Binder et al. 1
Heat shock protein-chaperoned peptides but not free peptides
introduced into the cytosol are presented efficiently
by MHC I molecules
Running Title: Chaperones in the cytosol facilitate antigen presentation
Robert J. Binder1, Nathalie E. Blachere1,2 and Pramod K. Srivastava*
Center for Immunotherapy of Cancer and Infectious Diseases
University of Connecticut School of Medicine
Farmington, CT 06030
* To whom correspondence should be sent at :
Pramod K. Srivastava, Ph.D.
University of Connecticut School of Medicine
MC1601, Farmington, CT 06030-1920
Tel: 860 679 4444; Fax: 860 679 4365
E-mail : [email protected]
1These authors contributed equally.
2Present address : Memorial Sloan Kettering Cancer Center, New York, NY 10021.
Character count: 51,209
Abbreviations used: BFA, brefeldin A; DSG, deoxyspergualin; ER, endoplasmic reticulum;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.
Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on March 8, 2001 as Manuscript M011547200 by guest on M
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Summary
The studies reported here bear on the events in the cytosol which lead to trafficking of
peptides during antigen processing and presentation by 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 which 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.
Keywords : antigen presentation / chaperone / cytosol / deoxyspergualin / proteasomes
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Introduction
Cellular proteins undergo degradation in the cytosol and the resulting peptides are
transported into the endoplasmic reticulum (ER), 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). As heat shock proteins (HSPs) are known to
chaperone a wide array of molecules (3) and as immunological and structural evidence
exists that HSPs chaperone antigenic peptides (see 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.
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Experimental Procedures
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 (6.0µg/ml for 3 hrs) and to maintain the block (0.6µg/ml for up to 12
hrs). Maintenance of the BFA block did not affect CTL function during the CTL assay.
EL4 cells, untreated or treated with Brefeldin A (BFA) at these concentrations were
analyzed by FACScan to show maximal decreases (~40%) in surface expression of MHC
I after 20hrs (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 unless otherwise specified. HL-1 and
RPMI media, together with pyruvate, glutamine, penicillin-streptomycin and non essential
amino acids were purchased from GIBCO BRL. RPMI containing 5%FCS (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
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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 mAbs were obtained from Pharmigen, (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)} (Boeringer-Mannheim) at a 3:2 ratio (microgram amounts)
for 15 minutes at room temperature. In all loading experiments, 1.5x106 cells (EL4, RMA
or RMA-S) were washed three times with serum-free HL-1 media and then incubated in
1ml HL-1 media with protein:DOTAP combination for 4 - 4.5 hours at 37oC. Control cells
were either mock loaded by incubating 1.5x106 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 2X103 51Chromium
(supplied as Na2CrO4; ICN) labeled target cells in 100µl of complete RPMI were added to
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various dilutions of T-Ag or VSV8-specific CTL in 100µl of complete RPMI, as indicated
by the effector to target (E:T) ratios. Effectors and targets were cultured in v-bottom 96
well plates for 4hours. Supernatants (50µl) 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:
Experimental 51Cr release - spontaneous 51Cr release X 100% Maximum 51Cr release - spontaneous 51Cr release
Maximum and spontaneous releases were measured by culturing 2X103 labeled
target cells in lysis buffer (0.5%NP40, 10mM Tris, 1mM EDTA, 150mM NaCl) and
complete RPMI, respectively, for 4hours. VSV8-specific CTL were obtained by dual
immunizations of C57BL/6 mice, one week apart, with N1 cells. Spleen cells were
harvested one week after the second immunization, restimulated in culture with irradiated
N1 cells and cloned by limiting dilution (14). 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 9mer peptide (NH2-AINNYAQKL-COOH),
previously named epitope 1 (11).
Flow cytometry analysis of DSG treated cells. N1 cells were irradiated (5,000
rads) and allowed to recover in AIM V medium with or without DSG for 48 hours at 37°C
or 25°C. Half of the cells incubating at 25°C were then placed at 37°C for an additional 8
hours. One group of the cells not treated with DSG but incubated at 25°C for 48 hours
was placed at 37°C in the presence of DSG for 8 hours. Cells (1x106) were then stained at
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4°C for 40 minutes with anti-Kb, anti-Db, anti-Dd, or anti-LFA fluorescein-conjugated Abs
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 Zeiss LSM confocal microscope.
