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 : srivastava@nso2.uchc.edu

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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Binder et al. 18

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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Binder et al. 19

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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Binder et al. 20

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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Binder et al. 21

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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Binder et al. 22

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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Binder et al. 23

to TAP, occur in a single dynamic multi-molecular assembly, which we termed the

presentosome (4).

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Binder et al. 24

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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Binder et al. 25

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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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Binder et al. 30

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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Binder et al. 33

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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