ags, including
We are thankful to Drs. M. Gerloni for help with the proliferation assay and
P. Lenert for the gift of CpG ODN 1826, S. Farber-Katz for preparing the
RNA samples, and Jennifer Lapira at the University of California San
Diego Biomedical Genomics Microarray laboratory for Illumina Beadarray
The authors have no financial conflict of interest.
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