Immunity, Vol. 18, 429–440, March, 2003, Copyright 2003 by Cell Press
The Cytosolic Endopeptidase, Thimet Oligopeptidase,
Destroys Antigenic Peptides and Limits the Extent
of MHC Class I Antigen Presentation
membrane for display to T cells. This process allows
the immune system to monitor for the presence of cells
bearing foreign peptides—for example, ones derived
from viral or mutated proteins.
It is unclear whether antigenic peptides (or longer
precursors) released from proteasomes are protected
ation with heat shock proteins (Srivastava et al., 1998)
or in other high molecular weight complexes (Paz et
al., 1999). It has been proposed that such interactions
protect these peptides from destruction by cytosolic
enzymes until they are translocated into the ER. It has
also been suggested that proteasomes discharge their
products directly into the TAP transporter (Hirsch and
Ploegh, 2000). Such a mechanism would also protect
peptides from hydrolysis in the cytosol. However, there
is presently no evidence for either mechanism, each
of which has been proposed to account for the high
efficiency of peptide presentation that has been re-
ported for certain peptides (Villanueva et al., 1994).
Saric et al. (2001) found that a variety of antigenic
peptides were rapidly degraded when added to cyto-
solic extracts. Although cell extracts contained many
exo- and endopeptidases capable of degrading oligo-
peptides, one endopeptidase, thimet oligopeptidase
(TOP, EC22.214.171.124), was particularly active in degrading
most of the antigenic peptides tested (Saric et al., 2001).
TOP is a cytosolic metallopeptidase that hydrolyzes oli-
gopeptides (typically of 6–17 residues) but not larger
polypeptides or proteins (Barrett et al., 1995). A variety
of functions for TOP have been proposed that involve
its attacking substrates in the extracellular space, e.g.,
degrading neurotransmitters or amyloid ? peptide (Vin-
cent et al., 1997; Yamin et al., 1999). Recent findings
by the proteasome (T.S. et al., unpublished data). How-
ever, it is unknown whether TOP can actually destroy
antigenic peptides or their longer precursors in intact
cells or whether those peptides that are presented on
MHC class I molecules are protected from this enzyme
and other cytosolic peptidases. The present studies
were undertaken to test whether antigenic peptides re-
leased by proteasomes in the cytosol are susceptible
to destruction by cytosolic peptidases, especially TOP.
Ian A. York,1Annie X.Y. Mo,1,4
Kristen Lemerise,1,5Wanyong Zeng,1,6
Yuelei Shen,1Carmela R. Abraham,3
Tomo Saric,2,7Alfred L. Goldberg,2
and Kenneth L. Rock1,*
1Department of Pathology
University of Massachusetts Medical School
Worcester, Massachusetts 01655
2Department of Cell Biology
Harvard Medical School
Boston, Massachusetts 02155
3Department of Biochemistry and Medicine
Boston University School of Medicine
Boston, Massachusetts 02118
Most antigenic peptides presented on MHC class I
molecules are generated by proteasomes during pro-
tein breakdown. It is unknown whether these peptides
are protected from destruction by cytosolic pepti-
dases. In cytosolic extracts, most antigenic peptides
gopeptidase (TOP). We therefore examined whether
TOP destroys antigenic peptides in vivo. When TOP
was overexpressed in cells, class I presentation of
antigenic peptides was reduced. In contrast, TOP
generated in the endoplasmic reticulum or endo-
somes. Conversely, preventing TOP expression with
siRNA enhanced presentation of antigenic peptides.
TOP therefore plays an important role in vivo in de-
grading peptides released by proteasomes and is a
significant factorlimiting theextent ofantigen presen-
Cellular proteins are continuously degraded by protea-
somes into oligopeptides ranging in size from about 3
to 24residues (Kisselevet al.,1999; Rockand Goldberg,
1999; Rock et al., 2002). Most of these oligopeptides
are rapidly hydrolyzed by cytosolic peptidases to amino
acids (Goldberg et al., 2002), which are then re-utilized
in protein synthesis or energy production. However, in
mammalian cells, a small fraction of these peptides are
transported into the endoplasmic reticulum (ER), where,
if they have the correct motif and are 8–10 residues
long, they become tightly bound by specific MHC class
I molecules (Rock and Goldberg, 1999). These peptide-
MHC complexes are then transported to the plasma
Effect of TOP Overexpression on the Presentation of
Peptides Expressed in the Cytosol from Minigenes
we initially studied the effects of transiently overex-
pressing this peptidase in vivo on the presentation of
SIINFEKL expressed in the cytoplasm from a synthetic
minigene. In this situation, the peptide is produced by
ribosomes, rather than proteasomes, and therefore we
could test if an antigenic peptide in the cytosol is sus-
4Present address: Antigenics Inc., Woburn, Massachusetts 01801.
5Present address: Viacell Inc., Boston, Massachusetts 02116.
6Present address: Dana-Farber Cancer Institute, Boston, Massa-
7Present address: ATABIS GmbH, Joseph-Stelzmann Str. 50, 50931
Figure 1. TOP Overexpression Inhibits MHC Class I Presentation of Cytosolic SIINFEKL
(A) The activities of TOP, aminopeptidases (AP), prolyl oligopeptidase (POP), or trypeptidyl peptidase II (TPPII) were measured as described
in Saric et al., 2001, in extracts from COS-Kbcells transfected for 48 hr with pTracer or pTracer-TOP or E36-Kbor DAP-Kbinfected for 3 hr
with vaccinia virus TOP (VV-TOP) or vaccinia virus-?gal (VV-?gal). The data are expressed as the fold-increase in activity in TOP-transfected
cells compared to control cells. The coefficient of variation in all groups was ?10%.
