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Division of Molecular Immunology, National Institute for Medical Research, London, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Studies of the processing and loading of antigenic peptides onto MHC-II molecules to date leave unexplained how peptides evade complete proteolysis and survive to sufficient length (typically 9–16 aa) to associate with MHC-II molecules (7). Furthermore, although H2-M can accelerate the association of peptides with MHC-II molecules (8), it possesses no peptide binding properties and cannot therefore function directly as a peptide loading factor. Both of these functions would benefit from chaperone molecules, which are exemplified by heat shock proteins (HSPs). These are a conserved group of protein families, of which the HSP60 and -70 families share the properties of promiscuous binding of unfolded peptides and ATP hydrolysis-coupled peptide release (9, 10, 11, 12). HSPs could conceivably behave as Ag scavengers in degradative compartments, protecting peptides from catabolic death and transferring them rapidly to vacant MHC-II molecules, using the energy of ATP hydrolysis. Experiments in which APCs have been subjected to heat shock showed evidence for modulation of MHC-II Ag presentation capacity (13, 14) and an increase in the generation of antigenic peptides in recovered endosomal fractions (14), as well as increased recovery of SDS-stable Ab MHC-II dimers (15). However, the presumed influence of up-regulation of HSPs in these experiments was difficult to distinguish from other possible effects of heat shock, such as up-regulation of lysosomal enzymes. Abs against hsp70 family members have also been shown to affect Ag processing (16, 17).
In the present study, we examined the role of a single defined HSP, the constitutively expressed 73-kDa heat shock cognate protein (HSC73), as a candidate for interaction with antigenic peptides and/or MHC-II loading in APCs. HSC73 is expressed in the cytosol (18), but is also present in lysosomes, as demonstrated by confocal (19) and electron microscopy (20), and by subcellular fractionation techniques (21). The subcellular localization of HSC73, therefore, could place it in proximity to MHC-II molecules in MIICs. Also known as HSC70, the physiological activities of this protein thus far characterized include: uncoating of clathrin-coated pits (22) and transport of cytoplasmic proteins to lysosomes for degradation (23, 24). Also, interaction of HSC73 with HLA-DR molecules has been demonstrated (25).
In this paper, we investigate the involvement of HSC73 in Ag presentation within the MHC-II presentation pathway. Overexpression of HSC73 in macrophages results in enhancement of Ag processing. We show that endocytosed Ag interacts with HSC73 and that this interaction is ATP-sensitive and inhibited by the immunosuppressive drug 15-desoxyspergualin (DSG), which was previously shown to specifically interact with HSC73.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The HSC73 cDNA was obtained from Dr. J. Fred Dice (Tufts University, Medford, MA) and subcloned into the ß-actin expression vector (26) at the SalI site downstream of the IVS-1 intron to generate the ß73/2.7 construct. 97.2 cells were transfected with the ß73/2.7 construct using the Fugene-6 reagent (Boehringer Mannheim, Indianapolis, IN), as detailed in the manufacturer’s protocol. Transfectants were selected for G418 resistance and individual clones picked and amplified under selection.
Cell lines and culture
The cell line 97.2 (27) is a bone marrow-derived
macrophage cell line of the haplotype H-2k,
generated by R. Palacios in the Basel Institute for Immunology (Basel,
Switzerland). Expression of MHC-II molecules was induced by culture
with 400 U/ml IFN-
for 48 h. The hen egg lysozyme
(HEL)-specific T cell hybridoma 2G7.1 (28) recognizes
epitope 1–18 of HEL presented in the context of
H-2Ek, whereas 3A9 recognizes HEL epitope 46–61
in the context of H-2Ak (29). The
A18 hybridoma recognizes epitope 106–121 of the fifth component of
mouse complement (C5) (30). Cells were maintained in IMDM
medium (Sigma, St. Louis, MO) containing 5% FCS, 2 x
10-3 M L-glutamine, 100 U/ml
penicillin, 100 µg/ml streptomycin, and 5 x
10-5 M ME (all Sigma). The IL-2-dependent cell
line CTLL was maintained in medium as above supplemented with IL-2.
Dendritic cells were generated ex vivo from bone marrow as previously
described (31).
Western blotting
Untransfected 97.2 cells and HSC73 transfectants were lysed at 5 x 106 cells/ml using 1% Brij-96 containing buffer (10 mM Tris-HCl, 1 mM EDTA, 0.15 M NaCl) supplemented with 200 µM PMSF, 5 mM iodoacetamide, 5 µM leupeptin, and 1 µg/ml pepstatin A (all from Sigma). A total of 25 µl of each lysate was separated on an 8% SDS-PAGE gel, followed by transfer onto nitrocellulose membrane and immunoblotting with the anti-HSC73 Ab 13D3 (Maine Biotechnologies, Portland, ME) and HRP-conjugated anti-mouse Ig (Southern Biotechnology Associates, Birmingham, AL), using enhanced chemiluminescence (Amersham Life Science, Buckinghamshire, U.K.) for detection, as detailed in the manufacturer’s protocol.
