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The Journal of Immunology, 2007, 178: 2241-2248.
Copyright © 2007 by The American Association of Immunologists, Inc.

The Role of Endoplasmic Reticulum-Associated Aminopeptidase 1 in Immunity to Infection and in Cross-Presentation1

Elke Firat*, Loredana Saveanu{dagger}, Peter Aichele{ddagger}, Peter Staeheli§, Jisen Huai*, Simone Gaedicke*, Ahmed Nil*, Gilles Besin, Benoît Kanzler, Peter van Endert{dagger} and Gabriele Niedermann2,*

* Clinic for Radiotherapy, University Hospital of Freiburg, Freiburg, Germany; {dagger} Institut National de la Santé et de la Recherche Médicale Unité 580, Paris, France and Université Paris Descartes, Paris, France; {ddagger} Institute for Medical Microbiology and Hygiene, Department of Immunology, University of Freiburg, Freiburg, Germany; § Institute for Medical Microbiology and Hygiene, Department of Virology, University of Freiburg, Freiburg, Germany; and Max-Planck Institute of Immunobiology, Freiburg, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Endoplasmic reticulum-associated aminopeptidase 1 (ERAP1) is involved in the final processing of endogenous peptides presented by MHC class I molecules to CTLs. We generated ERAP1-deficient mice and analyzed cytotoxic responses upon infection with three viruses, including lymphocytic choriomeningitis virus, which causes vigorous T cell activation and is controlled by CTLs. Despite pronounced effects on the presentation of selected epitopes, the in vivo cytotoxic response was altered for only one of several epitopes tested. Moreover, control of lymphocytic choriomeningitis virus was not impaired in the knockout mice. Thus, we conclude that lack of ERAP1 has little influence on antiviral immunohierarchies and antiviral immunity in the infections studied. We also focused on the role of ERAP1 in cross-presentation. We demonstrate that ERAP1 is required for efficient cross-presentation of cell-associated Ag and of OVA/anti-OVA immunocomplexes. Surprisingly, however, ERAP1 deficiency has no effect on cross-presentation of soluble OVA, suggesting that for soluble exogenous proteins, final processing may not take place in an environment containing active ERAP1.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Major histocompatibility complex class I molecules present peptides derived from endogenous proteins to MHC class I-restricted CD8+ CTLs. Most presented peptides are between 8- and 11-aa residues in length. Proteolysis of protein Ags in the classical pathway begins within proteasomes, which generate peptides between 3 and 20 aa in length. Proteasomes thus produce peptides of optimal length for class I binding as well as longer precursor peptides. The latter can be trimmed by aminopeptidases, whereas C-terminal trimming by exopeptidases seems not to be relevant (1, 2, 3). The peptide loading of MHC class I molecules takes place in the endoplasmic reticulum (ER)3 after translocation of optimally sized or precursor peptides by TAP through the ER membrane. TAP efficiently transports peptides of 8–15 aa (4). Trimming of precursor peptides can take place in the cytosol and in the ER, but the relative importance of cytosolic vs ER-based trimming is unclear to date.

CD8+ T cells also respond to viruses that do not directly infect dendritic cells (DCs) and to tumor Ags. In these cases, activation is based on the so-called cross-presentation of exogenous Ags, a pathway specific for DCs. Cross-presentation, also relevant in transplant rejection and in the maintenance of self-tolerance of MHC class I-restricted cells, requires that peptides derived from internalized Ags gain access to the MHC class I molecules. Up to now, little is known about the molecular processes and organelles involved in cross-presentation of phagocytosed and soluble Ags, which may involve distinct pathways (5), and of endocytosed Ag/Ig complexes. An involvement of the ER in cross-presentation of phagocytosed Ags, postulated by some authors (6), is a matter of particularly intense debate (7, 8). Given that ER-associated aminopeptidase 1 (ERAP1) is specifically located in the ER (9, 10) and possibly also in early phagosomes formed by recruiting ER membranes (5), ERAP1 knockout (KO) mice should be valuable for the analysis of ER-processing factors implicated in potential cross-presentation pathways.

In contrast to the uncertainties regarding their role in cross-presentation, the role of ER peptidases in the classical pathway is clearer, albeit not yet fully understood. ER trimming enzymes have recently been identified. The metallopeptidase ERAP1 is involved in peptide processing in mice as well as in humans (10, 11, 12). In humans, a second homologous peptidase (ERAP2) has been identified. ERAP1 and ERAP2 are encoded in a cluster of three M1 aminopeptidase genes on chromosome 5 designated the oxytocinase subfamily after its third member. In human cells, ERAP1 and ERAP2 form complexes that appear to be important for the processing of at least some precursor peptides (13).

Concerning the role of ERAP1 in MHC class I Ag processing, small interfering RNA knockdown experiments yielded contradictory results. One study reported reduced surface expression of H-2Kk and Ld (10). Another reported increased surface expression of human and monkey MHC molecules, whereas H2-Kb surface expression was not found to differ in HeLa-Kb cells. Cell surface expression of human MHC class I and of mouse H2-Kb molecules was only reduced when cells were pretreated with IFN-{gamma} (12). Similar RNA knockdown experiments in HeLa cells by us showed a moderate down-regulation of human cell surface MHC class I molecules in the absence of IFN-{gamma}, but no significant effect when the cells were pretreated with IFN-{gamma} (13).

