Endoplasmic Reticulum Targeting Alters
Regulation of Expression and Antigen
Presentation of Proinsulin
This information is current as
of June 18, 2017.
Hsiang-Ting Hsu, Linda Janßen, Myriam Lawand, Jessica
Kim, Alicia Perez-Arroyo, Slobodan Culina, Abdel Gdoura,
Anne Burgevin, Delphine Cumenal, Yousra Fourneau, Anna
Moser, Roland Kratzer, F. Susan Wong, Sebastian Springer
and Peter van Endert
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Copyright © 2014 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2014; 192:4957-4966; Prepublished online 28
April 2014;
doi: 10.4049/jimmunol.1300631
http://www.jimmunol.org/content/192/11/4957
The Journal of Immunology
Endoplasmic Reticulum Targeting Alters Regulation of
Expression and Antigen Presentation of Proinsulin
Hsiang-Ting Hsu,*,†,‡,1 Linda Janßen,x,1 Myriam Lawand,*,†,‡ Jessica Kim,*,†,‡
Alicia Perez-Arroyo,*,†,‡ Slobodan Culina,*,†,‡ Abdel Gdoura,*,†,‡ Anne Burgevin,*,†,‡
Delphine Cumenal,*,†,‡ Yousra Fourneau,*,†,‡ Anna Moser,*,†,‡ Roland Kratzer,*,†,‡
F. Susan Wong,{ Sebastian Springer,x and Peter van Endert*,†,‡
S
election, recruitment, and processing of cellular Ags broken down to peptides presented by MHC class I (MHC-I)
molecules have been studied extensively in the last 15 y (1).
After the initial demonstration that proteasome inhibitors reduced
peptide supply to MHC-I molecules dramatically (2), a vast body
of evidence in support of a central role of this large cytosolic protease
in the breakdown of class I–presented Ags has been accumulated (3).
However, although a general-purpose backup proteolytic system
probably does not exist, presentation of some epitopes and peptide
loading of some MHC-I alleles is not affected, or even enhanced, in
the presence of proteasome inhibitors (reviewed in Ref. 4).
For a number of epitopes, such “proteasome-independent”
presentation has been shown to involve tripeptidyl peptidase II
*INSERM, Unité 1151, 75015 Paris, France; †Centre National de la Recherche
Scientifique, Unité 8253, 75015 Paris, France; ‡Université Paris Descartes, Sorbonne
Paris Cité, 75015 Paris, France; xBiochemistry and Cell Biology, Molecular Life
Science Center, Jacobs University Bremen, 28759 Bremen, Germany; and {Centre
for Endocrine and Diabetes Science, Cardiff University School of Medicine, Cardiff
CF14 4XN, United Kingdom
1
H.-T.H. and L.J. contributed equally to this work.
Received for publication March 7, 2013. Accepted for publication March 21, 2014.
Address correspondence and reprint requests to Prof. Peter van Endert, INSERM
U1151, Hôpital Necker, 149 Rue de Sèvres, 75015 Paris, France. E-mail address:
[email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: ATF4, activating transcription factor 4; BiP, binding Ig protein; CHOP, C/EBP homologous protein; CHX, cycloheximide; Doxy,
doxycycline; Epo, epoxomicin; ER, endoplasmic reticulum; ERAD, ER-associated
degradation; HA, hemagglutinin; HT, HeLa tet-on; IRE-1, inositol-requiring protein
1; MHC-I, MHC class I; Pen/Strep, penicillin/streptomycin; PERK, PKR-like ER
kinase; PH, proinsulin-hemagglutinin; PPH, preproinsulin-hemagglutinin; PPI, preproinsulin; PPS, preproinsulin-SIINFEKL; pT, pTRE-Tight; qPCR, quantitative PCR;
RIDD, regulated IRE-1–dependent decay; RT, room temperature; T1D, type 1 diabetes; TPPII, tripeptidyl peptidase II; UPR, unfolded protein response; XBP-1, Xbinding protein 1.
Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300631
(TPPII), another large cytosolic protease (reviewed in Ref. 5), and,
more recently, insulin-degrading enzyme (6). Partial proteasome
inhibition that favors cleavage at the epitope C terminus whereas
inhibiting destructive internal cleavage is a second mechanism
likely to underlie some cases in which presentation is enhanced or
unchanged in the presence of proteasome inhibitors (7). However,
these mechanisms may not be sufficient to explain the number of
MHC-I ligands produced in a proteasome-independent manner,
and additional mechanisms may remain to be discovered. Moreover, considering the strong effects of proteasome inhibition on
numerous cellular pathways (8), inhibition of epitope presentation
by proteasome inhibitors is not sufficient to prove that the source
Ag is indeed broken down by the proteasome, so that the number
of epitopes produced by the proteasome may be overestimated.
Recent studies have provided strong evidence that many MHC-I
ligands are derived from rapidly degraded proteins (9). Many of
these presumably correspond to defective ribosomal products that
fail to become functional proteins because of incomplete synthesis,
folding, posttranslational processing, or assembly. Current concepts
hold that cytosolic, nuclear, and endoplasmic reticulum (ER)–targeted proteins are indiscriminately recruited for production of MHC-I
ligands. Recruitment of the latter proteins involves the ER-associated
degradation (ERAD) pathway through which proteins unable to pass
the ER quality-control checkpoints are retrotranslocated into the
cytosol for proteolysis by the proteasome (10). However, the nature
and stringency of a possible link between ER quality control and
production of MHC-I ligands remain unclear. Although one study has
demonstrated a correlation between the folding capacity available in
the ER and production of an MHC-I ligand (11), other studies have
suggested that alterations such as removal of N-glycosylation sites or
disulphide bridges, which both perturb protein stability and folding,
do not affect Ag presentation (12).
