Background
The ultimate role of the immune system in host defense is to eliminate infected and transformed cells [
1,
2]. A fundamental feature of infected and neoplastic cells is that they are stressed cells [
3‐
5]. In line with this, the innate immune system uses receptors such as NKG2D to recognize stressed cells [
4,
6,
7]. One key question, however, is whether cellular stress can influence recognition of transformed or infected cells by the adaptive immune system [
4,
8].
The single feature uniting different stress stimuli (heat shock, hypoxia, viral replication, abnormal proteins, starvation or transformation) is that they all ultimately lead to accumulation of unfolded or misfolded proteins in the lumen of the ER [
4,
5]. Infection and neoplastic transformation increase protein translation and thereby the folding demand on the ER [
9,
10]. This is particularly true for cells submitted to hypoxia, nutrient deprivation or low pH in poorly vascularized bulky tumors, metastases and sites of inflammation [
11,
12]. Moreover, acquisition of numerous mutations during tumor progression leads to accumulation of abnormal proteins with an increased propensity to misfolding that further raises the ER folding burden [
3,
13].
The ER responds to the accumulation of unfolded proteins by activating intracellular signal transduction pathways, collectively called the unfolded protein response (UPR) [
14,
15]. The UPR is a highly conserved adaptive response that allows survival to limited stress but leads to apoptosis in the presence of overwhelming stress [
16,
17]. Mammalian UPR acts through three main transducers (PERK, ATF6 and IRE1) that are activated by dissociation of the master chaperone BiP/GRP78 [
5,
15]. Activation of PERK leads to phosphorylation of the translation initiation factor eIF2α and attenuation of cap-dependent translation [
18]. The endonuclease activity of IRE1 generates a frameshift splice variant of XBP-1 encoding an active transcription factor that activates genes involved in protein degradation and controls the transcription of chaperones [
19‐
21]. Targets of the cleaved active form of ATF6 include the chaperones BiP and GRP94, and the transcription factors XBP-1 and CHOP [
17,
19]. Activation of these UPR transducers has pervasive effects on cellular protein economy: i) attenuation of protein translation, ii) increased degradation of ER proteins by ER-associated degradation (ERAD), iii) transcriptional activation of genes involved in the folding machinery of the ER and iv) increased degradation of ER-localized mRNAs [
14,
22].
Presentation of MHC I-associated peptides to CD8 T cells is tightly linked to protein economy. MHC I peptides are preferentially generated from newly synthesized but rapidly degraded polypeptides relative to slowly degraded proteins [
23,
24]. Following proteasomal degradation, peptides are translocated into the ER where they undergo N-terminal trimming, loading onto MHC I/β
2-microglobulin (β2m) heterodimers and export at the cell surface [
25‐
29]. Since the UPR regulates the two key processes that shape MHC I peptide processing (protein translation and degradation) we reasoned that ER stress should impinge on MHC I peptide presentation. We addressed this question and found that MHC I presentation was impaired during ER stress induced by palmitate or glucose starvation. Moreover, ER stress differentially affected presentation of peptides derived from a protein localized in the ER vs. the cytosol.
Discussion
The ER stands at the crossroad of two fundamental cellular processes: MHC I antigen presentation and UPR activation during ER stress. The UPR regulates protein synthesis and degradation, chaperoning and decay of ER mRNAs [
14,
15]. Thus, it has enormous potential to impinge on MHC I antigen processing which relies on all these processes. Here, we assessed the effect of ER stress on the final outcome of antigen processing and presentation: MHC I-peptide abundance. We demonstrated that ER stress induced by tunicamycin, palmitate or glucose deprivation, decreases peptide presentation by MHC I molecules. This finding is consistent with prior studies reporting reduced MHC I surface levels in human cells expressing a mutant HFE protein or overexpressing transcriptionally active isoforms of UPR-activated transcription factors ATF-6 and XBP-1 [
51,
52]. Recently, reduced expression of MHC I molecules was also observed in antigen presenting cells during palmitate treatment [
53]. Thus, diminution of MHC I surface expression upon UPR activation appears to be a generalized phenomenon occurring during ER stress induced by a variety of stimuli (pharmacological agents, mutant proteins, glucose starvation and saturated fatty acid).