(B) COS-Kbcells were cotransfected with a SIINFEKL minigene in pCDNA3 (or empty pCDNA3 [No Ova]) and a pTracer CMV plasmid encoding
GFP (Vector), GFP ? human TOP (TOP), or GFP ? the control protein ORP150 (ORP150), and after 48 hr stained for SIINFEKL ? Kbcomplexes
with 25.D1.16 or an isotype control (Bkg). For clarity the background fluorescence of only one group is shown because it was essentially
identical for all others (mean fluorescence intensity [MFI] ? SD for the background on all groups ? 14.6 ?/? 1.6). MFI were Bkg, 15.8; No
OVA,16.8; TOP, 214; Vector, 875; and ORP150, 922 fluorescent units.
(C) E36-KbAPCs were infected with vaccinia-T7 polymerase and either vaccinia-TOP or vaccinia-?gal and then transfected with a SIINFEKL
minigene under the control of a T7 promoter in a pBluescript plasmid. After 1.25 hr the cells were fixed with 1% paraformaldehyde and the
indicated number incubated with the SIINFEKL ? Kbspecific T cell hybrid, RF33.70. IL-2 production by RF33.70 is displayed.
(D) Similarto (B)except E36-Dbcellswere usedinstead of E36-Kb,vaccinia-T7 polymerasewas omitted, andafter 1.5hr cells weresuperinfected
for 1.5 hr with a vaccinia recombinant expressing ASNENMETM (a gift from J. Yewdell, NIH) instead of being transfected with a SIINFEKL
minigene, and the 12.3 hybridoma (Deckhut et al., 1993) (gift from D. Woodland, Trudeau Institute, NY) was used instead of RF33.70.
(E) Similar to (B) except that a KVVRFDKL minigene was used instead of SIINFEKL, the 1G8 hybridoma (Cole et al., 1994) (gift from J. McCluskey,
Flinders Medical Center) was used instead of RF33.70, and the transfection time was 2.5 hr.
expression vectors encoding SIINFEKL and TOP plus
green fluorescent protein (GFP) (which allowed trans-
fected cells to be identified) or control plasmids (GFP
alone; or an irrelevant protein, ORP150, plus GFP) were
transiently cotransfected into H-2Kb-expressing COS7
cells (COS-Kb). The presentation of SIINFEKL on H-2Kb
molecules was then detected by staining with a SIIN-
gador et al., 1997) and quantified by flow cytometry.
In the TOP-transfected COS-Kbcells, TOP activity was
increased 16-fold (Figure 1A), and the generation of SI-
INFEKL-Kbcomplexes was markedly reduced to levels
below those detected in cells expressing control pro-
teins GFP or ORP150 (Figure 1B).
We sought to confirm and extend these results in
other APCs. Since most APCs (unlike COS7 cells) do
not replicate plasmids episomally, we constructed a re-
combinant vaccinia virus expressing TOP and used the
vaccinia T7-polymerase system to express antigens
from the transfected plasmids. E36 and L cells (DAP)
were chosenbecause they are readilyinfected with vac-
cinia virus and present antigens well to T cell hybrido-
mas ((Michalek et al., 1993; our unpublished data).
H-2Kb-expressing E36 cells (E36-Kb) were infected with
vaccinia-TOP and vaccinia-T7 and transfected with a
plasmid containing the SIINFEKL minigene under the
control of a T7 promoter. The presence of SIINFEKL on
tion of IL-2 from a specific T cell hybridoma. In cells
infected with the TOP-encoding vaccinia vector the en-
zymatic activity of TOP was increased about 4- to 5-fold
(Figure 1A). The presentation of SIINFEKL on H-2Kbwas
pared to cells infected with a vaccinia control (express-
ing lacZ) (Figure 1C). Similar results were obtained in
experiments performed with DAP cells (see Figure 4C
below). Therefore, TOP in at least three different cell
types from different species can limit the presentation
of SIINFEKL expressed in the cytoplasm.
We next investigated whether TOP overexpression
would affect the presentation of other antigenic pep-
tides. Overexpression of TOP in intact cells also inhib-
Endopeptidase Limits Antigen Presentation
Figure 2. TOP Overexpression Does Not Affect Cell Viability, Protein Synthesis, or Expression of HA
(A) E36-Kbwere infected with vaccinia encoding ?gal (VV-?gal), TOP (VV-TOP), or uninfected (none), or COS-Kbcells were untransfected
(none), or transfected with pTracerCMV (GFP), or pTracerCMV-TOP (TOP) as described in Figure 1, and viability was determined by exclusion
of trypan blue dye. Data are shown as the mean viability ? SEM for multiple experiments.
(B) COS-Kbcells were transfected, or E36-Kbcells were infected, as described in Figure 1, and incorporation of35S-methionine into protein
was measured as described in the Experimental Procedures. Data are displayed as the mean percent inhibition ? SEM for multiple experiments
of cells expressing TOP compared to a control protein (?gal or ORP150).
