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*
Department of Immunohematology and Blood Bank, Leiden University Medical Center, Leiden, The Netherlands;
Division of Tumor Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands; and
Institute of Biochemistry, Charité, Humboldt University, Berlin, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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D) immediately flanking the
C-terminal anchor residue of the epitope. Proteasome-mediated digestion
analysis of a synthetic 26-mer peptide derived from the Friend sequence
shows that cleavage takes place predominantly C-terminal of D, instead
of V as is the case for the Moloney MuLV sequence. Therefore, the C
terminus of the epitope is not properly generated. Epitope-containing
peptide fragments extended with an additional C-terminal D are not
efficiently translocated by TAP and do not show significant binding
affinity to MHC class I-Kb molecules. Thus, a potential CTL
epitope present in the Friend virus sequence is not properly processed
and presented because of a natural flanking aspartic acid that
obliterates the correct C-terminal cleavage site. This constitutes a
novel way to subvert proteasome-mediated generation of proper antigenic
peptide fragments. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-stimulated cells, the
proteasome appears to be the rate-limiting factor in the cascade of
degradation, TAP translocation, and MHC class I peptide loading
(7). Two types of proteasomes are now recognized: the
constitutive (household) proteasome, expressing the ß subunits X,
Y, and Z, and the IFN--inducible immunoproteasome, expressing LMP2,
LMP7, and MECL-1 (8, 9, 10). Purified 20S proteasomes have been used for digestion analysis of several (small) proteins and long synthetic peptides (9, 11, 12, 13, 14), and a direct relationship between fragments generated by the proteasome in vitro and in vivo has been shown (15, 16). In vitro proteasome-mediated digestion analysis (12, 13, 15, 17) and proteasome inhibitor studies (17, 18) have shown that in particular the C terminus of the CTL epitope is precisely determined by the proteasome, whereas the N terminus is often elongated by one or two amino acids. The optimal MHC class I presentable peptide length is most likely generated by N-terminal trimming by ER resident (19, 20, 21) or cytosolic amino peptidases, as has recently been suggested for the OVA CTL epitope (17, 22). Several studies have shown that residues either within (13, 23) or in the flanking regions of the CTL epitope can have strong influence on the processing of the epitope (12, 15, 24, 25, 26, 27).
Evasion mechanisms that viruses and tumors have exploited to circumvent recognition by CD8 T lymphocytes operate at many different levels of the MHC class I processing and presentation pathways (reviewed in Ref. 28). At the level of proteolytic processing, EBV was found to interfere via an internal repeat of the EBNA-1 protein (29) and adenovirus 12 is able to eliminate expression of both LMP2 and LMP7 (30). We have previously shown that murine leukemia virus variants can evade immune recognition by a single amino acid change within the CTL epitope sequence that resulted in premature destruction of the epitope by proteasome-mediated cleavage (13). This shows that single-point mutations can result in alterations in the primary amino acid sequence that affects viral epitope processing by the proteasome.
We have studied the processing and presentation of a Moloney murine leukemia virus (MuLV) env gp70-encoded CTL epitope SSWDFITV (31) that was shown to be a subdominant epitope in C57BL/6 mice but can induce significant protective immunity upon DNA vaccination (32). The closely related Friend and Rauscher MuLV, but also the distinct, endogenous AKV-type MuLV, share a high degree of sequence homology in the epitope region. The Rauscher MuLV amino acid sequence comprising the epitope is identical to that of Moloney virus and CTL recognize cells infected with both virus types. However, CTL cannot recognize cells that express Friend MuLV. In this study, we show that obliteration of the required precise C-terminal proteasome-mediated cleavage of the homologous Friend MuLV env sequence results in failure of TAP translocation and MHC class I binding. Consequently, the Friend env sequence ceases to serve as an epitope, despite the presence of an intact, strongly MHC class I-binding sequence.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peptides were synthesized on a multiple peptide synthesizer (Abimed AMS 422;Abimed Analysen-Technik, Langenfeld, Germany) as described elsewhere (33). Peptides were purified by reversed phase-HPLC in an acetonitrile-water gradient and lyophilized from acetonitrile-water overnight. Before use, peptides were dissolved in proteasome digestion buffer as described previously (15).