Infection of Cells with Vesicular Stomatitis Virus (VSV). Vesicular Stomatis
Virus (VSV) was obtained from Advanced Biotechnology (Columbia, MD). Meth A or
EL4 cells were incubated with 10 plaque forming units (PFU) of VSV per cell for 1 hour
at 37oC in plain RPMI and allowed to recover in RPMI with 10% FCS for 4 hours. Cells
were washed three times in PBS (10mM phosphate buffer, 150mM NaCl, 2.7mM 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 9mer: NH2-AINNYAQKL-COOH
T-Ag 20mer (N-terminus extended): NH2-FFLTPHRHRVSAINNYAQKL-COOH
T-Ag 20mer (N+C termini extended): NH2-RHRVSAINNYAQKLCTFSFL-COOH
T-Ag 20mer (C-terminus extended): NH2-AINNYAQKLCTFSFLICKGV-COOH
Peptides were synthesized by Genemed to >95% purity as determined by HPLC. The
unextended MHC I binding 9mer peptide is identical to epitope I of the T-Ag (11). The T-
Ag 9mer stabilized MHC I molecules on RMA-S cells and sensitized targets for lysis by
the T-Ag specific CTL. All three T-Ag 20mer peptides failed to bind MHC H-2Db as
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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 700µl PBS for 10 mins at 50oC and incubated for
a further 30min at RT. The peptide concentration used for complexing was 10-6 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 300µl PBS at 37oC 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 Corporation). 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-PAGE and autoradiography (data not
shown). The efficiency of gp96, hsp70, hsp90 or SA to complex peptides was
comparable.
Inhibition of proteasome function. EL4 cells (107) in complete RPMI were
treated for 2 hours with 100µM of the proteasome inhibitor N-acetyl-L-leucinyl-L-leucinal-
L-norleucinal (LLnL) in DMSO or with 0.002%DMSO alone. In other experiments, EL4
cells were treated with 100µM lactacystin dissolved in DMSO for 1 hour. In both cases,
the treated cells were constantly in the presence of the inhibitor during loading with
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protein. FACScan analysis of inhibitor- treated cells showed greater than 35% decrease in
cell surface MHC I expression after 20hrs 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, 100,000g supernatants were
obtained from cell lysates and applied to a Mono Q column (Mono Q HR 16/10, was
purchased from Pharmacia Biotech and attached to the BIOCAD Perseptives Biosytems).
Treatment of MLTC and N1 cells with 15-deoxyspergualin (DSG). 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 -130°C. Twenty microgram per ml of DSG, with or without
peptide (final concentration of 1 µM), was added to the MLTC of VSV CTL clones. After
a five 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 four-hour 51Cr release assay.
Results
Demonstration of the experimental system to introduce molecules into the
cytosol. The cationic liposome, N-[-(2,3-Dioleoyloxy)propyl]-N,N,N,trimethylammonium
methylsulfate (DOTAP) was used to introduce HSP-peptide complexes or free peptides
into the cytosol. Distinct properties of the detergents NP-40 and Saponin were used to
demonstrate that DOTAP-loaded gp96 enters the soluble, non-vesicular, cytosolic
compartment of the cells (Figure 1). Although the cytosolic HSPs are of primary interest
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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% % NP-40 leads to solubilization of all non-nuclear membranes, while 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 NP40-lysates of non-loaded cells (Figure 1, lane 1) but not in the Saponin-
lysed non-loaded cells (Figure 1, lanes 2 and 3), as 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 (Figure 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 soluble, non-vesicular, cytosolic compartment of the cells (data not shown).
