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The Journal of Immunology, 2006, 177: 983-990.
Copyright © 2006 by The American Association of Immunologists

Role of Immunoproteasomes in Cross-Presentation1

Michael J. Palmowski*, Uzi Gileadi*, Mariolina Salio*, Awen Gallimore*, Maggie Millrain{dagger}, Edward James{dagger}, Caroline Addey{dagger}, Diane Scott{dagger}, Julian Dyson{dagger}, Elizabeth Simpson{dagger} and Vincenzo Cerundolo*,2

* Tumour Immunology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom; and {dagger} Transplantation Biology Group, Clinical Sciences Centre, Imperial College, Hammersmith Hospital, London, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The evidence that proteasomes are involved in the processing of cross-presented proteins is indirect and based on the in vitro use of proteasome inhibitors. It remains, therefore, unclear whether cross-presentation of MHC class I peptide epitopes can occur entirely within phagolysosomes or whether it requires proteasome degradation. To address this question, we studied in vivo cross-presentation of an immunoproteasome-dependent epitope. First, we demonstrated that generation of the immunodominant HY Uty246–254 epitope is LMP7 dependent, resulting in the lack of rejection of male LMP7-deficient (LMP7–/–) skin grafts by female LMP7–/– mice. Second, we ruled out an altered Uty246–254-specific T cell repertoire in LMP7–/– female mice and demonstrated efficient Uty246–254 presentation by re-expressing LMP7 in male LMP7–/– cells. Finally, we observed that LMP7 expression significantly enhanced cross-priming of Uty246–254-specific T cells in vivo. The observations that male skin grafts are not rejected by LMP7–/– female mice and that presentation of a proteasome-dependent peptide is not efficiently rescued by alternative cross-presentation pathways provide strong evidence that proteasomes play an important role in cross-priming events.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Dendritic cells (DC)3 have the ability to capture cellular tissue Ags and present them on MHC class I molecules to Ag-specific CD8+ T cells. This process, called cross-presentation, is an important pathway in controlling T cell priming in vivo (1). Although various mechanisms have been proposed to explain cross-presentation (as reviewed in Ref. 2), many questions remain to be answered. In particular, it is still unclear as to whether MHC class I cross-presented proteins enter the cytosol of DC, making them available for proteasomal degradation, transport into the endoplasmic reticulum (ER), and presentation on MHC class I molecules. Experiments conducted using cells deficient in the peptide transporter (TAP) complex have provided compelling evidence for a role of TAP in cross-presentation (3, 4). These results are consistent with a model in which peptides generated from the processing of exogenous proteins are translocated by TAP into the lumen of a cross-presentation compartment for association to MHC class I molecules. Recent studies (5, 6) have shown that fusion of ER with the endosomal-phagosomal system delivers the machinery required for peptide loading of MHC class I molecules directly into the phagosome. Although the ER-phagosome model offers important mechanistic insights into the MHC class I cross-presentation pathway, contribution of the ER to phagosome formation remains controversial (7, 8).

Although the evidence demonstrating the role of proteasomes in processing proteins synthesized by infected APC (i.e., direct presentation) is compelling (9, 10, 11, 12, 13, 14, 15), it remains unclear whether proteasomes play a role in generating peptides derived from cross-presented proteins. Recent studies (16, 17) have shown that the products of short-lived proteins are not able to be cross-presented, hence demonstrating that the efficiency of cross-presentation of exogenous Ags depends on the transfer of proteasome substrates, rather than proteasome products, from Ag donor cells to DC. However, the evidence that proteasomes are involved in cross-presenting exogenous Ags remains indirect and only based on in vitro data using proteasome inhibitors (1, 4, 18, 19, 20).

Exposure of cells to IFN-{gamma} induces the synthesis of three proteolytic proteasome subunits (LMP2, LMP7, and MECL-1), which are incorporated into an alternative form of proteasome, called immunoproteasome, displacing the constitutive subunits beta1, beta2, and beta5, respectively (21, 22, 23, 24). We and others (9, 10, 12, 13, 14) have previously demonstrated that defined T cell epitopes are exclusively generated by immunoproteasomes and fail to be generated by the constitutive proteasomes.

Because constitutive and immunoproteasomes are localized in the cytosol and are not found in the ER and endolysosomes, demonstrating that cross-presented proteins are processed by either form of proteasomes would provide compelling evidence supporting a model in which exogenous cross-presented proteins intersect the cytosol during cross-presentation. This reasoning led us to study cross-presentation of epitopes that are exclusively generated by immunoproteasomes and assess whether during cross-presentation they remain immunoproteasome dependent. Furthermore, to avoid the use of viral Ags to prevent the possibility that viral particles could infect recipient cells and be directly presented by recipient cells, we focused our analysis on the processing and presentation of MHC class I-restricted determinants derived from the ubiquitously expressed male-specific minor histocompatibility Ag genes Uty and Smcy (25, 26). The H-2b-restricted UTY and SMCY responses are specific for the epitope Uty246–254 (HYDbUty;25) and Smcy738–746 (HYDbSmcy;27). The observation that the presentation of the HYDbUty epitope is LMP7 dependent provided us with the opportunity to test the importance of immunoproteasomal degradation for cross-priming of HYDbUty-specific CTL in LMP7–/– mice using a cell-associated Ag expressed at physiological concentrations. The results of these experiments demonstrated the in vivo role of immunoproteasomes in the generation of cross-presented peptide fragments. We also observed that LMP7–/– female mice failed to reject LMP7–/– male skin grafts, demonstrating the importance of immunoproteasomes in immune surveillance.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
In vivo killing assays

