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/ß, Enhances the Presentation of an Immunodominant Lymphocytic Choriomeningitis Virus T Cell Epitope1
*
Research Department, Cantonal Hospital St. Gall, St. Gallen, Switzerland;
Institute of Experimental Immunology, Department of Pathology, University Hospital Zürich, Zürich, Switzerland;
Max-Delbrück Center for Molecular Medicine, Berlin-Buch, Germany; and
§
Institute for Biochemistry, Medical Faculty (Charité), Humboldt University, Berlin, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, three constitutively expressed
subunits of the 20S proteasome are replaced by the inducible subunits
LMP2 (low-molecular mass polypeptide 2), LMP7, and MECL-1
(multicatalytic endopeptidase complex-like-1) to form so-called
immunoproteasomes. We show in this study that overexpression of these
three subunits in triple transfectants led to a marked enhancement in
the H-2Ld-restricted presentation of the immunodominant
nonameric epitope NP118, which is derived from the nucleoprotein (NP)
of lymphocytic choriomeningitis virus. Overexpression of the 
and
ß subunits of the IFN--inducible proteasome regulator PA28, in
contrast, did not have a comparable effect. In vitro, immunoproteasomes
as compared with constitutive proteasomes generated higher amounts of
11- and 12-mer fragments containing the NP118 epitope. These are likely
to be cytosolic precursors of NP118, as a proline anchor residue in the
second position of NP118 may interfere with TAP-mediated transport of
the nonameric epitope itself. In conclusion, we provide evidence that
up-regulation of the three inducible subunits, LMP2, LMP7, and MECL-1,
can result in a marked improvement of Ag presentation and that,
depending on the epitope, PA28 and immunoproteasomes may differentially
affect Ag processing. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Experiments employing inhibitors of the proteasome have meanwhile shown
that it is the proteasome system (6) that generates most
of the class I ligands and their precursors (7, 8, 9, 10). This
notion has been further corroborated by the discovery of two
IFN--inducible proteasome subunits named LMP2 and LMP7, which are
encoded in the MHC class II locus. Upon their transcriptional
induction, LMP2 and LMP7 replace two constitutive ß-type subunits
designated 
and MB-1, respectively, in newly assembled 20S
proteasomes. The exchange of these proteasome subunits was shown to
alter the cleavage specificity of the 20S proteasome in vitro.
Replacement of the subunit by LMP2, for instance, dramatically
reduced the proteasomal cleavages C terminal of glutamic acid, while
the exchange of LMP7 for MB-1 was found to increase the cleavage C
terminal of hydrophobic residues in one (11), but not in
other studies (12, 13, 14). The in vivo effects of LMP2 and
LMP7 were analyzed in gene-targeted mice and mutant cell lines.
LMP7-deficient mice displayed a slight reduction of MHC class I
molecules on lymphocytes and macrophages and a reduced ability to
present the HY male Ag (15). The reduction in class I
expression could be rescued by administration of exogenous peptides,
indicating that the defect was due to a lack of appropriate peptide
ligands. LMP2-deficient mice, in contrast, were not reduced in class I
cell surface expression, but had a slightly reduced level of
CD8+ T lymphocytes and generated fewer CTL
precursors to an influenza NP epitope, while the response to Sendai
virus was normal (16). LMP2 and LMP7 double-deficient
lymphoblastoid cell lines showed no significant reduction in class I
cell surface expression (17, 18) and no apparent defect in
the class I-restricted presentation of several virus-derived T cell
epitopes (19, 20). Only in a mouse cell line selectively
deficient for LMP2 it could be shown that this subunit was required for
presentation of two epitopes of influenza hemagglutinin
(21). Taken together, the impact of LMP2 and LMP7 on Ag
processing and presentation was evident for certain epitopes, but not
very prominent for the bulk production of MHC class I ligands.
Recently, a third pair of subunit exchanges in the 20S proteasome has
been discovered. The subunit MECL-1, which, in contrast to LMP2 and
LMP7, is not encoded in the MHC, is inducible by IFN- and replaces a
subunit that was designated Z in the human and MC14 or LMP9 in the
mouse (22, 23, 24). The function of MECL-1 in Ag presentation
has not been investigated to date, and cell lines or mice that are
deficient for MECL-1 are not yet available. Interestingly, the
incorporation of MECL-1 into the 20S proteasome is strictly dependent
on the subunit LMP2 (25) and is accelerated in the
presence of LMP7 (26), suggesting that these subunits are
preferentially coincorporated, leading to the formation of so-called
immunoproteasomes. As the constitutively expressed subunits , MB-1,
and MC14 bear the three putative active centers of the proteasome, it
appears that all three subunits have an IFN--inducible homologue and
are amenable to modulation during the immune response.