Initial conclusive information on the impact of ERAP1 deficiency on class I epitope presentation was very recently obtained in KO mice (14, 15, 16). In this study, we describe the generation of an independent line of ERAP1 KO mice, the impact of ERAP1 deficiency on control of viral infections, and the analysis of cross-presentation of cell-associated and of soluble Ag as well as of Ag/Ig complexes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Generation of ERAP1 KO mice

We constructed a targeting vector containing 7.6-kb sequence homologous to the region of the ERAP1 locus. The homology region was amplified from genomic DNA of W4 embryonic stem cells (Taconic Farms) using the Expand long template PCR system (Roche) with the forward primer 5'-GGGACAGGGCTAGATTCTTTCTACCATTAG-3' and the reverse primer 5'-CTTGACTGTCTACCTTTCCATTACCTCCTC-3'. The PCR product was then digested with SwaI and Psp1406I and assembled in a pBluescript KSII+/– vector together with a single loxP site (introduced into intronic sequence between exons 4 and 5) and a neomycin resistance cassette flanked by two FRT sites and one loxP site (introduced into intronic sequence between exons 3 and 4). The targeting vector was linearized with KpnI and electroporated into W4 embryonic stem cells (129/SvEv). G418-resistant clones were screened by PCR. Positive clones were confirmed by Southern blot using 5' and 3' external probes and the PCR DIG Labeling Mix from Roche. For the amplification of the 5' probe, the forward primer 5'-CTAGCCACGGTTACACAGCA-3' and the reverse primer 5'-CTGACAGTTCCCTCCCATGT-3' were used. For the amplification of the 3' probe, the forward primer 5'-CAAGGCTTAATGGTTGAGAG-3' and the reverse primer 5'-GGCCAACAACTAGAAACAAC-3' were used. Chimeric mice were generated by blastocyst injection, and ERAP1 KO mice were generated by crossing the chimeras with CMV-FLP and CMV-Cre transgenic mice on the C57BL/6 background. ERAP1-deficient mice were further backcrossed with wild-type (wt) C57BL/6 mice.

Analysis of ERAP1 expression by RT-PCR

Total RNA was isolated from spleen cells and subjected to reverse transcription using the Sensiscript RT Kit (Qiagen). The ERAP1 cDNA was amplified using the forward primer 5'-GTGAATGTTGCTGAAGGACT-3' and the reverse primer 5'-GCCAAGCTTACTAGATGCAG-3', flanking the deleted ERAP1 gene fragment, thus producing a 245-bp fragment for the KO mice and a 366-bp fragment for the wt mice.

TCR transgenic mice and T cells

Transgenic Rag2–/– mice expressing TCRs recognizing the OVA peptide SIINFEKL (OT1) and the male Ag Smcy-3 (HY) on a C57BL/6 background were provided by B. Rocha (Institut National de la Santé et de la Recherche Médicale Unité 591, Paris, France), and TAP–/– mice, also on a BL6 background, were a gift from O. Lantz (Curie Institute, Paris, France). Rag+/+ OT1 mice were purchased from Charles River Laboratories. T cells from the latter strain were always used as effectors, generated by stimulation of naive OT1 splenocytes with an equal number of peptide-pulsed (1 µM SIINFEKL), irradiated splenocytes, followed by incubation for 6–9 days in RPMI 1640 medium with 10% FCS and 10% supernatant from Con A-stimulated rat spleen cells.

Generation of bone marrow (BM)-DCs and ear fibroblast cell lines

To produce BM-DCs, BM cells were seeded in petri dishes at 5 x 105/ml in IMDM with 10% FCS and 20% supernatant of J558 cells secreting GM-CSF (assays with OT1 and HY cells), or in RPMI 1640 supplemented with 20 ng/ml rGM-CSF. Cells were used between days 7 and 8. Ear fibroblasts from ERAP1 KO and wt mice were immortalized by serial passage in vitro.

Abs and flow cytometry

Spleen cells and DCs were incubated with anti-FcR Ab (clone 2.4G2) before staining for flow cytometry. The anti-CD4, anti-CD8{alpha}, anti-CD19, anti-CD11c, anti-H-2Kb, anti-H2-Db, and anti-H-2Ab Abs were obtained from BD Biosciences; the anti-IFN-{gamma} Ab and mouse rIFN-{gamma} were from eBioscience. Cells were analyzed by flow cytometry on a FACScan (Beckman Coulter).

Virus infection of mice

Lymphocytic choriomeningitis virus (LCMV) strain WE and recombinant vaccinia virus expressing LCMV nuclear protein (NP) (rVVNP) were propagated on L929 or BSC-1 cells, respectively. Mice were infected i.v. with either 200 PFU of LCMV or 2 x 106 PFU of rVVNP. LCMV-specific CTL responses were analyzed at day 8 of infection using splenocytes directly ex vivo. For influenza virus infection, mice were infected intranasally with a sublethal dose (300 PFU) of influenza A virus A/PR8/34 in 50 µl of PBS containing 0.3% BSA. Influenza-specific CTL responses were analyzed at day 7 of infection using lymphocytes isolated from the lungs of infected animals. Infections with rVVOVA (a gift from J. Yewdell, National Institutes of Health-National Institute of Allergy and Infectious Diseases, Bethesda, MD) were performed using 5 x 106 PFU i.p., and splenocytes were analyzed at day 7 of infection.

