From the Center for Immunotherapy of Cancer and Infectious Diseases, University of Connecticut School of Medicine, Farmington, Connecticut 06030
Received for publication, December 21, 2000, and in revised form, February 20, 2001
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ABSTRACT |
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The studies reported here bear on the events in
the cytosol that lead to trafficking of peptides during antigen
processing and presentation by major histocompatibility complex (MHC) I
molecules. We have introduced free antigenic peptides or antigenic
peptides bound to serum albumin or to cytosolic heat shock proteins
hsp90 (and its endoplasmic reticular homologue gp96) or hsp70 into the cytosol of living cells and have monitored the presentation of the
peptides by appropriate MHC I molecules. The experiments show that (i)
free peptides or serum albumin-bound peptides, introduced into the
cytosol, become ligands of MHC I molecules at a far lower efficiency
than peptides chaperoned by any of the heat shock proteins tested and
(ii) treatment of cells with deoxyspergualin, a drug that binds hsp70
and hsp90 with apparent specificity, abrogates the ability of cells to
present antigenic peptides through MHC I molecules, and introduction of
additional hsp70 into the cytosol overcomes this abrogation. These
results suggest for the first time a functional role for cytosolic
chaperones in antigen processing.
Cellular proteins undergo degradation in the cytosol, and the
resulting peptides are transported into the endoplasmic reticulum (ER),1 generally through
transporter associated with antigen processing (TAP). Within the ER,
the peptides are charged onto MHC I molecules. One of the key
unresolved questions in this scheme pertains to the mechanism through
which peptides are channeled to the TAP or other transporters. Although
peptides are generated in the cytosol, there is little evidence that
the cytosol harbors free peptides. It has been proposed that the
peptides exist in association with peptide-binding proteins in the
cytosol and the ER (1, 2). Because heat shock proteins (HSPs) are known
to chaperone a wide array of molecules (3) and because immunological
and structural evidence exists that HSPs chaperone antigenic peptides (see Ref. 4 for review), it was suggested that HSPs are the peptide-binding proteins that transport peptides (1, 2). This view has
received little formal attention in the form of support or rejection,
although no alternative mechanisms of peptide traffic have been
suggested. Nonetheless, evidence has continued to accumulate that
(a) HSPs are associated with peptides from a wide spectrum
of antigens, including tumor antigens (5, 6), viral antigens (7), model
antigens (8-10), and minor H antigens (8), and that (b) the
repertoire of peptides associated with the HSP of the ER is dependent
upon the functional status of TAP (9).
In this report, we address the issue functionally and ask if the
chaperoning of peptides in the cytosol by HSPs confers on the
HSP-chaperoned peptides any advantage not available to unchaperoned peptides in terms of their presentability by MHC I molecules.
Brefeldin A Treatment--
EL4 cells were treated with brefeldin
A (BFA) at two different concentrations in succession to respectively
block the MHC I pathway of antigen presentation (6.0 µg/ml for 3 h) and to maintain the block (0.6 µg/ml for up to 12 h).
Maintenance of the BFA block did not affect CTL function during the CTL
assay. EL4 cells, untreated or treated with BFA at these
concentrations, were analyzed by FACScan to show maximal decreases
(~40%) in surface expression of MHC I after 20 h (data not
shown). BFA-treated cells were loaded with protein and used as targets
in the CTL assay, as described, in the presence of BFA.
Cell Lines, Mice, and Reagents--
The T-Ag-transformed cell
lines SVB6 and PS-C3H were obtained from Prof. S. S. Tevethia and
have been previously described (11) The VSVNP-transfected EL4 cell
line, N1, was obtained from Dr. Lynn Puddington and has been previously
described (12). EL4 cells and the TAP-dysfunctional cell line, RMA-S,
were obtained from Prof. S. Nathenson. The RMA cell line has been
previously described (13).
All chemicals were purchased from Sigma Chemical Co. unless otherwise
specified. HL-1 and RPMI media, together with pyruvate, glutamine,
penicillin-streptomycin, and non-essential amino acids were purchased
from Life Technologies, Inc. RPMI containing 5% fetal calf serum
(Intergen) and 1% each of pyruvate, glutamine, penicillin-streptomycin, and non-essential amino acids is subsequently referred to as complete RPMI.
Antibodies--
HSPs were detected by immunoblotting with
specific antibodies: gp96 (rat monoclonal antibody SPA-850, clone
9G10); cytosolic hsp70 (mouse monoclonal antibody SPA-820, clone
N27F3-4 recognizes constitutive hsp73 and inducible hsp72); hsp90 (rat
monoclonal antibody SPA-845, clone 1R2D12p90). All these antibodies
were purchased from StressGen Biotechnologies Corp., Victoria, Canada. Anti-Kb, anti-Db, anti-Dd, or
anti-LFA-1 (clones AF6-88-5, KH95, 34-2-12, and 2D7,
respectively)-fluorescein-conjugated monoclonal antibodies were
obtained from PharMingen (San Diego, CA).