The 20S proteasome must be viewed as a proteolytic core complex that
needs to associate with regulatory complexes that control the cleavage
activity of the proteasome and the access of substrates to the lumen of
the 20S proteasome. The PA700 complex (or 19S regulator) consists of at
least 17 different subunits, and is thought to be required for the
binding and unfolding of ubiquitinated proteins (6). The
PA28 complex (or 11S regulator), in contrast, is constituted from two
different subunits, PA28 and PA28ß, which both are inducible by
IFN- (27). They form rings of seven subunits with about
equal stoichiometric amounts of and ß, which bind to the 20S
proteasome (28, 29, 30). Both the PA28 and ß subunits
have been shown to enhance peptide hydrolysis by the 20S proteasome,
but the respective functional contributions of and ß are still
elusive (31, 32, 33). With respect to PA28 function, we
proposed that it is involved in Ag processing, as overexpression of the
PA28 subunit markedly enhanced the Ag presentation of two different
virus-derived epitopes (34). Moreover, PA28 accelerated
the processing of nonameric MHC ligands from polypeptide precursors by
the 20S proteasome in vitro (35). Mechanistically, it was
proposed that PA28 induces the proteasome to perform dual cleavages
that immediately would liberate fragments of the appropriate size from
polypeptides precursors rather than performing consecutive cleavages
(35, 36). Although these experimental findings obtained in
several models systems support the proposed role for PA28 in Ag
processing, it remains to be shown whether all T cell epitopes
similarly benefit from PA28 expression.
In this work, we aimed at dissecting the effects of IFN- on Ag
processing by the proteasome system by the means of overexpressing
either mouse PA28 and ß or the subunits LMP2, LMP7, and MECL-1 in
double and triple transfectants of mouse fibroblasts, respectively. As
a model system for Ag presentation, we chose the infection of
BALB/c-derived fibroblast lines with the lymphocytic choriomeningitis
virus (LCMV). The cytotoxic immune response to LCMV is essential for
elimination of the virus from infected mice. In BALB/c mice, this
response is strongly dominated by CTLs specific for the
H-2Ld-restricted nonameric epitope NP118
(37), which consists of residues 118–126 of the LCMV-NP
and is generated in a proteasome-dependent manner (38). We
report in this study that the presentation of NP118 is markedly
enhanced through LMP2/LMP7/MECL-1 coexpression, and that the putative
11- and 12-mer precursors of this epitope are produced in vitro by
immunoproteasomes much more efficiently as compared with constitutive
proteasomes. Overexpression of PA28/ß, in contrast, had little
effect on Ag presentation in this system. Taken together, our data
demonstrate that the concerted expression of the IFN--inducible
proteasome subunits LMP2, LMP7, and MECL-1 may greatly enhance the
efficiency of the intracellular T cell epitope production.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c mice (H-2d) were purchased from the Institut für Labortierkunde, Tierspital Zürich (Zürich, Switzerland) and kept in a specific pathogen-free environment. LCMV-WE strain was originally obtained from F. Lehmann-Grube (Hamburg, Germany) (39). LCMV was propagated in the L929 fibroblast line, and viral stocks were kept at -70°C.
Synthetic peptides
The synthetic peptides were purchased from Echaz microcollections (Tübingen, Germany). The 25-mer encompassing LCMV-WE NP residues 108–132 KLKAKIMRTERPQASGVYMGNLTAQ contained the immunodominant 9-mer epitope (NP118–126), which is presented by H-2Ld.
Antibodies
For Western blot analysis, rabbit polyclonal Abs recognizing
mouse LMP2 (26), LMP7 (Affinity, Mamhead, U.K.), and MECL1
(25) as well as the PA28 and PA28ß subunits
(40) were used. The secondary Ab was a goat
anti-rabbit Ig HRP conjugate (Dako, Zug, Switzerland). For
immunoprecipitation of the proteasome, a rabbit anti-mouse
proteasome antiserum was used. The LCMV-NP was immunoprecipitated using
the rat mAb VL4 (41). For surface staining of the
H-2Ld, H-2Kd, and
H-2Dd molecules, the mAbs 28-14-81, 15-5-5S, and
19/191 were used and the LCMV-glycoprotein staining was performed with
the mouse mAb KL 25 (42), followed by a FITC-conjugated
sheep anti-mouse Ig (Silenus, Victoria, Australia). The
intracellular staining of the LCMV-NP was performed using mAb VL4,
followed by a PE-conjugated rabbit anti-rat IgG (Serotec, Oxford,
U.K.).
Cell lines
The B8 clone was obtained from Dr. U. Koszinowski (Munich,
Germany), and had been generated from the BALB/c-derived fibroblast
cell line C4 by cotrancfection with the IE1/pp89 gene of the mouse CMV
and a neomycin-resistance gene (13). B8 cells were grown
in complete IMDM (10% FCS, 2 mM L-glutamine, 100 U/ml
penicillin/streptomycin) supplemented with 250 µg/ml G418. The
stimulation of B8 cells with IFN- was always done for 2 days with 20
U/ml mouse rIFN- (Life Technologies, Basel, Switzerland). Triple
transfection of B8 cells with the BALB/c-derived LMP2, LMP7, and MECL1
cDNAs yielded the clones B27 M2 (25), B27 M6, and B27 M9.