The 20S proteasomes were purified from RMA cells as described elsewhere (34). RMA proteasomes contain relatively high amounts of the immunosubunits LMP2, LMP7, and MECL-1 (13). Peptides (26 mer, 20 µg) were incubated with 1 µg of purified RMA proteasomes in 300 µl at 37°C for 1, 4, and 24 h. Trifluoroacetic acid (30 µl) was added to stop the digestion. Peptide digestions were stored at -70°C.
Mass spectrometry (MS)
Electrospray ionization mass spectrometry was performed on a hybrid quadrupole time-of-flight mass spectrometer, a Q-TOF (Micromass, Manchester, U.K.), equipped with an on-line nanoelectrospray interface (capillary tip 20 µm internal diameter * 90 µm outer diameter) with an approximate flow rate of 250 nl/min. This flow was obtained by splitting of the 0.4 ml/min flow of a conventional high-pressure gradient system using an Acurate flow splitter (LC Packings, Amsterdam, The Netherlands). Injections were done with a dedicated micro/nano HPLC autosampler, the FAMOS (LC Packings), equipped with two additional valves for phase system switching experiments. Digestion solutions were diluted five times in water-methanol-acetic acid (95:5:1, v/v/v), and 1 µl was trapped on the precolumn (MCA-300-05-C8; LC Packings) in water-methanol-acetic acid (95:5:1, v/v/v). Washing of the precolumn was done for 3 min to remove the buffers present in the digests. Subsequently, the trapped analytes were eluted with a steep gradient going from 70% B to 90% B in 10 min, with a flow of 250 nl/min (A, water-methanol-acetic acid (95:5:1, v/v/v); B, water-methanol-acetic acid (10:90:1. v/v/v)). This low elution rate allows for a few additional MS/MS experiments if necessary during the same elution.
Mass spectra were recorded from mass 50–2000 Da every second with a
resolution of 5000 FWHM. The resolution allows direct determination of
the monoisotopic mass, also from multiple charged ions. In MS/MS, mode
ions were selected with a window of 2 Da with the first quadrupole, and
fragments were collected with high efficiency with the orthogonal
time-of-flight mass spectrometer. The collision gas applied was argon
(4*10–5 mbar), and the collision voltage 
30 V.
The peaks in the mass spectra were searched in the digested precursor peptide using the Biolynx/proteins software (Micromass, Manchester, U.K.) supplied with the mass spectrometer.
The intensity of the peaks in the mass spectra was used to establish the relative amounts of peptides generated after proteasome digestion. The relative amounts of the peptides are given as a percentage of the total amount of peptide digested by the proteasome. The percentage of the F and Y peptides were used to calculate the ratio between these two types of peptides. We found a ratio between 100-and 1000-fold more F than Y peptide.
Cell lines and culture conditions
RMA is a Rauscher MuLV-induced T lymphoma cell line; FBL-3 is a Friend MuLV-induced erythroleukemia cell line; RMA-S is an RMA-derived TAP2 mutant cell line; and EL-4 is a chemically induced (non-MuLV) thymoma cell line. B6 m29 is a fibroblast cell line derived from a primary culture of mouse embryo cells. All murine cell lines are of C57BL/6 (H-2b) origin. HeLa-Kb is a stable transfectant of the human HeLa cell line expressing the mouse H-2Kb MHC class I molecule. CTL clone 10B6 is derived from a Moloney MuLV-immunized B6.CH-2bm13 mouse and cultured as described elsewhere (31).
All cell lines were cultured in Iscove’s modified Dulbecco’s medium (BioWhittaker, Verviers, Belgium) supplemented with 8% heat-inactivated FCS (Greiner, Frickenhausen, Germany), 2 mM L-glutamine, 100 U/ml penicillin, and 20 µM 2-ME at 37°C in 5% CO2.