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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 study. 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 1) to the cytosolic chaperone
hsp90, and (iii) considerable immunological and structural information on gp96-peptide
interaction is already available (see 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 in the
Experimental Procedures. The T-Ag derived peptides were present in gp96 preparations
and were observed to be efficiently re-presented in this assay (Figure 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 25µg 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 (Figure 2A), indicating that similar to gp96, hsp70
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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
(approximately 40%) of HSP-loaded cells, 100µg, 250µg and 500µg of gp96, hsp70 and
hsp90, respectively, were required. Approximate amounts of HSP, below which no lysis
was detected were 25µg, 180µg and 400µg for gp96, hsp70 and hsp90 respectively.
A second, well characterized antigenic system, the Vesicular Stomatitis Virus
(VSV) 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 (Figure 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 (Figure 2B). Further, HSP preparations not carrying VSVNP derived peptides
(EL4-derived HSPs) (Figure 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 peptide specific, and require
intracellular processing of the HSP-peptide complexes.
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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. In order 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 9mer epitope I of the SV40 T-Ag protein (NH2-AINNYAQKL-COOH), or
20mer peptides extended on the amino terminus, carboxy terminus or both termini
(Figure 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
(approximately 10-8 M with respect to peptide concentration), or free peptides (10-6 or 10-
4M) 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 approximately 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 (Figure 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
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carrying the peptides, and (iii) MHC I epitopes are generated from peptides chaperoned
by HSPs regardless of whether they are extended on the N, C or both termini.
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 20,21). Since 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. As
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 hour prior to and during loading with either the endoplasmic
reticulum (ER) chaperone gp96 or the cytosolic chaperone hsp70 derived from N1 cells.
Suprisingly, re-presentation of VSVNP-peptides chaperoned by gp96 or hsp70 was
inhibited by LLnL (Figure 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.
Since LLnL has been shown to have inhibitory effects on proteases other than the
proteasome (22), we replaced LLnL with the proteasome-specific inhibitor, lactacystin. In
order to examine the proteasome dependence of HSP-chaperoned peptide re-presentation
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more precisely, we used HSP-peptide complexes reconstituted in vitro instead of the
naturally-derived complexes. The four T-Ag derived peptides used earlier (Figure 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 1) 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 (Figure 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 (Figure 4B). RMA cells pulsed
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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, while 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 an hour
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 (Figure 4C). This inhibition was reversible by incubating BFA-treated EL4 cells in
the absence of BFA for 3hours prior to loading with HSP-peptide complexes (Figure 4C).
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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 if 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, ref. 12), and anti-N1 CTL clones. The MLTCs generated in 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 (Figure 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. In order to determine if DSG was acting at the
level of the CTLs or the APC, the CTLs were purified and were cultured in medium with
or without DSG and were tested for their ability to lyze N1 cells. DSG treatment for as
long as ~100 hours had no discernible effect on the CTLs (Figure 5B).
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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 as
even a very small number of MHC I-peptide complexes are capable of stimulating
activated CTLs (32), a system was sought where no pre-formed specific MHC I-peptide
complexes exist. EL4 cells were treated (or not treated) with DSG for 24 hours 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 Figure 5A). The DSG-treated cells were observed to be unable
to stimulate the CTLs at all, while the control cells stimulated them as expected (Figure
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 ability to stimulate CTLs.
Treatment with DSG was found to have no effect on the antigen presenting ability of
VSV8 pulsed cells (Figure 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 (Figure 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 (Figures 1-3) demonstrate how it is
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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, ref. 15), into the DSG-treated cells one hour
after infection with VSV. The cells were used to stimulate the anti-VSV CTLs as before.
The experiment showed (Figure 7) that introduction of hsp70 but not SA could relieve
completely, 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-β2 microglobulin
complexes devoid of peptides are unstable on the cell surface at 37oC but are stable at
25oC (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 37oC lead to a nearly 5 fold reduction in the number
of Kb-peptide complexes as determined by the specific antibody Y3 (Figure 8). At 25oC,
no such inhibition was observed. Interestingly, if the EL4 cells kept at 25oC were now
shifted to 37oC, 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 25oC 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 25oC.
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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.