Freshly isolated male and female splenocytes were incubated in RPMI 1640 medium (Sigma-Aldrich) with 1 µM peptide for 1 h or in medium alone and labeled with different concentrations of fluorescent dye (CFSE or chloromethyl-benzoyl-aminotetramethyl-rhodamine; Molecular Probes). For a detailed description of the protocol, see Ref. 28 . Labeled cells were injected at 107 cells/mouse into the tail vein. Disappearance of peptide/fluorochrome-labeled cells was tracked using FACS analysis of freshly isolated PBL 72 h after injection. WinMDI 2.8 software (J. Trotter, The Scripps Research Institute; <http://facs.scripps.edu>) and CellQuest 3.3 software (BD Biosciences) were used to analyze the FACS data. Percentage of killing was calculated using the formula: 100 – ((percentage of immune mice Ag-positive cells/percentage of immune mice Ag-negative cells)/(average percentage of naive mice Ag-positive cells/average percentage of naive mice Ag-negative cells) x 100).

Mice and immunization

LMP7–/– (29) and B6 control female mice were bred in the local animal facility under specific pathogen-free conditions and used at 6–8 wk of age. For the in vivo killing assay (see Fig. 6), LMP7–/– and wild-type (WT) mice were immunized by i.m. injection of 50 µg plasmid DNA encoding the immunodominant MHC class I H2Db-restricted minimal Uty246–254 epitope WMHHNMDLI (Uty minigene) and boosted 2 wk later with 100 µl of PBS/106 PFU of recombinant vaccinia virus encoding the Uty minigene. The plasmid pSG2 has been described previously (30). For cross-presentation experiments, WT C57BL/6 naive mice received 2 x 107 CBA male splenocytes i.p. Ten days later, mice were boosted by i.v. injection of 106 PFU of recombinant vaccinia virus encoding the Uty minigene in 100 µl of PBS. Blood samples were taken 8 days after the booster immunization. For other experiments (e.g., for results, see Fig. 2C) mice were primed with minigene HYDbUty vaccinia and boosted by i.v. injection with 106 peptide-pulsed LMP7–/– female bone marrow (BM)-derived DC in PBS. BM DC were prepared as described previously (31).


Figure 6
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  		FIGURE 6. LMP7–/– male splenocytes are not lysed in vivo by HYDbUty-specific CTL. Five WT or five LMP7–/– female mice either naive (A) or primed with plasmid DNA followed by vaccinia virus both encoding the minimal Uty246–254 epitope (B and C) were used as recipients for in vivo killing of fluorochrome-labeled syngeneic splenocytes. Insets in B and C, Tetramer analysis of PBL from mice used for this assay. Mice were injected with a mixture of fluorescently labeled syngeneic spleen cell targets (top left panel). Male cells pulsed with HYDbUty peptide were labeled with the lowest concentration of CFSE, female cells with intermediate and male unpulsed cells with the highest concentration. WT mice were injected with splenocytes from WT males and females, whereas LMP7–/– females were injected with splenocytes from LMP7–/– donor mice accordingly. FACS plots of fresh PBL were conducted 9 days after vaccinia boosting, which was the day of injection of labeled cells. Efficient elimination from the PBL samples of male and Uty246–254 peptide-pulsed cells (UTY pep) can be observed in WT mice (B). In vaccinated LMP7–/– female animals, only peptide-pulsed cells were killed, whereas LMP7–/– male splenocytes were not lysed (C). A representative plot is shown for each group (A–C). D, Percentage of HY-specific in vivo killing is shown. The error bars indicate SD of five immunized LMP7–/– ({square}) and WT mice ({blacksquare}). E, UTY ({blacksquare}) and SMCY ({square}) mRNA levels were measured in total RNA extracts from splenocytes. Average quantities of UTY mRNA relative to 18S RNA content is shown from WT and LMP7–/– samples. Error bars indicate mean SD of triplicate measurements.

 

Figure 2
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  		FIGURE 2. Uty246–254 encoding vaccinia generates HYDbUty-specific and vaccinia-specific CD8+ T cell responses in LMP7–/– female mice. Twenty-five naive WT and 25 LMP7–/– female mice were injected with vaccinia virus encoding the HYDbUty epitope. A, Five mice from each group were sacrificed to measure by ELISPOT IFN-{gamma} responses to several vaccinia epitopes (peptides used in the assay are identified in Materials and Methods). HYDbUty-specific (B) and VACKbB8R-specific (C) CD8 T cell responses were measured by tetramer staining of freshly isolated PBL samples from the remaining mice. The bars (B and C) indicate percentages of tetramer-positive cells of total CD8+ cells. D, Vaccinia HYDbUty-primed LMP7–/– and WT female mice were split up into groups of five mice and boosted with LMP7–/– DC pulsed with different concentration of HYDbUty peptide epitope or unpulsed (No pep). Expansion of HYDbUty-specific cells in PBL ex vivo was measured; the percentage of tetramer-positive cells of CD8-positive cells is shown. Error bars indicate SD of 5 mice (A) and 20 mice (B and C).