Intracellular cytokine staining

Lung lymphocytes or splenocytes were pulsed with synthetic peptides at a final concentration of 1 µM. After 1 h, brefeldin A (Sigma-Aldrich) was added to a final concentration of 10 µg/ml and the incubation continued for another 4 h. Thereafter, the cells were incubated with anti-CD8{alpha} for 20 min at 4°C. Then cells were washed and fixed with 1% paraformaldehyde, washed and stained with anti-IFN-{gamma} in PBS with 0.5% saponin at 4°C overnight, and analyzed by flow cytometry.

Tetramer staining

Lymphocytes were resuspended in PBS containing 2% FCS at a concentration of 106 cells/ml and stained for 30 min at 4°C in 100 µl of Ab working solution. Before analysis, RBC were lysed using FACS lysing solution (BD Pharmingen). mAb specific for CD8 (clone 53-6.7) was purchased from BD Pharmingen. H-2Db MHC class I tetramers complexed with streptavidin-PE and containing the LCMV gp33–41 or NP396–404 peptide were prepared and used according to standard protocols.

Cytotoxicity assay

Single spleen cell suspensions from mice infected with LCMV-WE (day 8 of infection) were prepared, and the cytolytic activity was determined in a 51Cr release assay. MC57G or EL-4 target cells were pulsed with LCMV peptides gp33–41 or NP396–404 or with an irrelevant adenovirus E1A 234–243 peptide at a concentration of 10–6 M, and labeled with 250 µCi of 51Cr for 2 h at 37°C. Target cells were washed and incubated for 5 h with a serial dilution of spleen effector cells. Spontaneous release was <20%.

Determination of virus titers

LCMV titers in infected organs were determined using a focus-forming assay, as described (17). Titers were expressed as PFU per organ.

Ovary protection assay

Mice were primed with 200 PFU of LCMV-WE and challenged with 2 x 106 PFU rVVNP 25 days after primary infection. Four days after challenge infection, VV titers were determined in the ovaries of immunized and naive mice by plating serial dilutions of the organ homogenates on BSC-1 cell monolayers and staining with 0.1% crystal violet solution after 48 h (18).

Direct presentation of LCMV-NP396–404 by DCs

DCs were generated from BM with GM-CSF. At day 7, DCs were isolated with anti-CD11c beads (Miltenyi Biotec) and infected with LCMV at a multiplicity of infection of 0.01 for 2 days. During this time, the DCs were also matured with LPS (10 µg/ml). Mature, infected DCs were then incubated with NP396–404-specific T cells overnight. Thereafter, cells were spun and IFN-{gamma} was measured in the supernatant using an ELISA (BD Biosciences). An NP396–404-specific T cell line was generated from splenocytes of a mouse infected 4 wk before with a low dose of LCMV.

Direct presentation and cross-presentation assays of OVA in vitro

To study direct presentation, BM-DCs were infected for 1 h in serum-free RPMI 1640 with VV encoding full-length OVA or the antigenic peptide SIINFEKL at a multiplicity of infection of 30 or 10, respectively. (Viruses were provided by J. Yewdell, National Institutes of Health-National Institute of Allergy and Infectious Diseases, Bethesda, MD.) Infected cells were incubated in RPMI 1640 for 6 h, fixed with 1% formaldehyde for 1 min at room temperature to inactivate the virus, and neutralized by washing in PBS with 0.2 M glycine. Fixed BM-DCs were then added in various ratios to 100,000 OT1 effector cells overnight, and IFN-{gamma} production was measured by sandwich ELISA. To study cross-presentation of soluble OVA, BM-DCs were incubated for 6 h with graded amounts of ultracentrifuged OVA in complete medium, fixed with 0.002% glutaraldehyde for 1 min at room temperature, neutralized as described above, and added to 100,000 naive or effector OT1 T cells for overnight incubation before measurement of IL-2 or IFN-{gamma} production by ELISA. For cross-presentation of immunocomplexes, soluble OVA at 10 µg/ml was incubated for 1 h with anti-OVA rabbit polyclonal IgG (Sigma-Aldrich). The immune complexes were added to fresh or prefixed BM-DCs. Eight hours later, the BM-DCs were fixed and 100,000 naive OT1 were added. IL-2 secretion was measured 24 h later by ELISA.

Direct presentation of the Smcy Ag and in vivo cross-presentation assays

Direct presentation of the HY Ag was examined by incubating 105 naive transgenic anti-HY T cells for 24 h with 105 male BM-DCs, followed by measuring IL-2 secretion by ELISA. To measure cross-presentation in vivo, 106 naive HY T cells, labeled with 5 µM CFSE, were injected i.v. in ERAP1 KO mice or wt littermates. Between 16 and 24 h later, 107 irradiated male BALB/c splenocytes were injected i.v. Five days later, splenocytes of recipient mice were stained with an anti-clonotype mAb recognizing the anti-HY TCR (T3.70; eBioscience). The proliferative response of the anti-HY T cells was calculated as the division index (the number of mitotic events divided by the number of precursors), as described (19). For in vivo cross-presentation assays with soluble and cell-associated OVA, 2 x 106 naive CFSE-labeled OT1 cells were injected i.v., followed 16 h later by i.v. injection of the Ag: 20 µg of soluble OVA or 1 x 106 OVA-loaded, irradiated BALB/c splenocytes. BALB/c splenocytes were loaded with OVA by electroporation of 4 x 106 cells in 200 µl of OVA solution (1.5 mg/ml). Seventy hours later, the proliferation of OT1 cells was analyzed by FACS using anti-CD8 Ab (clone 53-6.7; BD Pharmingen) and anti-Vbeta5.1 Ab (clone MR9-4; BD Pharmingen).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Generation of ERAP1 KO mice

