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International Immunology Advance Access originally published online on March 28, 2006
International Immunology 2006 18(5):679-687; doi:10.1093/intimm/dxl005
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© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

The ubiquitin–proteasome system plays essential roles in presenting an 8-mer CTL epitope expressed in APC to corresponding CD8+ T cells

Xuefeng Duan1, Hajime Hisaeda1, Jianying Shen1, Liping Tu1, Takashi Imai1, Bin Chou1, Shigeo Murata2, Tomoki Chiba2, Keiji Tanaka2, Hans Jörg Fehling3, Takaomi Koga4, Katsuo Sueishi4 and Kunisuke Himeno1

1 Department of Microbiology and Immunology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
2 Department of Molecular Oncology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
3 Department of Immunology, University of Ulm, Ulm, Germany
4 Department of Pathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka

Correspondence to: K. Himeno; E-mail: himeno{at}parasite.med.kyushu-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
MUT1 is an H-2Kb-restricted 8-mer CTL epitope expressed in Lewis lung carcinoma (3LL) tumor cells derived from C57BL/6 (B6) mice. We constructed a chimeric gene encoding ubiquitin-fused MUT1 (pUB-MUT1). By using a gene gun, B6 mice were immunized with the gene prior to challenge with 3LL tumor cells. Tumor growth and lung metastasis were prominently suppressed in mice immunized with pUB-MUT1 but only slightly in those immunized with the MUT1 gene (pMUT) alone. CD8+ T cells were confirmed to be the final effector by in vitro experiments and in vivo removal of the cells with a corresponding antibody. Anti-tumor immunity was profoundly suppressed in mice deficient in an immuno-subunit of proteasome, LMP7. Furthermore, mice deficient in a proteasome regulator, PA28{alpha}/ß, failed to acquire protective immunity. Thus, application of the ubiquitin-fusion degradation pathway was useful even in immunization with genes encoding a single CTL epitope for induction of specific and active CD8+ T cells.

Keywords: antigen presentation, ubiquitin-fusion degradation pathway


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Since the 1980s, several tumor-associated antigens (TAAs) and CD8+ T cell epitopes in those antigens have been identified and cloned (1–4). However, it has been hard to induce active CD8+ CTL responses by conventional peptide/epitope-based vaccines (5). Host immunity, including CTL response to TAAs, is hyporesponsive or has developed immunological tolerance since most TAAs are poorly immunogenic self-antigens (6, 7). Although CTL responses are readily generated by live/live-attenuated vaccines (8–10), such vaccines have several problems that preclude their widespread use. Recently, vaccines using genes encoding TAAs have been developed by many investigators, and these have been expected to mimic the effect of live-attenuated vaccines in their ability to induce MHC class I-restricted CD8+ T-cell responses (11–23). In such DNA vaccination, co-delivery of TAA genes with those that encode cytokines such as granulocyte macrophage colony-stimulating factor and IL-12 is usually required. However, these cytokines inevitably have side effects.

Antigen presentation to CD8+ T cells is mediated by MHC class I molecules expressed on antigen-presenting cells (APCs)/dendritic cells. Primarily, CD8+ T cells recognize MHC class I-associated peptides derived from endogenous antigens, such as oncogene products or viral antigens, located in the cytosol. Prior to antigen presentation by MHC class I molecules, cytosolic antigens must be polyubiquitinated and processed to CTL epitopes by the proteasome. This pathway is termed the ubiquitin–proteasome system (UPS) (24–26). Early studies have indicated that this pathway is divided into two steps: substrate recognition, brought about by the ubiquitin conjugation system, and degradation, catalyzed by the 26S proteasome. During the recognition step, the ubiquitin-activating enzyme E1 activates ubiquitin in an ATP-requiring reaction to generate a high-energy thiol ester intermediate. One of several E2 proteins (ubiquitin-carrier proteins or ubiquitin-conjugating enzymes) transfers the activated ubiquitin moiety from E1, via an additional high-energy thiol ester intermediate to the substrate that is specifically bound to a member of the ubiquitin–protein ligase family, E3. E3s catalyze the last step in the conjugation process; covalent attachment of ubiquitin to the substrate. The ubiquitin molecule is generally transferred to an {varepsilon}-NH2 group of an internal lysine residue in the substrate to generate a covalent isopeptide bond between the C-terminal Gly76 of ubiquitin and the {varepsilon}-NH2 group. Ubiquitin itself is often a substrate for further ubiquitination, and proteins modified by such multiubiquitin chains are preferentially targeted for degradation by the proteasome (27, 28).