Insulin, a hormone derived from ER-targeted preproinsulin (PPI)
by sequential proteolytic processing in pancreatic b cells, qualifies
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Peptide ligands presented by MHC class I (MHC-I) molecules are produced by degradation of cytosolic and nuclear, but also endoplasmic reticulum (ER)–resident, proteins by the proteasome. However, Ag processing of ER proteins remains little characterized. Studying processing and presentation of proinsulin, which plays a pivotal role in autoimmune diabetes, we found that
targeting to the ER has profound effects not only on how proinsulin is degraded, but also on regulation of its cellular levels. While
proteasome inhibition inhibited degradation and presentation of cytosolic proinsulin, as expected, it reduced the abundance of
ER-targeted proinsulin. This targeting and protein modifications modifying protein half-life also had profound effects on MHC-I
presentation and proteolytic processing of proinsulin. Thus, presentation of stable luminal forms was inefficient but enhanced by
proteasome inhibition, whereas that of unstable luminal forms and of a cytosolic form were more efficient and compromised by
proteasome inhibitors. Distinct stability of peptide MHC complexes produced from cytosolic and luminal proinsulin suggests that
different proteolytic activities process the two Ag forms. Thus, both structural features and subcellular targeting of Ags can have
strong effects on the processing pathways engaged by MHC-I–restricted Ags, and on the efficiency and regulation of their
presentation. The Journal of Immunology, 2014, 192: 4957–4966.
4958
Materials and Methods
Vaccinia viruses
Recombinant vaccinia viruses expressing human PPI and OVA have been
described previously (19) and were produced in HeLaS3 cells by standard
procedures. Considering a published report that proteasome inhibitors
prevent expression of late vaccinia-encoded proteins (20), we used a strong
synthetic early/late promoter contained in the pSC65 plasmid to produce
the PPI virus. A total of 5 3 105 HeLa tet-on (HT)-Kd cells (see later) were
seeded in six-well plates on day 21. The next day, the cells were treated
with or without 0.5 mM epoxomicin (Epo) for 2 h followed by virus infection at a multiplicity of infection of 2 in DMEM with 2.5% FBS for 2 h.
Then the medium was replaced with fresh complete DMEM with or
without the drug, and cells were incubated for another 2 h, harvested, and
analyzed by immunoblot.
HeLa Tet-on-Kd cells
An HT cell line expressing the reverse tetracycline-controlled transactivator
was purchased from Clontech (Mountain View, CA). HT cells were cultivated in DMEM (Sigma, St. Louis, MO) supplemented with 10% heatinactivated FBS (Hyclone, South Logan, UT), 2 mM L-glutamine (PAA,
Pasching, Austria), 1% MEM nonessential amino acids (Life Technologies
Invitrogen, Carlsbad, CA), and 200 mg/ml Geneticin-418 (PAA). HT cells
expressing the mouse class I molecules H2-Kd or H2-Kb were produced by
transfection with pcDNA3.1/hygro(-) plasmids (Invitrogen, Cergy Pontoise,
France) containing the corresponding full-length cDNAs that had been
cloned from bulk splenocyte cDNA by high-fidelity PCR, using primers
containing XbaI (59) and BamHI (39) restriction sites. Transfectants were
selected with 0.4 mg/ml hygromycin B, cloned with cloning cylinders, and
examined for MHC-I expression by flow cytometry. For each class I
molecule, one clone displaying high-level monoclonal class I expression
was selected for further experimentation.
Reagents
Epo (0.15–0.5 mM), MG-132 (2 mM), AAF-CMK (20–50 mM), and
bafilomycin A1 (50 mM) were purchased from BioMol (London, U.K.);
lactacystin (10 mM) was purchased from Calbiochem (San Diego, CA).
Tunicamycin (5 mg/ml), brefeldin A (10 mg/ml), and cycloheximide
(CHX; 20 mg/ml) were purchased from Sigma (St. Louis, MO). Butabindide oxalate (250 mM) was purchased from Tocris (Bristol, U.K.) and
added each 90–120 min following published protocols (21). The inhibitor
concentrations used are indicated in parentheses.
Abs
Anti-insulin Ab (H-86) was purchased from Santa Cruz Biotechnology
(Heidelberg, Germany). Anti–b-actin was from Sigma, and anti–b2 microglobulin was from Dako (Trappes, France). Secondary Abs for
immunoblots were goat anti-rabbit HRP and goat anti-mouse HRP
(Jackson Immunoresearch Laboratory, Suffolk, U.K.) used at 1:25,000.
Anti–CD8-allophycocyanin and anti–IFN-g–PE Abs were from BD Biosciences (Rockville, MD). Expression of H-2Kd was examined using Ab
20-8-4S.
pTRE-Tight constructs
PPI expression plasmids were constructed in the vector pTRE-Tight (pT;
Clontech) containing the Tet Response Element. Initially, the pT cloning
site was modified by insertion between the SalI and XbaI sites of complementary oligonucleotides encoding a hemagglutinin tag YPYDVPDYA,
or the OVA CD8+ T cell epitope SIINFEKL. Then cDNAs encoding PI or
PPI were amplified from a previously described cloned human PPI sequence (19), using primers containing BamHI (59) and SalI (39) sites, and
subcloned into modified or unmodified pT. Plasmids expressing PPI in
which one or several Cys residues were replaced by Ser were constructed by
site-directed mutagenesis using the QuikChange kit (Stratagene, La Jolla,
CA) and Phusion high-fidelity polymerase (New England Biolabs, Evry,
France). Correct sequences of all plasmids were confirmed by sequencing.
Transient transfection
HT-Kd cells were transfected with pT plasmids using a Gene Pulser Xcell
electroporator (Bio-Rad, Hercules, CA). A total of 6 3 106 cells in 250 ml
electroporation buffer, 10 mM HEPES in PBS, were electroporated with 20
mg endotoxin-free DNA at 250 V and 900 mF. Pulsed cells were recovered
in warm DMEM with 20% FBS. Protein expression was induced by addition of 0.5–1.0 mg/ml doxycycline (Doxy; Sigma) either immediately
or 16–18 h after transfection, as indicated. Where indicated, cells were
treated with drugs before, during, and/or after induction of protein expression. Cells were harvested using trypsin, washed once in PBS with
1 mM PMSF (Sigma), counted, and analyzed by immunoblot.