Cellular Loading of Proteins or Peptides--
To prepare
proteins (gp96, hsp70, hsp90, or SA; complexed or not) for loading, the
indicated amount of protein was incubated with DOTAP
(N-[-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (C43H83NO8S)) (Roche
Molecular Biochemicals) at a 3:2 ratio (microgram amounts) for 15 min
at room temperature. In all loading experiments, 1.5 × 106 cells (EL4, RMA, or RMA-S) were washed three times with
serum-free HL-1 media and then incubated in 1 ml of HL-1 media with a
protein:DOTAP combination for 4-4.5 h at 37 °C. Control cells were
either mock-loaded by incubating 1.5 × 106 cells in
the same amount of DOTAP alone or were incubated with protein alone in
the absence of DOTAP (pulsed cells). After loading (or mock loading for
controls), cells were washed three times with HL-1 media and once with
complete RPMI. Where indicated, loaded, mock-loaded, or pulsed cells
were used as targets in CTL assays. Loading efficiencies of gp96,
hsp70, hsp90, or serum albumin alone were the same, and
inter-experimental values did not vary significantly. Free peptides
were loaded into cells using the same protocol.
CTL Assays--
CTL assays were carried out as follows. Briefly
2 × 103 51chromium (supplied as
Na2CrO4; ICN) labeled target cells in
100 µl of complete RPMI were added to various dilutions of T-Ag or
VSV8-specific CTL in 100 µl of complete RPMI, as indicated by the
effector-to-target ratios. Effectors and targets were cultured in
v-bottomed 96-well plates for 4 h. Supernatants (50 µl) were
harvested, mixed with scintillation fluid, and counted in a 1450 MicroBeta Trilux liquid scintillation counter (Wallac Inc.). Percent
specific 51Cr release was measured as follows,
Maximum and spontaneous releases were measured by culturing
2 × 103 labeled target cells in lysis buffer (0.5%
Nonidet P-40, 10 mM Tris, 1 mM EDTA, 150 mM NaCl) and complete RPMI, respectively, for 4 h.
VSV8-specific CTL were obtained by dual immunizations of C57BL/6 mice,
1 week apart, with N1 cells. Spleen cells were harvested 1 week after
the second immunization, restimulated in culture with irradiated N1
cells, and cloned by limiting dilution (14). The specificity of the CTL
clone was tested by cold target inhibition and antibody blocking
experiments. This CTL clone was shown to be specific for the VSV8
peptide (NH2-RGYVYQGL-COOH) bound to Kb
molecules. A similar strategy, with SVB6 cells, was used to obtain the
T-Ag-specific CTL clone. This clone was shown to be specific for the
9-mer peptide (NH2-AINNYAQKL-COOH), previously named
epitope 1 (11).
Flow Cytometry Analysis of DSG-treated Cells--
N1 cells were
irradiated (5000 rads) and allowed to recover in AIM V medium with or
without DSG for 48 h at 37 °C or 25 °C. Half of the cells
incubating at 25 °C were then placed at 37 °C for an additional
8 h. One group of the cells not treated with DSG but incubated at
25 °C for 48 h was placed at 37 °C in the presence of DSG
for 8 h. Cells (1 × 106) were then stained at
4 °C for 40 min with anti-Kb, anti-Db,
anti-Dd, or anti-LFA fluorescein-conjugated antibodies and
analyzed on a FACScan flow cytometer purchased from Becton Dickinson
(San Jose, CA).
Immunofluorescence--
Cells were fixed with 4%
paraformaldehyde, permeabilized with 0.5% saponin and probed with
anti-VSV glycoprotein Cy3-coupled antibody (Sigma). Cells were
visualized using a Zeiss LSM confocal microscope.
Infection of Cells with Vesicular Stomatitis
Virus--
Vesicular stomatis virus (VSV) was obtained from Advanced
Biotechnology (Columbia, MD). Meth A or EL4 cells were incubated with
10 plaque forming units of VSV per cell for 1 h at 37 °C in
plain RPMI and allowed to recover in RPMI with 10% fetal calf serum
for 4 h. Cells were washed three times in PBS (10 mM
phosphate buffer, 150 mM NaCl, 2.7 mM KCL, pH
7.4). gp96 was then purified from these cells as described below.
In Vitro Reconstitution of Protein·Peptide
Complexes--
The following peptides were used (underlined
sequences represent the precise MHC I binding epitope):
unextended MHC binding 9-mer, NH2-AINNYAQKL-COOH; T-Ag
20-mer (N terminus extended), NH2-FFLTPHRHRVS- AINNYAQKL-COOH; T-Ag 20-mer
(N+C termini extended), NH2-RHRVSAINNYAQKLCTFSFL-COOH;
T-Ag 20-mer (C terminus extended), NH2-AINNYAQKLCTFSFLICKGV-COOH.
Peptides were synthesized by Genemed to >95% purity as determined by
high pressure liquid chromatography. The unextended MHC I binding 9-mer
peptide is identical to epitope I of the T-Ag (11). The T-Ag 9-mer
stabilized MHC I molecules on RMA-S cells and sensitized targets for
lysis by the T-Ag-specific CTL. All three T-Ag 20-mer peptides failed
to bind MHC H-2Db as determined by their inability to
stabilize empty MHC molecules on the surface of RMA-S cells and their
inability to sensitize target cells for lysis by T-Ag-specific CTL.
Purified gp96 or hsp90 was incubated, at the indicated amount, with
peptide at a protein-to-peptide molar ratio of 1:50 in 700 µl of PBS
for 10 min at 50 °C and incubated for a further 30 min at room
temperature. The peptide concentration used for complexing was
10 Inhibition of Proteasome Function--
EL4 cells
(107) in complete RPMI were treated for 2 h with 100 µM of the proteasome inhibitor
N-acetyl-L-leucinyl-L-leucinal-L-norleucinal (LLnL) in Me2SO or with 0.002% Me2SO alone. In
other experiments, EL4 cells were treated with 100 µM
lactacystin dissolved in Me2SO for 1 h. In both cases,
the treated cells were constantly in the presence of the inhibitor
during loading with protein. FACScan analysis of inhibitor-treated
cells showed greater than 35% decrease in cell surface MHC I
expression after 20 h confirming inhibition of MHC I trafficking.