One fascinating strategy for enhancing CTL responses to low antigenic peptides may be to increase the efficiency of antigen processing by, for example, using a gene that encodes a ubiquitin-fused version of the target peptide. Previous work has shown that artificially fused proteins bearing a non-removable N-terminal ubiquitin moiety, replacing the C-terminal Gly76 of ubiquitin with Ala or Val (29, 30), are readily degraded by UPS. The proteolytic system involved is termed the ubiquitin-fusion degradation pathway (UFD) (27, 31). In our previous experiment, we constructed a chimeric DNA encoding a fusion protein linking murine ubiquitin to the N-terminus of a full-length murine melanocyte antigen, tyrosinase-related protein 2 (TRP-2), to introduce into the UFD. C57BL/6 (B6) mice immunized with the DNA acquired potent anti-tumor immunity against B16 melanoma mediated by specific CTL (32). Vaccination activated CD8+ T cells specific for autologous TRP-2, although conventional DNA vaccination with the gene encoding only TRP-2 failed to break the tolerance to self-TRP-2 and to develop anti-tumor immunity.

CTL epitopes in TAA peptides are 8–10 amino acids long and are presented to CTL by MHC class I molecules on the APC. There have been some reports on the use of edited epitope genes in DNA vaccines (33–37), but these vaccines have not always been successful in inducing active CTL and/or protective immunity. It is immunologically and clinically important to clarify whether vaccination with a gene encoding only a single CTL epitope in TAAs can induce potent CTL activity when the gene has been fused with a ubiquitin gene. In other words, it should be established whether DNA vaccines with genes for 8–10 amino acid peptides can effectively induce specific CTLs if they have been directed toward UFD, as was the case with a full-length peptide TRP-2, as described above.

The eight-amino acid-long TAA (4) MUT1 is a mutant with a substitution of a highly conserved Cys 54 to Gln 54 in the mouse gap junction protein connexin 37 (Con 37) expressed in vascular endothelial cells, and this mutant MUT1 gene has been confirmed to be a CTL epitope of TAAs expressed in Lewis lung carcinoma (3LL) derived from B6 mice (38). In the present study, we constructed a fusion gene encoding an 8-mer MUT1 and ubiquitin. B6 mice immunized with the fusion gene acquired potent anti-tumor immunity against 3LL tumor cells despite the fact that vaccination with the gene encoding only MUT1 was not effective. We demonstrated that UFD plays an essential role in inducing activated CD8+ T cells even when a single CTL epitope is used as an immunogen, as was the case in the system with a full-length TRP-2 and melanoma. In the present study, mice deficient in proteasome-associated gene, LMP7 or PA28{alpha}/ß, were used to confirm this conclusion.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Animals and tumors
Female B6 mice aged 7 weeks were purchased from Seac Yoshitomi (Fukuoka, Japan) and experiments were performed in accordance with the institutional guidelines of Kyushu University, Japan. Proteasome activator PA28 knockout (PA28{alpha}–/–/ß–/–) and immunoproteasome subunit LMP7 knockout (LMP7–/–) mice of B6 background were established by our group (31, 39). 3LL, a highly metastatic and poorly immunogenic tumor cell line, was a generous gift from Cell Resource Center for Biomedical Research Institute of Development, Aging and Cancer, Tohoku University (Sendai, Japan) and was maintained in RPMI 1640 media supplemented with 10% fetal bovine serum, 100 IU ml–1 penicillin, 100 µg ml–1 streptomycin, 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 50 mM NaHCO3 and 2 mM L-glutamine.