Immunoblot
Cell pellets were resuspended in loading buffer with 100 mM DTT at
a concentration of 1 3 107 cells/ml. Ten microliters of cell lysate was
loaded on 12.5% SDS-PAGE gels, transferred to polyvinylidene fluoride
membranes, blocked with 5% skim milk, and stained with anti-insulin Abs
used at 260 ng/ml. Abs to b2-microglobulin or b-actin (both used at
1:5,000) were used as loading controls. HRP-coupled secondary Abs were
used at 1:25,000. Loading controls were stained on the same membrane as
insulin after membrane stripping at 50˚C for 30 min in 100 mM 2-ME, 2%
SDS, and 62.5 mM Tris-HCl, pH 6.7. An ECL detection system, Immobilon Western HRP (Millipore, Guyancourt, France), was used for developing the membranes. Images were taken with a CCD camera (Fujifilm,
Tokyo, Japan).
Microscopy
HT cells were electroporated with plasmids encoding PPI or PI. Cells were
seeded on coverslips in a 24-well plate, and protein expression was induced
immediately with 1 mg/ml Doxy. After 24 h, cells were washed in PBS and
fixed in 4% paraformaldehyde in PBS for 20 min at room temperature
(RT). Fixation was stopped using 0.2 M glycine, pH 7.4 for 5 min, and
followed by permeabilization with 0.2% saponin (Sigma), 2% FBS in PBS
for 20 min. Cells were stained with anti-insulin Ab diluted at 1:50 in PBS
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as an interesting example of a donor of MHC-I ligands in light of
several considerations. Insulin is a critical autoantigen in human
type 1 diabetes (T1D), as well as in the principal rodent model of
T1D, the NOD mouse, in which insulin appears to trigger autoimmunity (13). In NOD mice, a significant population of CD4+
and CD8+ T cells found in early islet infiltrates recognizes insulin,
and both cell types can transfer disease. Insulin-specific autoreactive T cells are also detected in the blood of T1D patients (14),
and insulin-specific CD8+ T cells can kill b cells in humanized
NOD mice (15).
Given that insulin is expressed in thymic epithelial cells and that
insulin is constantly present in the peripheral circulation, autoreactive T cells that recognize insulin should be exposed to strong
pressure resulting in negative selection or induction of peripheral
tolerance (13). Indeed, immunodominant CD8+ and CD4+ T cell
insulin epitopes display low MHC binding affinity, resulting in
low-avidity interactions between peptide–MHC complexes and
T cells (16). As noted by other authors, it is surprising that such
low-avidity interactions are sufficient to trigger autoimmunity.
One possibility, proposed by Suri et al. (17), is that very high
concentrations of b cell proteins or debris are released during T1D
pathogenesis, thereby both overcoming low T cell avidity and
explaining the tissue specificity in the face of systemic presence of
soluble insulin. Another possibility relates to the exclusive production of PPI, comprising a leader and the C peptide in addition
to insulin, by b cells (13). However, the immunodominant CD4+
and CD8+ T cell epitopes reside in the insulin B chain, which
argues against this hypothesis. Alternatively, Ag processing of
ER-targeted PPI in b cells may differ from that of internalized
insulin, resulting in quantitatively or qualitatively distinct presentation of insulin epitopes. The fact that b cells display a
constitutive activation of the unfolded protein response (UPR),
a tripartite cellular response to high protein load in the ER that
includes upregulation of the ERAD pathway, might also be of
interest in this context (18).
We have studied the processing and Ag presentation of insulin,
and we report that ER targeting has a strong effect both on the
expression of insulin in the presence of proteasome inhibition and
on the proteolytic pathways involved in its processing for MHC-I
presentation. These results contribute to our understanding of the
processing of ER-targeted Ags, but they may also be of relevance
for the role of insulin as primary autoantigen in T1D.
ER TARGETING AND MHC CLASS I Ag PRESENTATION
The Journal of Immunology
with 0.1% saponin and 2% FBS for 30 min at RT followed by Alexa 594–
coupled goat anti-rabbit Abs diluted 1:200 for 30 min at RT. Stained cells were
again fixed for 10 min at RT, and the reaction stopped with 50 mM NH4Cl in
PBS for 5 min. Finally, coverslips were incubated briefly with 50 ng/ml DAPI
(Invitrogen), mounted with DABCO (Sigma), and sealed with nail polish.
Analysis of gene expression
Analysis of protein synthesis and degradation
To analyze general protein or insulin synthesis, or insulin degradation, we
transfected 107 HT cells with 24 mg plasmid DNA using 120 mg branched
polyethylenimine (Sigma-Aldrich), followed immediately by induction of
protein expression by addition of 1 mg/ml Doxy and cell incubation at
37˚C for 18 h. Then 0.5 mM Epo or an equivalent volume of DMSO was
added to the cells. After incubation for 2 h, the cells were detached with
trypsin, washed 23 in PBS, resuspended at 5 3 106 cells/ml in modified
RPMI 1640 labeling medium (Sigma-Aldrich) substituted with 2% FCS,
glutamine, and penicillin/streptomycin (Pen/Strep) and DMSO or Epo as
before, and starved for 30 min at 37˚C. After the starvation period, the cells
were pelleted, resuspended in fresh labeling medium, split into several
tubes, and labeled using [35S] methionine/cysteine (Expre35S35S Protein
Labeling Mix, 259 MBq; Perkin-Elmer). When protein or insulin synthesis
was examined, labeling was followed immediately by counting of incorporated radioactivity or insulin immunoprecipitation. Cells pretreated in
the same manner were labeled as described for 15 min, followed by
resuspension in chase medium (DMEM containing 10% FCS, 100 mg/ml
methionine and 500 mg/ml cysteine, glutamine, Pen/Strep; PAA) and
chasing for the indicated times, to study insulin degradation. At each chase
time point, an aliquot was taken and put on ice.
The labeled cells were washed 23 in ice-cold PBS and lysed in 500 ml
PBS containing 1% Nonidet-P40, 200 mM PMSF, and 500 mM iodoacetamide at 4˚C for 1 h to monitor insulin synthesis or degradation. When
general protein synthesis was examined, 5 ml of the lysate was removed at
this point and added to 3 ml liquid scintillation mixture (Ultima Gold,
Packard Bioscience), incubated for 1 h at RT, and counted in a Tri-Carb
Liquid Scintillation Counter (Packard-Bioscience). The (total or remaining) lysate was centrifuged for 5 min at 4˚C and 16,400 3 g, and the
supernatant was precleared for 30 min at 4˚C using 50 ml packed Staphylococcus aureus (Sigma-Aldrich). The precleared lysate was transferred
to Protein A beads (Calbiochem) previously coated with anti-insulin Ab.