To generate ERAP1 KO mice, we used the Cre-loxP and the FLP-FRT recombinase technologies (Fig. 1). The homology region for the targeting vector consisted of 7.6 kb covering the murine ERAP1 genomic sequence from exons 3 to 9. A single loxP site and an FRT-flanked neomycin resistance cassette bearing an additional loxP site were introduced in introns 3 and 4, respectively. Cre- as well as FLP-mediated recombination was performed by crossing floxed mice to Cre- or FLP-transgenic mice, resulting in the deletion of exon 4 and the neomycin resistance cassette, respectively. ERAP1 KO mice were born at mendelian frequencies and appeared normal upon gross physical examination.


Figure 1
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  		FIGURE 1. Generation of ERAP1 KO mice. A, Schematic diagram of the relevant Erap1 genomic region, targeting vector, generation of the floxed allele by homologous recombination and of the KO allele by Cre- and FLP-mediated recombination. The 5' and 3' external probes used in the Southern blot analysis are indicated. B, Southern blot analysis of a representative embryonic stem cell clone confirming homologous recombination. C, RT-PCR confirming deletion of exon 4. Total RNA from spleen cells of KO (–/–) and wt (+/+) mice was reverse transcribed and amplified with ERAP1-specific primers, resulting in a 245-bp fragment for the KO and a 366-bp fragment for the wt cells.

 
MHC class I presentation levels in ERAP1 KO mice

Similar to recent reports (14, 15, 16), we observed a significant down-regulation of the overall cell surface levels of H-2Kb and H-2Db peptide complexes in ERAP KO mice. As determined by flow cytometry, levels of H-2Kb and H-2Db were reduced by 14–42% and 3–40% (n = 5 experiments), respectively. This was observed for various types of freshly isolated splenocytes (Fig. 2A) as well as for DCs generated from BM precursors in vitro (Fig. 2B). As expected, cell surface levels of MHC class II-peptide complexes were normal (Fig. 2, A and B). IFN-{gamma} induces the transcription of a variety of genes related to Ag processing and presentation, including ERAP1. To our surprise, but similar to a recent report (15), we found no significant differences in H-2Kb and Db expression on fibroblasts from ERAP1 KO mice compared with those from wt mice upon treating with IFN-{gamma} (Fig. 2C). In contrast, when immature BM-derived DCs were matured by adding IFN-{gamma}, pronounced differences in H-2Kb and Db expression levels were observed (Fig. 2B).


Figure 2
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  		FIGURE 2. MHC class I, but not MHC class II expression on the cell surface is reduced in ERAP1-deficient mice. Different types of primary cells from wt (+/+) and KO (–/–) mice were stained with Abs directed against MHC class I H-2Kb, H-2Db, or MHC class II H-2Ab and analyzed by flow cytometry. A, MHC expression by splenocytes (one representative experiment of five is shown). B, MHC expression by DCs induced by GM-CSF from BM precursors and incubated overnight with 250 ng/ml mouse IFN-{gamma}. C, MHC class I expression by cultured mouse ear fibroblasts treated or not treated with 200 U/ml IFN-{gamma} for 30 h.

 
CTL responses and immunity to viruses

Infection with LCMV strongly activates the immune system and is almost exclusively controlled by CD8+ CTLs, which expand massively upon LCMV infection (20). Surprisingly, despite the pronounced decrease of MHC class I expression levels in ERAP1 KO mice, we found no difference, upon acute LCMV infection, in the proportions of splenic CD8+ T cells in ERAP1 KO mice compared with wt littermates (Fig. 3A). In addition, we determined the in vivo CD8+ CTL frequencies to three H-2Db-restricted LCMV epitopes, using three different methods (chromium release as well as intracellular cytokine and tetramer staining), which all gave similar results. The CTL frequencies to the ER leader sequence-derived immunodominant (ID) epitope gp33–41 and to the subdominant (SD) gp276–286 epitope were comparable in wt and KO mice (Fig. 3, B–D). In contrast, the response against the ID NP396–404 epitope was substantially reduced in KO mice, to the level of the SD epitope gp276–286 in wt mice (Fig. 3, B, E, and F).


Figure 3
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  		FIGURE 3. CD8+ T cell responses to LCMV are affected for the NP396–404 CTL epitope. Mice were infected i.v. with LCMV-WE, and 8 days later splenocytes were analyzed for the presence of LCMV-specific CD8+ T cells. A, Proportion of CD8+ T cells in spleens of LCMV-infected mice (n = 6 per group). B, Frequencies of epitope-specific CTLs were determined by intracellular IFN-{gamma} staining after a 5-h restimulation with the respective peptides in vitro (n = 6). C, Frequency of gp33–41-specific CTLs determined by staining with H-2Db/gp33–41 tetramers directly ex vivo (n = 6). D, Lytic activity of gp33–41-specific CTLs determined directly ex vivo in a chromium release assay; gp33–41 peptide-pulsed cells were used as targets, and splenocytes from infected ERAP1 KO mice (open symbols) and wt littermates (closed symbols) as responders. Dotted lines show controls using target cells pulsed with an irrelevant peptide. Three independent experiments are shown. E, Frequency of NP396–404-specific CTLs determined with H-2Db/NP396–404 tetramers (n = 6). F, Lytic activity of NP396–404-specific CTLs determined analogously to the experiments described in D using NP396–404-pulsed target cells. G, Impaired presentation of the LCMV-NP396–404 epitope by LCMV-infected DCs. Mature ERAP1+/+ and ERAP1–/– BM-DCs infected with LCMV were incubated with NP396–404-specific CTLs overnight. The activation of the CTLs was determined by measuring IFN-{gamma} secretion in an ELISA. DCs pulsed with the NP396–404 peptide were used as positive control.