Transfection of the MECL-1 transfectant BME13 (25) with a
LMP2 expression vector yielded the cell line B2 M1. Double transfection
of B8 cells with the cDNA of mouse PA28 and ß (40)
generated the clones BPß2 and BPß13. These cells were
cultured in selection medium consisting of complete IMDM supplemented
with 2.5 µg/ml puromycin (Sigma, Buchs, Switzerland), 400 µg/ml
hygromycin B (Roche, Basel, Switzerland), and 250 µg/ml G418 (Life
Technologies). T2-Ld is the TAP-deficient human
lymphoblastoid line T2 cell line (43) transfected with
H-2Ld.
CTL lines and T cell hybridoma
CTL lines recognizing the LCMV-NP epitope NP118 were generated
from spleen cell suspensions of LCMV memory mice that had been infected
i.v. with 200 PFU LCMV-WE at least 1 mo before. Splenocytes were plated
in complete IMDM supplemented with 5 x
10-5 M 2-ME and 10% rat
Con A supernatant. Cells were restimulated every 7–10 days with NP118
peptide-loaded and irradiated (80 Gy) T2-Ld cells
at a ratio of CTL to APC of 5:1. Loading with synthetic peptides was
for 1 h at room temperature with
10-7 M of peptide.
Generally, the CTL line was found to be exclusively specific for
NP118/H-2Ld after three rounds of restimulation.
For the ex vivo CTL assay, mice were infected i.v. with 200 PFU
LCMV-WE, and after 8 days, the mice were sacrificed and a spleen cell
suspension was prepared for immediate use as effector cells in a
standard chromium release assay. The NP-118-specific hybridoma clone
HNP-118 resulted from a fusion of an LCMV NP118-specific CTL line with
the TCR-deficient lymphoma BWZ36.1 (44) expressing CD8
and a lacZ reporter construct under the control of the IL-2
promotor/enhancer (38). These cells were grown in complete
IMDM/hypoxanthine, aminopterin, thymidine (HAT; Sigma) supplemented
with 0.5 mg/ml hygromycin B.
Generation of rVV-NP118
We cloned the synthetic DNA sequence encoding the nonameric NP118–126 epitope plus one additional methionine at the N terminus (MRPQASGVYM) into the vaccinia vector pSC11.3OR2 (45). The plasmid DNA was used to cotransfect thymidine kinase-negative 143B fibroblasts (American Type Culture Collection (ATCC), Manassas, VA) after their infection with wild-type vaccinia virus. Successful homologous recombination of the vaccinia vector with the vaccinia virus was assessed by selection for 5-bromo-2'-deoxyuridine-resistant, ß-galactosidase-expressing virus plaques. Plaques were selected and replaqued three times until one isolated plaque was chosen for amplification on BS-C-40 (ATCC) cells.
Purification of 20S proteasome
The lysis, purification, and quantitation of the 20S proteasome from B8 cells were performed exactly as described (13).
Metabolic labeling and immunoprecipitation
Cells grown to confluence were starved in cysteine/methionine-free RPMI 1640, 10% dialyzed FCS for 1 h at 37°C and labeled with 0.2 mCi/ml Tran35S label (ICN, Eschwege, Germany) for the indicated time. Labeling medium was removed and cells were washed with PBS, harvested, and lysed for 30 min at 4°C in 50 mM Tris/HCl, pH 8, 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 0.75 µM aprotinin, 10 µM leupeptin, 2.8 µM pepstatin, and 0.85 mM PMSF. The postnuclear lysates were counted for 35S incorporation, and equal aliquots were used for immunoprecipitation. The lysate was precleared for 1 h at 4°C with preimmune serum coupled to protein G-Sepharose CL-4B (Pharmacia, Uppsala, Sweden), followed by immunoprecipitation with the indicated Ab bound to protein G-Sepharose for 3 h at 4°C. The precipitates were washed with PBS, pH 7.5, 0.1% Triton X-100, and separated on IEF and/or SDS-PAGE (performed as described in Ref. 23). The proteins were visualized by autoradiography on x-ray films or by using a BAS 1500 radioimager (Fuji, Tokyo, Japan).
Western blot analysis
Cells were lysed in 50 mM Tris/HCl, pH 7.5, 5 mM MgCl2, 1 mM EDTA, 0.5% Triton X-100, 0.75 µM aprotinin, 10 µM leupeptin, 2.8 µM pepstatin, and 0.85 mM PMSF for 30 min at 4°C. The postnuclear supernatant was quantified by OD, and aliquots of 130 µg protein were applied to a 12% SDS-PAGE. The proteins were blotted onto nitrocellulose membrane (Schleicher und Schuell, Dassel, Germany), blocked with PBS/10% horse serum/5% (w/v) low fat dry milk/0.4% Tween-20, and agitated overnight at 4°C with the indicated Ab in PBS/2% low fat dry milk/0.1% Tween-20. The blots were washed and incubated for 1 h with the HRP-conjugated secondary Ab. After extensive washing with PBS/0.2% Tween-20, proteins were visualized on x-ray films by enhanced chemiluminescence.