CTL bioassays and proteasome inhibition
Chromium-51 release cytotoxicity assays were performed as described (13). The mean percentage of specific lysis of triplicate wells was calculated as follows: % specific lysis = ([cpm experimental release - cpm spontaneous release]/[cpm maximum release - cpm spontaneous release]) x 100.
Measurement of secreted TNF-
by stimulated CTL was performed with a
bioassay using WEHI 164 clone 13 cells as described previously
(35). Percentage of TNF- release of triplicate wells
was calculated as follows: % TNF- release =
[(A550-650
experimental wells - A550–650
wells containing medium
only)/(A550–650 wells containing 500
pg/ml TNF- - A550–650 wells
containing medium only)] x 100.
Production of IFN- by the CTL was measured by a sandwich ELISA
performed in maxisorp plates (Nunc, Roskilde, Denmark) using
anti-mouse IFN--specific mAbs (clones R4–6A2 and biotinylated
XMG1.2; PharMingen, San Diego, CA), streptavidin-conjugated poly-HRP
(CLB, Amsterdam, The Netherlands), and 2,
2'-azino-bis(3-ethylbenzthiazoline)-6-sulfonic acid (Sigma, St. Louis,
MO) as a substrate. In every test titrated amounts of recombinant mouse
IFN- (PharMingen) served as a standard. OD at 415 nm was measured
using kineticalc 2.12 software in an EL312e Biokinetics ELISA plate
reader (Biotek Instruments, Winooski, VT).
The proteasome-specific inhibitors lactacystin (Calbiochem, Breda, The Netherlands) (36) and 4-hydroxy-5-iodo-3-nitrophenylacetyl-leu-leu-leu-vinylsulfone (NLVS; Calbiochem) (37) were used to block proteasome activity. B6 m29 cells were infected with Abelson Moloney MuLV by incubation of the cells for 3–4 days in complete culture medium as described previously (38). These cells were used as targets in CTL cytokine secretion assays in the presence or absence of 20 µM lactacystin or 20 µM NLVS in complete culture medium for 4 h at 37°C. Cells were washed three times with exceeding amounts of culture medium. RMA tumor cells were treated using a peptide-stripping procedure as described elsewhere (18): cells were incubated with 10 µM lactacystin for 2 h at 37°C, followed by a 1–2 min mild acid treatment (pH 3.0) according to Storkus et al. (39) and incubated for another 2 h with lactacystin. After extensive washing, the cells were used as targets in 51Cr release assays.
Transient transfections
Plasmid DNA was transfected into HeLa.Kb cells and seeded onto flat-bottom 96-well plates, using the DEAE-dextran method, as described previously (40). Two days after transfection, culture medium was replaced and 5000 CTL were added in each well. Expression constructs used were minigene construct-encoding 8-mer peptide SSWDFITV directly behind the signal sequence of E3 protein of adenovirus type 5 (41); Moloney MuLV p-gag/pol-gtp (ScaI-NaeI fragment); and Moloney MuLV p-env (BglII-NheI fragment) driven by the Moloney MuLV long terminal repeat.
TAP translocation assays
The TAP-dependent translocation assay was performed as described elsewhere (42). In short, peptides of interest were tested for their ability to compete for TAP-dependent translocation of a 125I-iodinated model peptide in streptolysin O-permeabilized EL-4 cells.
MHC class I peptide-binding assay
The RMA-S peptide-binding assay was performed as described previously (13, 43). In short, RMA-S cells were cultured for 36 h at 26°C and were added to serial dilutions of peptide. After 4 h of incubation at 37°C, cells were washed and stained with an H-2Kb-specific Ab, and specific fluorescence was determined using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA).
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Results |
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CTL raised against Moloney MuLV are cross-reactive to Rauscher MuLV sharing the epitope sequence SSWDFITV, as described before (31). In contrast, these CTL do not recognize cells transformed by the closely related Friend MuLV, despite the high degree of sequence homology in the epitope SSWDYITV that differs in only one residue that constitutes a well-defined alternative anchor residue at this position (44). Indeed, the Y-containing synthetic peptide can efficiently bind to the MHC class I-Kb molecule and upon loading onto Kb-positive target cells can be recognized by the CTL (Ref. 31 and below).