(C) COS-Kbcells were transfected with pTracerCMV expressing TOP ? GFP (TOP), ORP150 ? GFP (ORP150), or GFP alone (Vector), together
with pJFE14 expressing influenza HA. After 48 hr the cells were stained with anti-HA or an irrelevant antibody. The Bkg traces are cells stained
with an irrelevant antibody. In this same experiment, TOP inhibited the presentation of SIINFEKL (data not shown).
ited the presentation of the influenza-derived peptide
ASNENMETM on H-2Dbclass I molecules (Figure 1D),
as well as the presentation on H-2Kbof a subdominant
epitope from ovalbumin, KVVRFDKL (Figure 1E). Thus
TOP can reduce MHC class I presentation of at least
three antigenic peptides expressed from minigenes.
bility, as judged by their exclusion of trypan blue (Figure
2A) and their normal rate of protein synthesis, as as-
sessed by incorporation of35S-methionine/cysteine into
proteins (Figure 2B). Furthermore, when TOP was overex-
pressed with influenza hemagglutinin (HA), the amount of
HA expressed on the cell surface was not decreased
below control levels (Figure 2C). Therefore, TOP overex-
presentation without interfering with plasmid transfec-
protein trafficking, or exocytosis.
coexpressed in COS-Kbcells with a plasmid that ex-
gene and GFP (data not shown), TOP overexpression
reduced the generation of SIINFEKL-Kbcomplexes (Fig-
ure 3D) but did not decrease the expression of GFP
(Figure 3E), further demonstrating that TOP overexpres-
sion specifically affected MHC antigen presentation
ple antigenic peptides that are generated by protea-
somes from full-length proteins.
We have also compared the effects of overexpress-
ing TOP to those of overexpressing another cytosolic
peptidase. In cytosolic extracts, puromycin-sensitive
genic peptides (though not SIINFEKL or several of its
N-extended precursors) (Saric et al., 2001), and PSA
was reported to trim certain N-extended precursors to
the proper size for presentation (Stoltze et al., 2000).
COS-Kbcells were transfected with ovalbumin together
with a vector encoding PSA and GFP, and SIINFEKL-Kb
of PSA mRNA (data not shown) but did not reduce the
presentation of SIINFEKL-Kbcomplexes on the cell sur-
face (Figure 3F), which is consistent with findings in cyto-
solic extracts (Saric et al., 2001). Similar results were ob-
tained when PSA was cotransfected with the SIINFEKL
minigene instead of full-length ovalbumin (Figure 3G).
Therefore, overexpression of any cytosolic peptidase
does not inevitably reduce antigen expression, but in-
stead the effects depend on the particular enzyme (and
perhaps the antigenic peptide).
Effect of TOP Overexpression on the Presentation
of Peptides Generated from Full-Length Proteins
Most MHC class I-presented peptides or their precur-
sors are generated by proteasomes during breakdown
of intracellular proteins (Cascio et al., 2001), and it is
possible that proteasome-derived peptides are deliv-
ered directly to the peptide transporter TAP on the ER
membrane (Hirsch and Ploegh, 2000), that components
of the proteasome complex protect antigenic peptides
from cytosolic peptidases, or that the proteasome-gen-
erated peptides might be preferentially bound by chap-
erones or other proteins that target them to the TAP
tation of SIINFEKL and KVVRFDKL, which were gener-
ated by proteasomes from whole ovalbumin, were af-
fected by the expression of TOP. TOP overexpression
markedly inhibited the presentation of both peptides
from ovalbumin in E36-Kbcells (Figures 3A and 3B) and
length influenza nucleoprotein (Figure 3C), which also
requires proteasomes (Mo et al., 1999). When TOP was
Effect of TOP on the Presentation of Peptides
in the ER
TOP is a cytosolic enzyme and is not detected in the
ER even after transfection (Saric et al., 2002; data not
shown). To test if the inhibition of antigen presentation
was in fact due to rapid hydrolysis of antigenic peptides
Figure 3. TOP Transfection Inhibits MHC Class I Presentation of Peptides from Full-Length Antigen Constructs
(A) Similar to Figure 1C except cells were transfected with pCDNA3 encoding a full-length ovalbumin cDNA instead of the SIINFEKL minigene.
These data come from the same experiment as in Figure 1C. Although it has previously been reported that higher numbers of peptide-MHC
complexes are generated from minigenes than whole protein antigens (Anton et al., 1997), we have consistently failed to observe this
phenomenon in our system, possibly because of the longer times of antigen expression or different APCs.
(B) Similar to Figure 1E except cells were transfected for 2 hr with full-length ovalbumin cDNA (5 ?g) instead of SIINFEKL. These data come
from the same experiment as in Figure 1E.
(C) Similar to Figure 1D except cells were coinfected for 6 hr with vaccinia recombinants encoding TOP or ?gal and full-length influenza
nucleoprotein (gift from J. Yewdell, NIH) instead of ASNENMETM.
(D) Similar to Figure 1B except that ovalbumin was expressed instead of SIINFEKL, and the plasmid and inserts were reversed (ovalbumin
was expressed from pTracerCMV, and TOP, ORP150, or no insert was expressed from pcDNA). Expressing ovalbumin from pcDNA, and TOP,
ORP150, or no insert from pTracer, gave essentially identical effects on SIINFEKL-H-2Kbgeneration. MFI were Bkg, 3.84; Vector, 90.3; ORP150,
107; and TOP, 28.2.
(E) The same experiment as Figure 3D, showing the GFP fluorescence of the total population. With pTracerCMV, about half the transfected
cells express high levels of GFP. Transfection efficiency, about 30%, was low in this experiment. The curves for TOP, ORP150, and Vector
are superimposed on one another.