Purification and Identification of HSPs--
hsp70 and gp96 were
purified from cells according to previously described methods (16, 17).
hsp90 was purified according to the protocol of Denis (18) with minor
modifications. Briefly, 100,000 × g supernatants were
obtained from cell lysates and applied to a Mono Q column (Mono Q HR
16/10, purchased from Amersham Pharmacia Biotech and attached to the
BIOCAD, Perseptive Biosytems).
Treatment of MLTC and N1 Cells with
15-Deoxyspergualin--
15-Deoxyspergualin (DSG) was a gift from Dr.
S. Nadler at Bristol-Myers Squibb Co. (Wallingford, CT). Lyophilized
DSG was dissolved in PBS and stored in aliquots at a concentration of 10 mg/ml at Demonstration of the Experimental System to Introduce Molecules
into the Cytosol--
The cationic liposome, DOTAP, was used to
introduce HSP·peptide complexes or free peptides into the cytosol.
Distinct properties of the detergents Nonidet P-40 and Saponin were
used to demonstrate that DOTAP-loaded gp96 enters the soluble,
non-vesicular, cytosolic compartment of the cells (Fig.
1). Although the cytosolic HSPs are of
primary interest in this study, gp96 was used as a test case, because
its distinct non-cytosolic localization (in the ER) permitted
determination of the compartment into which the HSP·peptide complexes
were being introduced, as will become clear from the following. The
gp96/DOTAP-loaded cells were lysed with each of two detergents. Lysis
of live cells with 0.5% Nonidet P-40 leads to solubilization of all
non-nuclear membranes, whereas lysis with 0.01% Saponin results in
solubilization of plasma membranes but not internal membranes (19). The
lysates were centrifuged to obtain the solubilized components, which
were analyzed for the presence of gp96 by immunoblotting: The internal
resident gp96 is detected in the Nonidet P-40-lysates of non-loaded
cells (Fig. 1, lane 1) but not in the Saponin-lysed
non-loaded cells (Fig. 1, lanes 2 and 3), because
gp96 is a luminal component of the ER compartment, which remains
impervious to Saponin. gp96 is not detected in cells treated with DOTAP
without gp96 (lane 2) or gp96 without DOTAP (lane
3). The only instance where gp96 is detectable in the
Saponin-solubilized cells is if it has been introduced along with DOTAP
into cells (lane 4), i.e. from an exogenous
source. As additional controls, all samples tested predictably positive
for the cytosolic chaperone hsp70 (Fig. 1, bottom panel, lanes 1-4). Thus, DOTAP-mediated delivery of gp96 (and by
deduction other proteins) into cells introduces them into the cytosolic compartment. Similar results were obtained with introduction of labeled
peptides by DOTAP. Quantitative analysis of exogenously introduced
radiolabeled proteins through DOTAP indicated that ~5% of the
DOTAP-loaded protein is introduced into the cytosol and that >96% of
this 5% is detected in a soluble, non-vesicular, cytosolic compartment
of the cells (data not shown).
HSP-chaperoned Peptides Introduced into the Cytosol Become Ligands
for MHC I--
As discussed in the previous section, the cytosolic
chaperones hsp90 and hsp70 are of primary interest for the studies
described all through this report. However, the ER chaperone gp96 was
also used in all studies, primarily because (i) gp96 was used for the demonstration that DOTAP introduces proteins into the cytosol, (ii)
gp96 is highly homologous (protein sequence homology of 50%) (see Ref.
1) to the cytosolic chaperone hsp90, and (iii) considerable immunological and structural information on gp96-peptide interaction is
already available (see Ref. 4).
gp96, purified from the T-Ag-transformed cell line SVB6, and
chaperoning T-Ag-derived peptides was loaded into EL4 cells by DOTAP.
Presentation of T antigen-derived peptides by MHC I molecules of EL4
cells was monitored by specific lysis of DOTAP-loaded cells using a CTL
clone specific for epitope 1 of the T-Ag (11) as described under
"Experimental Procedures." The T-Ag-derived peptides were present
in gp96 preparations and were observed to be efficiently re-presented
in this assay (Fig. 2A).
Percentage of lysis of the loaded cells increased with increasing
amounts of gp96 loaded into the cells. No lysis was observed with less
than 25 µg of gp96. In parallel control experiments, EL4 cells were
pulsed, in the absence of DOTAP (as opposed to loaded) with gp96, to
determine if there is extracellular exchange of peptides between gp96
and surface MHC I molecules on EL4 cells. No surface charging was detected.
SVB6-derived hsp70 or hsp90 preparations were also loaded into EL4
cells in increasing doses. Antigen-specific recognition of the loaded
cells by CTLs was observed when either hsp70 or hsp90 was loaded (Fig.
2A), indicating that similar to gp96, hsp70 or hsp90 donate
their chaperoned peptides to become ligands for MHC I molecules. Again,
lysis of cells was dependent on the amount of HSP loaded by DOTAP.
Although peptides chaperoned by all three HSPs could become ligands for
re-presentation by MHC I molecules, the efficiency of doing so was
different for each HSP. For comparable lysis (~40%) of HSP-loaded
cells, 100, 250, and 500 µg of gp96, hsp70, and hsp90, respectively,
were required. Approximate amounts of HSP, below which no lysis was
detected were 25, 180, and 400 µg for gp96, hsp70, and hsp90, respectively.