After a 1-h incubation at 4˚C, the beads were washed 53 in PBS containing 0.1% Nonidet-P40, dried with a syringe, and boiled in 45 ml sample
buffer at 95˚C for 10 min. Finally, 15 ml of the sample was separated on
a 15% SDS-PAGE and blotted on PDVF membrane. Autoradiographs
taken with Fuji imaging plates and a Fuji FLA-2000 apparatus were analyzed by densitometry using ImageJ.
Ag presentation assay
CD8+ T cell lines from peptide-immunized HLA-A2 transgenic HHD
mice have been described previously (19). G9C8 TCR-transgenic T cells
were obtained as frozen spleen cells and cultured in Click’s medium with
complete supplements (10% heat-inactivated FBS, 2 mM L-glutamine, 1%
Pen/Strep, 10 mM HEPES, and 50 mM 2-ME). A total of 106 G9C8
splenocytes was stimulated with 107 irradiated NOD splenocytes and 1026
M peptide insulin B15–23 LYLVCGERG (Schafer-N, Copenhagen, Denmark). On days 2 and 3, 10% of T cell growth factor obtained by stimulating rat splenocytes for 24 h with Con A was added. Cells were
restimulated in the same manner three times. On day 7 of the fourth
(or subsequent) stimulation(s), the G9C8 T cells were used for Ag presentation assays. For this, HT-Kd cells were transfected with 30 mg
PPI-encoding or 15 mg PI-encoding plasmids by electroporation, as
described earlier. Expression of protein was induced with 1 mg/ml Doxy
for 24 h, and cells were treated subsequently with drugs, as indicated.
Where appropriate, surface peptide–MHC-I complexes were dissociated in
acid (163 mM citric acid, 320 mM NaH2PO4, pH 2.6) for 1 min. Acid
stripping reduced expression of H-2Kd by 94% and stimulation of G9C8
cells by transfectants by 60–90%. Acid-stripped cells were incubated with
a lower (0.2 mM) Epo concentration to avoid toxicity. Cells were harvested
and counted, and added at a ratio of 10:1 (105 HT and 104 T cells) to G9C8
cells in 96-well conical bottom plates for a maximum of 12 h at 37˚C and
in the presence of 5 mg/ml BFA. Cells were stained with anti–CD8aallophycocyanin Ab diluted 1:100 in FACS buffer (2% FBS in PBS) on ice
for 30 min, washed, and fixed and permeabilized with the Cytofix/Cytoperm
kit (BD Biosciences). Finally, cells were stained with anti–IFN-g–PE Ab
used at 1:30 on ice for 30 min. IFN-g expression of CD8+ cells was analyzed
using a FACS CANTOII (BD Biosciences). Data were analyzed with FlowJo
(Tree Star, Ashland, OR).
Results
Effect of proteasome inhibition on presentation and expression
of vaccinia virus–encoded PPI
To study Ag processing of PPI, we first used previously described
(19) cytotoxic T cell (CTL) lines produced in HLA-A2–expressing transgenic mice that recognize epitopes in the three parts of
the proinsulin (PI) molecule (A and B chains, C peptide). PI expression was induced by infection of HeLa APCs with a vaccinia
virus encoding PPI (Fig. 1A). Incubation of APCs with a proteasome inhibitor before virus infection reduced presentation of all
virus-encoded epitopes substantially (range 40–95%), whereas
presentation of the corresponding synthetic peptides was not affected. This suggested that presentation of PI epitopes is proteasome dependent.
To examine a possible effect of proteasome inhibition on Ag
abundance, we analyzed PPI expression in virus-infected cells.
APCs were preincubated with inhibitors and washed before addition of virus to avoid possible inhibitor effects on cell infection.
Presentation of an epitope encoded by a vaccinia virus–encoded
minigene was not affected by proteasome inhibitors, providing
further evidence against an effect of inhibitors on infection or
general protein expression (data not shown). Surprisingly, the
nonreversible proteasome inhibitor Epo, when present either before virus infection only or present both before and postinfection,
abolished PPI expression almost completely (Fig. 1B). Addition of
the inhibitor after virus infection had the opposite effect and
resulted in increased PI detection in immunoblots. Experiments
with a second drug (lactacystin) had equivalent effects, demonstrating reduced and increased PI detection upon proteasome inhibition before or after vaccinia infection, respectively (Fig. 1C).
These experiments allowed for two conclusions: 1) compromised
proteasome function antagonizes cellular PI accumulation, possibly because of inhibition of synthesis; and 2) in cells synthesizing
PPI, proteasome inhibition results in PI accumulation, suggesting
a role of the proteasome in PPI degradation.
Effect of proteasome inhibition and drugs inducing ER stress
on tetracycline-regulated expression of PPI
Vaccinia virus infection has strong effects on cellular metabolism
that may affect Ag presentation by MHC-I molecules. We therefore
established a second regulated expression system, using transient
transfection of HeLa cells expressing the tetracycline regulator,
which had previously been transfected with the murine H-2Kd
MHC-I molecule and in this article is designated HT-Kd cells. The
insulin constructs expressed (Fig. 2A) included a cytosolic form
devoid of the signal peptide (PI), cytosolic PI extended by
a hemagglutinin (HA) tag (proinsulin-HA [PH]), wild type PPI,
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
For analysis of gene expression in vaccinia-infected cells, 5 3 106 HT-Kd
cells were infected with insulin or OVA-expressing viruses at a multiplicity
of infection of 30 and treated with drugs as described earlier. Cells transiently transfected with pT plasmids were rested for 16 h after transfection
before simultaneous induction with 1 mg/ml Doxy and treatment with
drugs for 2 (two experiments) or 5 h (one experiment). RNA was extracted
from cells using Illustra RNAspin Mini kit (GE Healthcare), following the
manufacturer’s instructions. One microgram RNA was transcribed to
cDNA using the Improm-II Reverse Transcription System (Promega,
Charbonnieres, France). Quantitative PCR (qPCR) analysis of gene expression was performed using primers specific for activating transcription
factor 4 (ATF4), binding Ig protein (BiP), C/EBP homologous protein
(CHOP), spliced X-binding protein 1 (XBP-1), insulin and OVA, SYBR
Green Mix (Eurogentec, Angers, France), and ABI7900 equipment (Applied Biosystems). GAPDH was used as housekeeping gene for normalization, except for spliced XBP-1 where unspliced XBP-1 was used.
qPCRs were performed using 2 ng/ml cDNA. qPCR results are expressed
as ratios between the normalized expression of test samples and that of
reference samples, which are indicated in the figure legends.