Fig. 1
A shows the lack of
recognition of Friend MuLV-induced FBL-3 tumor cells by the
Moloney-specific CTL clone 10B6. Loading of FBL-3 cells with the
synthetic peptide SSWDFITV resulted in efficient recognition, showing
that these cells can be lysed by CTL. Even in a more sensitive TNF
release assay (Fig. 1B), Friend virus-induced FBL-3 tumor
cells are not recognized in contrast to RMA tumor cells endogenously
expressing Rauscher MuLV Ags. In the same experiment, CTL can recognize
Moloney MuLV env and the minimal epitope sequence introduced as a
minigene by transient transfection, whereas Moloney MuLV
gag-pol-transfected cells are not recognized. FBL-3 cells have normal
levels of MHC class I expression and FBL-3 can be killed by other
MuLV-specific CTL recognizing the endogenously presented gagL CTL
epitope (data not shown), indicating normal expression of MuLV-encoded
proteins. These data indicate that the potential Friend MuLV-encoded
CTL epitope sequence is not properly processed and presented for CTL
recognition.
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C
shows significant inhibition of cytotoxicity of CTL clone 10B6 by
lactacystin treatment of RMA cells. CTL recognition could be rescued by
addition of the exogenous synthetic peptide SSWDFITV. Similar
experiments were performed with B6-derived fibroblast cells that were
infected with Moloney MuLV. Both lactacystin (36) and the
vinyl sulfone inhibitor NLVS (37) completely inhibited
epitope processing in these MuLV-infected cells (Fig. 1D). Proteasome-mediated digestion indicates altered C-terminal cleavage
The naturally occurring viruses Moloney, AKV, and Friend MuLV
share high sequence homology in the region of the Moloney CTL epitope.
Only two residues differ (Table I). The
phenylalanine (F) residue within the epitope at the anchor position 5
(Moloney) is exchanged for a tyrosine (Y) (AKV and Friend), which is an
allowed anchor residue for Kb binding. The
residue just flanking the C-terminal cleavage site of the epitope is
different for all three virus types. The Moloney sequence contains an
asparagine (N), AKV, a serine (S), and Friend, an aspartic acid (D), at
this position. To investigate whether CTL recognition is related to the
amino acid sequence differences discussed, the processing of the
Moloney, AKV, and Friend MuLV epitopes from their natural context was
tested. To this end, we performed in vitro proteasome digestions of the
respective 26-mer synthetic peptides harboring the epitope flanked with
its natural amino acid sequences. Table II shows the results of the digestion
experiments for the three synthetic peptides. The 20S
proteasome-mediated digestion of the Moloney peptide results in four
major fragments after 1 h of digestion and two major fragments
after 4 h. The proteasome precisely determines the C-terminal
valine (V) of the epitope. The minimal 8-mer CTL epitope fragment
SSWDFITV could not be detected in the digests. The two major fragments
were the 7-mer SWDFITV and the 10-mer PSSSWDFITV. The most likely
relevant epitope precursor peptide found is the 10-mer fragment,
requiring N-terminal trimming to generate the CTL epitope SSWDFITV.
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show that the
cleavage products generated are not influenced by the presence of
either F or Y at position 5 of the epitope core sequence. Instead,
cleavage is dominantly influenced by the aspartic acid. The presence of
this negatively charged residue next to the valine abolishes the
precise C-terminal cleavage site of the epitope.
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We have previously shown that the SSWDFITV peptide is efficiently
translocated into the ER in a TAP-dependent translocation assay
(42). We now report the efficiency of TAP translocation in
vitro of the peptides, as generated by the 20S proteasome. Table IV shows that both the Moloney 8-mer
SSWDFITV and the Friend homologue SSWDYITV are very efficiently
translocated by TAP as synthetic peptides. However, no detectable TAP
translocation was displayed by the peptides flanked by D at the C
terminus for all length variants tested of both the Moloney and Friend
sequences. Apparently, the negatively charged residue at the C terminus
disturbs murine TAP-dependent ER translocation of these peptides
(45). No detectable translocation was measured with the
7-mer variants SWDFITV and SWDYITV, nor with the 10-mer length variants
PSSSWDFITV and PSSSWDYITV. Thus, the most likely candidate for TAP
transport is the efficiently translocated exact MHC-binding sequence
SSWDFITV (or SSWDYITV, if generated).