A second, well characterized antigenic system, the Vesicular Stomatitis
Virus (VSV) system, was used to test the generality of the observation
in the T-Ag system. VSV nucleoprotein (VSVNP) derived peptides
chaperoned by gp96 or hsp70 (purified from the VSVNP-transfected cell
line N1 (12) are effectively re-presented and recognized by
VSVNP-specific CTL after the respective HSPs are introduced into the
cytosol of EL4 cells by DOTAP (Fig. 2B). To demonstrate that
lysis by VSVNP-specific CTL, of cells loaded with HSPs, is
peptide-dependent, equivalent amounts of peptide-free hsp70, obtained by ATP treatment of N1-derived hsp70 preparations (15),
were delivered into EL4 cells. No lysis of EL4 cells loaded with
peptide-depleted hsp70 preparations was observed (Fig. 2B). Furthermore, HSP preparations not carrying VSVNP-derived peptides (EL4-derived HSPs) (Fig. 2B), did not render loaded cells
susceptible to VSVNP-specific CTLs, with any amount of HSP loaded. The
results imply that presentation and consequent cell lysis are both
peptide-dependent and -specific and require intracellular
processing of the HSP·peptide complexes.
HSP-chaperoned Peptides Are Presented >100-fold More Efficiently
Than Free Peptides--
It is difficult to monitor and quantify
presentation of specific antigenic peptides in naturally derived
HSP·peptide complexes. To quantitate the efficiency of
re-presentation of specific HSP-chaperoned peptides, HSPs reconstituted
in vitro with known quantity of antigenic peptides or their
extended versions were used. The Db-restricted 9-mer
epitope I of the SV40 T-Ag protein (NH2-AINNYAQKL-COOH), or
20-mer peptides extended on the NH2 terminus, COOH
terminus, or both termini (Fig.
3A) were complexed to HSPs
gp96, hsp90, or hsp70, or a control peptide-binding protein serum
albumin (SA) (15 and "Experimental Procedures"). Peptides thus
complexed (~10 Re-presentation of HSP-chaperoned Peptides Requires Functional
Proteasomes, Is TAP-dependent, and Is Brefeldin
A-sensitive--
The cytosolic proteasomes have been implicated as the
primary producers of peptide ligands for MHC I molecules (for review see Refs. 20, 21). Because DOTAP-mediated loading of cells with the
HSP·peptide complexes results in presentation the peptides by MHC I,
we tested the requirement for proteasomal activity for re-presentation
of HSP-chaperoned peptides. Because HSPs are purified from cells after
the peptides have been generated through protease activity and also
have been shown to chaperone precise MHC I peptide epitopes (6, 7, 10),
we expected that re-presentation of HSP-chaperoned peptides would not
require further proteasomal action. EL4 cells were treated with the
proteasome inhibitor, N-acetyl-Leu-Leu-norleucinal (LLnL)
for 1 h prior to and during loading with either the endoplasmic
reticulum (ER) chaperone gp96 or the cytosolic chaperone hsp70 derived
from N1 cells. Surprisingly, re-presentation of VSVNP peptides
chaperoned by gp96 or hsp70 was inhibited by LLnL (Fig.
4A), suggesting that
re-presentation of HSP-chaperoned peptides requires functional protease
activity. Control, LLnL-untreated EL4 cells loaded with gp96 or hsp70
in an identical manner were able to re-present VSVNP-derived
peptides.
Because LLnL has been shown to have inhibitory effects on proteases
other than the proteasome (22), we replaced LLnL with the
proteasome-specific inhibitor, lactacystin. To examine the proteasome
dependence of HSP-chaperoned peptide re-presentation more precisely, we
used HSP·peptide complexes reconstituted in vitro instead
of the naturally derived complexes. The four T-Ag-derived peptides used
earlier (Fig. 3A) were complexed separately to gp96, hsp70,
hsp90, or the non-HSP, SA. HSP·peptide complexes, reconstituted in vitro, were loaded independently but identically, into
EL4 cells, not treated or treated with lactacystin prior to loading. It
was observed (Table I) that (i) treatment
with lactacystin inhibited re-presentation of all the
extended peptides, (ii) surprisingly, treatment of cells
with lactacystin inhibited re-presentation of even the
precise unextended MHC I-binding peptides when chaperoned by
hsp70 or hsp90; (iii) in another surprise, re-presentation of the
precise MHC I binding peptide complexed to gp96 was not inhibited by
lactacystin. These observations suggest that, during re-presentation,
proteasomes may contribute function(s) other than proteolytic
degradation of extended peptides. They also suggest that peptides
chaperoned by the ER HSP, gp96, are processed by a different mechanism
from that of peptides chaperoned by the cytosolic hsp70 and hsp90. The
structural basis for this difference is not yet clear.
Peptides generated in the cytosol are transported predominantly by TAP
into the endoplasmic reticulum for association with MHC I molecules
(23-27). The requirement for TAP in re-presentation of HSP-chaperoned
peptides was tested by comparing peptide re-presentation in
TAP-expressing cells RMA and in TAP-dysfunctional cells RMA-S. RMA-S
cells were not lysed by VSVNP-specific CTL after being loaded with
N1-derived gp96 or hsp70 at any dose of HSP used (Fig. 4B). In comparison, RMA cells, expressing functional TAP molecules, did
re-present the HSP-chaperoned peptides as measured by the effective
lysis of HSP-loaded RMA cells (Fig. 4B). RMA cells pulsed with HSPs in the absence of DOTAP were not susceptible to lysis, indicating that lysis was not due to extracellular exchange of peptides.