Proteasomal fragmentation of polypeptides and analysis of peptide products
A total of 80 µg of a synthetic 25-mer peptide derived from
the sequence of the LCMV-NP (NP108–132) was incubated with 4 µg
purified 20S proteasome in a total volume of 1200 µl of digestion
buffer (30 mM Tris/HCl, pH 7.5, 10 mM KCl, 2 mM DTT) at 37°C. At
indicated time points, aliquots of 300 µl were removed and frozen to
stop the reaction. These cleavage products were separated on a µRPC
C2/C18 SC 2.1/10 reverse-phase column using a SMART System (Pharmacia).
Eluent A, 0.1% trifluoroacetic acid (TFA); eluent B, 70% acetonitrile
+ 0.1% TFA. Gradient 10–30% B in 55 min, flow rate 100 µl/min.
Peak fractions were collected, vacuum dried, and resolved in 60%
acetonitrile, 0.1% TFA. For the identification of peptides, the
samples were coprecipitated with a matrix of
-cyano-4-hydroxycinnamic acid in acetone and analyzed by MALDI-MS
(VG-TofSpec; Fison Instruments, Manchester, U.K.). For microsequence
analyses of the HPLC-separated peptide samples, a Procise protein
sequencer system (Applied Biosystems, Foster City, CA) was
used.
Cytolytic assays
Target cells (B8 fibroblasts and transfecants thereof) were infected either for 24 h with LCMV-WE at a multiplicity of infection of 0.01 or for 1 h with rVV-NP118 with a multiplicity of infection of 3. One million cells were labeled in 200 µl with 100 µCi Na251CrO4 for 1 h at 37°C before washing and incubation of 104 cells/well in a 96-well plate with effector CTL at indicated E:T ratios at 37°C. Chromium release was measured after 4 h in 70 µl of culture supernatant. For determining the spontaneous release, the supernatant of target cells without adding effector CTL was measured. The total release was determined by adding 0.1% Nonidet P-40 (final concentration) to labeled targets. Specific lysis was calculated as (experimental release - spontaneous release)/(total release - spontaneous release) x 100%. All samples were measured in triplicates.
lacZ assay
The LCMV-NP118-specific hybridoma clone HNP-118 was cocultured overnight with 5 x 104 LCMV-infected target cells in 96-well plates at an E:T ratio of 10:1, 5:1, and 1:1. The cultures were washed once with PBS and lysed by addition of 100 µl of Z buffer (0.15 mM chlorophenol red ß-galactoside (CPRG; Roche), 100 mM 2-ME, 9 mM MgCl2, 0.125% Nonidet P-40 in PBS). After 4 h of incubation at 37°C, the absorbance at 570 nm (reference wavelength at 620 nm) was read using a SpectraFluor Plus plate reader (Tecan, Gröding, Austria).
Viability assay
A total of 20 µl Cell Titer 96 AQueous One Solution Reagent was added to 5 x 104 cells in 100 µl medium in 96-well plates. The plates were incubated for 1 h at 37°C before the absorbance was recorded at 450 nm using the SpectraFluor Plus plate reader.
Flow cytometry
For surface staining, aliquots of 4 x 105 cells in PBS + 2% FCS were incubated for 15 min in a round-bottom 96-well plate on ice with the indicated Ab, washed three times, and subsequently stained by FITC-conjugated secondary Ab. The LCMV-NP was detected by intracellular staining. In brief, aliquots of LCMV-infected cells were permeabilized with 0.1% saponin (Sigma) in PBS + 2% FCS for 10 min, and the staining was performed in this buffer until completion. The NP was stained for 30 min with the VL4 Ab, washed twice, and subsequently stained by PE-conjugated Ab for an additional 30 min. After two washing steps, the staining was analyzed on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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was estimated to induce up
to 400 genes, many of which can be expected to influence the MHC class
I-restricted Ag presentation pathway (46). In this work,
we wanted to separately investigate how an incorporation of the
subunits LMP2, LMP7, and MECL-1 into the proteasome as it occurs
subsequent to IFN- stimulation would affect Ag processing and
compare it with the effect of PA28 up-regulation. To this end, we
created transfectants overexpressing either the and ß subunits of
PA28 or the three IFN--inducible subunits of the
immunoproteasome.