4959
4960
and PPI variants lacking one (PPI-C20S) or two (PPI-C72S)
disulphide bridges, or extended by carboxyterminal tags that introduce an N-linked glycosylation site (preproinsulin-HA [PPH],
preproinsulin-SIINFEKL [PPS]), combined with disulphide bridge
deletion (data not shown) or not. Transiently transfected HT-Kd
cells incubated with the tetracycline analog Doxy showed insulin
staining that overlapped with the ER marker KDEL for PPI, but
not for the cytosolic PI form, as expected (Supplemental Fig. 1).
HT-Kd cells induced 16 h after PPH transfection with Doxy expressed
large amounts of the protein, whereas the protein was undetectable in
uninduced cells. In contrast, Doxy-induced cells expressing PH
contained hardly detectable amounts of the protein (Fig. 2B).
We then tested the effect of proteasome inhibition on Doxyregulated expression of PPH and PH. PPH expression was reduced after preincubation with Epo and when the drug was
continuously present, whereas addition of the drug after 8 h of
induction had no effect (Fig. 2B). Similar effects were obtained
FIGURE 2. Tetracycline-regulated PPI expression system. (A) PPI variants are expressed in pT. The position of epitope insulin B15–23 recognized
by G9C8 cells is indicated. Stars indicate an N-glycosylation site at the
transition between the A chain and hemagglutinin (HA) and SIINFEKL
(S8L) tags. Thin lines indicate disulphide bridges. (B) Insulin immunoblots
and b2-microglobulin loading controls of HT cells transfected with pTPPH or pT-PH. Sixteen hours after transfection, cells were incubated with
0.5 mM Epo or solvent for 2 h (Pre-incub.). Then protein expression was
induced by incubation with 1 mg/ml Doxy for 6 h except in the lane labeled
nil. Finally, the cells were incubated for another 2-h period with Epo or
Doxy alone, or simultaneously with both drugs (Post-incub.). One of three
experiments is shown.
with lactacystin and MG132, irreversible and reversible proteasome inhibitors, respectively (data not shown). These results
confirmed those obtained with virus-infected cells and demonstrated that reduced PPI/PPH abundance was not limited to the
vaccinia virus expression system. In striking contrast with the
results obtained with PPH, proteasome inhibition before, after, or
simultaneously with induction strongly increased the amount of
detectable PH, such that even uninduced cells accumulated greater
amounts than induced cells in the absence of Epo (Fig. 2B). This
suggested that PI that is ectopically expressed in the cytosol is
rapidly degraded by the proteasome. Importantly, the inhibitory
effect of proteasome inhibition on PPI and PPH abundance did not
extend to PH and, therefore, was related to the presence of a signal
peptide mediating ER targeting.
Seeking to explain the effect of ER targeting on protein expression in the presence of proteasome inhibition, we considered
the possibility that ER stress could have affected synthesis of PPH
and PH differentially. The UPR is a physiologic response to high
protein load in the ER that relies on three ER sensor proteins that
are activated on consumption of ER chaperones by unfolded
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 1. Effect of proteasome inhibition on expression and presentation of vaccinia-encoded PPI. (A) Killing of HeLa-HHD cells infected
for 1 h with a vaccinia virus–encoding PPI, or pulsed with 10 mM cognate
peptide, was tested in a standard 51Cr release assay, using specific CD8+ T
cell lines produced by immunization of HLA-A2 transgenic mice. Where
indicated, cells were incubated with lactacystin before vaccinia infection,
first at 100 mM for 1 h and then overnight at 1 mM. (B) HT cells were
treated in duplicate with 1 mM Epo or DMSO (solvent) for 2 h, then
infected with virus for another 2 h, and finally left for a third 2-h period
with solvent or Epo. The gel images in this and the following panels were
cropped. (C) HT cells were treated with 10 mM lactacystin or solvent for
2 h, washed, and infected with PPI- or OVA-expressing vaccinia viruses for
4 h in the presence of 10 mM lactacystin or solvent. The stripped blot was
stained for b2-microglobulin as loading control. Insulin expression was
detected in (B) and (C) by immunoblot. One of two (A) or three (B and C)
experiments is shown. M, m.w. marker; rPI, 5 ng recombinant proinsulin.
ER TARGETING AND MHC CLASS I Ag PRESENTATION
The Journal of Immunology
FIGURE 3. Effect of drugs on different PPI forms. (A–D) HT cells were
transfected with pT plasmids encoding PPH, PPI, or PPH-C72S, as shown.
Sixteen hours later, cells were preincubated with the indicated drugs or
solvent for 2 h before adding 1 mg/ml Doxy for 6 h, and a final period (Post
incub.) with drugs, also as indicated. Expression of insulin and of actin and
b2-microglobulin loading controls was assessed by immunoblot. One of
two experiments is shown.
reason underlying reduced abundance of plasmid-encoded PPI/H
remained unclear.