(F) COS-Kbcells were cotransfected with full-length ovalbumin in pCDNA3, and a pTracer CMV plasmid encoding GFP (Vector), or GFP ?
human puromycin-sensitive aminopeptidase (PSA), and after 48 hr stained for SIINFEKL?Kbcomplexes with 25.D1.16 and analyzed by flow
cytometry. In this experiment pTracerCMV-TOP was also tested and inhibited antigen presentation by approximately 80%, as expected (data
not shown). MFI were Bkg, 6.2; PSA, 527; and Vector, 405 fluorescent units.
(G) Similar to Figure 3F except SIINFEKL minigene was expressed from the pcDNA3 vector. MFI were Bkg, 5.61; Vector, 233; PSA, 454
in the cytosol, we examined the effect of TOP transfec-
delivered directly into the ER, thus avoiding exposure to
cytosolic enzymes. A SIINFEKL minigene was expressed
causes the SIINFEKL peptide to be cotranslationally
transported into the ER (Anderson et al., 1991; Craiu et
al., 1997). The leader sequence then is removed by the
signal sequence peptidase acting by itself, or together
with the ER-resident aminopeptidase ERAP1 (Saric et
al., 2002; Serwold et al., 2002; York et al., 2002). Over-
Endopeptidase Limits Antigen Presentation
Figure 4. Overexpression of TOP Does Not
Reduce the Presentation of Peptides Gener-
ated in the ER or in Endosomes
(A) Similar to Figure 1B except cells were
transfected with a SIINFEKL minigene with
an N-terminal signal sequence instead of the
unmodified SIINFEKL minigene. These data
come from the same experiment as in Figure
1B. MFI were Bkg, 15.1; No OVA, 17; Vector,
1337; ORP150, 1395;and TOP ? ER-targeted
SIINFEKL, 1301 fluorescent units.
or vaccinia ?gal (?gal), incubated with the
indicated concentrations of ovalbumin for 4
hr, and then fixed with paraformaldehyde.
The presence of ovalbumin ? I-Adcomplexes
was detected by measuring the production
kevitz et al., 1983).
(C) Similar to Figure 1B except DAP-Kbcells
were used instead of E36-Kband the incuba-
tion with antigen was 4 hr instead of 1.5 hr.
targeted SIINFEKL (Figure 4A), even though it markedly
reduced the presentation of SIINFEKL generated in the
cytoplasm (Figures 1B and 1C). Thus, TOP transfection
specifically inhibits the presentation of cytoplasmically
derived peptides and does not nonspecifically affect
as MHC class I synthesis, assembly, or exocytosis (see
also Figures 4A, 5B, and 5E, described below).
Consequently, conditions that limit the supply of pep-
tides to MHC class I molecules (e.g., inhibition of the
proteasome [Rock et al., 1994] or of the TAP transporter
[Townsend and Trowsdale, 1993])) reduce MHC appear-
ance on the cell surface. If TOP is destroying large num-
MHC class I molecules on the cell surface. We therefore
analyzed the effect of TOP transfection on the total ex-
pression of MHC class I molecules on the cell surface.
We cotransfected into COS7 cells cDNAs for an MHC
class I molecule (either H-2Kbor HLA-A3) together with
TOP or a control vector, and after 48 hr measured the
expression of the MHC class I molecule on the trans-
fected cells. Overexpression of TOP caused a marked
reduction in the levels of H-2Kb(Figure 5A) and HLA-A3
(Figure 5C) molecules on the cell surface. To confirm
that this decreasein surface MHC classI molecules was
due to a reduction in the supply of antigenic peptides,
construct used in Figure 4A, which bypasses TOP in the
cytoplasm. In this situation, in which peptide supply to
the SIINFEKL binding H-2Kbis restored, TOP overex-
pression no longer reduces the levels of H-2Kbon the
cell surface (Figure 5B), whereas the levels of HLA-A3
(which does not bind SIINFEKL) remain low (Figure 5D).
These results indicate that most MHC-presented pep-
tides are susceptible to digestion by cytoplasmic pepti-
dases and again confirm that high intracellular levels of
TOP do not cause any general defects in MHC class I
assembly or transport.
In these experiments, MHC class I expression is re-
duced on the cell surface presumably because TOP
limits the supply of peptides needed to stabilize MHC
class I molecules and allow their movement from the
ER to the cell surface. To further test this point, we
investigated whether TOP overexpression reduced the
Effect of TOP on the Presentation on MHC Class II
Molecules of Peptides Generated
in Endosomal Compartments
presentation. In this pathway, peptides are generated
by proteolysis in the endocytic compartment (Germain
and Margulies, 1993), which does not contain TOP. L
cells (DAP) expressing the MHC class II molecule I-Ad,
and the p41 isoform of invariant chain (Peterson and
Miller, 1992), were both infected with vaccinia recombi-
with different concentrations of ovalbumin added to the
medium. It has previously been shown that transfected
L cells internalize exogenous ovalbumin, degrade it in
endocytic compartments, and present ovalbumin-derived
peptides on the transfected class II molecules (Diment,
1990; A. Sant, personal communication). Overexpres-
sion of TOP had little or no effect on the presentation
to a specific T cell hybridoma of a class II-presented
peptides generated from ovalbumin (Figure 4B), even
though in L cells it strongly inhibited the presentation
on MHC class I molecules of SIINFEKL from full-length
ovalbumin or a minigene (Figure 4C).