 
Cell lines and media

The fibrosarcoma lines MC57 (H-2b) (32) were grown in RPMI 1640 supplemented with 10% FCS, 10 mM HEPES, 100 IU/ml penicillin, 100 µg/ml streptomycin, 5 x 10–5 M 2-ME, and 2 mM glutamine (R10). MC57 cells constitutively express LMP7, detectable by the Western blot test (data not shown).

Tetramer staining and ELISPOT assay

PBL were prepared from tail vein blood samples using RBC lysis buffer (Invitrogen Life Technologies). Nucleated cells were resuspended in R10. PBL samples were stained with 0.5 µg tetramers in 20 µl of RPMI 1640 with 10% FCS for 20 min at 37°C, then cells were costained with FITC-conjugated anti-CD8-{alpha} (Caltag Laboratories) on ice, washed twice in PBS at room temperature, and analyzed on a FACSCalibur (BD Biosciences) using CellQuest software. Tetramers were made as described previously (30). Briefly, for the production of HYDbUty tetramers, we refolded H2-Db H chain with human beta2-microglobulin and the HYDbUty peptide WMHHNMDLI, and for VACKbB8R tetramers we refolded the H2-Kb H chain with human beta2-microglobulin and the B8R vaccinia peptide TSYKFESV (33).

For ex vivo measuring of other vaccinia responses, mice were injected with 106 PFU of vaccinia virus (Western Reserve (WR) strain), 9 days later spleens were harvested and suspended in R10, 105 spleen cells were incubated in a 96-well IFN-{gamma} ELSISPOT plate overnight in R10 containing 1 µM peptide B8R20–27, A19L47–55, A47L138–146, or A42R88–96 (33). The assay was developed according to the manufacturer’s instructions (Mabtech) and analyzed using an automated ELISPOT reader (Autoimmun Diagnostika).

In vitro stimulation assay

CD8+ clones specific for HYDbUty (WMHHNMDLI), clones CTL10 (34), U2D6 (35), 4.1A1M1 (35), and HYDbSmcy (KCSRNRQYL)-specific clone S1FP (35) were maintained in bulk cultures restimulated once every 10–30 days, with irradiated spleen cells expressing male-specific minor H Ag, in R10 supplemented with 10 U per ml of human rIL-2 (hu-rIL-2). For the proliferation assays, the T cell clones were harvested from bulk cultures at least 10 days after the last restimulation and incubated at 104 clone cells per well in flat-bottom 96-well plates with 5 x 105 irradiated spleen cells. For the duration of the proliferation assay, the medium was supplemented with 1 unit per ml of hu-rIL-2.

For the TNF-{alpha} and IFN-{gamma} release assays, 5 x 104 target cells were incubated with the same number of T cells for 4 h in 96-well round-bottom plates in 200 µl of R10 supplemented with 10 U per ml of hu-rIL-2 at 37°C. Supernatant IFN-{gamma} or TNF-{alpha} concentration was quantified using sandwich ELISA kits (PeproTech). For proteasome inhibition, target cells were incubated in 1 µM proteasome-specific inhibitor epoxomicin (36) (Affinity Research Products) for 30 min before infection with vaccinia virus using 5 PFU per cell. Infected targets were washed three times in R10 before they were added to the effector cells. For IFN-{gamma} treatment, cells were treated overnight with 300 U IFN-{gamma} per ml (PeproTech). Cells were infected with a range of vaccinia viruses, washed three times in R10, and added to the effector cells.

Skin grafting

Mice were grafted as described by Billingham (37) with syngeneic male and female tail skin grafts placed adjacent in the same graft bed, on one side of the thorax. The grafts were held in place by plaster casts, which were removed at 10 days. The grafts were thereafter monitored for signs of rejection. Rejection was designated as the day when <10% viable graft remained. This progressed to replacement by white scar tissue, whereas the adjacent female control graft remained intact, pigmented, and grew hairs.

HY-specific CD8+ T cell responses were monitored in skin-grafted animals by tetramer staining of PBL samples. Blood was obtained from the tail vein of grafted animals and stained for FACS analysis as described above.

Murine embryonic fibroblast (MEF) lines and Lenti virus transductions

WT and LMP7–/– MEF isolated from 14-day-old embryos were passaged four to six times before transduction with a lentiviral construct expressing murine LMP7 and GFP separated by an internal ribosomal entry site (IRES). Full-length (FL) LMP7 was amplified from C57BL/6 spleen cDNA using 5'-CGGGATCCACCATGGCGTTACTGGATCTGTGCGGTGCCGC-3' and 5'-CATCCGCTCGAGTCACAGAGCGGCCTCTCCGTACTTG-3' and cloned into the PHR-SIN lenti vector containing an IRES-GFP construct that was provided by M. Collins (Windeyer Institute, University College, London, U.K.); the production of lentiviral particles was described previously (31).