Reduced protein abundance can primarily be because of a lower
rate of synthesis and/or a higher rate of degradation. We studied
the rates of synthesis and degradation of the different plasmidexpressed insulin forms using immunoprecipitation of metabolically labeled insulin. Pretreatment of transfected cells with Epo,
present during cell starvation or not, did not alter the rate of global
protein synthesis, as reflected by label incorporation during shortterm pulses (Fig. 4B). Epo pretreatment also had no significant
effect on synthesis of four ER-targeted insulin forms (Fig. 4C and
4D). Synthesis of PI and PPI-C72S could not be monitored because sufficient amounts of these forms could not be immunoprecipitated. Examination of insulin degradation confirmed the
anticipated effects of the different protein modifications used
(Fig. 4E). Thus, removal of the insulin signal peptide resulted in
an extremely short half-life, whereas addition of any C-terminal
tag with an N-glycosylation site increased the half-life of ERtargeted insulin. Removal of disulphide bridges from HA-tagged
insulin slightly reduced half-life, whereas the difference between
the half-lives of PPH and PPS suggests a modest effect of the tag
sequence independent of glycosylation (Fig. 4E). Presumably as
a result of different rates of degradation and possibly also synthesis, the steady-state levels of the different insulin forms dis-
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proteins and that downregulate general protein synthesis, upregulate levels of proteins involved in protein folding and degradation,
and trigger apoptosis if the stress cannot be resolved (22). Mild ER
stress can also be triggered by proteasome inhibition through illdefined mechanisms (23, 24). In addition, ER stress can result in
rapid downregulation of the synthesis of a subset of ER-targeted
proteins via inositol-requiring protein 1 (IRE-1)–mediated cleavage of the corresponding mRNAs (25), providing a potential
mechanism for specific downregulation of ER-targeted, but not
cytosolic PI in the presence of proteasome inhibitors.
If reduced expression of PPH was due to activation of the UPR,
then other drugs inducing this condition were expected to have
a similar effect. We studied the effect of tunicamycin and brefeldin
A, two drugs inducing massive ER stress by blocking N-linked
glycosylation and protein export from the ER, respectively. At
the same time, we tested drug effects on additional PPH/I forms
devoid of the nonphysiologic HA tag or lacking two disulphide
bridges. The latter experiments were designed to reveal whether
protein modifications that presumably alter stability modify the
effect of drugs on protein abundance. Surprisingly, and in contrast
with Epo, tunicamycin had only a minor effect on PPH levels,
although it abolished glycosylation of PPH, which demonstrated its
pharmacological activity (Fig. 3A). In contrast, brefeldin A, which
retains secretory pathway proteins such as b2-microglobulin within
the cell (Fig. 3B), abolished PPH expression. Tunicamycin and
Epo had the same contrasting effects on abundance of PPI, demonstrating that the artificial glycosylation site and HA tag were not
responsible for the observed effects (Fig. 3C). Expression of a PPH
mutant lacking two disulphide bridges was also abolished by Epo,
but in this case, tunicamycin strongly reduced expression (Fig. 3D).
These results allowed for several conclusions: 1) a state of compromised proteasome function reduces abundance of all ER-targeted
PPI/H forms irrespective of the presence of N-glycans or disulphide bridges; 2) reduced stability of ER-targeted PPH may correlate
with stronger reduction of expression in the presence of ER stress
inducers; and 3) different agents known to induce ER stress have
distinct effects on abundance of ER-targeted PPI/H.
To directly study activation of the UPR, we quantified cellular
mRNA levels for several proteins that are upregulated in its
presence including IRE-1 (also called ERN1), XBP-1, BiP, CHOP,
and ATF4. The latter three proteins are all induced by the PKR-like
ER kinase (PERK) branch of the UPR. As expected, tunicamycin
alone, or in combination with vaccinia infection or plasmid
transfection, induced BiP and CHOP mRNA levels strongly and
IRE-1 (ERN1) and splicing XBP-1 mRNA levels modestly, thus
activating both the IRE-1 and PERK branches of the UPR.
Moreover, high-level expression of vaccinia virus–encoded PPI,
but not OVA, resulted in upregulation of several UPR markers,
whereas transfection of PI/PPI-encoding plasmids had a modest
effect, if any (Supplemental Fig. 2). However, Epo treatment alone,
or in combination with cell infection or transfection, did not result
in any noticeable activation of UPR markers. Therefore, the effect
of Epo on PPI/H expression is not due to activation of the IRE-1
and/or PERK branches of the UPR at the transcriptional level.
In the initial wave of the UPR, regulated IRE-1–dependent decay
(RIDD) of specific ER-targeted mRNAs acts as a rapid mechanism
for reducing the protein load in the ER (26). Indeed, in vaccinia
virus–infected cells, Epo treatment reduced PPI mRNA levels by
90% (Fig. 4A), whereas the effect on OVA mRNA levels was
modest (reduction by 40%). However, Epo treatment had no significant effect on the level of PH or PPH mRNA in plasmidtransfected cells (Fig. 4A, right panel). These results suggested
that the effect of Epo on the abundance of vaccinia-expressed PPI
was at least partly due to reduced protein translation, whereas the
4961
4962
ER TARGETING AND MHC CLASS I Ag PRESENTATION
played substantial differences, with high abundance of tagged
ER forms, intermediate abundance of physiologic PPI, and low
abundance of PPI-C72S lacking two disulphide bridges (Fig. 4F).
Assay measuring T cell stimulation by transfectants expressing
PPI and PI
The results described so far indicated that reduced presentation
of MHC-I–restricted epitopes by vaccinia virus–infected APCs
(Fig. 1A) in the presence of proteasome inhibitors most likely had
been caused by inhibitor effects on PPI synthesis. To identify the
proteolytic pathways involved in PI and PPI degradation for presentation by MHC-I molecules, we used experimental conditions
in which cells were first allowed to synthesize the antigenic protein before inhibitors were added (Fig. 5A). This was achieved by
24-h incubation with Doxy followed by a brief acid treatment to
remove cell-surface Kd-insulin B15–23 complexes. The generation
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FIGURE 4. Effect of drugs on Ag mRNA levels and rates of synthesis and degradation. [(A), left panel] HeLa cells were treated for 2 h with solvent, 0.5
mM Epo, or 5 mg/ml tunicamycin; then cells were either infected with PPI- or OVA-encoding vaccinia viruses for 4 h or incubated for 4 h in medium before
harvesting, RNA extraction, and examination by qPCR. All values obtained were first normalized with respect to the housekeeping gene GAPDH. Then
normalized ratios were calculated using values obtained for noninfected cells as calibrator. [(A), right panel] Results of an equivalent experiment in which
HT cells were transfected and treated with drugs or solvent. One of three experiments is shown. (B) HT cells were preincubated for 2 h in medium with
DMSO (solvent) or with 0.5 mM Epo, then starved for 30 min in the absence (Epo) or presence (Epo + starvation) of Epo before labeling for the indicated
times with [35S]-methionine/cysteine and measuring of incorporated radioactivity. The average of three experiments is plotted. (C) Plasmid-transfected
HT cells were pretreated similarly with solvent or Epo and then metabolically labeled for the indicated periods before immunoprecipitation of insulin. The
immunoprecipitates were separated by SDS-PAGE, and precipitated insulin was quantified by film exposure and densitometry. (D) One of the two
experiments whose average is plotted in (C). (E) Transfected HT cells were similarly labeled for 15 min followed by a chase for the indicated lengths of
time. The amount of immunoprecipitated insulin was determined as in (C) and (D), and is expressed as ratio of signal at each time point divided by the
signal at the end of the 15-min pulse. The average of two experiments for PPS and three for the other forms is plotted. (F) HT cells were transfected with the
indicated constructs, expression was induced overnight with Doxy, and insulin levels were assessed by immunoblot.