Ag presentation assays
Class II-restricted presentation to T cell hybrids was tested by
coculture of 5 x 104/well 97.2 cells (all
macrophages were pretreated with 400 U/ml IFN- for 48 h) with
5 x 104/well T hybridoma cells in
flat-bottom 96-well plates (Costar, Cambridge, MA). Ag was given in
serial dilution as synthetic peptide corresponding to HEL[1–18],
HEL[46–61], or C5[106–121], or HEL protein (Sigma) or C5 protein.
Alternatively, Ag was kept at a constant concentration and methyl-DSG
was given in serial dilution from a maximal concentration of 20 µg/ml
(DSG was obtained from Nippon Kayaku, Tokyo, Japan). All experiments
involving DSG were performed in medium as described previously, with
the substitution of 1x Nutridoma (Boehringer Mannheim) for 5% FCS.
Presentation was allowed to occur for 24 h in culture, following
which, 50 µl of supernatant was harvested and transferred to a fresh
plate for measurement of IL-2 by coincubation with 5 x
103/well CTLL cells. CTLL proliferation was
measured by [3H]thymidine incorporation over
18 h. All values given are means of triplicate cultures, and all
experiments were performed at least twice.
Immunofluorescence microscopy
Untransfected 97.2 and HSC73 transfectants, induced for MHC-II
expression by IFN- (400 U/ml for 48 h) were grown on Lab-Tek
chamber slides (Nalge Nunc, Naperville, IL) to semiconfluence and fixed
with acetone at 4°C. Cells were permeabilized with 0.5% Triton X-100
(Sigma) and nonspecific binding blocked with 0.5% aqueous gelatin
(Sigma). Cells were then stained with the H2-E-specific mAb 14.4.4S
(American Type Culture Collection, Manassas, VA; no. HB-32) conjugated
with FITC and the HSC73-specific mAb 13D3 (Maine Biotechnology). 13D3
staining was visualized with anti-IgM Bio (Jackson ImmunoResearch,
West Grove, PA) and streptavidin-Texas Red (PharMingen, San Diego, CA).
Cells were mounted in CitiFluor mountant. Images of cells were acquired
at the Confocal and Image Analysis Lab (CIAL) at the National Institute
for Medical Research (London, U.K.) using an Olympus IX70 fluorescence
microscope and a Photometrix cooled-CCD camera. Image deconvolution was
performed using DeltaVision software to correct for out-of-focus light.
Analytical flow cytometry of transfectants was performed using a
FACScan (Becton Dickinson, Mountain View, CA), and the date were
processed using Cellquest software (Becton Dickinson). Macrophages were
stained with FITC-labeled anti-H2-Ek Ab
14.4.4, either untreated or after 48 h incubation with 400 U/ml
IFN- to induce MHC-II synthesis.
Immunoprecipitation of HSC73 on latex beads
A total of 10 mg of HEL was added to 97.2 cells in culture, and uptake of the Ag was allowed to occur for 12 h. For DSG inhibition experiments, 5 µg/ml DSG was given in culture for 6 h before addition of Ag, and left in culture during Ag uptake. Cells were then washed extensively and subsequently lysed at 5 x 106 cells/ml using 1% Brij-96 containing buffer supplemented with protease inhibitors, as detailed above. Lysates were incubated with mAb-coupled latex beads at 4°C for 8 h rotating. The anti-HSC73 mAb 13D3 and the isotype-matched control mAb 6-68 (specific for Thy1.2) were coupled to LB8 0.8-µm diameter latex beads (Sigma), as described previously (32). Supernatant was removed and latex beads washed extensively with lysis buffer and resuspended in IMDM as a 1:1000 stock suspension. Beads were used in Ag presentation assays as above, using 2 x 104/well bone marrow-derived dendritic cells as APC. Beads were given in serial dilution from a maximal starting concentration of 5% of stock suspension.
ATP treatment of HSC73 immunoprecipitates
Anti-HSC73 and control mAb immunoprecipitates, made as described above, were pelleted to remove IMDM and resuspended to 0.1% suspension in 100 mM Tris-HCl, 50 mM MgSO4 buffer, with or without 50 mM ATP added. Treatment was performed for 2 h at room temperature, after which beads were extensively washed with IMDM and resuspended in IMDM at 1:1000 stock suspension. Beads were then used in bone marrow-derived dendritic cell Ag presentation assays, as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To study the involvement of HSC73 in the MHC-II Ag processing
pathway, this molecule was transfected into the macrophage cell line
97.2. 97.2, like fresh bone marrow macrophages, show relatively
suboptimal Ag processing capacity for soluble exogenous Ag
(33), so that changes in Ag presentation capacity are
easily detected. HSC73 was placed under the control of the constitutive
human ß-actin promoter (26), and a panel of independent
clones stably transfected with this construct was generated. 97.2 cells
stably transfected with the empty ß-actin vector only were used as a
control. HSC73 transfectants were shown to overexpress the protein
compared with vector-only transfected cells and untransfected cells
(Fig. 1
A).