Plasmid construction
The plasmid pUB-MUT1 introducing the ubiquitin-fused MUT1 antigen was constructed by directly designing the MUT1 sequence into the anti-sense primer 5'-ATGGATCCCGGCTGGGCTGTGTTCTGCTCAAAGGCACCTCTCAGG-3' and using the sense primer 5'-TCGAATTCGTTAACAGGTCAAAATGCAG-3' to amplify the pUB vector (32) that we constructed. The gene was then inserted into EcoRI and BamHI sites of pcDNA3.1(–). The gene encoding the MUT1 epitope without ubiquitination was amplified by PCR using sense 5'-TCGGTACCATGGTTTTTGAGCAGAACACAGCCCAGCCGGACTGTGCCTTC-3' and anti-sense 5'-AGCTCCCGGGAGCTTTTTGCAAAA-3' primers, using pcDNA3.1(–) as the template. The gene was then inserted into the KpnI and XmaI sites of pcDNA3.1(–).

In vivo gene transfer and implantation of 3LL tumor cells
We used a Helios Gene Gun (BioRad, NY, USA) as described previously (32, 40–42). B6 mice were immunized three times with 6 µg plasmid at 1-week intervals using a control vector, pUB, pMUT1 or pUB-MUT1. Ten days after the final vaccination, 1 x 105 3LL tumor cells in 50 µl PBS were implanted into the left footpad of B6 mice, and tumor growth was evaluated by footpad swelling. The swelling size was calculated by subtracting the thickness of the right footpad from that of the left footpad after measuring with a caliper twice weekly. For the experiment on lung metastasis, B6 mice were intravenously challenged with 2 x 105 3LL tumor cells in 200 µl PBS. Twenty-eight days after challenge, mice were sacrificed and then all lobes of both lungs were dissected out and weighed.

Cytotoxicity assay
Mice were sacrificed at the time of tumor challenge and their spleen cells (4 x 107) were co-cultured with MUT1 peptide (4 µg ml–1) in six-well culture plates in complete RPMI 1640 medium. After 3 days culture, graded numbers of viable effector cells were placed into round-bottomed 96-well plates with 1 x 104 [3H]thymidine-labeled EL4 cells, which were pulsed with MUT1 peptide (4 µg ml–1) for 2 h. After 6 h incubation, the cells were harvested onto glass-fiber filters and radioactivity was counted using a ß scintillation counter, and specific killing was calculated as described previously (43).

Measurement of IFN-{gamma} production
Ten days after the third immunization, mice were sacrificed and spleen cells were isolated. Twenty million splenocytes were co-cultured with or without MUT1 epitope for 48 h at 20 µg ml–1. Then the supernatant was collected and IFN-{gamma} production was measured by ELISA.

In vivo depletion of T cell subsets
Anti-CD4 mAb (clone GK1.5) or anti-CD8 mAb (clone 2.43) was injected intra-peritoneally at 0.5 mg per mouse on days –3 and –1 of tumor challenge. Tumor cells were inoculated on day 0. Depletion of each T cell subset was confirmed by flow cytometry; >95% of the appropriate cell subset was depleted.

In vitro transfection and western blotting
One hundred thousand COS-7 cells in a 2.5-cm-diameter dish (Nunc, Roskilde, Denmark) were transfected with 2 µg pMUT1 or pU-MUT1 by using Lipofectamine (Invitrogen, Carlsbad, CA, USA) with or without the proteasome inhibitor epoxomicin (Sigma, St Louis, MO, USA). Twenty-four hours after transfection, cell lysates were prepared by adding 200 µl lysis buffer (50 mM Tris–HCl, 1% Nonidet P-40/1% SDS, 1 µM leupeptin/100 µM phenylmethylsulfonylflouride, 1 µM pepstatin A and 100 µM EDTA), and 15 µg protein was used for western blotting with anti-ubiquitin (Medical & Biological Laboratories, Japan) as the first antibody. Peroxidase-conjugated anti-mouse IgG (H + L) (Zymed Laboratories, San Francisco, CA, USA) was used as the second antibody. Binding antibody was detected by using enhanced chemiluminescence reagents (Amersham Life Science, Buckinghamshire, UK).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Anti-tumor immunity against 3LL tumor cells in mice immunized with pUB-MUT1 expression vectors
As a candidate gene for a DNA vaccine against murine 3LL tumor, we prepared an expression vector termed pMUT1 encoding the MUT1 epitope, which is a mutant with a substitution of a highly conserved Cys 54 to Gln 54 in mouse gap junction protein Con 37 (aa 52–59) in mouse 3LL tumor cells. We also constructed pUB-MUT1 encoding modified ubiquitin (G76A) at the N-terminus of the MUT1 epitope so as not to be cleaved by ubiquitin C-terminal hydrolases (Fig. 1), and we examined the effect of DNA vaccination on the 3LL in vivo and in vitro. In our study, B6 mice immunized with pMUT1 showed the same level of tumor growth as that in mice immunized with the control vector or pUB (Fig. 2A). In contrast, tumor growth in the mice immunized with pUB-MUT1 was much slower compared with that in the other three groups. More than 15% of pUB-MUT1-immunized mice remained tumor free until the end of the experiment (data not shown). Lung metastasis was almost completely suppressed in the pUB-MUT1-immunized group as evaluated by lung weight (Fig. 2B). Histological analysis also demonstrated that there was no sign of metastasis in these mice. In sharp contrast, multiple metastatic lesions were observed in mice in the other groups (Fig. 2C). Immunization of B6 mice with pUB-MUT1 did not affect growth of B16F1 melanoma cells, which are derived from B6 mice but do not express MUT1 antigen (Fig. 2D), indicating that anti-tumor immunity induced by pUB-MUT1 immunization is highly antigen specific. Thus, immunization with pUB-MUT1, but not pMUT1, induces strong anti-tumor immunity in vivo against 3LL tumor cells.