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The peptides found to be generated by the proteasome were tested
for MHC class I binding and CTL recognition. Fig. 2 shows the results of MHC-binding
experiments using synthetic peptides of different length variants. Fig. 2A shows the F-containing peptides (Moloney) and Fig. 2B the Y-containing peptides (Friend). The 8-mer peptide
SSWDFITV, previously defined as the optimally binding and recognized
peptide (28), was reproducibly the peptide with the most
optimal MHC class I-Kb-binding ability. In most
proteasome digestion experiments, the amount of this peptide was below
the detection level (Tables II and III). The most abundantly generated
7-mer peptide SWDFITV did not exhibit significant binding to the MHC
class I-Kb molecule. The precursor peptide
fragment PSSSWDFITV binds to the MHC class I-Kb
molecule, but with significantly less efficiency than the 8-mer. The
presence of an aspartic acid as the C-terminal residue of the peptides
strongly decreases the binding capacity to the MHC class
I-Kb molecule. Similar results were obtained with
the Y-containing peptides (Fig. 2B). The same synthetic
peptides were used in cytotoxicity assays with CTL clone 10B6 (Fig. 3). The recognition by the CTL is
completely abolished when the D is present at the C terminus. We
conclude that the presence of this negatively charged residue next to
the C-terminal anchor of the CTL epitope strongly disrupts TAP
translocation as well as binding to the MHC class I groove and thereby
CTL recognition.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5-fold lower than that of the F peptide.
However, the affinity of this peptide is still very high and within the
range of other well-defined Kb- and
Db-presented CTL epitopes as published earlier
(46). The affinity of the CTL clone used (10B6, originally
raised against the F-containing epitope) for the Y-containing peptide
is lower than that for the F-containing peptide, but still within the
range of the difference in MHC-binding affinity (31). Our
results also show no measurable difference between F and Y peptide in
TAP translocation (Table IV). We have calculated the difference in F-
vs Y-containing epitope generation from Moloney vs Friend 26-mer
peptide substrates in the proteasome-digestion assays. In these digests
between 100- and 1000-fold more F than Y peptide can be detected. The
much greater difference in peptide formation by the proteasome
indicates that the proteasome fine specificity rather than the
difference in MHC binding is causally related to the lack of epitope
presentation of the Friend virus epitope. The proteasome determines the C terminus of the epitope, but frequently the N terminus of the peptide fragments generated is a few residues longer than the optimal MHC-presented peptide. The need for precise C-terminal cleavage of CTL epitopes by the proteasome has been reported for different epitopes (17, 18), and its relevance is illustrated in our study. Next to MuLV, we have performed 20S proteasome digestion analysis on long synthetic peptides harboring several defined CTL epitopes such as Sendai virus (47) and human papilloma virus epitopes (43). Although the relevance of in vitro proteasome digestion analysis has been discussed (2), we have applied this method on several epitope-containing sequences and found very distinct cleavage specificities and a strong correlation of the cleavage products with the presented epitopes. In most cases tested, the proteasome cleaved immediately next to the C terminus of the epitope, whereas the N terminus was not precisely defined (our unpublished results). This indicates that the generation of the correct C terminus is a crucial step in CTL epitope generation as also deduced from studies of the OVA Kb-presented epitope SIINFEKL (17), and suggests that cytosolic carboxypeptidase activity cannot compensate for imprecise C-terminal anchor residue cleavage.