Since the UPR provokes the degradation of ER-localized mRNAs [
22], accelerated decay of MHC I mRNA might have been responsible for the reduction of cell surface MHC I expression. However, the presence of normal levels of MHC I and β2m transcripts allowed us to exclude this possibility. During ER stress, transducers of the UPR seek to decrease the ER burden by suppressing translation initiation through phosphorylation of eIF2α by activated PERK [
14,
15]. We demonstrated that inhibition of protein synthesis and phosphorylation of eIF2α did occur in EL4 cells treated with palmitate or deprived of glucose. Of note, the effect of these two treatments on phosphorylation of eIF2α was similar, yet inhibition of overall protein synthesis was more severe in glucose-deprived than in palmitate-treated cells. We presume that this discrepancy was due to brisk inhibition of the mammalian target of rapamycin (mTOR) pathway during glucose starvation [
54]. Inhibition of mTOR blocks phosphorylation of p70 ribosomal S6 kinase and eukaryotic initiation factor 4E binding protein 1 and thereby leads to inhibition of protein synthesis. Given the dramatic inhibition of protein synthesis during glucose starvation, it was notable that the translation rate of the two HEL variants was not affected. That feature of our HEL variants is not unique as there are several proteins whose synthesis is unaffected during ER stress [
9,
54].
We found that ER stress-induced inhibition of overall protein synthesis curtails the synthesis of new MHC I molecules. Nevertheless, we do not believe that decreased synthesis of MHC I proteins per se was a leading factor responsible for decreased levels of MHC I molecules at the cell surface. Our assertion is based on three lines of evidence: i) a 5% decline of MHC I synthesis in palmitate-treated cells was not commensurate with a drop of 30–40% of surface MHC I, ii) during ER stress, cell surface levels of MHC I proteins were decreased much more than those of other glycoproteins that must also pass through the same maturation process and quality control in order to be exported at the cell surface, and iii) the total amount of intracellular MHC I proteins was not decreased during stress suggesting that MHC I molecules did not reach the cell surface mainly because they were sequestered in the ER. In addition, de Almeida et al. showed that a partial UPR signaling induced by overexpression of ATF-6 or XBP-1 in the absence of genuine stress stimulus also resulted in decreased MHC I surface expression [
52]. MHC I heavy chains and β2m are present in excess in the ER. The limiting factor in the assembly and presentation of MHC I-peptide complexes is peptide delivery to the ER [
28,
45]. Moreover, peptides presented by MHC I molecules derive mainly from proteins that are degraded a few seconds or minutes after their synthesis as opposed to stable proteins with a slow turnover. Thus, generation of MHC I peptide ligands is tightly coupled to ongoing protein synthesis and inhibition of translation rapidly decreases the amount of cell surface MHC I-peptide complexes [
55]. Our favorite hypothesis is therefore that decreased MHC I presentation during ER stress is due mainly, albeit not exclusively, to restriction of peptide availability. Given that MHC I molecules preferentially sample polypeptides that are being actively translated [
55], we posit that global attenuation of protein synthesis caused by palmitate and glucose starvation limits the amount of a vast repertoire of peptides available for insertion in MHC I molecules. Nevertheless, we do not exclude the possibility that defective synthesis of MHC I and other possible mechanisms such as inappropriate loading of peptides, contribute to diminution of MHC I-peptide presentation. This would be mainly the case of peptides deriving from proteins whose synthesis is not curtailed upon ER stress. For instance, our results show that ER stress diminished presentation of K
b-SIINFEKL complexes even though the synthesis of the proteins source of this particular peptide (HEL variants) was not affected.