Recombinant vaccinia viruses

Vaccinia viruses (WR strain) encoding the HYDbUty peptide MWMHHNMDLI, or the FL-Uty gene were made by cloning each insert into the thymidine kinase locus of vaccinia virus using the vector pSC11 as previously described (38). Vaccinia virus was stored at 108 PFU per ml in PBS at –80°C.

Western blotting

Lysed cells were separated by SDS-PAGE. The gel was blotted onto a Hybond-C membrane. Detection of LMP7 was performed using anti-LMP7 antiserum as described elsewhere (39).

Quantitative RT-PCR

RNA was prepared from mouse spleen cells using TRIzol reagent (Invitrogen Life Technologies) and treated with RNase-free DNase using RNeasy MinElute spin columns (Qiagen). Concentrations were determined spectrophotometrically by measuring the absorbance at 260 nm. Relative concentrations of 18S ribosomal RNA, Smcy, and Uty transcripts were determined using the Quantitect SYBR Green RT-PCR kit from Qiagen, following the manufacturer’s recommendations. Reactions were performed on a DNA Engine Opticon (MJ Research) system. Primer pairs were assessed by melting curve analysis and gel electrophoresis to ensure only specific products were generated. The Smcy and Uty primer pairs were chosen from regions with low homology to Smcx and Utx, respectively, and did not produce a PCR product with female mouse RNA. The primer sequences were as follows: 18S forward, 5'-CCGCAGCTAGGAATAATGGAAT-3'; 18S reverse, 5'-CGAACCTCCGACTTTCGTTCT-3'; Smcy forward, 5'-CATGTAAAGGAGATAAGGAACT-3'; Smcy reverse, 5'-ATGAATGCGCTCAGATTGGG-3'; Uty forward, 5'-AGTGTCCAGACAGCTTCACAT-3'; and Uty reverse, 5'-TGAGCATCCCCTTTTGAATTCT-3'. Each primer pair produced a single product of ~200 bp. To prevent genomic DNA amplification, the primers were designed to anneal upstream and downstream of intron sequences. Test samples were assayed in triplicate. Gel electrophoresis and melting curve analysis confirmed the absence of primer dimers. Expression values were determined from standard curves generated using dilutions of male RNA extracted from a male B cell line and plotting cycle threshold values against log quantity. Normalized Smcy and Uty values were obtained by division with the corresponding 18S value.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
LMP7–/– female mice fail to reject LMP7–/– male skin grafts

Initial experiments were conducted to assess whether female LMP7–/– mice could reject LMP7–/– male skin grafts. Syngeneic male and female skin was grafted into the same graft bed on WT and LMP7–/– female mice. Mice were monitored for graft rejection. We observed that LMP7–/– mice were unable to reject syngeneic male LMP7–/– skin grafts, whereas WT female mice rejected their male grafts within 20–40 days (Fig. 1A).


Figure 1
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  		FIGURE 1. LMP7–/– female mice do not reject LMP7–/– male skin grafts. Two groups of six WT and six LMP7–/– female mice were grafted with syngeneic male and female skin grafts. Mice were monitored for male-specific graft rejection (A) and HY-specific CD8 T cell responses (B). Percentage of surviving male grafts is shown ({blacktriangledown}, LMP7–/– male grafts on LMP7–/– female mice; {blacksquare}, WT male grafts on WT female mice; A). The percentage of HYDbUty- and HYDbSmcy-specific T cell expansion was measured by ex vivo tetramer analysis at day 45 after grafting is shown in B. The average percentage of tetramer-positive cells of total CD8+ is shown for each group. Error bars indicate mean SD of six mice.

 
Because male-specific skin graft rejection involves recognition of the HY-encoded gene products Uty and Smcy by CD8+ T cells (35), frequency of HYDbUty- and HYDbSmcy-specific T cells was monitored in skin-grafted mice (Fig. 1B). Ex vivo tetramer analysis was conducted in these grafted mice from freshly isolated PBL on days 20 and 45 after grafting (Fig. 1B and data not shown). At both time points, ex vivo CD8+ responses specific for HYDbUty were detected in WT but not in LMP7–/– mice grafted with syngeneic male grafts (Fig. 1B). In contrast, HYDbSmcy-specific CD8+ levels were similar in WT and LMP7–/– mice. These results demonstrate that the generation of HYDbUty-specific CD8+ T cells is impaired in LMP7–/– female mice. Furthermore, lack of rejection of male skin grafts in LMP7–/– female mice, in the presence of significant numbers of HYDbSmcy-specific T cells, demonstrates that the HYDbSmcy-specific CD8+ T cell response observed is not able to mediate graft rejection in LMP7–/– mice.