Figure 1
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  		Fig. 1. (A) Amino acid sequences of Con 37 (aa 52–59) and MUT1. The eight-amino acid-long TAA peptide MUT1 is derived from mutation of a highly conserved Cys 54 to Gln 54 in the mouse gap junction protein Con 37 from the spontaneous C57BL/6 3LL. (B) Sketch map of normal mono-ubiquitin, pUB-MUT1 and pMUT1.

 

Figure 2
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  		Fig. 2. Induction of antigen-specific anti-tumor immunity by vaccination with pUB-MUT1. B6 mice were immunized with the indicated plasmid, and 1 x 105 3LL tumor cells were implanted into the left footpad of each mouse. Tumor growth was evaluated by footpad swelling (A). Each value is the average ± SD from six mice in each group. Asterisks indicate statistical significance at P < 0.02 by the t-test. Vaccinated mice were intravenously challenged with 2 x 105 3LL tumor cells, and lung metastasis was analyzed by weighing lungs (B) and by histological examination (C). Asterisks indicate statistical significance at P < 0.05 by the t-test. Arrowheads indicate metastatic tumor lesions. (D) B6 mice that had been vaccinated with the indicated plasmid were challenged with 1 x 105 B16F1 melanoma cells, which do not express MUT1 antigen. Tumor growth was evaluated by footpad swelling. Each experiment was repeated at least three times and gave similar results.

 
Activation of CD8+ T cells in mice immunized with pUB-MUT1
To determine the effector cells in the observed protective immunity, mice immunized with pUB-MUT1 were treated with anti-CD4 or anti-CD8 antibody to deplete the corresponding T cell subset, and then 3LL tumor cells were implanted. As shown in Fig. 3(A), anti-CD8 treatment completely abolished the anti-tumor immunity induced by pUB-MUT1 immunization. In contrast, treatment with control IgG or anti-CD4 antibody did not alternate the anti-tumor immunity, indicating that the immunity induced by pUB-MUT1 immunization is mediated by MUT1-specific CD8+ T cells.


Figure 3
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  		Fig. 3. Critical roles of CD8+ T cells in anti-tumor immunity induced by pUB-MUT1. (A) Effect of depletion of CD8+ T cells on anti-tumor immunity. Mice that had been immunized with pUB-MUT1 were treated with anti-CD4 or anti-CD8 antibody on days –3 and –1 of tumor challenge. Tumor growth was evaluated by footpad swelling. Each value is the average ± SD from six mice in each group. Complete depletion of the corresponding T cells was confirmed by flow cytometry (data not shown). Splenocytes isolated from mice 10 days after the final vaccination were analyzed for lytic activity (B) and production of IFN-{gamma} (C). (B) Radio-labeled EL4 cells (1 x 104) pulsed with MUT1 were cultured with spleen cells at different effector-to-target ratios. CTL activity was not observed in the absence of MUT1 (data not shown). (C) Amounts of IFN-{gamma} in supernatants from splenocytes cultured with (filled bars) or without (open bars) MUT1 epitope were determined by the ELISA method. Each value is the average ± SD from triplicate culture. Asterisks indicate statistically significant decreases compared with the control group.