A main conclusion of our work is that ER stress-induced attenuation of MHC I-peptide presentation is more severe when the source protein is localized in the cytosol than in the ER. The difference between proteins in these two cell compartments was UPR-specific because it did not occur in the low glucose condition in which no UPR markers were significantly induced. Our cell lines expressing HEL-Cyto-SIINFEKL and HEL-ER-SIINFEKL displayed identical responses to palmitate treatment or glucose starvation. The two cell lines showed similar upregulation of UPR markers and equivalent reduction in cell surface levels of H2K
b and H2D
b during ER stress. Despite the fact that the translation rates and degradation profiles normally differ in both cell lines, they displayed similar levels of K
b-SIINFEKL complexes under steady-state conditions. On the contrary, presentation of K
b-SIINFEKL complexes was differentially affected in these cell lines during ER stress. Only 1–2 out of every 10,000 peptides generated by the proteasome bind to MHC I molecules [
28]. Our data therefore beg the question: how would an ER-retained protein generate more peptides than a cytosolic protein during ER stress? We showed that this difference was not due to variations in the translation rate of each precursor protein during ER stress. This suggests that differences in peptide presentation resulted from discrepancies in the degradation of ER vs. cytosolic proteins during ER stress. UPR transducers specifically enhance degradation of proteins in the secretory pathway in order to decrease the ER folding load. During ER stress, cotranslational protein translocation is inhibited and newly-synthesized ER proteins are triaged for degradation (ERAD) [
38,
56,
57]. Furthermore, retrotranslocation of ER-resident proteins in the cytosol for proteasomal degradation is enhanced [
58]. Based on this, we expected to see an increased degradation of the ER-retained HEL variant during ER stress. However, the stability of the ER-retained protein remained unchanged while the stability of the cytosolic HEL variant increased during ER stress. The most parsimonious explanation for the latter findings would be that during ER stress, proteasomes focus primarily on degradation of ER as opposed to cytosolic proteins. This would be consistent with the fact that the primary role of the UPR is to decrease the folding burden in the stressed ER. We therefore propose that regulation of proteasomal degradation during ER stress leads to a reduction in MHC I peptide ligands generated from cytosolic precursors. Further studies will be needed to determine whether this concept can be generalized to other proteins and other MHC I-associated peptides.
What might be the impact of the UPR on immune recognition of infected and neoplastic cells? Paradoxically, if the decreased generation of MHC I-peptide complexes results mainly from inhibition of translation, it could facilitate recognition of virus-infected cells. Phosphorylation of eIF2α hampers canonical cap-dependent translation initiation which regulates synthesis of 95–98% of cellular mRNAs [
9]. However, some viruses can use internal ribosomal entry sites in their 5' noncoding region to initiate cap-independent translation [
9,
59]. Thus, by preferentially repressing presentation of self peptides, the UPR could facilitate recognition of viral peptides (the needle in the haystack [
60]). The potential impact of the UPR on recognition of neoplastic cells is not inherently obvious. On the one hand, by repressing production of MHC I-peptide complexes, the UPR may hinder presentation of tumor antigens to CD8 T cells. Indeed, generation of optimal CD8 T cell responses is promoted by high epitope density on antigen presenting cells [
61,
62]. However, an elegant study by Schwab
et al. has shown that upon induction of eIF2α phosphorylation by ER stress, cells can generate MHC I-associated peptides derived from cryptic translational reading frames [
63]. Expression of such cryptic peptides by neoplastic cells might trigger recognition of stressed cells by CD8 T lymphocytes. Finally, a high fat diet rich in saturated fatty acids such as palmitate, could potentiate the conditions of ER stress found in tumour cells and lessen even more MHC I-peptide presentation. In fact, obesity has been associated with increased susceptibility to infection and impaired immune responses [
53,
64]. We anticipate that high-throughput sequencing of MHC I-associated peptides [
33] will be necessary to comprehensively evaluate how ER stress molds the peptide repertoire (in terms of both abundance and diversity), and to gain further insights into the global impact of the UPR on recognition of stressed cells by CD8 T lymphocytes.
Methods
Cell lines
EL4 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (GIBCO Burlington, ON, Canada) supplemented with 5% fetal bovine serum (FBS) and antibiotics. EL4 stable transfectants were grown in the same medium supplemented with 1000 μg/ml of G418.