 
The decreased response to NP396–404 in the ERAP1 KO mice suggested impaired generation of the epitope in the classical Ag-processing pathway of professional APCs. To test this, DCs were generated from BM precursors of ERAP1 KO mice and wt littermates and were infected with LCMV. IFN-{gamma} secretion by NP396–404-specific CTLs was significantly lower after incubation with DCs from KO mice compared with DCs from wt mice. In contrast, peptide-pulsed KO or wt DCs stimulated the CTLs to a similar extent (Fig. 3G).

Based on the data in Fig. 3, it was possible that, owing to an impaired CTL response to selected epitopes, the control of LCMV is compromised in ERAP1-deficient mice. However, there was no difference in the clearance of LCMV between infected wt and KO mice. In immunocompetent mice, LCMV titers reach levels of ~107 PFU/g spleen at day 4–5 of infection. Thereafter, virus is eliminated, and virus titers fall to the limit of detection between days 8 and 10 (21). As seen in Fig. 4A, the LCMV titers of both wt and KO mice were similar in all organs tested at day 8 after infection.


Figure 4
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  		FIGURE 4. Organ virus titers in ERAP1 KO mice (open symbols) and wt littermates (filled symbols). A, Organ titers at day 8 after infection with 200 PFU of LCMV strain WE. Points represent individual mice. B, Titers of a rVV expressing LCMV-NP in the ovaries of mice. To induce memory CTLs, ERAP1 KO mice and wt littermates were initially infected with LCMV. Then, 25 days later, the mice were challenged with rVVNP, and 4 days later the rVVNP titers were determined in the ovaries of the mice. Each point represents one individual mouse. Controls are naive mice not primed with LCMV, indicated by the stars. C, Mice were primed with 100 µg of synthetic NP396–404 peptide in IFA or only with IFA, and 10 days later challenged with 200 PFU of LCMV-WE. Four days after the challenge, spleen LCMV titers (C) and NP396–404-specific CTL frequencies (see text) were determined. The dotted line represents the detection limit of the assay (100 PFU/organ).

 
To test whether the reduced numbers of NP396–404-specific CTLs in LCMV-infected ERAP1 KO mice affected protective immunity when this was restricted to viral NP, mice were first infected with LCMV and challenged several weeks later with rVVNP. Four days after the challenge, the rVVNP titers were determined in the ovaries (an organ with very high virus replication). In LCMV-primed wt mice, the challenge with the rVVNP should lead to rapid expansion of NP-specific memory CTLs, which cause efficient elimination of rVVNP-infected cells. As seen in Fig. 4B, 4 days after the challenge, rVVNP was completely eliminated in the ovaries of both LCMV-primed wt and ERAP1 KO animals. In contrast, naive control mice had high rVVNP titers in their ovaries. Thus, the reduced frequencies of NP396–404-specific CTLs induced in LCMV-primed mice do not impair a subsequent elimination of an rVV expressing the LCMV-NP. However, in this scenario, CTLs specific for SD NP epitopes may also contribute to virus elimination. Interestingly, when the mice were primed with the NP396–404 peptide itself, and the challenge was performed with LCMV, the KO mice cleared LCMV less efficiently (Fig. 4C). Under these conditions, the same, but low numbers of NP396–404-specific CTLs are induced in both types of mice (frequencies at day 4 after LCMV challenge: wt mice, 1.21 ± 0.26; KO mice, 1.3 ± 0.21). The experiment thus assesses presentation of the NP396–404 epitope in vivo and demonstrates that virus elimination can be affected when immunity is exclusively restricted to an epitope whose presentation is compromised by ERAP1 deficiency, at least when the specific CTL frequencies are low and the challenge virus is rapidly replicating. These data also confirmed the in vitro Ag presentation data using LCMV-infected DCs (see Fig. 3G).

We also studied the CTL responses against two other well-characterized mouse pathogenic viruses (VV and influenza virus), which, in contrast to LCMV, provoke only a moderate stimulation of the CD8+ T cell compartment. We used a rVV expressing OVA (rVVOVA) for infection of the mice and determined the CTL response against five recently described H-2Kb/Db-restricted VV epitopes (22) and against the ID OVA epitope SIINFEKL. As seen in Fig. 5A, no substantial differences were found in the frequencies of epitope-specific CTLs in ERAP1 KO mice compared with wt littermates. This includes the response to the SIINFEKL peptide despite the fact that the presentation of this epitope is substantially reduced on rVVOVA-infected DCs in vitro (see below). Frequencies of influenza-specific CTLs were determined 1 wk after intranasal infection using lymphocytes isolated from the lungs of infected mice. Also, in this study, no statistically significant differences were found between wt and KO mice for the two dominant Db-restricted epitopes (23) tested (Fig. 5B). Together, these data show that ERAP1 deficiency cannot only be compensated in the strong CTL response to LCMV, but also has no effect on the CTL responses to VV and influenza virus.