Characterization of transfectants expressing LMP2/LMP7/MECL-1 and
PA28/ß
For the generation of stable transfectants, we chose the mouse
fibroblast line B8, which endogenously expresses little LMP2, LMP7, and
MECL-1. Expression constructs encoding these three subunits were
transfected, and after isolation of drug-resistant cells, three
different clones named B27 M2, B27 M6, and B27 M9, which according to
Northern analysis strongly overexpressed the respective mRNAs (data not
shown), were chosen for further characterization. The 20S proteasomes
of these transfectants and, for comparison, of IFN--stimulated and
unstimulated B8 cells were immunoprecipitated under stringent
conditions, and the composition of proteasome subunits was analyzed on
two-dimensional IEF/PAGE. As shown on autoradiographies in Fig. 1
, the three triple transfectants
expressed LMP2 and MECL-1 in amounts that were comparable with those of
IFN--stimulated B8 cells, while in untreated B8 cells these subunits
were not detectable. The constitutively expressed proteasome subunits
and MC14 were replaced to a large extent by LMP2 and MECL-1,
respectively, in the triple transfectants and in IFN--treated cells
in accordance with previous reports (22, 23, 24).
Unfortunately, the LMP7 subunit has a basic isoelectric point, and
hence migrates out of the IEF gel of our two-dimensional
electrophoretic system. Therefore, we examined the expression level of
LMP7 as well as of LMP2 and MECL-1 in Western analysis. As shown in
Fig. 2, the inducible subunits are found
in the triple transfectants in similar amounts as in IFN--treated B8
cells, while in unstimulated B8 cells neither LMP2 nor MECL-1 and only
little LMP7 were detected. Thus, the Western results are congruent with
the immunoprecipitation analysis (Fig. 1) and a previous examination of
purified 20S proteasomes from the triple transfectant B27 M2 and
IFN--treated and untreated B8 cells on Coomassie-stained
nonequilibrium pH-gradient gel electrophoresis (NEPHGE)/PAGE
two-dimensional gels (13, 23, 25). From these gels, it was
apparent that the exchange of LMP7 for MB-1 was complete in the B27 M2
transfectant and B8 cells after treatment with IFN- for 3
days.
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and ß subunits. Two clones designated
BPß2 and BPß13 were chosen for further characterization, as
they strongly expressed both mRNAs in Northern analysis (not shown).
Western analysis revealed that the PA28 expression in these clones
was comparable with IFN--treated B8 cells and exceeded that of the
low basal expression in B8 recipient cells by a factor of about 3 (Fig. 2
). The PA28ß protein was barely detectable in B8 cells, but
prominently expressed in the BPß2 and BPß13 transfectants to
an amount that was equal or even higher than found in IFN--treated
cells.
The overexpression of LMP2, LMP7, and MECL-1 or PA28 and ß
does not alter class I cell surface expression
A potential function that is discussed both for the inducible
subunits of the immunoproteasome and for PA28 is an enhancement in the
production of appropriate peptide ligands for class I presentation. As
MHC ligands are required for the stabilization and cell surface
transport of class I molecules, we investigated whether their surface
expression would be altered by LMP2/LMP7/MECL-1 or PA28/ß
overexpression. The B8 fibroblast line was originally derived from a
BALB/c mouse, and hence expresses the MHC class I molecules
H-2Ld, H-2Dd, and
H-2Kd. A flow-cytometric analysis revealed that
neither the joint overexpression of LMP2, LMP7, and MECL-1 nor of
PA28 and ß had a significant effect on the surface expression of
these class I molecules (Fig. 3). Only
the treatment of B8 cells with IFN- resulted in an about 10-fold
increase in class I surface expression. This result indicates that if
there is a change in Ag processing in the transfectants, this cannot be
attributed to a general alteration of MHC class I cell surface
expression.
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The cytotoxic immune response to LCMV in mice of the
H-2d haplotype is predominantly directed against
a H-2Ld-restricted peptide epitope called NP118,
which is constituted from the LCMV-NP residues 118–126
(37). We decided to compare the presentation of the NP118
epitope of LCMV-infected B8 cells with that of LMP2/LMP7/MECL-1 and
PA28/ß transfectants. The cells were LCMV infected and used after
24 h as targets in a cytolytic chromium release assay employing
NP118/H-2Ld-specific CTLs as effectors.
Remarkably, the LMP2/LMP7/MECL-1 triple transfectants showed a strong
enhancement of cytolysis by NP118-specific T cells when compared with
parental B8 cells. As can be seen in Fig. 4A, two to three times more
LMP2/LMP7/MECL-1-transfected cells compared with B8 cells were lysed by
the same effectors in several independent experiments, and this
difference could not be overcome by increasing the amounts of effector
cells to higher E:T ratios. Interestingly, the PA28/ß double
transfectants were lysed by NP118-specific CTLs to a similar extent as
untransfected B8 cells. The latter finding differs from previous
results obtained with PA28 single transfectants of B8 using CTLs
specific for the influenza NP or the mouse CMV immediate early protein
pp89 (34), suggesting that the effects of PA28 may not
promote Ag presentation in different systems to the same
degree.