The Journal of Immunology
4963
Next, we studied the effect of proteasome inhibition on the presentation of the different Ag forms. This revealed striking and unexpected differences (Fig. 6B). Whereas presentation of cytosolic PI
was inhibited by MG132, presentation of all ER-targeted forms was
increased. Moreover, the magnitude of the increase correlated with
protein half-life, so that presentation of PPH was tripled, whereas
that of PPI-C72S, the least stable ER-targeted form, was almost
unchanged (Fig. 6B). This correlation applied to ER-targeted forms
carrying two different C-terminal tags (Fig. 6B and 6C). The small
effect of the deletion of the C72 disulphide bridge also correlated
with the slightly reduced stability of this PPH variant (Figs. 4E and
6B); although we did not measure the stability of the PPS-C20S
variant, it is reasonable to assume that the smaller increase in presentation upon proteasome inhibition as compared with unmodified
PPS reflected reduced stability. In conclusion, protein modifications
increasing protein half-life and steady-state abundance correlated
negatively with efficacy of presentation and positively with enhanced presentation in the presence of proteasome inhibitors.
Effect of proteasome inhibitors on presentation
The enhancing effect of proteasome inhibitors on the presentation
of ER-targeted PPI forms suggested that other proteases might
process these Ags. AAF-CMK, which inhibits both cytosolic TPPII
and lysosomal TPPI, but also the cytosolic aminopeptidases
bleomycin hydrolase and puromycin-sensitive aminopeptidase
(29), reduced presentation of several ER-targeted Ag forms substantially (Fig. 6C), whereas bafilomycin, an inhibitor of vacuolar
H+-ATPase, had a smaller inhibitory effect on presentation of
several ER-targeted forms and of cytosolic PI (Fig. 6B). Considering that TPPII is known both to act as an enzyme trimming
epitope precursors (30) and to generate some epitopes (reviewed
in Ref. 5), we tested butabindide, a more specific TPPII inhibitor.
We next studied the efficiency of presentation of different PPI forms,
as well as the effect of protease inhibitors on their presentation.
Transfectants expressing PI or PPI both stimulated G9C8 cells
(Fig. 5B), but intriguingly, cells expressing cytosolic PI consistently
had higher stimulatory capacity than those expressing any
ER-targeted Ag form (Figs. 5B and 6A). Moreover, there were
marked differences between the different ER-targeted forms:
N-glycosylation reduced presentation, whereas deletion of disulphide
bridges had the opposite effect (Fig. 6A). Considering the results
shown in Fig. 4C–F, we concluded that protein half-life and steadystate abundance were inversely correlated with presentation efficacy.
FIGURE 5. Stimulation of G9C8 T cells by transfected HT cells. (A) HT-Kd cells transfected as indicated
and induced with Doxy for 24 h were treated or not for
2 h with 0.5 mM Epo for 2 h, acid stripped, and reincubated for 8 h with or without 0.2 mM Epo. Lanes are
mounted from two different blots and show the PI content of equivalent cell numbers. (B) HT-Kd cells transfected with pT-PPI or pT-PI were treated with 1 mg/ml
Doxy for 24 h and incubated with G9C8 T cells for 12 h
followed by flow cytometric analysis of intracellular
IFN-g staining in CD8+ cells. Control: G9C8 cells alone.
HT-Kd cells incubated with 1026 M peptide insulin B15–23
were used as positive control. (C) Decay of Kd/insulin
B15–23 complexes. HT-Kd cells were pulsed with 1026 M
peptide insulin B15–23 and incubated for the indicated
time. Then cells were added to G9C8 T cells for an additional 12 h followed by flow cytometric analysis of
IFN-g production. (D) Recovery of Kd/insulin B15–23 complexes after acid stripping. PPI-expressing HT-Kd cells
were induced for 6 h with Doxy and then acid stripped.
After an additional 2- to 6-h incubation, G9C8 cells were
added for a 12-h incubation in the presence of BFA followed by intracellular IFN-g staining and flow cytometric
analysis. Asterisk indicates cells continuously incubated
with BFA after acid stripping; box indicates cells not subjected to acid stripping. One of two (C and D), four (A), or
eight (B) experiments is shown.
Possible role of alternative pathways in presentation of PI and
PPI
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of new Kd-insulin B15–23 complexes from previously synthesized
PPI, or from newly synthesized PI, in the presence of protease
inhibitors was then monitored.
Production of antigenic epitopes was read out using TCRtransgenic G9C8 CD8+ T cells with specificity for the H2-Kd–
restricted T cell epitope insulin B15–23 (27), an epitope with
immunodominant status in the NOD mouse that is recognized by
diabetogenic T cells found in pancreatic islet infiltrates (28).
In vitro activated and restimulated G9C8 T cells secrete IFN-g
upon stimulation with 1026 M cognate peptide and H2-Kd APCs
(Fig. 5B); IFN-g secretion was not detectable at ,1028 M peptide
(data not shown). Cell-surface H2-Kd–insulin B15–23 complexes
had a half-life of ∼11.5 h (Fig. 5C), which was compatible with
the use of an IFN-g secretion assay carried out in the presence of
brefeldin A as readout for T cell stimulation. Moreover, acidtreated H2-Kd cells transfected with PPI (or PI, data not shown)
recovered full stimulatory capacity for G9C8 T cells 5 h after
removal of cell-surface pMHC complexes (Fig. 5D). Therefore,
acid-stripped transfectants were incubated for a minimum of 6 h
before addition of T cells in subsequent experiments.