Effect of Overexpressing TOP on the Levels of Surface
MHC Class I Molecules
Newly synthesized MHC class I molecules are retained
in the ER until they bind peptides (York and Rock, 1996).
Figure 5. TOP Inhibits MHC Class I Assembly and Transport
(A) COS7 cells were cotransfected with H-2Kbin pcDNA3 and a pTracerCMV plasmid encoding GFP (Vector), OPR150 ? GFP (ORP150), or
TOP ? GFP (TOP). The H-2Kbtransfection was omitted in the background group (Bkg). After 48 hr the cells were stained with anti-H-2Kb(Y3)
and analyzed as described in Figure 1. MFI were Bkg, 22.7; Vector, 1922; ORP150, 1948; and TOP, 708 fluorescent units.
(B) Same as (A) except cells were also transfected with an ER-targeted signal sequence-SIINFEKL minigene. MFI were Bkg, 25.7; Vector,
1922; and TOP, 2151 fluorescent units.
(C) Similar to (A) except cells were transfected with pJFE14-HLA-A3 instead of H-2Kband were stained with anti-HLA-A3 instead of anti-H-
2Kb. MFI were Bkg, 25.6; TOP, 89.9; ORP150, 211; and Vector, 237 fluorescent units.
(D) Same as (C) except cells were also transfected with a signal sequence-SIINFEKL minigene. MFI were Bkg, 25.6; TOP, 93.4; and vector,
244 fluorescent units. All panels are from the same experiment.
(E) DAP-Kbcells infected with vaccinia-TOP (TOP), vaccinia-?gal (?gal), or uninfected (none) were labeled with35S-methionine and their MHC
class I molecules were immunoprecipitated from detergent lysates with an antibody that detects all free heavy chain (leftmost three lanes),
or with antibodies that detect only the indicated assembled MHC class I complexes (remaining lanes).
(F) The autoradiographs shown in (E) were scanned and densitometric analysis was performed.
assembly of stable MHC class I molecules. DAP-Kbcells
infected with recombinant vaccinia virus expressing
TOP were also found to have a marked reduction in their
surface expression of MHC class I molecules (data not
shown). These cells were infected with vaccinia virus
tosidase, and were then labeled with35S-cysteine and
methionine. The extent of assembly of the labeled MHC
class I molecules was investigated by immunoprecipita-
tion using antibodies specific for assembled MHC class
I molecules or the component heavy and light chains.
Compared to cells infected with control vaccinia virus,
TOP overexpression minimally reduced the synthesis of
MHC class I heavy chains (Figure 5E, leftmost three
lanes, and Figure 5F) or light chains (data not shown),
but it did cause a marked reduction in assembled class
I H-2Dk, H-2Kk, and H-2Kbheterodimers (Figure 5E and
5F). The extent of the reduction in assembly varied for
different alleles, ranging from approximately 35% of
control assembly for H-2Dkto about 55% of control
for H-2Kk. In other words, TOP overexpression (without
affecting the synthesis of MHC class I heavy or light
Endopeptidase Limits Antigen Presentation
chains) reduces the assembly of stable MHC class I
molecules, presumably by causing the destruction of
many antigenic peptides in the cytosol. The difference
differing susceptibility of the peptides that bind to the
trol or TOP siRNA and examined the synthesis and
assembly of MHC class I molecules in a pulse-chase
experiment. Although the siRNA did not affect the syn-
thesis of MHC class I heavy chain (Figure 7A, upper
panel), TOP siRNA increased the assembly of stable
and Figure 7B).
Elimination of TOP from HeLa-Kbcells with siRNA in-
creased the levels on the cell surface of mouse H-2Kb
(Figure 7C) by about 80%, and of human HLA-A, B, and
C molecules (Figure 7D) about 2-fold, as measured by
flow cytometry. This increase in surface expression cor-
related well with the 2-fold increase in MHC class I as-
sembly measured in immunoprecipitations (Figures 7A
and 7B). Again, this effect was precisely opposite to
that observed when TOP was overexpressed in cells
(Figure 5). Therefore, under physiological conditions
TOP must destroy many antigenic peptides and gener-
ally limit antigen presentation.
Effect of Preventing TOP Expression
on MHC Class I Presentation
These results indicate that increasing the cellular con-
sol reduces MHC class I presentation. In HeLa cell ex-
tracts, the endogenous TOP is the peptidase primarily
responsible for the rapid destruction of many synthetic
antigenic peptides (Saric et al., 2001), and 9–17-residue
proteasome products (T.S. et al., unpublished data). To
determine whether this enzyme, at the levels found nor-
mally in cells, also hydrolyzes antigenic peptides and
thus limits antigen presentation in vivo, we tested in
Hela-Kbcells whether selective elimination of TOP with
small interfering RNA (siRNA) oligonucleotides (Elbashir
et al., 2001) would increase antigen presentation. As a
control, we used siRNA directed against mouse TOP
whose sequence in this region is different from that of
human TOP. Transfection of this control siRNA had no
effect on expression of human TOP. However, when
human TOP siRNA was transfected into HeLa-Kbcells,
TOP mRNA was reduced by 85%–90% compared to
control siRNA-treated cells (Figure 6A) and TOP protein
was reduced at least by 90% (the limit of sensitivity of
the antibody), while several other assayed mRNAs (e.g.,
?-actin and ERAP1 [data notshown]) and proteins (such
as calreticulin [Figure 6B]) were not affected. The trans-
fection efficiency of siRNA was estimated at 95% (data
not shown), so that even if TOP was completely elimi-
nated from transfected cells, 5% of control TOP levels
would still be present in protein or RNA preparations.