The lack of detectable levels of HYDbUty-specific T cells in LMP7–/– female mice grafted with male LMP7–/– skin is consistent with the possibility that processing and presentation of the HYDbUty epitope is impaired in LMP7–/– male cells, indicating that immunoproteasomes play an important role in the generation of this epitope. Alternatively, lack of HYDbUty-specific tetramer-positive T cell responses in female LMP7–/– mice could be accounted for by an altered T cell repertoire in LMP7–/– female mice. The latter possibility is consistent with data showing that adoptively transferred T cells from LMP2-deficient mice respond to influenza virus infection with an altered hierarchy, compared with T cells from WT mice (14). To distinguish between these two possibilities, LMP7–/– and WT female mice were immunized with recombinant vaccinia virus encoding the minimal HYDbUty epitope. Using ELISPOT analysis of freshly isolated splenocytes, we observed that the ranking order and overall intensity of a range of vaccinia-specific CD8 responses in LMP7–/– mice is very similar to the ranking order and T cell frequency seen in WT mice (Fig. 2A). We also used tetramer staining to monitor ex vivo the HYDbUty-specific T cell response (Fig. 2B) and the response to the recently described (33) dominant vaccinia epitope derived from the B8R Ag (hereafter referred to VACKbB8R) (Fig. 2C). We found that ex vivo HYDbUty tetramer-positive PBL ranged between 0.7 and 1.2% of CD8+ cells in both LMP7–/– and WT-vaccinated mice (Fig. 2B). We also found that the response to the immunodominant vaccinia VACKbB8R epitope was comparable in both strains of mice: ~15% of blood CD8+ T cells (Fig. 2C). These results suggest that the T cell repertoire of LMP7–/– mice is similar to the repertoire of WT C57BL6 mice. To confirm further this possibility, we injected i.v. both HYDbUty vaccinia-primed LMP7–/– and WT female mice with BM-derived LMP7–/– female DC pulsed with a wide range of Uty246–254 peptide concentrations (1µM to 1 pM) and observed equal expansions of HYDbUty-specific CD8 cells in PBL of WT and LMP7 mice (Fig. 2D). Combined, these results suggest that lack of LMP7 expression does not significantly alter the repertoire of Db- and Kb-restricted CD8+ T cells. The observation that primed WT and LMP7–/– mice respond similarly to the preprocessed Uty246–254 peptide across a wide range of concentrations strongly suggest that the processing of the UTY protein, rather than the HYDbUty-specific T cell repertoire, is impaired in LMP7–/– mice.

Presentation of the HYDbUty epitope is immunoproteasome and TAP dependent

To assess in vitro the presentation of the HYDbUty epitope by LMP7–/– male cells, we tested whether splenocytes from LMP7–/– male mice are able to stimulate proliferation of the HYDbUty-specific CD8+ clone 4.1A1M1 (Fig. 3). We found that although splenocytes from WT male mice efficiently induced this clone to proliferate, LMP7–/– male splenocytes failed to sensitize the clone 4.1A1M1 to proliferate (Fig. 3A). In contrast, HYDbSmcy-specific clones were equally efficiently stimulated to proliferate by LMP7–/– and WT male splenocytes (Fig. 3B). Further control experiments showed that the CD4+ clone B9 specific for HYAbDby (40) responded equally efficiently to splenocytes from WT and LMP7–/– male mice (data not shown; clones are identified in Materials and Methods).


Figure 3
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  		FIGURE 3. LMP7–/– splenocytes fail to present the HYDbUty epitope to a HYDbUty-specific T cell clone. Fresh spleen cells from WT male or female mice (WT) or LMP7–/– male or female mice (LMP7–/–) were incubated with either the CD8+ clone 4.1A1M1 recognizing the HYDbUty peptide (A), or clone S1FP recognizing the HYDbSmcy epitope (B). Proliferation of the responding clones is shown as cpm after thymidine incorporation. Error bars indicate mean SD of quadruplicate measurements.

 
To address whether the inability of LMP7–/– male cells to present the HYDbUty epitope could be restored by expressing LMP7 protein in LMP7–/– male cells, we engineered a lentiviral vector encoding the mouse LMP7 protein (Fig. 4) and transduced freshly established MEF cultures from LMP7–/– male mice (Fig. 4). We demonstrated that although LMP7–/– male MEF were unable to induce the HYDbUty-specific CD8 clone 4.1A1M1 to secrete IFN-{gamma} or TNF-{alpha} (Fig. 4 and data not shown), expression of LMP7 protein in the LMP7–/– male MEF restored the male-specific cytokine response (Fig. 4). This result shows that expression of LMP7 in LMP7–/– male cells restores HYDbUty presentation.


Figure 4
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  		FIGURE 4. LMP7–/– expression in LMP7–/– cell line results in stimulation of a HYDbUty-specific T cell clone. A, MEF lines from male and female WT mice and LMP7–/– male mice were used to stimulate HYDbUty-specific T cell clone 4.1A1M1. MEF were transduced with lentiviral vectors encoding GFP, LMP7, or were mock-transduced (Neg). Bars indicate concentration of IFN-{gamma} in the supernatants of the stimulations. Error bars indicate mean SD of quadruplicate measurements. B and C, Lentivirus-encoded LMP7 expression in B16F10 and MEF. Cells were transduced using lentiviral particles encoding a GFP construct (IRES GFP), or a mouse FL-LMP7 construct (LMP7 IRES GFP), or were mock transduced (Mock). LMP7-negative B16F10 cells were transduced, LMP7 expression was monitored by the Western blot (B). Efficiency of lentiviral transduction was monitored in MEF used in the experiment shown (A) by assessing GFP expression by FACS analysis (C).