 
We next examined the function of CD8+ T cells from mice immunized with MUT1-expressing plasmids by assessing CTL activity and IFN-{gamma} production. We isolated splenocytes from various groups of B6 mice that had been immunized with pUB-MUT1, pMUT1, pUB or the control vector. Splenocytes from mice immunized with pUB-MUT1 vaccine showed prominent CTL activity against syngeneic EL4 target cells pulsed with MUT1 epitopes (Fig. 3B), and generated a remarkable amount of IFN-{gamma} when cultured with 20 µg ml–1 MUT1 epitope compared with the other groups of mice (Fig. 3C).

Polyubiquitination and proteasomal processing of ubiquitin-fused MUT1 epitope
We previously reported that a full-length melanoma/melanocyte peptide, TRP-2, must be processed by UPS prior to the activation of TRP-2-specific CD8+ T cells. In the present study, we employed the MUT1 gene, which encodes only a single 8-mer epitope for CD8+ T cells, as a vaccine candidate. Immunization with pMUT1 scarcely induced activated CD8+ T cells. On the other hand, immunization with pUB-MUT1 induced potent anti-tumor immunity mediated by CD8+ T cells specific for the epitope, suggesting that processing via UPS is a critical step, even for a CD8+ T cell-specific 8-mer epitope, prior to presentation on specific cells. To elucidate the difference between pMUT1 and pUB-MUT1 in the polyubiquitination and the processing via proteasomes, COS-7 cells transfected with pMUT1 or pUB-MUT1 were cultured in the presence or absence of proteasome inhibitor epoxomicin. Bands containing ubiquitin were not detected in cells transfected with pMUT1. One possibility is that polyubiquitination did not occur in these cells because MUT1 does not include lysine residues, acceptors of ubiquitination (28). COS-7 cells transfected with pUB-MUT1 contained not only a ubiquitin–MUT1 band but also additional upper bands consisting of polyubiquitinated products (Fig. 4A). Epoxomicin increased protein expression 1.5-fold (Fig. 4C). These results suggest that fusion of ubiquitin to the 8-mer MUT1 epitope made it susceptible to polyubiquitination, and processing by UPS.


Figure 4
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  		Fig. 4. Fusion of ubiquitin made the MUT1 epitope susceptible to polyubiquitination and processing by UPS. Plasmid-transfected COS-7 cells were cultured in the presence or absence of a proteasome inhibitor, epoxomicin. Twenty-four hours after transfection, cells were harvested and analyzed by western blotting using an anti-ubiquitin antibody (A, upper panel) or anti-HSP90 antibody as an internal control (A, lower panel). (B) Expression of MUT1 and UB-MUT1 were densitometrically quantified. Results represent the relative ratio of MUT1 level in COS-7 cells transfected with pMUT1 in the absence of epoxomicin after normalization with HSP90 expression.

 
Defective induction of anti-tumor immunity by immunization with pUB-MUT1 in LMP7–/– and PA28{alpha}–/–/ß–/–
In order to clarify whether ubiquitin-fused CD8+ T cell epitope requires to go through the proteasome to efficiently activate precursor CD8+ T cells, we used immunoproteasome subunit LMP7-deficient and proteasome regulator PA28{alpha}/ß complex-deficient mice. After challenging with 3LL tumor cells, the same tendency was seen in LMP7 knockout mice, although it was not always in a completely dependent manner (Fig. 5).


Figure 5
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  		Fig. 5. Protective effect induced by the pUB-MUT1 was just partly cancelled in the LMP7–/– mice. B6 mice and LMP7–/– mice that had been vaccinated with pUB-MUT1 were challenged with 1 x 105 3LL tumor cells implanted into the left footpad. Asterisks indicate statistical significance at P < 0.05. This experiment was repeated at least three times and gave similar results.

 
Anti-tumor immunity was almost completely abolished in PA28-deficient mice compared with wild-type B6 mice (Fig. 6A). Furthermore, PA28-deficient mice completely failed to acquire anti-tumor immunity, as evaluated by either tumor growth inhibition or prevention of lung metastasis after immunization with pUB-MUT1 (Fig. 6B).