4964
ER TARGETING AND MHC CLASS I Ag PRESENTATION
Discussion
FIGURE 6. Effect of drugs on direct and indirect presentation of
epitope insulin B15-23. (A–C) HT-Kd cells were transfected with different constructs as indicated and treated with 1 mg/ml Doxy for 24 h.
Then cells were treated with the indicated drugs for 2 h, acid stripped,
and reincubated with the same compounds at lower concentration for an
additional 8 h. Drug concentrations were: MG132, 2 mM/2 mM; bafilomycin: 0.1 mM/0.1 mM; AAF-CMK: 50 mM/20 mM; Epo: 0.5 mM/0.2
mM. Stimulation of G9C8 cells was evaluated like in Fig. 3 and is
expressed as percentage change relative to stimulation in the absence of
inhibitors in (B) and (C). One of two (C), three (B), or four (A) experiments is shown.
Butabindide had no effect on presentation (data not shown), arguing against a role of TPPII in degradation of ER-targeted Ag.
If the processing pathways acting on cytosolic and ER-targeted
PI differ, the peptides produced in them also might be distinct;
although peptide insulin B15–23 is the optimal peptide recognized
by G9C8 cells (16), N- or C-terminally extended variants could be
stimulatory. We reasoned that if different peptides result from
degradation of PI versus PPI, the stability of the complexes
formed with H-2Kd molecules might differ. To address this, we
incubated HT-Kd cells with CHX, an inhibitor of protein synthesis
blocking production of new complexes, and monitored the decay
of H-2Kd/insulin B15–23 complexes using G9C8 cells. We first
tested the effect of CHX on expression of H2-Kd and observed
a reduction by 33% after a 4-h incubation (Fig. 7A). Under the
same conditions, presentation of PPI was reduced by 30%, whereas
that of PI was reduced by 75% (Fig. 7B). Thus, pMHC complexes
formed upon degradation of PPI were significantly more stable than
those formed upon degradation of PI, consistent with production of
distinct peptides from the two Ag forms.
We have demonstrated that ER targeting of PI affects three
parameters: protein abundance in the presence of proteasome inhibition, efficacy of presentation, and effect of proteasome inhibition
on Ag presentation. Moreover, in the case of ER-targeted Ag forms,
we find that the two latter parameters correlate with modifications
altering protein half-life. Finally, we report evidence suggesting that,
for a given Ag, subcellular localization may affect the exact nature of
an epitope presented. Collectively, these results highlight the important role of subcellular Ag localization in MHC-I Ag processing.
Searching for a mechanism explaining reduced PI expression in
the presence of proteasome inhibitors, the fact that the phenomenon
was limited to ER-targeted PI forms led us to speculate that ER
stress was involved. The UPR has a physiologic and dual role in
production of insulin by b cells: functional PERK and IRE-1
branches of the UPR are required for b cell survival and function; however, chronic activation of IRE-1 can compromise insulin
production (31, 32). Both IRE-1–dependent cleavage of PPI
mRNA and global attenuation of protein translation mediated
by the PERK-dependent pathway were candidate mechanisms
explaining reduced PPI synthesis in the presence of proteasome
inhibitors (22). However, we did not find evidence for significant
IRE-1–dependent XBP-1 splicing or for PERK-dependent upregulation of BiP, CHOP, or ATF4 in Epo-treated cells. Moreover, the
fact that tunicamycin, which caused significant activation of both
the IRE-1 and the PERK-dependent pathways, did not reduce PPI
levels argued against involvement of these pathways.
Nevertheless, the only significant effect of Epo treatment was the
reduction of PPI mRNA levels by 90% in vaccine-infected cells.
PPI mRNA can become a substrate of the RIDD pathway upon
chronic IRE-1 hyperactivation or when IRE-1 is overexpressed
(32, 33). Given that this pathway involves the nuclease activity
of activated IRE-1, which is also responsible for the cleavage of
XBP-1 mRNA, absence of spliced XBP-1 mRNA in Epo-treated
cells at first sight argues against an involvement of RIDD. However, dissociation between XBP-1 splicing and IRE-1–dependent
decay of other mRNAs, which seems to require ill-defined other
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FIGURE 7. (A) Effect of CHX on H-2Kd expression. HT-Kd cells were
treated with 20 mg/ml CHX for 4 h. (B) PPI expression was induced in
HT-Kd cells, 16 h after transfection, for 6 h before treatment with 2 mM
MG132 or 20 mg/ml CHX for 4 h. One of two experiments is shown.
The Journal of Immunology
forms with distinct cleavage specificities, as previously discussed by Golovina and associates (37).
More complex pathways may also underlie the observations
described in this article. Comprehensive investigations of larger
epitope panels suggest that the standard model of endogenous Ag
processing for MHC-I molecules may be inappropriately reductionist in the case of many epitopes, and that Ag processing may
involve additional compartments and intracellular transport events
(38). For example, the results obtained with AAF-CMK, butabindide, and bafilomycin in this study suggest the implication of
a lysosomal protease. Although we have not been able to obtain
consistent results with pharmacological induction and inhibition
of macroautophagy (data not shown), a role for this pathway in the
presentation of ER-targeted PPI also cannot be ruled out. Two
mechanisms that might be involved in such a pathway, donation of
autophagosome membranes from ER-like compartments, and engulfment of ER membranes by autophagosomes, have been described previously (39, 40). A pathway in which Ags escape from
autophagosomes into the cytosol where they tend to be degraded
by the proteasome, similar to that described by English et al. (41)
for an HSV protein, might also handle ER-targeted PI.
In summary, we report that ER targeting can have a profound
impact on the processing of an MHC-I–restricted epitope. This
observation first underlines the importance of monitoring subcellular Ag targeting and Ag expression in the presence of proteasome inhibitors in Ag processing studies. Furthermore, our results
highlight the complexity of Ag processing pathways, which
remains to be fully characterized. Lastly, our findings may be of
importance in autoimmune T1D. It has been shown using mice
expressing B7 in b cells that triggering of T1D by vaccination with
insulin-expressing plasmids requires ER targeting of insulin (42).
Disclosures
The authors have no financial conflicts of interest.
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ER TARGETING AND MHC CLASS I Ag PRESENTATION