The presentation of the SSWDFITV epitope requires TAP translocation
(48) and has been reported previously (42) to
be optimally translocated as the minimal 8-mer, although the 9-mer
SSSWDFITV is also translocated quite efficiently. However, these
fragments are not generated by the 20S proteasome. We could not detect
any of the 8-mer or 9-mer fragments. The most abundant
epitope-containing precursor peptide found was the 10-mer
PSSSWDFITV. However, this 10-mer peptide was not efficiently
translocated by TAP (Table IV). The formation of the optimal MHC class
I-binding 8-mer peptide apparently requires N-terminal trimming by
aminopeptidases. These enzymes are present in both the cytosol and in
the ER lumen. Both activities can play a role in the generation of MHC
class I-presented peptides. ER trimming has been shown to be important
for other epitopes (19, 20, 21). For example, peptides with
proline at position 3 are poorly translocated by TAP (42, 13) and therefore require N-terminal extensions for proper
translocation to the ER. For these epitopes, N-terminal aminopeptidase
trimming in the ER is most likely required to generate the CTL epitope.
However, for epitopes inefficiently translocated by TAP, like in our
study, the Moloney 10-mer precursor peptide PSSSWDFITV, cytosolic
N-terminal trimming is most likely the enzymatic activity required to
generate peptides that will be efficiently translocated by TAP.
Recently, a cytosolic leucine aminopeptidase was described to be
IFN--inducible and involved in the generation of the OVA CTL epitope
SIINFEKL under in vitro conditions (22). The role of
cytosolic peptidases in vivo has not been firmly established yet. On
the other hand, we cannot firmly exclude that low numbers of 10-mer
precursor peptides of the Moloney epitope enter the ER that after
N-terminal trimming by ER-resident aminopeptidases yields sufficient
epitope formation for CTL recognition. MHC class I-dependent N-terminal
trimming is the major activity required to generate the proper MHC
class I binding minimal peptide of the OVA epitope, as recently shown
by Paz et al. (49).
The proteasomal cleavage specificity could be a potential target mechanism for viral or tumor immune escape. In an earlier study, we showed that a single residue exchange within another MuLV CTL epitope led to premature proteasome-mediated destruction of the potential CTL epitope (13). This mechanism finally led to a similar outcome, namely, nonpresentation of the epitope and thereby evasion on the immune system. Recently, a similar finding was shown for a p53 CTL epitope, in which a tumor-associated single residue mutation (R to H) C-terminally flanking the CTL epitope led to the absence of epitope-containing peptides (50). In this study, the exact mechanism of the lack of epitope generation was not elucidated, although it appears that the flanking residue not only influenced the generation of precursor peptides but also the formation of the minimal epitope. This might indicate that in this case the flanking R influences cleavage inside the epitope, leading to destruction of the epitope. In our study, the flanking D apparently does not influence cleavage within the epitope. The presence of D rather causes a shift in the location of the cleavage site. As a result, precursor peptides are generated that cannot enter the ER because of the negatively charged D that abolishes TAP translocation. A single residue exchange at a site crucial for epitope excision can be a way to evade CTL recognition. In general, these findings represent potential CTL evasion mechanisms at the processing level defined by only a single amino acid mutation.
This MuLV epitope is a subdominant CTL epitope in C57BL/6 mice, whereas
the dominant epitope is the recently described
Db-presented gagL epitope (51). The
underlying mechanism(s) determining the dominance or hierarchy of CTL
epitopes has not been clarified yet, but the relationship with
processing mechanisms leading to lower epitope densities at the cell
surface has been suggested in several studies (12, 22).
The subdominance of the Moloney Kb-presented
epitope SSWDFITV might be regulated by processing events,
involving either the suboptimal formation of TAP-transportable
precursor peptides by the proteasome, or by the efficiency of
aminopeptidase activity in cytosol or ER. These arguments are
underlined by our observations that CTL killing of RMA cells,
endogenously expressing the epitope, is always intermediate and can be
increased by adding exogenous synthetic peptide (Fig. 1C).
This suggests that cell surface presentation of this epitope is not
very high and might be suboptimal for inducing high frequency CTL
responses.