Figure 5
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  		FIGURE 5. CD8+ T cell responses to rVV-OVA and influenza virus are not affected in ERAP1-deficient mice. A, Mice were infected i.p. with rVV-OVA, and 7 days later splenocytes were analyzed for the frequencies of VV and SIINFEKL epitope-specific CTLs. Frequencies of IFN-{gamma}-producing CD8+ T cells were determined by intracellular IFN-{gamma} staining after a 5-h restimulation with the respective VV-derived peptides or with SIINFEKL. The results for six wt and six KO mice are shown. B, Frequencies of CTLs specific for two major influenza virus-specific CTL epitopes are not substantially altered in ERAP1-deficient mice. Mice were infected intranasally with influenza A virus. After 7 days, lung lymphocytes were isolated and, after restimulation with the H-2Db-restricted influenza epitopes NP366–374 and PA224–233, analyzed flow cytometrically for intracellular IFN-{gamma}. The results for six wt and six KO mice are shown.

 
Cross-presentation of soluble and cell-associated Ags

It is a matter of controversy whether presentation of phagocytosed material involves Ag shuttling into the endogenous pathway involving the perinuclear ER, or, alternatively, fusion of ER membranes with phagosomes, resulting in formation of a specialized, processing-autonomous ER phagosome (5, 6, 8, 24). In any case, all the present models of cross-presentation envisage peptide loading in an environment of ER components. This includes cross-presentation of soluble Ags, which has been suggested to involve retrograde Ag access to the perinuclear ER (5). Therefore, we expected ERAP1 deficiency to affect cross-presentation both of particulate and soluble Ags.

Initially, we tested the role of ERAP1 for presentation of endogenously expressed and of exogenously added soluble OVA protein. Processing of endogenous OVA, synthesized upon DC infection with a rVV, was compromised by ERAP1 deficiency, although this effect could be overcome by increasing the number of DCs added to OT1 T cells (Fig. 6A). As expected, ERAP1 deficiency had no effect on presentation of the preprocessed SIINFEKL epitope, transcribed from a VV-encoded minigene (Fig. 6B). Surprisingly, ERAP1 deficiency also had no effect on cross-presentation of soluble exogenous OVA, neither in vitro nor in vivo (Fig. 6, C and D), although proteasome inhibition reduced cross-presentation of soluble OVA significantly (data not shown). Thus, a pathway requiring the proteasome, but not ERAP1, may contribute to cross-presentation of soluble proteins. Interestingly, however, ERAP1 deficiency had a considerable effect on cross-presentation of OVA complexed to anti-OVA mAbs (Fig. 6E). Contamination of OVA protein preparations with antigenic peptide could be excluded in experiments with fixed DCs, which were not at all recognized by OT1 T cells when pulsed with soluble OVA protein, but were recognized as efficiently as unfixed cells when pulsed with SIINFEKL peptide (Fig. 6, E and F).


Figure 6
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  		FIGURE 6. ERAP1 deficiency compromises generation of SIINFEKL from endogenously expressed and receptor-internalized OVA, but not from soluble protein. BM-DCs from ERAP1 KO mice or wt littermates were infected with VV encoding OVA (A) or with VV encoding the antigenic peptide SIINFEKL (B) and added at different ratios to OT1 effector cells. Antigenic stimulation of OT1 T cells was evaluated by measuring IFN-{gamma} production by ELISA. C, BM-DCs pulsed for 6 h with graded amounts of soluble OVA were fixed and incubated overnight with naive OT1 T cells. D, The cross-presentation of soluble OVA injected i.v. was examined by quantitating the proliferation of CFSE-labeled OT1 cells injected i.v. in ERAP1 KO or wt mice; the division indices obtained for four KO and four wt mice are shown. E, Soluble OVA (10 µg/ml) was preincubated with anti-OVA polyclonal rabbit IgG, and the immune complexes were incubated with fresh or prefixed BM-DCs, followed by addition of naive OT1 cells 8 h later. F, Shows that paraformaldehyde-fixed BMDCs efficiently presented the SIINFEKL peptide (10–10 M), but not soluble OVA (0.5 mg/ml). Readout of T cell activation in C, E, and F was IL-2 secretion measured by ELISA. All of the experiments shown were performed at least twice.

 
To study cross-presentation of a cell-associated Ag, we chose the male HY Ag Smcy-3, for which mice expressing a specific transgenic TCR are available (25). ERAP1-deficient mice have been shown recently to respond poorly to immunization with syngenic male splenocytes expressing this Ag; it was speculated that this may be due to an altered TCR repertoire or to compromised cross-presentation (14). Initially, we tested direct presentation of the Smcy Ag by male BM-DCs, and found it to be strongly diminished by ERAP1 deficiency and abrogated by TAP deficiency (Fig. 7A). Thus, similar to a second male Ag, Uty, studied by Hammer et al. (14) and Yan et al. (15), endogenous presentation of Smcy is ERAP1 dependent. To test cross-presentation of cell-associated Smcy, we performed cotransfer experiments, in which wt or ERAP1 KO mice were injected successively with MHC class I-mismatched splenocytes and CFSE-labeled naive CD8+ T cells expressing the specific TCR. ERAP1 deficiency reduced the proliferative response of cross-primed CD8+ T cells by about two-thirds (Fig. 7, B and C). Therefore, ERAP1 was required for efficient cross-presentation of cell-associated male Ag. Similar results were obtained with a second cell-associated Ag (OVA-electroporated MHC-mismatched cells; Fig. 7D).