 
Although proteasome inhibitors have been shown to inhibit a broader range of proteases (41, 42), to provide complementary evidence for a proteasome requirement for the presentation of the HYDbUty epitope, we demonstrated that incubation of MC57 cells (LMP7+/+) with the proteasome inhibitor epoxomicin, before infection with a vaccinia virus encoding FL-Uty vaccinia, abolished presentation of the HYDbUty epitope (Fig. 5A). These results provide complementary evidence for a proteasome requirement for the presentation of the HYDbUty epitope. As a control, MC57 cells infected with vaccinia virus encoding the optimal length HYDbUty epitope construct in the presence or absence of epoxomicin were capable of presenting the HYDbUty epitope (Fig. 5A). We extended these results by demonstrating that male spleen cells from TAP-1–/– B6 mice, unlike those from WT males were unable to stimulate the HYDbUty-specific clone 4.1A1M1 (Fig. 5B).


Figure 5
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  		FIGURE 5. Generation of the HYDbUty epitope is epoxomicin sensitive and TAP dependent. TNF secretion by the HYDbUty-specific clone 4.1A1M1 was measured after stimulation with MC57 cells infected with FL-Uty encoding vaccinia (FL-UTY), irrelevant vaccinia (VAC), or vaccinia encoding the HYDbUty minigene (Mini UTY). A, Vaccinia-infected MC57 cells were used in the absence ({blacksquare}) or in the presence ({square}) of epoxomicin. B, TNF secretion was measured from the supernatant of HYDbUty-specific CD8+ clone 4.1A1M1 incubated with freshly isolated WT ({diamond}), LMP7–/– ({square}), and TAP–/– ({blacktriangleup}) male splenocytes. Control cultures with peptide-pulsed TAP–/– male or female spleen cells triggered TNF secretion of >5000 pg/ml TNF (data not shown). Error bars indicate mean SD of triplicate measurements.

 
Expansion of HYDbUty-specific tetramer-positive T cells in LMP7–/– female mice by vaccinia virus encoding the optimal length HYDbUty epitope (Fig. 2) provided an opportunity to assess whether HYDbUty-specific CD8+ T cells in LMP7–/– females could reject LMP7–/– male target cells in vivo. We found that CFSE-labeled WT male splenocytes were rapidly lysed in HYDbUty-primed WT females, as compared with naive control mice (Fig. 6, A and B). Similar results were obtained with HYDbUty peptide-pulsed targets, which were rapidly eliminated in both vaccinated LMP7–/– and WT mice (Fig. 6, C and B). However, LMP7–/– male cells were not rejected in the presence of HYDbUty-specific CD8 T cells, confirming that LMP7–/– male cells are incapable of efficiently presenting the HYDbUty peptide in vivo (Fig. 6, C and D). To address whether the expression of UTY is impaired in LMP7–/–, we performed quantitative RT-PCR on RNA extracted from fresh splenocytes. The results of these experiments demonstrated no significant differences in the levels of UTY and SMCY mRNA in WT or LMP7–/– male splenocytes (Fig. 6E).

Cross-presentation of the HYDbUtyepitope is greatly enhanced by the presence of immunoproteasomes in recipient mice

Demonstrating that the presentation of the HYDbUty epitope is dependent on LMP7 expression provided us with the opportunity to assess whether uptake of the UTY protein in vivo could result in priming of HYDbUty-specific T cells in female LMP7–/– mice. In vivo evidence that proteasomes are involved in cross-presenting exogenous proteins is lacking, and the only published data are based on the use in vitro of proteasome inhibitors. To study cross-presentation of the UTY protein, ruling out direct presentation events by donor cells, MHC-mismatched CBA male splenocytes were injected into WT or LMP7–/– female recipients and then boosted with recombinant vaccinia virus encoding the HYDbUty epitope minigene (Fig. 7A). Control groups of WT and LMP7–/– female mice were injected with the same number of female splenocytes. Tetramer staining of freshly isolated PBL showed strong HYDbUty responses in WT female mice, whereas a significant expansion of HYDbUty-specific T cells was detected in only one LMP7–/– female mouse. These results suggest that cross-priming of HYDbUty-specific T cells is significantly impaired in LMP7–/– mice (Fig. 7A).


Figure 7
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  		FIGURE 7. Cross-presentation of HYDbUty in LMP7–/– female mice is severely compromised. A, Ex vivo HYDbUty tetramer stains of freshly isolated PBL are shown from female WT or LMP7–/– animals injected with MHC-mismatched female (female CBA) or male (male CBA) spleen cells, followed by an injection of minigene HYDbUty vaccinia. Each bar corresponds to a measurement from one animal. The horizontal line separates responding mice from those that have frequencies of tetramer-positive cells similar to the control groups. B, Ex vivo HYDbUty tetramer stains of freshly isolated PBL are shown from female WT or LMP7–/– animals injected with TAP–/– male splenocytes (TAP–/– spleen), boosted with minigene HYDbUty vaccinia, or minigene HYDbUty vaccinia only (Mini UTY).