This study shows that a single amino acid, directly flanking the C-terminal residue of a CTL epitope, can strongly influence epitope formation. Precise definition of the C terminus of CTL epitopes by proteolytic machineries such as the proteasome is an essential step in proper peptide generation. Our findings suggest that abolition of precise C-terminal cleavage by a single point mutation can be a highly efficient mechanism of immune escape.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. F. Ossendorp, Department of Immunohematology and Blood Bank, Leiden University Medical Center, Building 1, E3-Q, P.O. Box 9600, 2300 RC Leiden, The Netherlands. E-mail address:
3 Abbreviations used in this paper: ER, endoplasmic reticulum; MuLV, murine leukemia virus; MS, mass spectrometry; NLVS, 4-hydroxy-5-iodo-3-nitrophenylacetyl-leu-leu-leu-vinylsulfone.
Received for publication July 8, 1999. Accepted for publication December 1, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-Interferon and expression of MHC genes regulate peptide hydrolysis by proteasomes. Nature 365:264.[Medline]
stimulation modulates the proteolytic activity and cleavage site preference of 20S mouse proteasome. J. Exp. Med. 179:901. (IFN )-inducible subunits. J. Exp. Med 187:97. can stimulate post-proteasomal trimming of the N terminus of an antigenic peptide by inducing leucine aminopeptidase. J. Biol. Chem. 273:18734. inducible 11S regulator (PA28) and the LMP2/LMP7 subunits govern the peptide production by the 20S proteasome in vitro. J. Biol. Chem. 270:23808.This article has been cited by other articles:
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A. Milicic, D. A. Price, P. Zimbwa, B. L. Booth, H. L. Brown, P. J. Easterbrook, K. Olsen, N. Robinson, U. Gileadi, A. K. Sewell, et al. CD8+ T Cell Epitope-Flanking Mutations Disrupt Proteasomal Processing of HIV-1 Nef J. Immunol., October 1, 2005; 175(7): 4618 - 4626. [Abstract] [Full Text] [PDF]
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F. Ossendorp, N. Fu, M. Camps, F. Granucci, S. J. P. Gobin, P. J. van den Elsen, D. Schuurhuis, G. J. Adema, G. B. Lipford, T. Chiba, et al. Differential Expression Regulation of the {alpha} and {beta} Subunits of the PA28 Proteasome Activator in Mature Dendritic Cells J. Immunol., June 15, 2005; 174(12): 7815 - 7822. [Abstract] [Full Text] [PDF]
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Y. Kimura, T. Gushima, S. Rawale, P. Kaumaya, and C. M. Walker Escape Mutations Alter Proteasome Processing of Major Histocompatibility Complex Class I-Restricted Epitopes in Persistent Hepatitis C Virus Infection J. Virol., April 15, 2005; 79(8): 4870 - 4876. [Abstract] [Full Text] [PDF]
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T. Furutsuki, N. Hosoya, A. Kawana-Tachikawa, M. Tomizawa, T. Odawara, M. Goto, Y. Kitamura, T. Nakamura, A. D. Kelleher, D. A. Cooper, et al. Frequent Transmission of Cytotoxic-T-Lymphocyte Escape Mutants of Human Immunodeficiency Virus Type 1 in the Highly HLA-A24-Positive Japanese Population J. Virol., August 15, 2004; 78(16): 8437 - 8445. [Abstract] [Full Text] [PDF]
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T. M. Allen, M. Altfeld, X. G. Yu, K. M. O'Sullivan, M. Lichterfeld, S. Le Gall, M. John, B. R. Mothe, P. K. Lee, E. T. Kalife, et al. Selection, Transmission, and Reversion of an Antigen-Processing Cytotoxic T-Lymphocyte Escape Mutation in Human Immunodeficiency Virus Type 1 Infection J. Virol., July 1, 2004; 78(13): 7069 - 7078. [Abstract] [Full Text] [PDF]