A requirement for TAP for presentation of VSVNP may appear inconsistent
with the earlier findings of Bevan and colleagues (28) who showed that
VSVNP can be presented by MHC I in the absence of functional TAP2
molecules in RMA-S cells. However, a closer scrutiny of the previous
data and our results shows that the differences are not inconsistent.
Essentially, TAP2-negative cells such as RMA-S can still re-present
VSVNP, whereas TAP1-negative cells cannot, thus suggesting that TAP1
homodimers may still be able to transport peptides into the ER. It is
conceivable that, under limiting quantities of antigenic peptides, such
as those created by introduction of HSP·VSVNP complexes, the relative
efficiencies of the TAP1/TAP2 heterodimer vis-à-vis the TAP1
homodimer, become more evident. A second possibility may be envisaged
where the VSVNP peptides generated in N1 cells are transported by
anomalous TAP-independent means, whereas direct introduction of the
same peptides with HSPs introduces them into the classical
TAP-dependent pathway.
After MHC I molecules are loaded with peptides in the ER, they are
transported to the cell surface via the Golgi by vesicular traffic.
Brefeldin A (BFA) is a known inhibitor of post-ER vesicular traffic
(29). EL4 cells were not treated or treated with BFA for 1 h prior
to and during DOTAP-mediated loading of SVB6-derived gp96, hsp70, or
hsp90. The loaded cells were then tested for lysis by T-Ag-specific
CTLs. It was observed that BFA completely inhibited re-presentation of
peptides chaperoned by gp96, hsp70, and hsp90 (Fig. 4C).
This inhibition was reversible by incubating BFA-treated EL4 cells in
the absence of BFA for 3 h prior to loading with HSP·peptide
complexes (Fig. 4C).
Sequestration of Endogenous Cytosolic HSPs Abrogates Presentation
of Antigenic Peptides by MHC I Molecules--
Deoxyspergualin (DSG) is
a small molecular weight immunosuppressive drug shown to interact
specifically with hsp70 and hsp90 (30, 31). The drug can enter cells
and interact with endogenous hsp70. We sought to exploit the
HSP-binding property of DSG to test whether binding of DSG will lead to
sequestration of hsp70 and hsp90, which will now be unable to chaperone
the newly generated antigenic peptides into the endogenous presentation
pathway. This idea was tested in a series of experiments. As a first
measure, DSG was added to mixed lymphocyte tumor cultures (MLTC) of N1 cells (EL4 cells transfected with the gene encoding VSV NP (12)) and
anti-N1 CTL clones. The MLTCs generated in the presence and absence of
DSG were tested in a cytotoxicity assay for activation and
proliferation of antigen-specific CTLs. Treatment with DSG was observed
to inhibit dramatically the activation/proliferation of VSV NP-specific
CTLs (Fig. 5A). However, this
inhibition could be reversed completely if VSV NP-derived peptide VSV8
were added to the MLTC. Addition of an irrelevant peptide
(corresponding to an epitope from SV40 T antigen with the same
restriction element as the VSV epitope), did not reverse the
inhibition. These data indicate that treatment with DSG resulted in a
limitation in the quantity of the VSV epitope on N1 cells. To determine
if DSG was acting at the level of the CTLs or the antigen-presenting
cell, the CTLs were purified and were cultured in medium with or
without DSG and were tested for their ability to lyse N1 cells. DSG
treatment for as long as ~100 h had no discernible effect on the CTLs
(Fig. 5B).
The effect of DSG on N1 cells was monitored directly. As N1 cells
already contain a population of specific MHC I·peptide complexes, which have a certain half-life, and because even a very small number of
MHC I·peptide complexes are capable of stimulating activated CTLs
(32), a system was sought where no preformed specific MHC I·peptide
complexes exist. EL4 cells were treated (or not treated) with DSG for
24 h so as to allow sequestration of hsp70 and hsp90 molecules.
Cells were then infected with VSV. The virus-infected and viral
antigen-expressing cells, which had or had not been exposed to DSG
pre-infection, were used to stimulate anti-VSV NP CTLs, as described in
a previous experiment (see Fig. 5A). The DSG-treated cells
were observed to be unable to stimulate the CTLs at all, whereas the
control cells stimulated them as expected (Fig.
6A). As an additional control
in these studies, EL4 cells, with or without prior treatment with DSG,
were pulsed with VSV8, and these were tested for the ability to
stimulate CTLs. Treatment with DSG was found to have no effect on the
antigen-presenting ability of VSV8 pulsed cells (Fig. 6A).
Prior treatment of cells with DSG had no effect on viral infection and
expression of viral proteins as determined by staining of infected
cells with anti-G protein antibody coupled to a photochrome (Fig.
6B). These results show clearly that treatment with DSG
interferes with a step in the antigen-presenting cell, which is
required for generation of the specific MHC I·peptide complex,
although the block is not in generation of MHC I molecules per
se.
Although DSG has been shown to interact specifically with hsp70 and
hsp90 (30), the possibility that the effects observed are not due to
the HSPs but due to interaction of DSG with an unknown intracellular
pathway, the role of hsp70 was tested more directly. Experiments shown
in some of the previous figures (Figs. 1-3) demonstrate how it is
possible to introduce molecules into the cytosol of living cell with
the help of DOTAP. This method was now used to introduce hsp70, or as a
control, SA (which has been shown previously to bind peptides
efficiently (15)), into the DSG-treated cells 1 h after infection
with VSV. The cells were used to stimulate the anti-VSV CTLs as before.