Figure 7
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  		FIGURE 7. Direct presentation of the HY Ag Smcy and cross-presentation of cell-associated Ags require ERAP1. A, Naive HY-specific T cells were incubated for 24 h with BM-DCs from male ERAP1 KO mice or DCs from male wt littermates; BALB/c or TAP-deficient C57/BL6 BM-DCs were additional controls. IL-2 secretion of stimulated T cells was measured by ELISA. Cross-presentation of cell-associated Ags was measured by injecting CFSE-labeled T cells specific for Smcy (B and C) or OT1 (D) and male BALB/c splenocytes (B and C) or OVA-electroporated BALB/c splenocytes (D) into ERAP1 KO or control mice. B, Shows representative FACS plots for proliferation of CFSE-labeled, clonotype-positive (T3.70+), Smcy-specific T cells 5 days later. C and D, Show the division indices (number of mitotic events/number of precursors) for Smcy-specific T cells (C) or OT1 cells (D) obtained for three or six KO and wt mice, respectively.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The results presented in this work are consistent with a substantial contribution of ERAP1 in the cellular processing and presentation of MHC class I-restricted Ags, leading to a reduction of up to 40% in overall MHC class I surface expression. However, owing to the pronounced plasticity of the CTL response, these alterations do not translate into a significant modulation of antiviral immunohierarchies and compromised antiviral immunity in the three virus infections studied in this work. We also find that ERAP1 is required for cross-presentation of cell-associated Ag and of OVA-immune complexes, but not of soluble OVA Ag, a surprising result that suggests that final processing of soluble exogenous Ags may not occur in an environment containing active ERAP1.

On cells derived from KO mice, the decrease in the cell surface expression was similar for H-2kb and H-2Db. Thus, our study and the others on ERAP1 KO cells, as well as two small interfering RNA-knockdown studies suggest that the net effect of ERAP1 is an increase in peptide supply for MHC class I presentation, even though ERAP1 leaves some class I ligands untouched and destroys some others (10, 13, 14, 15). Our results and those of Yan et al. (15) suggest furthermore that the positive effect of ERAP1 on MHC class I expression by several cell types, such as fibroblasts, splenocytes, and HeLa cells, is diminished upon treatment with IFN-{gamma} (13, 15). However, we found that MHC class I levels on ERAP-deficient DCs that had been matured with IFN-{gamma} were rather dramatically reduced compared with DCs from wt littermates.

We have determined directly ex vivo the CTL responses against a panel of epitopes elicited upon three different viral infections. Surprisingly, we found substantial deviations in the frequencies of CTLs for only one epitope, the ID LCMV-NP396–404 peptide. Because LCMV efficiently infects professional APCs, including DCs, and efficiently replicates in secondary lymphoid organs (26), it is conceivable that the induction of LCMV-specific CTLs is largely based on direct Ag presentation by DCs. Accordingly, the lower CTL frequencies to NP396–404 correlate with impaired generation of the epitope by LCMV-infected DCs (see Fig. 3G). Direct presentation of the gp33–41 epitope on LCMV-infected DCs was increased in the absence of ERAP1 expression (15). Because our results show no increase in CTL frequencies, it is possible that the enhanced presentation is insufficient to lead to detectable increases in clonal expansion of CTLs. CTL frequencies for the third major Db-restricted LCMV epitope gp276–286, which induces a moderate CTL response, were not affected in our KO mice.

Due to the plasticity of the T cell response, only drastic differences in epitope presentation seem to translate into significantly different CTL frequencies. It was therefore of interest whether the observed impaired presentation of LCMV NP396–404 in ERAP1 KO mice is associated with decreased resistance to viral infection. We did not find evidence for immunodeficiency, because the ERAP1 KO mice controlled LCMV during acute infection just as fast and as efficiently as wt littermate mice. In addition, LCMV-primed ERAP1 KO mice controlled a secondary infection with a rVV expressing the LCMV-NP. Only when we used a more stringent system in which resistance is mediated exclusively by memory CTLs against the defectively presented LCMV NP396–404 epitope itself, accomplished by peptide immunization, was control of a secondary LCMV infection compromised in the KO mice.

Yan et al. (15) recently reported slightly reduced CTL responses against several influenza-derived epitopes, but this did not reach statistical significance. We determined the cytotoxic responses against two of these epitopes, which are dominant during acute influenza virus infection, but similarly did not find any statistically significant differences between the responses of KO mice and wt littermates. Thus, both studies jointly suggest that ERAP1 deficiency does not lead to strong alterations in the CTL response to influenza A virus.

York et al. (16) recently concluded from a comparison of CTL frequencies to LCMV and rVV-OVA epitopes between wt and ERAP1 KO mice that there were marked shifts in the hierarchy of immunodominance in viral infections. However, their results and ours for the three major LCMV epitopes, as assessed by cytokine production, are similar, because the modest increase in CTLs against the gp276–286 epitope found by York et al. in ERAP1 KO mice was not always statistically significant in their experiments. Alterations in CTL frequencies against the dominant VV-derived epitope B8R reported by York et al. (16) could not be detected in the analysis of our ERAP1 KO mice.