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R. Draenert, S. Le Gall, K. J. Pfafferott, A. J. Leslie, P. Chetty, C. Brander, E. C. Holmes, S.-C. Chang, M. E. Feeney, M. M. Addo, et al. Immune Selection for Altered Antigen Processing Leads to Cytotoxic T Lymphocyte Escape in Chronic HIV-1 Infection J. Exp. Med., April 5, 2004; 199(7): 905 - 915. [Abstract] [Full Text] [PDF]
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M. Altfeld, M. M. Addo, R. Shankarappa, P. K. Lee, T. M. Allen, X. G. Yu, A. Rathod, J. Harlow, K. O'Sullivan, M. N. Johnston, et al. Enhanced Detection of Human Immunodeficiency Virus Type 1-Specific T-Cell Responses to Highly Variable Regions by Using Peptides Based on Autologous Virus Sequences J. Virol., July 1, 2003; 77(13): 7330 - 7340. [Abstract] [Full Text] [PDF]
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J. Hu, N. M. Cladel, M. D. Pickel, and N. D. Christensen Amino Acid Residues in the Carboxy-Terminal Region of Cottontail Rabbit Papillomavirus E6 Influence Spontaneous Regression of Cutaneous Papillomas J. Virol., October 25, 2002; 76(23): 11801 - 11808. [Abstract] [Full Text] [PDF]
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B. Mommaas, J. Kamp, J.-W. Drijfhout, N. Beekman, F. Ossendorp, P. van Veelen, J. den Haan, E. Goulmy, and T. Mutis Identification of a Novel HLA-B60-Restricted T Cell Epitope of the Minor Histocompatibility Antigen HA-1 Locus J. Immunol., September 15, 2002; 169(6): 3131 - 3136. [Abstract] [Full Text] [PDF]
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K. Yusim, C. Kesmir, B. Gaschen, M. M. Addo, M. Altfeld, S. Brunak, A. Chigaev, V. Detours, and B. T. Korber Clustering Patterns of Cytotoxic T-Lymphocyte Epitopes in Human Immunodeficiency Virus Type 1 (HIV-1) Proteins Reveal Imprints of Immune Evasion on HIV-1 Global Variation J. Virol., July 29, 2002; 76(17): 8757 - 8768. [Abstract] [Full Text] [PDF]
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M. P. Velders, S. Weijzen, G. L. Eiben, A. G. Elmishad, P.-M. Kloetzel, T. Higgins, R. B. Ciccarelli, M. Evans, S. Man, L. Smith, et al. Defined Flanking Spacers and Enhanced Proteolysis Is Essential for Eradication of Established Tumors by an Epitope String DNA Vaccine J. Immunol., May 1, 2001; 166(9): 5366 - 5373. [Abstract] [Full Text] [PDF]
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J. H. Kessler, N. J. Beekman, S. A. Bres-Vloemans, P. Verdijk, P. A. van Veelen, A. M. Kloosterman-Joosten, D. C.J. Vissers, G. J.A. ten Bosch, M. G.D. Kester, A. Sijts, et al. Efficient Identification of Novel Hla-A*0201-Presented Cytotoxic T Lymphocyte Epitopes in the Widely Expressed Tumor Antigen Prame by Proteasome-Mediated Digestion Analysis J. Exp. Med., January 1, 2001; 193(1): 73 - 88. [Abstract] [Full Text] [PDF]
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T. van Hall, A. Sijts, M. Camps, R. Offringa, C. Melief, P.-M. Kloetzel, and F. Ossendorp Differential Influence on Cytotoxic T Lymphocyte Epitope Presentation by Controlled Expression of Either Proteasome Immunosubunits or Pa28 J. Exp. Med., August 21, 2000; 192(4): 483 - 494. [Abstract] [Full Text] [PDF]
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) are not recognized by 10B6 CTL in a 51Cr
release assay, but can be lysed by addition of synthetic peptide
SSWDFITV (•). Rauscher MuLV-induced lymphoma cells RMA (
) are
recognized and killed independently of the presence of exogenous
peptide (
). B, Specificity of CTL clone 10B6 was
measured by TNF-
and 
, RMA acid
stripped and lactacystin treated. Addition of the synthetic peptide
SSWDFITV shows the viability of the treated cells (•, 