Discussion
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 to 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 be able to
presented 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, as 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 and
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
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requirements observed for hsp90 and hsp70 versus gp96 (Table 1). In addition since
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. Thirdly, 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 C-terminal
EEVD sequence (34). Since DSG localizes almost exclusively in the cytosol (35) and
since 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 (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 genes 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
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proposal that in addition to generating peptides from intact proteins, proteasomes are
involved in delivery of the peptides into the TAP pathway through a multi-molecular
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
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to TAP, occur in a single dynamic multi-molecular assembly, which we termed the
presentosome (4).
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Acknowledgements
This work was supported by NIH Grants CA64394 and CA44786, DARPA grant
BAA96024 and a research agreement with Antigenics, Inc, in which company one of us
(PKS) has a significant financial interest. Finally, the authors thank Dr. Sreyashi Basu of
our laboratory for critically reading the manuscript.
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Table 1.
Influence of inhibition of functional proteasomes by lactacystin on re-presentation of
precise and extended peptides chaperoned by HSPs.
Percent Inhibition of Re-presentation *
Peptides
Chaperone
N-extended
20mer
N+C-extended
20mer
C-extended
20mer
Db-binding
9mer
Gp96 60.1 96.0 81.0 0
Hsp90 61.2 98.0 98.2 71.1
Hsp70 90.1 98.3 65.3 46.1
* EL4 cells were treated with lactacystin prior to loading with HSP-peptide complexes
reconstituted in vitro as described in the Experimental Procedures. Re-presentation was
monitored in a 51Cr release assay by a T-Ag specific CTL clone. Data were obtained in a
cytotoxicity assay done at an effector : target ratio of 5:1. Percent inhibition was
calculated, assuming as 100% the specific cytotoxicity obtained in the absence of
lactacystin. This was approximately 60%.
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Legends to Figures
Figure 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
(lane 3 and 4) in the absence (lane 3) or presence (lane 1, 2 and 4) of DOTAP as indicated
on the top of the gel. Cells were completely lysed with 0.5 %NP-40 (lane 1) or the plasma
membranes were selectively lysed with 0.01% saponin (lane 2-4). Supernatants of the cell
lysates (100,000g, 90min) from each sample were resolved by SDS-PAGE and the blots
for each sample was probed with antibodies against gp96 or the cytosolic hsp70.
Figure 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.
Figure 3. T-Ag derived peptides chaperoned by HSPs, but not by SA or unchaperoned,
can be re-presented. (A) 20mer peptides extended from the precise MHC I H-2Db binding
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Binder et al. 31
epitope (grey) on the amino, carboxy 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.
Figure 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 3hours were also loaded in parallel. Loaded cells were used as targets for T-Ag
specific CTL in a 51Cr release assay.
Figure 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 VSV NP 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
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Binder et al. 32
with VSV Kb epitope (circles) or T-antigen (triangles) peptides. The CTLs recovered were
tested for their ability to lyse N1 cells in a four-hour 51Cr release assay. (B) Cytotoxic
activity of CTLs is not affected by treatment with DSG. CTLs incubated for five days
without (open square) or with 20 µg/ml of DSG (closed square) were tested for
cytotoxicity against 51Cr-labeled N1 cells.
Figure 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 hours. 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 four-hour 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 Fluorescent Scanning Microscopy
(Zeiss LSM 410 invert). Dual channel imaging was performed and both images were
overlaid. Channel 1 (green) transmittance image and channel 2 (red) fluorescence image.
Figure 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 hours. EL4 cells were washed and infected with VSV for
1hr prior to introduction of PBS (circles), 50µg of hsp70 (squares) or 50µg mouse serum
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albumin (triangles) by the transfection reagent DOTAP. Cells were then tested for their
ability to be recognized by VSV CTLs in a four-hour 51Cr release assay.
Figure 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 25oC or
37oC for 48 hours. One group of cell, as indicated, was changed at 40 hours from 25oC to
37oC for eight additional hours. Cells were then stained with fluorescein-conjugated anti-
Kb, anti-Db, or anti-LFA antibodies and analyzed by flow cytometry.
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Robert J. Binder, Nathalie E. Blachere and Pramod K. Srivastavacytosol are presented efficiently by MHC I molecules
Heat shock protein-chaperoned peptides but not free peptides introduced into the
published online March 8, 2001J. Biol. Chem.
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