The experiment showed (Fig. 7) that
introduction of hsp70 but not SA could completely relieve the
inhibition in antigen-presenting ability in DSG-treated cells.
The ability of DSG to block the trafficking of peptides destined for
loading the cell surface MHC I molecules was tested by an independent
assay. MHC I·
These observations provide strong support for the idea that HSPs are
necessary for transport of antigenic peptides in the cytosol and that
DSG interferes with this step.
The studies reported here shed light on trafficking of peptides in
the cytosol, leading to presentation of peptides by the MHC I
molecules. First, free peptides introduced into the cytosol are
presented quite inefficiently as compared with HSP-chaperoned peptides.
This observation supports the idea that the trafficking of peptides in
the cytosol does not occur by passive diffusion but by active
mechanisms, including chaperoning (1, 2, 4). This is particularly
relevant, because of quantitative considerations. The quantity of
peptides available naturally in a cell (in the order of
sub-femtograms/cell for an epitope derived from a moderately expressed
protein) is too low to allow presentation by MHC I molecules, if the
peptide were to diffuse passively. The same quantity of peptide has a
significantly higher likelihood of getting presented if it were
chaperoned by an HSP molecule, as shown here. Second, chaperoning
(i.e. being carried by a larger molecule) is necessary but
insufficient for peptide re-presentation, because SA-chaperoned peptides are not presented any more efficiently than free peptides. The
structural rules that define the requirement for and efficiency of
chaperoning in presentation for each HSP, such as binding affinities, number of peptide binding sites, peptide-dissociation rates,
association with other peptide-recipient proteins are not yet
known; however, our studies provide an assay through which they
could be divined. These structural rules may account, at least
partially, for the differences in proteasome requirements observed for
hsp90 and hsp70 versus gp96 (Table I). In addition, because
hsp70 and hsp90 are cytosolic proteins, their delivery, with DOTAP,
places them in the appropriate environment to mediate their chaperoning
function. In contrast, the normally ER-resident gp96 may direct its
peptides into an alternate route when delivered into the cytosol, for
example, by docking with different peptide-recipient proteins. Third,
and complementary to the first two sets of results, our studies show that sequestration of intracellular, native hsp70 (and hsp90) interferes with transport of antigenic peptides to MHC I molecules. DSG
binds to cytosolic HSPs, hsp70 and hsp90 via the COOH-terminal EEVD
sequence (34). Because DSG localizes almost exclusively in the cytosol
(35) and because the ER hsp gp96 does not have the EEVD sequence, the
effects of DSG appear primarily restricted to the cytosolic chaperones.
Binding of DSG to HSPs does not affect the ability of peptides to bind
to or be released from these HSPs and thus may mediate its effect on
HSPs indirectly (Ref. 34 and data not shown). To the best of our
knowledge, this is the first demonstration of an obligatory role for
hsp70 molecules in antigen presentation by MHC I molecules. Our results
in this regard explain the observations of Wells et al. (36)
who showed that transfection of antigen presentation-defective B16
melanoma cells with hsp70 renders the cells presentation
competent. Finally, our results hint at a surprising role for
proteasomes, beyond cleavage of proteins or precursor peptides into
final-sized MHC I epitopes. HSP-chaperoned peptides, even though
generated from the processing events, including proteasomal activity in
the cells from which they are purified, require further proteasomal
action for re-presentation. Even when the peptides chaperoned by hsp70 or hsp90 are the exact MHC I-binding size, their re-presentation requires the presence of functional proteasomes. This observation is
consistent with the proposal that, in addition to generating peptides
from intact proteins, proteasomes are involved in delivery of the
peptides into the TAP pathway through a multimolecular assembly, the
presentosome (4). Treatment of cells with proteasome inhibitors may
abolish the ability of the proteasomes to dock or undock with various
members of the presentosome such as TAP and block peptide transport.
The proteasomes may, in addition or alternatively, be actively involved
in the release of peptides from the chaperone, a process that may
require protease activity. This however remains to be demonstrated in a
direct manner.
Our results may be viewed in light of some recent developments on the
cytosolic events in antigen processing. Bercovich et al.
(37) have shown recently that recruitment of hsc70 or hsp27 is required
for ubiquitination of certain protein substrates in studies in
vitro. Using a metabolically unstable form of the influenza virus
nucleoprotein (NP) as the antigen, Anton et al. (38) have recently identified specific sites in the cytosol where antigenic NP
peptides are generated. They propose that NP is chaperoned to these
sites by hsc70 and that the polyubiquitinylated NP undergo degradation
by proteasomes in situ at these sites, leading to generation
of antigenic peptides. In view of our results that free antigenic
peptides in the cytosol are presented extremely inefficiently and that
HSP-chaperoned peptides are presented effectively, we propose to extend
the model envisaged by Anton et al. (38) to suggest that
hsc70 (and hsp90) is involved not only in the afferent end of this
process by chaperoning partially or fully unfolded polypeptide chains
to this site, but also in chaperoning the resulting antigenic peptides
to the TAP complex. We have suggested previously that all of these
processes, including the transport of peptides to TAP, occur in a
single dynamic multimolecular assembly, which we termed the
presentosome (4).
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(Eq. 1)
6 M. Approximately 1% of the gp96 or hsp90
molecules was loaded with the exogenous peptides by this method (15).