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-stimulated B8 cells as targets and freshly isolated
CTLs from LCMV-infected mice as effectors. As it has been shown that in
a primary H-2d-restricted immune response to LCMV
no epitopes other than NP118 significantly contribute to cytolysis
(47), it is safe to assume that the results obtained in a
polyclonal ex vivo response reflect the recognition of NP118. Also, in
this set up, the LCMV-infected LMP2/LMP7/MECL-1 triple transfectants
were recognized much better than B8 cells or the PA28/ß
transfectants (Fig. 4B). Moreover, we found that the
treatment of B8 cells with IFN- enhanced the cytolysis to a similar
degree as the overexpression of LMP2, LMP7, and MECL-1, suggesting that
immunoproteasomes contribute considerably to this enhancement. To
confirm that the elevated NP118 presentation in LMP2/LMP7/MECL-1 triple
transfectants was due to an enhancement in epitope generation and not
to unrelated defects in the control cell lines, we generated the
vaccinia recombinant rVV-NP118 encoding the nonameric NP118 epitope as
a minigene. Infection with rVV-NP118 led to a high lysis by
NP118-specific CTLs, which was equal for the B8 recipient and the
transfectants, indicating that the enhanced NP118 presentation in
LCMV-infected LMP2/LMP7/MECL-1 triple transfectants was due to a better
efficiency in Ag processing (Fig. 4C). As LMP2 and MECL-1 on
the one hand and LMP7 on the other hand can incorporate into the 20S
proteasome independently, we decided to investigate how an
LCMV-infected LMP2/MECL-1 double transfectant of B8 would present the
NP118 epitope. As shown in Fig. 4D, also in the LMP2/MECL-1
double transfectant B2 M1, the NP118 epitope was presented at a higher
level, suggesting that expression of LMP2 and MECL-1 is sufficient to
achieve this enhancement. To ensure that the observed effects were not
due to clonal variation or transfection artefacts, we tested two B8
clones that were transfected with the empty expression vector in the
same experiment, and they, as expected, presented the NP118 epitope at
the same level as the B8 recipient cells (Fig. 4D).
To exclude that the increase in presentation of NP118 in the triple
transfectants was due to higher LCMV replication and NP synthesis in
these cells, we immunoprecipitated the NP from the same batch of cells
that were used as target cells in the ex vivo cytolytic assays (Fig. 4B). As shown in Fig. 5A, the NP content was not
significantly different in the LMP2/LMP7/MECL-1 triple transfectants,
the PA28/ß double transfectants, or B8 cells in the presence or
absence of IFN-. This result was further confirmed by
flow-cytometric analysis, which revealed identical levels of LCMV-NP
expression in LCMV-infected B8 cells and the transfectants (Fig. 5B). Thus, we conclude that the improved recognition of
LMP2/LMP7/MECL-1 triple transfectants must be due to a greater
efficiency in the intracellular generation of the NP118 epitope rather
than a higher level of NP synthesis in these cells.
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A), although the
cell number and viability (Fig. 6B) as well as LCMV-NP
expression (data not shown) were identical among stimulators.
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-inducible subunits is
believed to alter the peptide processing of the 20S proteasome, we
decided to investigate in vitro whether evidence for altered processing
can be obtained in this model and whether these changes are consistent
with the observed in vivo effects. Immunoproteasomes greatly favor the in vitro production of putative cytosolic precursors of the NP118 epitope
We purified 20S proteasomes both from B8 cells and from the triple
transfectant B27 M2 and monitored the fragmentation of a synthetic
25-mer polypeptide from the sequence of LCMV-NP that contained the
nonameric NP118 epitope. After several time periods, aliquots were
removed from the proteolytic reaction and the fragments were separated
by reversed phase HPLC (Fig. 7) and
identified by MALDI-MS and automated Edman degradation. It is apparent
that the same kind of fragments were produced by constitutive
proteasomes from B8 cells and immunoproteasomes isolated from B27 M2
cells. However, although the kinetics of 25-mer consumption was
comparable, the quantity of the respective peptide fragments produced
differed markedly between the two proteasome populations. These
fragments accumulated over the time of the digest, indicating that
these differences in fragment quantity were already apparent at early
time points of the cleavage reaction when the 25-mer substrate was
still in excess.
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) were produced by
immunoproteasomes in about 6- and 3-fold higher amounts, respectively.
A 10-mer containing NP118 was produced in double amounts by
constitutive proteasomes, but it was not a prominent fragment in either
of the reactions. Also, the NP118 nonamer epitope itself was not a
prominent peptide product, and quantitation by Edman degradation
required that the HPLC fraction containing NP118 (as identified by
MALDI-MS) was pooled from several runs. This analysis revealed that the
NP118 nonamer was produced by constitutive proteasomes to 1.5-fold
greater amounts than by immunoproteasomes. Given that the putative 11-
and 12-mer precursors, but not the NP118 nonamer, meet the criteria for
an efficient transport by TAP transporters (see Discussion),
these in vitro results are consistent with an increased presentation of
the NP118 epitope in cell lines overexpressing LMP2, LMP7, and
MECL-1.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-inducible proteasome
subunits LMP2 and LMP7 in the MHC class II locus 10 years ago had
raised great expectations with respect to the function of these
subunits in MHC class I-restricted Ag presentation. The phenotypes of
cell lines or mice deficient for LMP2 and/or LMP7, however, were subtle
and in many systems not apparent. In previous studies, we and others
have tried to characterize the impact of LMP2 and LMP7 on Ag
presentation by overexpressing these subunits, thus imitating their
induction by IFN- (13, 48). Despite strong
overexpression of LMP2, there was only little LMP2 protein incorporated
into the 20S proteasome compared with IFN--stimulated cells. The
reason for this is now known: the third IFN--inducible exchange of
MC14 (or Z) by MECL-1 is required for incorporation of LMP2 (25, 26), which explains why LMP2 could only be incorporated
efficiently into the proteasome in rare clones that displayed an
endogenous up-regulation of MECL-1 expression (49).