HeLa-Kbcells were initially transfected with control or
TOP siRNA and 3 days later were transfected with a
plasmid coexpressing a SIINFEKL minigene and GFP.
Quantification of SIINFEKL-Kbcomplexes on trans-
fected cells revealed that reducing TOP expression en-
hanced the generation of these complexes nearly 2-fold
(Figure 6C). Similarly, the presentation of SIINFEKL was
approximately doubled in TOP siRNA-treated cells
results, which are opposite to those seen when TOP
expression is increased (Figure 1), indicate that under
tation of SIINFEKL. Inhibition of TOP did not affect the
expression of transfected influenza hemagglutinin (Fig-
ure 6E), showing that the effect on SIINFEKL presenta-
tion was not due to general effects on protein ex-
Together, the present findings demonstrate that TOP
limits MHC class I antigen presentation by destroying
peptides in the cytoplasm. When overexpressed, TOP
inhibited only the presentation of peptides generated in
tion of a peptide delivered directly into the ER or the
presentation on MHC class II molecules of a peptide
generated in endocytic compartments. Also, TOP over-
expression did not reduce overall protein synthesis, the
production of MHC heavy or light chains, or the appear-
ance on the cell surface of an unrelated membrane pro-
tein, influenza hemagglutinin. Therefore, TOP’s effects
are highly selectiveand are not dueto some nonspecific
or toxic effect on the cells.
Increasing the cellular content of TOP inhibited the
presentation of three well-defined antigenic peptides. It
also reduced generally the assembly and surface ex-
pression of many class I molecules, and this was due
to a reduction in the supply of antigenic peptides be-
cause it was reversed when a MHC class I binding pep-
tide was targeted by a signal sequence into the ER. It
is very unlikely that TOP reduces peptide supply to the
ER by inhibiting the function of the TAP transporter di-
rectly because pure TOP only hydrolyzes oligopeptides
and not proteins (Barrett et al., 1995). Therefore, the
inhibition of antigen presentation when TOP levels rise
is almost certainly due to accelerated degradation of
proteasome products in the cytosol, which is consistent
with the finding that TOP rapidly degrades many pep-
tides in cell extracts (Saric et al., 2001). Moreover, rapid
destruction of antigenic peptides by TOP must occur
normally in cells, since eliminating TOP with siRNA, or
by introducing selective inhibitors of TOP enzyme (data
not shown), enhanced the assembly and transport to
the cell surface of multiple MHC class I molecules, as
well as the presentation of a specific antigenic peptide.
Elimination of TOP with siRNA did not affect cell growth
or viability (data not shown), endogenous proteins were
synthesized at normal rates (e.g., MHC class I heavy
chain: Figure 7A, upper panel), and expression of a
transfected protein (influenza hemagglutinin: Figure 6E)
Effect of Preventing TOP Expression on MHC Class I
Molecule Assembly and Surface Expression
If TOP at physiological levels constantly destroys anti-
genic peptides or their longer precursors, then eliminat-
ing TOP should increase the supply of antigenic pep-
tides, which should in turn result in increased assembly
and surface expression of MHC class I molecules. To
test this hypothesis, we treated HeLa-Kbcells with con-
Figure 6. Loss of TOP Expression Enhances the Presentation of SIINFEKL on H-2Kb
(A) Four days after treatment with siRNA directed against human TOP (hTOP1; hTOP1 ? 3) or with control siRNA (mTOP1: directed against
mouse TOP in a region not homologous to human TOP), mRNA was extracted from HeLa-Kbcells and cDNA was prepared. Real-time PCR
for human TOP, and for human ?-actin, was performed on the cDNA and on plasmids containing the appropriate genes for standardization.
The copy number of human TOP was normalized to ?-actin expression. Shown is the average of three experiments ?/? standard deviation.
(B) Hela-Kbcells were treated with siRNA as in (A) and detergent lysates were prepared. 3-fold serial dilutions of the lysates were immunoblotted
with antibodies specific for TOP (upper panel) or calreticulin (lower panel).
(C) HeLa-Kbcells were transfected with the siRNA hTOP1, or with mTOP1 as a control. Three days later, the cells were transfected with
pTracerSR? expressing the minigene SIINFEKL as well as GFP. After an additional day the cells were stained for SIINFEKL ? Kbcomplexes
(25.D1.16), and GFP-positive cells were analyzed by flow cytometry as described in Figure 1. No OVA: cells transfected with pTracerSR?
vector only and stained with 25.D1.16. Background fluorescence (based on staining with an irrelevant antibody) was very similar to that of
the No OVA group. MFI were Bkg, 12.2; TOP siRNA, 3826; and Ctrl siRNA, 2167 fluorescent units.
(D) Similar to (A) except that cells were transfected with pTracerSR? expressing ovalbumin. MFI were Bkg, 38.8; hTOP, 2549, and Ctrl siRNA,
1647 fluorescent units.