The indicated amount of purified hsp70 or SA was incubated with peptide
at a protein-to-peptide molar ratio of 1:50 in 300 µl of PBS at
37 °C for 1 h. Peptide concentration used for complexing was
10
6 M. To remove free, uncomplexed peptides,
complexes were washed extensively with PBS in an Ultrafree-4
centrifugal device, Biomax 10K NMWL membrane (Millipore Corp.). To
determine the efficiency of complexing, peptides were labeled with
125I (ICN) using IODO-BEADs (Pierce). In parallel with
unlabeled peptides, 125I-labeled peptides were complexed to
proteins and checked by SDS-polyacrylamide gel electrophoresis and
autoradiography (data not shown). The efficiency of gp96, hsp70, hsp90,
or SA to complex peptides was comparable.
130 °C. Twenty micrograms per ml of DSG, with or without peptide (final concentration of 1 µM), was added
to the MLTC of VSV CTL clones. After a 5-day incubation at 37 °C,
each well of the MLTC was harvested and tested for its ability to lyse 51Cr-labeled N1 and EL4 cells in a 4-h 51Cr
release assay.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (38K):
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Fig. 1.
Proteins loaded with DOTAP are localized in
soluble, non-vesicular compartments of the cell. EL4 cells were
incubated without (lanes 1 and 2) or with gp96
(lanes 3 and 4) in the absence (lane
3) or presence (lanes 1, 2, and
4) of DOTAP as indicated on the top of the gel.
Cells were completely lysed with 0.5% Nonidet P-40 (lane 1)
or the plasma membranes were selectively lysed with 0.01% saponin
(lanes 2-4). Supernatants of the cell lysates (100,000 × g, 90 min) from each sample were resolved by
SDS-polyacrylamide gel electrophoresis, and the blots for each sample
were probed with antibodies against gp96 or the cytosolic hsp70.
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Fig. 2.
Peptides chaperoned by HSPs are re-presented
by EL4 cells after cytosolic loading into the cytosol.
A, gp96, hsp70, or hsp90 purified from the T-Ag-transformed
cell line SVB6 was loaded into EL4 cells at different doses as
indicated. Closed crosses indicate EL4 cells pulsed with
HSPs without DOTAP. Cells were used as targets for T-Ag-specific CTL
clones in a 51Cr release assay. B, gp96 or hsp70
purified from the VSVNP-transfected cell line N1 or EL4 cells was
loaded into EL4 cells at different doses as indicated. Open
crosses indicate EL4 cells pulsed with HSPs without DOTAP.
ATP treated hsp70 indicates the N1-derived hsp70 preparation
treated with ATP to remove peptides. Loaded cells were used as targets
for VSVNP-specific CTL clones in a 51Cr release
assay.
8 M with respect to peptide
concentration) or free peptides (10
6 or 10
4
M) were loaded into EL4 cells with DOTAP. In parallel,
experiments using radiolabeled HSPs, SA, and each of the peptides were
used to determine how much of each moiety administered with DOTAP could be recovered in the cytosol of the cells. This exercise demonstrated that ~6-8% of the quantity of each moiety introduced in the cells by DOTAP could be recovered from the cytosol (data not shown). The
constancy of this number allows for valid comparisons among the results
with each antigenic moiety. The cells into which the HSPs, SA, or
peptides were introduced were then monitored for lysis by T-Ag-specific
CTLs (Fig. 3B). It was observed that (i) a concentration of
10
4 M free peptide was required for loading
to observe lysis of the EL4 cells comparable to that observed for
10
8 M concentration of peptide when
chaperoned by HSPs, (ii) peptides chaperoned by SA, which binds
peptides efficiently ("Experimental Procedures"), were not
re-presented by MHC I molecules, suggesting that HSPs play a role
different from simply carrying the peptides, and (iii) MHC I epitopes
are generated from peptides chaperoned by HSPs regardless of whether
they are extended on the NH2, COOH, or both termini.
View larger version (24K):
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Fig. 3.
T-Ag-derived peptides chaperoned by HSPs, but
not by SA or unchaperoned, can be re-presented. A,
20-mer peptides extended from the precise MHC I H-2Db
binding epitope (gray) on the NH2, COOH, or both
termini were synthesized and complexed to gp96, hsp90, hsp70, or SA.
B, peptides complexed to gp96, hsp90, hsp70, or SA or
unchaperoned peptide were loaded into EL4 cells. Control EL4 cells were
pulsed with these complexes without DOTAP. Loaded or pulsed cells were
used as targets for T-Ag-specific CTL in a 51Cr release
assay.
View larger version (25K):
[in a new window]
Fig. 4.
Re-presentation of peptides chaperoned by
HSPs requires functional proteasomes, is TAP-dependent, and
is brefeldin A-sensitive. A, EL4 cells were untreated
or treated with N-acetyl-Leu-Leu-norleucinal prior to
loading with N1-derived gp96 or N1-derived hsp70 preparations at the
indicated concentration. Loaded or unloaded cells were used as targets
in a 51Cr release assay with VSVNP-specific CTL as
described. B, RMA or RMA-S cells were loaded or pulsed with
N1-derived gp96 or hsp70. RMA-S cells, pulsed with VSV8, and RMA were
also used as controls. VSVNP-specific CTL were used as the effectors.
C, EL4 cells untreated or treated with brefeldin A were
loaded with gp96, hsp70, or hsp90 derived from SVB6 cells. Brefeldin
A-treated EL4 cells, allowed to recover for 3 h, were also loaded
in parallel. Loaded cells were used as targets for T-Ag-specific CTL in
a 51Cr release assay.