Accordingly, the coexpression of LMP2, LMP7, and MECL-1 yielded a good
incorporation of all three subunits into the 20S proteasome in
transfectants similar to immunoproteasomes from IFN--treated cells
(Fig. 1). This was the prerequisite to study their effect on Ag
presentation by means of overexpression.
A key finding of our study is that the presentation of the
immunodominant NP118 epitope by H-2Ld is markedly
enhanced in transfectants overexpressing LMP2, LMP7, and MECL-1. At
first sight, our in vitro results seemed to be at odds with this
finding, as the nonameric NP118 epitope was a minor product in
proteasome digests and was even generated by constitutive proteasomes
in 1.5-fold greater amounts compared with immunoproteasomes. However,
the NP118 epitope bears a proline as an anchor residue for binding to
the H-2Ld class I molecule in position 2. Mouse
and human TAP transporters hardly transport peptides with proline in
position 2 from the cytosol into the ER lumen, suggesting that
N-terminally elongated precursors need to be formed and transported
(3, 2). Interestingly, two potential cytosolic precursors
of this epitope (the 11 or 12 mer in the scheme of Fig. 8) were
produced in 3- and 6-fold greater amounts in vitro if LMP2, LMP7, and
MECL-1 were incorporated into the 20S proteasome. The 11 and 12 mer are
good candidates for precursors of the NP118 epitope because they have
the appropriate C terminus, which normally cannot be generated by
peptidases other than the proteasome (5) and because they
are not too long to be efficiently transported by TAP transporters.
The NP118 epitope (shown in Fig. 8) is directly preceded by a glutamic
acid residue at position 117. Proteasomal cleavage events at the C
terminus of glutamic acid have been unanimously shown to be
down-regulated by replacement of the constitutive subunit through
LMP2 when proteasome activity was measured with help of fluorogenic
peptides (11, 12, 13, 14) or polypeptides (36). This
finding would be consistent with our in vitro result that the
generation of the NP118 9 mer, which requires the cleavage C terminal
of Glu117, is generated less efficiently by
LMP2-containing immunoproteasomes as compared with constitutive
proteasomes. The generation of the 11- and 12-mer precursors, in
contrast, may have been enhanced by suppressing cleavages at
Glu117. If the NP118 nonamer cannot be
efficiently conveyed by the TAP transporter, then the suppression of
the cleavage after Glu117 would favor the
generation of the required precursors, and thus enhance Ag presentation
as we have observed. The suppression of cleavages C terminal of
glutamic acids may also explain earlier results by Sibille et al.
(21), who noted that LMP2 was required for the
presentation of two influenza hemagglutinin epitopes, HA11 (FEANGNLI)
and HA8 (IEGGWTGMI), both of which contain glutamic acid residues
within their sequence.
Another interesting residue in the NP118 epitope is the proline in the
second position serving as an anchor residue for
H-2Ld. A recent analysis of how the proteasome
may process polypeptides containing proline residues suggested that
proline within an epitope sequence may preserve the epitope from
destruction by proteasome-mediated internal cleavages
(50). This proposal would fit nicely with the fact that
proline is an anchor residue for several human and murine MHC class I
molecules. It has been shown by inhibitor studies (51, 52)
and the statistical analysis of proteasomal cleavage products
(53) that it is not only the P1 residue that defines
substrate binding and proteasomal cleavage, but that up to 5 aa to both
sides of the proteolytic site may codetermine whether a cleavage occurs
or not. While according to extrapolations from the three-dimensional
structure of yeast proteasomes the exchange of LMP2 for renders the
preference for the P1 residue from acidic to hydrophobic (54, 52), an obvious modification of the P1 pocket could not be
predicted for the exchanges of LMP7 for MB-1 and MECL-1 for MC14. It
would therefore be interesting to investigate whether LMP7 and MECL-1
introduce preferences for positions other than P1 and whether proline
could be one of them. Finally, it is noteworthy that the 11-mer
fragment that is produced in 6-fold excess by immunoproteasomes
compared with constitutive proteasomes is liberated by cleavage C
terminal of an arginine residue. Arginine is a prominent proteasome
cleavage site and is used by several human MHC class I molecules as a
C-terminal anchor residue. Interestingly, the constitutive homologue of
MECL-1, named MC14 in the mouse and PUP1 in yeast, was shown to be in
charge of cleavages C terminal of basic residues (55, 53, 56). It would therefore be interesting to test whether the
incorporation of human MECL-1 into the proteasome may enhance the
cleavages C terminal of arginine.