(E) HeLa-Kbcells were transfected with the siRNA hTOP1, or with mTOP1 as a control. Two days later, the cells were transfected with
pTracerCMV (Bkg), or with pTracerCMV expressing influenza hemagglutinin. After an additional 2 days the cells were stained for hemagglutinin
and analyzed by flow cytometry. MFI were Bkg, 6.01; Ctrl siRNA, 5981; and hTOP siRNA, 5848 fluorescent units.
was unaffected by TOP inhibition. Although TOP nor-
mally limits the availability of most peptides for antigen
presentation, other still-unidentified cytosolic endopep-
tidases and aminopeptidases can hydrolyze antigenic
peptides in cell extracts (Saric et al., 2001) (T.S. et al.,
unpublished data) and presumably invivo, and may also
help limit the efficiency of antigen presentation. Thus,
antigenic peptides (and their longer precursors) appear
to resemblethe greatmajority ofpeptides releasedfrom
proteasomes in that they are subject to further rapid
degradation by cytosolic peptidases.
Although TOP has been known and characterized
all mammalian cells (Carvalho and Camargo, 1981;
Orlowski et al., 1983; Camargo et al., 1997), its physio-
logical role(s) was unknown. Our data and related bio-
chemical studies on cell extracts (Saric et al., 2001) (T.S.
et al., unpublished data) clearly demonstrate that this
endopeptidase is responsible for hydrolyzing oligopep-
tides in the cytosol of cells. In the final steps in the
pathway for degradation of proteins, TOP appears to
cleave proteasome products of the sizes of antigenic
Endopeptidase Limits Antigen Presentation
Figure 7. Loss of TOP Expression Enhances Assembly and Surface Expression of Class I Molecules
(A) HeLa-Kbcells were transfected with the siRNAs hTOP1 and hTOP3, or with mTOP1 as a control. Three days later, the cells were radiolabeled
for 10 min and chased for 0, 15, or 45 min before lysis and immunoprecipitated with antibodies specific for unfolded heavy chain, or HLA-A,
B, and C assembled with ?2-m, as described in Experimental Procedures.
(B) The autoradiographs from (A) were scanned and analyzed by densitometry.
(C) Hela-Kbcells were transfected with the siRNAs hTOP1 and hTOP3, or with mTOP1 as a control. Four days later, the cells were stained
with anti-H-2Kb(Y3) and phycoerythrin-anti-Ig antibodies and analyzed by flow cytometry as described in Figure 1. Bkg, background fluores-
cence, based on staining with an irrelevant antibody. MFI were Bkg, 4; TOP siRNA, 189; and Ctrl siRNA, 102 fluorescent units. (C) Similar to
(B) except that cells were stained with anti-HLA-A, B, C (PA2.6). MFI were Bkg, 4; TOP siRNA, 413; and Ctrl siRNA, 205 fluorescent units.
peptides or their precursors (9–17 residues) to pieces,
most of which are rapidly hydrolyzed by exopeptidases
to amino acids.
An important implication of our findings is that the
extent to which any particular peptide is presented on
MHC class I molecules is a function both of its rate of
production and cytosolic destruction. A similar interplay
between peptide generation and destruction occurs in
the endocytic compartment for MHC class II presenta-
tion (Vidard et al., 1991). Therefore, blocking TOP or
mutating sequences of antigens so that they become
approach for enhancing antigen presentation and im-
possible that overproduction of TOP or other cytosolic
peptidases may be a mechanism by which some can-
cers or viruses evade immune destruction, or may even
represent a novel approach for suppressing immune
response in transplanted cells or organs.
These findingsargue that antigenic peptidesand their
precursors are not protected by molecular chaperones
from intracellular degradation in the cytosol or that if
such protective mechanisms do exist, they are not very
effective. How then do any antigenic peptides escape
destruction? In fact most probably don’t and are de-
stroyed before binding to MHC molecules. Thus after
release from proteasomes, antigenic precursors con-
front a kinetic competition between being cleaved by
cytosolic peptidases, such as TOP, and uptake into the
ER. It remains possible that there is some protection by
cytosolic binding proteins (e.g., heat shock proteins)
(Srivastava et al., 1998). If so TOP, by destroying the
free peptide, may cause dissociation of bound peptides
and then further hydrolysis. The present findings and
do not support the earlier report that TOP strongly binds
that TOP actually functions to facilitate peptide delivery
to TAP (Portaro et al., 1999; Silva et al., 1999). The rea-
sons for these discrepant earlier observations are still
not clear (for a critical discussion, see Saric et al., 2001).
While the destruction of antigenic peptides by TOP
and presumably other cytosolic peptidases can reduce
MHC class I presentation and thus may appear to be
deleterious, this degradative process is probably an un-
avoidable consequence of the immune system utilizing
a phylogenetically older catabolic pathway whose prin-
cipal function in all cells is to break down proteins into
amino acids. On the other hand, for efficient generation
of antigenic peptides, it may also be important that pro-
teasome products are free in the cytosol, since some
longer precursors to antigenic peptides have to be
trimmed by aminopeptidases to the MHC class I-pre-
sented epitopes (Beninga et al., 1998; Craiu et al., 1997;
Mo et al., 1999; Stoltze et al., 2000). Therefore, to the
precipitation, and Western blotting were performed as previously
described (York et al., 2002).
extent that this N-terminal trimming occurs in the cyto-
plasm, peptides need to be accessible to peptidases.
In addition, this rapid destruction of antigenic pep-
in the regulation of immune responses. Under normal
conditions most cells express very low levels of MHC
class I, presumably because high constitutive expres-
sion of these molecules might increase the danger of
autoimmunity. In the absence of infection, peptide de-
inflammatory mediators, especially interferon-?, MHC
class I presentation is enhanced, in part by inducing
new proteasome subunits and the proteasome activator,
PA28 (REG). The immunoproteasomes and complexes
containing PA28 tend to produce more N-extended ver-
sions of antigenic peptides than “non-immune” protea-
somes (Cascio et al., 2002). Because these precursor
peptides are present in greater amounts and tend to
be degraded more slowly by TOP and other cytosolic
peptidases than the mature epitopes, they are more
likely to reach the ER and after trimming be presented
on MHC class I molecules. (Goldberg et al., 2002; Saric
et al., 2001).