Influence of inhibition of functional proteasomes by lactacystin on
re-presentation of precise and extended peptides chaperoned by HSPs
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Fig. 5.
hsp70 is involved in the transport of
antigenic peptides to MHC I molecules. Treatment of cells with DSG
reduces their capacity to stimulate CTLs. A, CTL clone
against VSVNP and feeder cells were incubated in media without
(open symbols) or with 20 µg/ml DSG (solid
symbols) for 5 days in the presence of N1 (squares) or
EL4 pulsed with VSV Kb epitope (circles) or
T-antigen (triangles) peptides. The CTLs recovered were
tested for their ability to lyse N1 cells in a 4-h 51Cr
release assay. B, cytotoxic activity of CTLs is not affected
by treatment with DSG. CTLs incubated for 5 days without (open
square) or with 20 µg/ml of DSG (closed square) were
tested for cytotoxicity against 51Cr-labeled N1
cells.
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Fig. 6.
Pretreatment of cells with DSG blocks antigen
presentation by MHC I molecules. A, EL4 cells were
irradiated and incubated without (open symbols) or
with 40 µg/ml DSG (closed symbols) for 24 h. EL4
cells were washed prior to infection with VSV (squares) or
pulsing with VSV8 (circles) and were tested for their
ability to be recognized by VSV CTLs in a 4-h 51Cr release
assay. The asterisk denotes background lysis of EL4 cells in
the absence or presence of DSG. B, pretreatment of cells
with DSG does not effect infection of cells by VSV. EL4 cells were
fixed, permeabilized, and incubated with anti-VSV G protein antibody
conjugated to cyt3 photochrome. Cells were analyzed by a confocal
fluorescence scanning microscopy (Zeiss LSM 410 invert). Dual channel
imaging was performed, and both images were overlaid. Channel 1 (green), transmittance image; channel 2 (red),
fluorescence image.
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Fig. 7.
Recovery of antigen presentation in
DSG-treated cells by introduction of hsp70. EL4 cells were
irradiated and incubated without (open symbols) or with 40 µg/ml DSG (closed symbols) for 24 h. EL4 cells were
washed and infected with VSV for 1 h prior to introduction of PBS
(circles), 50 µg of hsp70 (squares), or 50 µg
of mouse serum albumin (triangles) by the transfection
reagent DOTAP. Cells were then tested for their ability to be
recognized by VSV CTLs in a 4-h 51Cr release assay.
2 microglobulin complexes devoid of peptides are
unstable on the cell surface at 37 °C but are stable at 25 °C
(33). The MHC I·peptide complexes can also be detected by
conformation and Kb-specific antibodies. These tools were
used to examine the presence of stable MHC I molecules on the cell
surface of DSG-treated and untreated cells. It was observed that
treatment of EL4 cells with DSG at 37 °C lead to a nearly 5-fold
reduction in the number of Kb-peptide complexes as
determined by the specific antibody Y3 (Fig. 8). At 25 °C, no such inhibition was
observed. Interestingly, if the EL4 cells kept at 25 °C were now
shifted to 37 °C, the DSG-treated cells showed a nearly 5-fold less
quantity of Kb moieties than the DSG-untreated cells,
indicating that a large proportion of Kb molecules of
DSG-treated cells at 25 °C were devoid of peptides and hence labile
in the DSG-treated cells. EL4 cells also express the LFA molecule whose
expression in DSG-treated and untreated cells was monitored and found
to be unaffected by treatment with DSG, indicating that DSG was not
affecting the secretory pathway per se, as also indicated by
the experiment carried out at 25 °C.
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Fig. 8.
Treatment of cells with DSG affects detection
of folded MHC I·peptide complexes on the cell surface. EL4 cells
were incubated with or without DSG at 25 °C or 37 °C for 48 h. One group of cell, as indicated, was changed at 40 h from
25 °C to 37 °C for 8 additional hours. Cells were then stained
with fluorescein-conjugated anti-Kb, anti-Db,
or anti-LFA antibodies and analyzed by flow cytometry.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENT |
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We thank Dr. Sreyashi Basu of our laboratory for critically reading the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants CA64394 and CA44786, Defense Advanced Research Project Agency Grant BAA96024, and a research agreement with Antigenics, Inc, in which company one of us (P. K. S.) has a significant financial interest.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Both authors contributed equally to this work.
§ Present address: Memorial Sloan-Kettering Cancer Center, New York, NY 10021.
¶ To whom correspondence should be addressed: School of Medicine, MC1601, University of Connecticut, Farmington, CT 06030-1920. Tel.: 860-679-4444; Fax: 860-679-4365; E-mail: srivastava@nso2.uchc.edu.
Published, JBC Papers in Press, March 8, 2001, DOI 10.1074/jbc.M011547200
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ABBREVIATIONS |
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The abbreviations used are: ER, endoplasmic reticulum; BFA, brefeldin A; DSG, deoxyspergualin; Deoxyspergualin (DSG), HSP, heat shock protein; MLTC, Mixed Lymphocyte Tumor Culture; NP, nucleoprotein; SA, serum albumin; TAP, transporter associated with antigen processing; VSV, vesicular stomatitis virus; RPMI, Roswell Park Memorial Institute; DOTAP, N-[-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (C43H83NO8S); MHC, major histocompatibility complex; PBS, phosphate-buffered saline; LLnL, N-acetyl-L-leucinyl-L-leucinal-L-norleucinal; CTL, cytotoxic T lymphocyte; T-Ag, large T-antigen protein from SV40; LFA, leukocyte function antigen-1..
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