New and convincing evidence that the incorporation of LMP2, MECL-1,
and/or LMP7 can determine the fate of an epitope have been reported
while this work was in revision. Sewell et al. (57)
reported that an HLA-A0201-restricted epitope from the N terminus of
HIV-1 reverse transcriptase was only presented when cells were
expressing LMP7. Also, an epitope from influenza matrix protein could
only be presented on HLA-A0201 when LMP7 was expressed in the infected
cells (58). Sijts et al. showed that an
HLA-Aw68-restricted epitope from hepatitis B virus core Ag could only
be presented under IFN- stimulation, and in vitro results suggested
that LMP2, LMP7, and MECL-1 were required for the epitope generation
(59). In contrast to these studies, the generation of
several tumor epitopes was recently shown to be abrogated by IFN-
treatment of tumor cells, and at least for the RU1 Ag of renal
carcinoma the overexpression of LMP2, LMP7, and MECL-1 was shown to
reduce the presentation of an HLA-B51-restricted RU1 epitope
(60). Taken together, it appears that although LMP2, LMP7,
and MECL-1 incorporation seems to slightly increase the total amount of
suitable class I ligands (15), the effect on the single
epitope can be either deleterious or beneficial. It is therefore quite
important to note whether a given CTL epitope was characterized in
presenting cells that do or do not express LMP2, LMP7, or MECL-1.
A somewhat surprising finding of this study was the fact that
overexpression of PA28 and PA28ß to the same or even higher degree
as obtained with IFN- stimulation did not alter the presentation of
the NP118 epitope. Previously, we have found that either the single
overexpression of PA28 (34) or the concerted expression
of PA28 and ß enhanced the presentation of T cell epitopes from
mouse CMV pp89 protein or the NP of influenza virus. Mechanistically,
the detailed analysis of in vitro digests suggested that PA28 may
induce the proteasome to perform concerted dual cleavages that would
liberate fragments of appropriate length for MHC class I binding
(35). It was proposed that PA28 may achieve these dual
cleavages by coordinating the cleavage reactions of two juxtaposed
active centers of the proteasome, which can be spanned by a nonameric
peptide in extended conformation. However, this effect should in theory
be of advantage for the generation of all epitopes. Thus, our results
suggest that PA28 functions in a different and more epitope-specific
way. Although it has meanwhile been confirmed by a number of groups
that PA28 markedly alters proteasomal cleavage reactions in vitro
(13, 35, 36, 50), the principles and mechanisms of PA28
function still need to be better defined.
IFN- induces a multitude of genes that affect Ag processing and also
have direct adverse effects on viral replication. It is hence not easy
to decide to which degree the different gene products contribute to the
elimination of LCMV in vivo. The application of neutralizing Abs to
IFN- in mice showed that this treatment enhanced the viral load in
the course of the immune response (61) and that
neutralization of IFN- could prevent elimination of the virus
(62). More recent experiments in gene-targeted mice
deficient for IFN- showed that in the absence of IFN-, LCMV could
be eliminated after an acute experimental infection, although the
cytolytic activity of LCMV-specific CTLs from these mice was reduced by
a factor of 2–3. Similarly, mice that lacked the IFN- receptor were
able to clear an acute infection with LCMV, but virus titers on day 10
postinfection remained 10 times higher in spleen and lung, but 1,000
and 10,000 times higher in liver and ovary, respectively
(63). Interestingly, the organs with the lowest titer of
LCMV in IFN-Ro/o as compared with wild-type
mice (spleen, lung) were organs of high constitutive expression of
LMP2, LMP7, and MECL-1 (64). Whether LMP2, LMP7, and
MECL-1 contribute to these in vivo effects should now be investigated
in gene-targeted mice deficient for LMP2 or LMP7.
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Acknowledgments |
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Footnotes |
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2 Current address: Departamento de Biología Celular y Molecular, Facultad de Ciencias Biologícas, Pontificia Universidad Católica de Chile, Casilla 114D, Santiago, Chile.
3 Address correspondence and reprint requests to Dr. Marcus Groettrup, Kantonsspital St. Gallen, Laborforschungsabteilung, Haus 09, CH-9007 St. Gallen, Switzerland.
4 Abbreviations used in this paper: ER, endoplasmic reticulum; IEF, isoelectric focusing; LCMV, lymphocytic choriomeningitis virus; LMP, low-molescular mass polypeptide; MALDI-MS, matrix-assisted laser desorption ionization-mass spectroscopy; MECL, multicatalytic endopeptidase complex-like; NP, nucleoprotein; TFA, trifluoroacetic acid; VV, vaccinia virus.
Received for publication July 30, 1999. Accepted for publication May 2, 2000.
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