 
Because we have demonstrated that presentation of the HYDbUty epitope is TAP dependent (Fig. 5B), we conducted additional experiments in a syngeneic model by injecting male TAP–/– splenocytes into syngeneic WT or LMP7–/– female mice (Fig. 7B). The expansion of HYDbUty-specific CD8+ T cells was measured using HYDbUty tetramers on freshly isolated PBL (Fig. 7B). We observed a significant expansion of HYDbUty-specific T cells in WT but not in LMP7–/– female mice primed with syngeneic TAP–/– male splenocytes.

Overall, by combining the results (Fig. 7, A and B), cross-primed HYDbUty responses could be identified in only 1 of 13 LMP7–/– female mice, whereas HYDbUty-specific cross-priming was detectable in a large proportion (9 of 13) of WT mice (Fig. 7). Although the proteolytic pathway responsible for the generation of the HYDbUty epitope in the single LMP7–/– responder needs to be identified, these results demonstrate that cross-presentation of the HYDbUty epitope is significantly impaired by the lack of immunoproteasomes (Fisher’s exact test, p = 0.0036). These findings provide strong evidence that the intersection between the cytosolic and phagolysosomal pathways is required for the generation of proteasome-dependent MHC class I epitopes derived from cross-presented proteins.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The results of the experiments presented in this study demonstrate the impact of LMP7 expression on immune surveillance and cross-priming, showing that cross-presentation of HYDbUty is enhanced by the presence of immunoproteasomes.

We have analyzed direct presentation of the HYDbUty epitope in WT- and LMP7-deficient splenocytes using either endogenous (Figs. 1, 3, 4, and Fig. 6, B and C) or vaccinia encoded (Fig. 5A) Uty protein. We have then compared indirect presentation of the HYDbUty epitope by assessing cross-priming of HYDbUty-specific responses in WT- and LMP7-deficient mice (Fig. 7, A and B).

The results of the first part of the study demonstrated that direct presentation of the HYDbUty epitope is LMP7 dependent. We showed that syngeneic male skin grafts fail to prime ex vivo detectable HYDbUty tetramer responses in LMP7–/– female mice (Fig. 1B), whereas responses to the subdominant HYDbSmcy epitope are unaffected, and LMP7–/– female mice respond to HYDbUty minigene vaccination (Fig. 2), but fail to reject CFSE-labeled male LMP7–/– splenocytes (Fig. 6). Additionally, we showed that LMP7–/– male splenocytes fail to stimulate proliferation of HYDbUty-specific clones, even though they are able to stimulate proliferation of HYDbSmcy-specific clones (Fig. 3). Finally, the expression of LMP7 in embryo fibroblasts isolated from LMP7–/– male mice restores HYDbUty presentation (Fig. 4).

Differences in the cleavage patterns of standard proteasome and immunoproteasomes have been demonstrated using purified proteasomes with synthetic peptides (43) and FL-proteins (13, 44). The general consensus is that 20S immunoproteasomes are more adept at producing peptides with hydrophobic and positively charged COOH-terminal residues. Schirle and colleagues (13) demonstrated that 20S immunoproteasomes have a preference for leucine and isoleucine at P1 and glycine at position P1'. The COOH flanking residue of the HYDbUty epitope (246WMHHNMDLI254 GDNT; P1 and P1' positions are in bold) is compatible with the amino acid preferences identified by in vitro digestions with purified immunoproteasomes (13) and suggests that the expression of the immunoproteasomes is essential for the generation of the HYDbUty epitope. Experiments conducted in the presence of proteasome inhibitors in the LMP7-positive cell line MC57 (Fig. 5) and the finding that expression of LMP7 in fibroblast lines derived from LMP7–/– male mice restores their ability to present the HYDbUty epitope to specific T cells (Fig. 4) confirmed that generation of the HYDbUty epitope is immunoproteasome dependent.

The role of immunoproteasomes in cross-presentation

Phagosomes have been shown to be a specialized organelle capable of mediating cross-presentation. TAP and other components of the peptide-loading complex, as well as the ER resident peptidase ER aminopeptidase-1, are present in purified phagosomes (1, 5, 6, 45). Furthermore, TAP has been shown to be involved in cross-presentation (3, 4) and proteasomes and ubiquitinated proteins have also been found to be associated with phagosomes (6). Although these results strongly suggest that a cytosolic step is necessary for cross-presentation, no definitive experiments showing export of internalized proteins from phagosomes to cytosol has been published.

In this study, we have demonstrated that HYDbUty-specific cross-priming is significantly compromised in LMP7–/– mice. Because proteasomes are not present in endocytic compartments, but are exclusively in the cytosol and nucleus of cells, our results add to the indirect evidence supporting a phagosome to cytosol pathway for cross-presentation.