Figure 6
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  		Fig. 6. Impaired pUB-MUT1 vaccination induced anti-tumor immunity in PA28{alpha}–/–/ß–/– mice. (A) B6 mice and PA28{alpha}–/–/ß–/– mice that had been vaccinated with pUB-MUT1 were challenged with 1 x 105 3LL tumor cells implanted into the left footpad. (B) Mice immunized with pUB-MUT1 were inoculated with 2 x 105 3LL tumor cells intravenously. Twenty-eight days after inoculation, mice were sacrificed and the lungs were weighed. Asterisks indicate statistical significance at P < 0.05 by the t-test. Each experiment was repeated at least three times and gave similar results.

 
These results suggest, again, that even the 8-mer MUT1 epitope used in our study must be further handled by the UPS prior to presentation on MHC class I molecules and activating specific CD8+ T cells, as in the case of the full-length TRP-2 (32).


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
We previously developed a DNA vaccine with an expressing plasmid encoding a fusion protein between full-length TRP-2, a melanoma antigen, and ubiquitin. Immunization with the DNA vaccine resulted in remarkable activation of CD8+ T cells specific for TRP-2 and induction of robust anti-melanoma immunity, presumably by preferential leading of ubiquitin-fused TRP-2 to the proteasomes and enhancement of its degradation (32). Thus, ubiquitination of tumor antigens could be an ingenious strategy for inducing anti-tumor immunity.

In the present study, we exploited a DNA vaccine for a lung cancer, 3LL, by constructing a gene encoding MUT1, a CTL epitope with an 8-mer peptide but not a full-length protein. The pMUT1 encoding only MUT1 could not induce immunity against 3LL tumor cells, as evaluated by tumor growth, survival rate, and lung metastasis. Interestingly, vaccination with pUB-MUT1 encoding MUT1 fused with ubiquitin at the N-terminus efficiently induced MUT1-specific CD8+ T cells (Fig. 3) and conferred mice with protective anti-tumor immunity (Fig. 2). Thus, the expression of MUT1 alone within cells appears not to be sufficient to induce CD8+ CTLs specific for the epitope, whereas CTLs are potently activated when the CTL epitope has been ubiquitinated, and then processed via UPS and presented to MHC class I molecules. The difference in anti-tumor immunity between pMUT1 and pUB-MUT1 may be that the gene product in the latter is efficiently manipulated by UPS, and the process should be a crucial step of induction of CTL even when a gene encoding single 8-mer epitope is used as an immunogen. It should be noteworthy that CD8+ T cells are also activated without support from CD4+ helper T cells in our study, as some authors reported (32, 44–46).

Recently, a new ubiquitination factor, E4, has been found (47). E4 protein does not participate in the ubiquitin–enzyme thioester cascade. Moreover, in striking contrast to E3s, E4 does not interact with the substrate directly, but apparently with the ubiquitin moieties of ubiquitin–substrate conjugates. It is a crucial switch, which triggers degradation by adding just one or two ubiquitin moieties to the conjugate and may be important for regulating degradation of proteins already primed for degradation by oligoubiquitination. In this escort pathway to the proteasome, UFD, a protein substrate is modified by one or two ubiquitin moieties using E1, E2 and E3 enzymes. Based on the recognition of the oligoubiquitinated substrate by CDC48UFD1/NPL4, E4 extends the ubiquitin chain by a few extra ubiquitin moieties. Subsequently, the ubiquitin–protein conjugate is delivered to the proteasome for degradation by the binding of RAD23 or DSK2 (48). In our system, ubiquitin-fused MUT1 antigen may be easily recognized by CDC48UFD1/NPL4 and therefore efficiently delivered to the proteasome.

The immunomodulatory cytokine IFN-{gamma}, which is produced by activated Th1, NK and CD8+ T cells, enhances antigen presentation by activating proteasome subunits and regulators in addition to up-regulating the generation of either the MHC or TAP gene. IFN-{gamma} alters proteasome activity by incorporation of three IFN-{gamma}-inducible catalytic subunits, LMP2, LMP7 and MECL-1, to replace the constitutive catalytic subunits (Y/{delta}, X/MB1 and Z, respectively) in the 20S core particle during proteasome biogenesis (25, 41, 49). These IFN-{gamma}-induced immunoproteasomes are speculated to be more favorable than constitutive proteasomes for antigen presentation because the subunits induced by IFN-{gamma} stimulate cleavage after hydrophobic, basic and branched chain residues instead of acidic ones (50–54). There have been several reports on the importance of immunoproteasomes for the production of various MHC class I epitopes, although certain antigenic peptides are known to be generated by standard (or constitutive) proteasomes (26, 53).