DNA constructs
pHYK/HEL-ER/myc and pCMV/HEL-Cyto/myc plasmids encoding ER-retained or cytoplasmic HEL, respectively, were provided by S. Ostrand-Rosenberg (University of Maryland, Baltimore, USA). The pHYR/HEL-ER plasmid contains the HEL gene (that includes a signal sequence) fused to the ER-retention signal KDEL, whereas pCMV/HEL-Cyto codes for HEL with a modified N-terminus and lacks ER-retention signal. These plasmids have successfully been shown to target HEL to the ER or to the cytosol [
34,
35]. pHYK/HEL-ER and pCMV/HEL-Cyto were sequenced to ascertain correct sequence and reading frame. Fragments coding for HEL-ER or HEL-Cyto were fused by PCR to the region coding for the ovalbumin-derived peptide SIINFEKL, flanked by a sequence of 18 bp (LEQLE-
SIINFEKL- TEWTS, here referred to as SIINFEKL) to ensure proteasome- and TAP-dependent peptide processing [
65,
66]. PCR amplification products were subcloned into the pPCR-Script Amp cloning vector (Stratagene, Cedar Creek, TX, USA). HEL-ER-SIINFEKL or HEL-Cyto-SIINFEKL were excised and cloned into the bicistronic pIRES-EGFP2 vector (Clontech, Mountain View, CA, USA) to generate pIRES-EGFP2/HEL-ER-SIINFEKL and pIRES-EGFP2/HEL-Cyto-SIINFEKL (Figure
1A). Both constructs were sequenced to ascertain correct sequence and reading frame.
Stable transfectants
EL4/HEL-ER-SIINFEKL and EL4/HEL-Cyto-SIINFEKL were generated by transfecting EL4 cells with the appropriate HEL-containing pIRES-EGFP2 plasmid. Transfections were done with Lipofectamine LTX Reagent (Invitrogen, Burlington, ON, Canada) as instructed by the manufacturer. 24 hours after transfection, single cells expressing the brightest signal of EGFP were sorted by fluorescence-activated cell sorting (FACS) on a FACSAria cell sorter (BD Biosciences, Mississauga, ON, Canada). Stable transfected clones were further selected by drug resistance (1000 μg/ml of G418) in combination with repeated cycles of FACS of EGFP-positive cells. Clones expressing similar levels of Kb-SIINFEKL at the cells surface were selected for use in further experiments.
Stress induction
ER stress was induced by incubating cells in fresh medium containing 0.25 mM of palmitate or 2.5 μg/ml of tunicamycin (Sigma-Aldrich, St. Louis, MO, USA) for the indicated times. Palmitate was prepared as described previously [
67] and delivered as a complex with fatty acid-free BSA. Glucose starvation was induced by culturing cells in glucose and sodium pyruvate-free or in low glucose (1000 mg/L) DMEM medium (GIBCO) supplemented with 5% dialyzed FBS and antibiotics for the indicated times. Control cells were grown in high glucose DMEM medium, containing 4500 mg/L of glucose and 110 mg/L of sodium pyruvate supplemented with 5% FBS and antibiotics.
Flow cytometry
MHC I molecules at the cell surface were stained with biotin-conjugated anti-H2K
b (clone AF6-88.5) and biotin-conjugated anti-H2D
b (clone KH95), followed by PeCy5 or APC-conjugated streptavidin. Other cell surface glycosylated proteins were stained with FITC-conjugated anti-CD45.2, FITC-conjugated anti-CD5 (Ly1), APC-conjugated anti-TCR-β and PE-conjugated anti-CD32. All antibodies were purchased from BD Biosciences. K
b-SIINFEKL levels were determined with the 25-D1.16 antibody [
36] followed by staining with APC-conjugated anti-mouse IgG
1 (Clone X56). Propidium iodide (BD Biosciences) was used to exclude cells in later apoptotic stages from the analysis. Cells were analyzed on a BD LSR II flow cytometer using FACSDiva (BS Biosciences) and FCS Express softwares (De Novo Software, Los Angeles, CA, USA) [
68,
69].
Acid strip assay
MHC I-peptide complexes were eluted with acid treatment as previously described [
47‐
49]. Briefly, cells (~5 × 10
5) were resuspended in 0.2 ml of citrate phosphate buffer at pH 3.3 (0.131 M citric acid/0.066 M Na
2HPO
4, NaCl 150 mM) for 1 minute, neutralized with appropriate medium pH 7.4 and either reincubated in fresh medium or stained for flow cytometry analysis.