Viability and Protein Synthesis Assays
Cell viability was determined by trypan blue exclusion. Protein syn-
thesis was measured by the incorporation of
TCA insoluble fractions as previously described (Rock et al., 1994).
Antigen Presentation Assays
Transfected and/or infected E36-Kb, E36-Db, or DAP-KbAPCs were
fixed with 1% paraformaldehyde and incubated with antigen-spe-
cific T cell hybridomas as described (Craiu et al., 1997). After 18 hr,
the production of IL-2 from the hybridomas was measured using
CTLL2 cells as described (Craiu et al., 1997). These APCs were
chosen because they present well to the murine T-T hybridomas
and are highly infectable with vaccinia virus. SIINFEKL?Kbcom-
plexes were quantified on COS-Kband Hela-Kbcells by immunofluo-
rescence using a specific mAb (25.D1.16) (Porgador et al., 1997)
because these xenogenic cells present poorly to our murine T-T
hybridomas but worked well in this staining assay.
The monoclonal antibody 15.5.5 recognizes ?2-microglobulin (?2-
m)-associated H-2Dk(Ozato et al., 1980); 16.1.1N recognizes ?2-
m-associated H-2Kk(Ozato et al., 1980); and Y3 recognizes ?2-m-
associated H-2Kb(Jones and Janeway, 1981). A rabbit antiserum
that recognizes unfolded mouse heavy chain was kindly provided
by Stan Nathenson (Albert Einstein College of Medicine). The mono-
clonal antibody W6/32 (Parham et al., 1979) recognizes HLA-A, B,
and C alleles only in association with ?2-m. HC10 (Stam et al., 1986)
recognizesunfolded HLA-BandC. Anti-TOPantiserumand theanti-
TOP monoclonal antibody IVD6 have been previously described
(Conn et al., 1996).
COS-Kbcells, HeLa-Kb, DAP-Kb, E36-Kb, and E36-Db cells were pre-
viously described (Michalek et al., 1993; Craiu et al., 1997; Mo et
chain were a gift from Jim Miller (University of Rochester).
mRNA was extracted from cells with an RNeasy kit (Qiagen, Valen-
cia, CA). cDNA was prepared using SuperScript II (Invitrogen). Real-
time PCR using Taq (Invitrogen) and an iCycler (BioRad, Hercules,
CA) was performed using primers directed against human TOP
(ACTTCCCCCTCCTGAAGAAA and CCTTGAGGATAGCGCAGTTC)
or ?-actin (CGAGGCCCAGAGCAAGAGAG and CGGTTGGCCTT
Transfections and Infections
COS7 or COS-Kbcells were transiently cotransfected with pTrac-
erCMV (Invitrogen) or pTracerSR? (pTracerSV40 [Invitrogen] with an
SR? promoter [Takebe et al., 1988]) and pCDNA3 (Invitrogen) or
pJFE14 (Elliott et al., 1990) (1–2 ?g of each plasmid) expressing
enzymes, antigens, or MHC class I molecules, using FuGene6
and incubated for 24–48 hr.
E36-Kb, E36-Db, or DAP-KbAPCs were infected with either vac-
cinia-TOP or, as a control, vaccinia-?gal (MOI 10) alone or in some
experiments together with vaccinia-T7 polymerase (MOI 10). Under
these conditions, 99 ? 0.28% of the cells were infected with the
vaccinia vectors. After 1.5–2 hr, antigen was introduced by infection
with a vaccinia recombinant. Alternatively, in the cells infected with
vaccinia-T7, antigen was introduced by transfection with an antigen
cDNA under the control of a T7 promoter in a pBluescript plasmid
(Stratagene, La Jolla, CA) (1 ?g) using Lipofectin liposomes (In-
vitrogen) as previously described (Craiu et al., 1997); in this system
the level of antigen expressed in replicate groups transfected with
a pBluescript antigen plasmid is almost identical (SD for the fluores-
cence intensity varying by less than 5% of the mean) and is propor-
tional to the amount of plasmid transfected (Craiu et al., 1997).
Enzymatic activities in cell extracts were measured as described
(Saric et al., 2001). The substrates used were, for TOP, Mcc-Pro-
Leu-Gly-Pro-D-Lys-Dnp-OH; for AP, Leu-Amc; for POP, Z-Gly-Pro-
Amc; and for TPP-II, Ala-Ala-Phe-Amc.
A.X.Y.M was supported by a training grant from the NIH. We thank
Dr. Shih-Chung Chang (Harvard Medical School) for performing
assays measuring TOP activity, and Drs. Lou Hersh and Michael
Thompson (University of Kentucky) for PSA cDNA. We are grateful
to SarahTrombley for expertassistance in preparation ofthis manu-
script and to Jennifer Keyes and Janice Favreau for technical assis-
Received: July 12, 2001
Revised: December 18, 2002
RNA oligonucleotides hTOP1 (directed to positions 645-663 of hu-
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(Elbashir et al., 2001). Hela-Kbcells were transfected as described
(York et al., 2002). In some experiments, hTOP1 and hTOP3 oligonu-
cleotides were cotransfected.
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