Although ERAP1 is induced by IFN-{gamma}, it had only little influence on antiviral immunity in our experiments, despite the fact that IFNs are induced during viral infections. However, our results do not exclude that ERAP1 deficiency has greater effects in other viral infections, particularly in cases in which the response depends only on a single or on very few epitopes whose presentation is strongly diminished by ERAP1, in which only small amounts of viral Ag are expressed, or low frequencies of specific CTLs are induced.

An example in which ERAP1 deficiency impressively correlates with a compromised CTL response is the minor histocompatibility Ag HY. In this example, Hammer et al. (14) found a strongly impaired CTL response against two known Db-restricted epitopes when female ERAP1 KO mice were primed with MHC-matched splenocytes from wt males, followed by restimulation with the male cells in vitro. These authors speculated that defective cross-presentation may account for the observed defect. However, in vivo CTL priming to these epitopes has been reported to depend only to 10% on cross-priming (27). In any case, because mice were primed with MHC class I-matched cells by Hammer et al. (14), the experimental setup was not suitable for studying cross-presentation.

First evidence for a role of ERAP1 in cross-presentation has been reported by Yan et al. (15), who performed in vitro studies with splenic DCs pulsed with latex bead-coated OVA. Considering the small effect of ERAP1 deficiency on antiviral CTL responses, we wondered whether a lack of ER trimming would have an effect on cross-priming in vivo. As model for a cell-associated Ag, we used the HY Ag Smcy-3. First, we established that direct presentation of the Db-restricted Smcy-derived HY epitope KCSRNRQYL requires ERAP1. We found a pronounced effect of ERAP1 deficiency on direct presentation of this epitope, similar to the findings for the second Db-restricted HY epitope WMHHNMLDI (derived from Uty) studied by both Hammer et al. (14) and Yan et al. (15). Moreover, in an in vivo assay, cross-priming of Smcy-specific T cells by male splenocytes was strongly compromised in ERAP1-deficient mice. Similarly, we found a significant effect on cross-presentation of cell-associated OVA. Therefore, ERAP1 plays an important role in the cross-presentation pathway handling cell-associated material. Processing of cross-presented phagocytosed Ags may involve a specialized phagosome that has recruited all required ER components, including ERAP1 (6), or may take place in association with the perinuclear ER (24). Whatever the correct model, both predict a role for ERAP1, which is confirmed by our results.

We also wondered whether ERAP1 deficiency affected cross-presentation of soluble proteins. To explain this poorly understood phenomenon, it has been proposed recently that soluble proteins including OVA gain retrograde access to the perinuclear ER, where they are processed by the ER-associated degradation pathway (5, 28). Although we and others find that efficient direct presentation of the OVA257–264 epitope requires ERAP1, we found cross-presentation of soluble OVA to be independent of ERAP1, but predominantly dependent on the proteasome. Different scenarios might explain this uncoupling of proteasome and ERAP1 requirements. According to previous observations (28, 29), Lamp1+ late endosome/lysosomal compartments are an important site for loading peptides derived from soluble OVA onto recycling MHC I molecules. These compartments could actually contain ERAP1 provided by ER fusion (30), but the acidic pH could well render it inactive (31). Vacuole-associated proteasomes may be involved through the ER-associated degradation pathway (28, 32, 33), and processed peptides could then be reimported by TAP (30, 34). Alternatively, proteasomes may be required for trafficking of peptide-loaded MHC class I molecules via the classical route, a prerequisite for MHC recycling. Another plausible explanation for the absence of an ERAP1 contribution in our experiments is simply that several different pathways may well operate redundantly in cross-presenting soluble proteins (5, 28, 29, 30, 35, 36).

In contrast to soluble OVA, cross-presentation of OVA/anti-OVA immune complexes requires ERAP1. It is conceivable that immune complex formation protects OVA from rapid proteolytic destruction in endosomes and/or favors its translocation to the cytosol, both of which would favor processing of OVA in an ERAP1-dependent, ER-type processing pathway, possibly identical with that handling phagocytosed Ags (37). Whatever the ultimate explanation for the discrepancy between the roles of ERAP in cross-presentation of, on the one hand, soluble, and, in contrast, phagocytosed or receptor-internalized material, our results underline the complexity of the cellular pathways involved in cross-presentation, which remains to be elucidated.


   Acknowledgments
 
We thank Drs. Klaus Eichmann and Hermann Frommhold for their generous support, Dr. Randy Cassada for critical reading of the manuscript, and Dr. Nilabh Shastri for sharing information before publication. We gratefully acknowledge the gift of rVV from Dr. Jon Yewdell, of OT1 and HY transgenic mice from Dr. Benedita Rocha, and of TAP-deficient mice from Dr. Olivier Lantz.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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.

1 This work was supported by a grant from the Forschungskommission of the Medical Faculty of the University of Freiburg (NIE346/04; to G.N.). Back

2 Address correspondence and reprint requests to Dr. Gabriele Niedermann, Clinic for Radiotherapy, University Hospital of Freiburg, D-79106 Freiburg, Germany. E-mail address: gabriele.niedermann{at}uniklinik-freiburg.de Back

3 Abbreviations used in this paper: ER, endoplasmic reticulum; BM, bone marrow; DC, dendritic cell; ERAP, ER-associated aminopeptidase; ID, immunodominant; KO, knockout; LCMV, lymphocytic choriomeningitis virus; NP, nuclear protein; SD, subdominant; VV, vaccinia virus; wt, wild type. Back

Received for publication August 28, 2006. Accepted for publication November 28, 2006.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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