It is of interest that in one LMP7–/– female mouse a strong UTY246–254 response was detected after injecting MHC-mismatched male cells (Fig. 7A). This result suggests that in a small proportion of cases proteasome-independent degradation of the UTY protein results in cross-presentation of the HYDbUty epitope. Several alternative possibilities may account for this result. First, proteasome-independent vacuolar cross-presentation pathways have previously been described (20); such a vacuolar pathway of cross-presentation was shown to be TAP independent and cathepsin S dependent. Second, we have previously shown (12) that events altering proper folding of the antigenic proteins may favor immunoproteasome-independent generation of MHC class I epitopes. Third, UTY degradation products could have been generated by the donor cells (i.e., male CBA splenocytes) and then taken up and cross-presented by the recipient LMP7–/– DC (46).

A proportion of WT mice failed to be primed by the injection of male CBA cells (Fig. 7A). Although it has recently been shown (47) that priming of HY-specific CD8+ T cell responses is operative in the presence of full MHC mismatch, we cannot rule out the possibility that the lack of HYDbUty epitope-specific responses in a proportion of WT female mice could be accounted for by the presence of immunodominant allospecific responses. However, to rule out the possibility that alloreactive responses could have out-competed the generation of HYDbUty epitope-specific CD8+ T cells in LMP7–/– mice, experiments were conducted by injecting syngeneic TAP–/– male cells into LMP7–/– female mice (Fig. 7B). The results of these experiments confirmed the lack of expansion of the HYDbUty epitope-specific response in LMP7–/– mice.

The role of immunoproteasomes in immune surveillance

In addition to the analysis of the role of immunoproteasome in cross-presentation, our results address the role of immunoproteasomes in immune surveillance. We showed that lack of LMP7 expression in peripheral tissues expressing a defined endogenous Ag (i.e., UTY protein) protects them from HYDbUty T cell lysis (Fig. 6) and results in skin graft survival (Fig. 1).

Although in skin-grafted WT females HYDbUty-specific T cells were abundant, no HYDbUty CD8+ T cell responses were detectable in LMP7–/– female mice that received LMP7–/– male skin grafts (Fig. 1B). In contrast, LMP7–/– female mice responded to the HYDbSmcy epitope following syngeneic male skin grafting (Fig. 1). The observation that an HYDbSmcy-specific response is incapable of mediating male skin graft rejection is consistent with the recent finding that immunization of WT mice with an HYDbSmcy epitope DNA vaccine does not influence the rejection time of WT skin male grafts (48). It is possible that either low avidity of the HYDbSmcy-specific TCR or inefficient presentation of the HYDbSmcy epitope on the target cells is responsible for the lack of skin graft rejection in the presence of HYDbSmcy-specific T cells (49). Alternatively, lack of male skin graft rejection by the HYDbSmcy-specific response could be accounted for by suboptimal generation of the HYDbSmcy epitope in male skin grafts, as a result of tissue-specific processing by proteasomes (50).

TCR repertoire in LMP7–/– mice

An alternative possibility to account for the lack of UTY246–254 response in LMP7–/– female mice injected with MHC-mismatched male cells and lack of male skin graft rejection is that LMP7–/– female mice have an altered TCR repertoire, resulting in lower affinity UTY246–254-specific T cells. The results of our experiments make this possibility very unlikely because HYDbUty-specific CD8 T cells were expanded equally in vaccinia-primed (Fig. 2B) and peptide-pulsed DC-boosted (Fig. 2D) WT and LMP7–/– female mice using a wide range of concentrations of the UTY246–254 peptide. Furthermore, the ranking order of vaccinia-specific responses in LMP7–/– mice is identical to the ranking order observed in WT mice (Fig. 2A). HYDbSmcy-specific responses (Fig. 1B), OVA257–264, and influenza NP366–374-specific responses were also shown to be comparable in WT and LMP7–/– mice (data not shown).

In conclusion, our results present in vivo evidence that immunoproteasomes play an important role in controlling cross-priming and support a phagosome-to-cytosol cross-presentation model. We also showed that lack of immunoproteasomes affects immune surveillance, as demonstrated by the lack of HY-specific skin graft rejection in LMP7–/– female mice, underscoring the importance of the observation that many tumors down-regulate basal expression of LMP7.


   Acknowledgments
 
We thank Marcus Groettrup for the donation of the LMP7-specific antiserum and Paul Klenerman for critical reading of this manuscript.


   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 funded by Cancer Research U.K. (C399-A2291), the U.S. Cancer Research Institute, and the U.K. Medical Research Council, and the European Commission DC VACC (503037). Back

2 Address correspondence and reprint requests to Dr. Vincenzo Cerundolo, Tumour Immunology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, OX3 9DS, U.K. E-mail address: vincenzo.cerundolo{at}imm.ox.ac.uk Back

3 Abbreviations used in this paper: DC, dendritic cell; ER, endoplasmic reticulum; MEF, murine embryonic fibroblast; IRES, internal ribosomal entry site; WT, wild type; FL, full length; BM, bone marrow; hu, human. Back

Received for publication November 17, 2005. Accepted for publication April 26, 2006.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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