Total RNA was isolated with TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Purified RNA was reverse transcribed using the High Capacity cDNA reverse transcription Kit with random primers (Applied Biosystems, Foster City, CA, USA) as described by the manufacturer. A reference RNA (Stratagene, La Jolla, CA, USA) was also transcribed in cDNA. Expression level of target genes was determined using primer and probe sets from Universal ProbeLibrary
https://www.roche-applied-science.com/sis/rtpcr/upl/index.jsp or Applied Biosystems (ABI Gene Expression Assays or SYBR green PCR Master Mix,
http://www.appliedbiosystems.com/). Primer sequences are given in Additional file 1. RT-qPCR assay for XBP-1 was designed to amplify both the normal and spliced forms. Pre-developed TaqMan
® assays for β-actin were used as endogenous controls. RT-qPCR analyses were performed as described using a PRISM
® 7900 HT Sequence Detection System (Applied Biosystems) [
70]. The relative quantification of target genes was determined by using the ΔΔCT (cycle threshold) method. Relative expression (RQ) was calculated using the Sequence Detection System (SDS) 2.2.2 software (Applied Biosystems) and the formula RQ = 2
-ΔΔCT.
To measure protein synthesis, EL4 cell lines were cultured in presence or absence of glucose (4.5 mg/ml) for 18 hours. [3H]Leucine (10 μCi/mL) was added during the last hour. Cells were washed twice with ice-cold PBS and fixed for 30 minutes on ice with 10% TCA. Cells were then rinsed with water and lysed with 0.1 N NaOH. Radioactivity incorporation was determined with a liquid scintillation analyzer Tri-CArb 2800TR (Perkin Elmer).
In vivo biosynthetic labeling experiments were carried out as described previously [
71]. Briefly, to evaluate the rate of synthesis of HEL and MHC I, EL4 cell lines were grown in control conditions or in the presence of 2.5 mM of palmitate or in the absence of glucose for 17 hours. After this period, 10
7 cells per condition were starved of methionine and cysteine for 30 min.
35S-labeled methionine and cysteine (220 μCi/mL) were then added for 1 hour. Cells were harvested and lysed in Triton X-100 buffer (50 mM Tris pH7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 40 mM β-glycerophosphate) supplemented with complete protease inhibitor cocktail (Roche Molecular Biochemicals, Laval, QC, Canada) and phosphatase inhibitors (1 mM Na
3VO
4 and 5 mM NaF). Immunoprecipitation of ER-retained HEL or cytosolic HEL and MHC I were performed using anti-HEL antibody purchased from Affinity BioReagents (Golden, CO, USA) or anti-H2K
b or anti-H2D
b hybridoma culture supernatans antibody [
49], according to the method described previously [
71]. Proteins were separated by SDS-PAGE and labeled proteins were detected by fluorography.
Immunoblotting
EL4 cell lines were cultured under control conditions or submitted to glucose deprivation or palmitate treatment (0.25 mM) for the indicated times. When indicated, 100 μg/mL of cycloheximide (Sigma-Aldrich) was used for various durations to measure the stability of HEL variants. Cells were harvested and lysed in Triton X-100 buffer. The lysates were cleared by centrifugation and the protein content was measured by the Bradford method (Biorad, Mississauga, ON, Canada). Samples were resolved by SDS-PAGE and immunoblotted with the following antibodies: anti-β-actin (AC-15) from Sigma-Aldrich, anti-HEL from Affinity BioReagents, anti-MHC class I (2G5) from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA), anti-α-tubulin, anti-phospho-eIF2α (Ser51), anti-eIF2α and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG from Cell Signaling Technology (Beverly, MA, USA), and HRP-conjugated goat anti-mouse IgG from BD Pharmigen (San Diego, CA, USA). Chemiluminescent signal was detected using a LAS3000 imaging system (Fujifilm, Tokyo, Japan) and quantification of band intensities was done using the Multi Gauge v3.0 (Fujifilm) and the ImageQuaNT v5.0 (Molecular Dynamics, Sunnyvale, CA, USA) softwares.
Statistical analysis
The means of normally distributed data were compared using the Student t test, with a P value of < 0.05 considered significant. Data are presented as the mean and SD. Whenever the results are expressed as a percentage of control, the statistical analysis was performed on the actual value.
Authors' contributions
DPG designed the study, carried out experiments and analyzed the data. PLT designed and carried out metabolic labeling experiments. MPH and DDV participated in flow cytometry experiments. EC participated in molecular cloning experiments. SM designed biochemical experiments. CP conceived and designed the study. DPG and CP drafted the manuscript, and all authors edited and approved the final manuscript.