It is reported that MECL-1 requires LMP2 for efficient incorporation into preproteasomes and LMP7 is required for efficient maturation of preproteasomes containing LMP2 and MECL-1 (54). On this basis, we employed LMP7-deficient mice to confirm the contribution of immunoproteasomes to protective immunity. In our study, anti-tumor immunity was profoundly attenuated in LMP7-deficient mice although not completely (Fig. 5). Thus, LMP7 appears to play an important role in the presentation of the MUT1 epitope on MHC class I molecules, probably synergistically with other immunoproteasome subunits such as LMP2 and/or MECL-1. Notably, we also found that a large proportion of ubiquitin-fused MUT1 underwent UFD after the serial attachment of ubiquitin (Fig. 4), despite the fact that MUT1 is a single 8-mer epitope for CD8+ T cells.

Polyubiquitinated substrates are deubiquitinated at the entrances before passing through proteasomes. A constitutive 19S proteasome regulator (PA700) is known to be responsible for detachment of polyubiquitin at the entrance of the proteasome. This regulator may play an essential role in opening the gate, which is thought to increase the rate of peptide output (55), and prevent excessive degradation of epitopes by quick retrieval of properly degraded peptides from catalytic ß-rings. As in the case with immunoproteasomes, IFN-{gamma} plays a crucial role in the expression of two proteasome regulators PA28{alpha} and ß, which form the heptameric proteasome activator complex PA28 (56). This heteromultimer has the potential to bind to the {alpha} rings of the 20S core particle, thereby enhancing proteolytic activity, although the mechanism of PA28-mediated enhancement of intrinsic core proteasome activity has not been fully elucidated (57). In vitro studies have shown that purified PA28{alpha}/ß can enhance coordinated dual cleavage by the 20S proteasome, leading to augmented liberation of epitopes (58). Furthermore, expression of PA28{alpha} in mouse fibroblasts is known to increase the sensitivity to lysis by virus-specific CTLs (59). We found previously that processing of an epitope derived from the tumor antigen TRP-2 of murine B16 melanoma is entirely dependent on PA28{alpha}/ß in vitro (33) and in vivo (34, 60). In this study, PA28{alpha}–/–/ß–/– mice also failed to acquire anti-tumor immunity after immunization with pUB-MUT1 (Fig. 6).

Since the sequence of the oligonucleotides used to generate the fusion UB-MUT1 does not contain the stop codon, the construct used in this study is a little longer than the minimal sequence of eight amino acids. Accordingly, even the 8-mer CTL epitope may need to be manipulated by UPS, including PA28{alpha}/ß, prior to presentation to MHC class I molecules and activating the corresponding CD8+ T cells.

Another fascinating possibility regarding the role of UPS in the present system is that proteasomes have functions other than proteolytic activity. Epitopes passing through proteasomes readily find their way to TAP and are consequently transported to the endoplasmic reticulum, where they bind to MHC class I molecules. The precise roles of UPS in our system are still under investigation. It should be noted that the mutant mice used in this study, either LMP7–/– or PA28{alpha}–/–/ß–/–, showed no alteration in the ratio of CD4+ and CD8+ T cells to whole spleen or lymph node cells (data not shown).

This is believed to be the first report suggesting that DNA vaccination with a gene encoding a single CD8+ T-cell epitope in tumor antigens potently induces anti-tumor immunity when the gene has been fused with the ubiquitin gene, depending on the UPS.


   Acknowledgements
 
We thank M. Sano for technical support. This work was supported by grants-in-aid from the Ministry of Education, Science, Sport, and Culture of Japan (15019075, 15025255, 15390136 and 15659265).


   Abbreviations
 
APC, antigen-presenting cell
Con 37, connexin 37
3LL, Lewis lung carcinoma
TAA, tumor-associated antigen
TRP-2, tyrosinase-related protein 2
UFD, ubiquitin-fusion degradation pathway
UPS, ubiquitin–proteasome system

   Notes
 
Transmitting editor: T. Sasazuki

revised 21 October 2005, accepted 31 January 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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