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B shows that HSC73 is localized in
spherical organelles in both HSC73 transfectants and untransfected APC;
the majority of these spherical organelles also stain positive for
MHC-II. Thus, a subset of HSC73 in transfected and control 97.2
macrophages colocalizes with MHC-II+
compartments. Overexpression of HSC73 enhances Ag processing
HSC73 transectants and vector-only control transfectants were
assayed for their ability to process and present Ag to three different
MHC-II-restricted T cell hybridomas. Ag was given either as
processing-dependent native protein, or as processing-independent
peptide. MHC-II expression on all transfectants was similar to control
transfectants, indicating that HSC73 overexpression does not influence
the levels of class II (Table I). HSC73
transfectants showed greatly improved capacity to present native HEL
protein to the HEL-specific H2-Ek-restricted T
cell hybrid 2G7.1, compared with control transfectants (Fig. 2A). Presentation of HEL
peptide was not significantly different between HSC73 transfectants and
vector-transfected controls (Fig. 2B). Similarly,
presentation of native C5 protein to the
H2-Ek-restricted T cell hybridoma A18 was
strongly enhanced in HSC73-transfected 97.2, compared with control
transfectants, whereas presentation of peptide was equivalent in all
APC (Fig. 2, C and D). The increase in
presentation was not only evident for Ag presented with
H-2Ek, but also for HEL presented in the context
of H-2Ak (Fig. 2, E and F;
HEL-specific T cell hybrid 3A9). Since peptide given exogenously
associates with cell surface MHC-II molecules, rather than
intracellular class II (34), the enhancement of Ag
presentation by HSC73-transfected macrophages most likely reflects a
change in intracellular processing events.
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If HSC73 fulfills a role in the MHC-II processing pathway, one
would expect it to interact with Ag internalized by the APC. We used an
immunoprecipitation strategy to examine whether antigenic activity
could be coimmunoprecipitated with HSC73 from APCs fed with Ag.
HSC73-transfected and untransfected 97.2 cells were fed HEL in culture
and then lysed, followed by immunoprecipitation of HSC73 with
anti-HSC73 mAb coupled to latex beads. As a control, parallel
immunoprecipitations were conducted using an irrelevant isotype-matched
mAb coupled to latex beads. Immunoprecipitates were then added to
cultures of bone marrow-derived dendritic cells and the HEL-specific T
cell hybridoma 2G7.1. HSC73 immunoprecipitates from both transfected
and untransfected 97.2 cells contained antigenic activity, as seen in
activation of the HEL-specific T cell hybrid by dendritic cells exposed
to latex beads carrying HSC73 immunoprecitate; control
immunoprecipitates contained insignificant amounts of antigenic
material (Fig. 3). To exclude the
possibility that association of HSC73 and HEL occurred post lysis, a
control experiment was performed with a mixture of lysate from HEL-fed
untransfected macrophages and lysate from unfed HSC73 transfectant.
This would provide the overexpressed HSC73 present in the lysate of
transfectants with a source of HEL in vitro. As shown in Fig. 3D, whereas HEL coimmunoprecipitated with HSC73 from Ag-fed
transfectants resulted in presentation by dendritic and T cell
activation, no such interaction was detected in the lysate mixtures; in
this experiment, no antigenic material was coimmunoprecipitated from
lysate of Ag-fed untransfected macrophages. This emphasizes that HSC73
interacts with exogenously sourced Ag in vivo in a form that can be
processed and presented by dendritic cells. Furthermore,
HSC73-overexpressing transfectants consistently yielded high amounts of
antigenic material, whereas untransfected macrophages did not always do
so, indicating that greater abundance of HSC73 in the transfectants
allows increased binding of internalized Ag.
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), whereas control-treated control
complexes retained their antigenic activity. Thus, the antigenicity
associated with HSC73 was shown to be lost in an ATP-dependent
manner.
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DSG is an immunosuppressive agent that was shown to specifically bind to HSC73 (35, 36). DSG has a peptidomimetic structure and is thought to bind and preclude peptide binding to HSC73. It has previously been shown to affect MHC-II Ag processing without affecting MHC-II expression (37). We therefore used DSG as a reagent to probe the involvement of HSC73 in MHC-II processing.
To investigate directly whether binding of Ag to HSC73 could be
prevented by DSG, HSC73-transfected and untransfected 97.2 cells were
incubated with DSG before and during exposure to exogenous HEL protein.
Cells were subsequently lysed and HSC73 immunoprecipitated, as
described above. Immunoprecipitates were fed to dendritic cells for
presentation to HEL-specific hybridoma 2G7.1. It was found that
incubation with DSG during Ag uptake abolished antigenic activity from
HSC73 immunoprecipitates of both HSC73-transfected and untransfected
97.2 cells, whereas immunoprecipitates from cells incubated with Ag in
the absence of DSG were antigenic (Fig. 5A). To test whether the
absence of a response was indeed due to a direct effect of DSG on
binding of HEL peptides by HSC73, rather than an inhibitory effect on
processing by DC, an experiment was set up mixing DSG containing
HSC73.14 precipitates with precipitate from HSC73.14 fed with HEL in
the absence of DSG. Stimulation by the mixture of precipitates did not
markedly reduce T cell activation (Fig. 5C), confirming that
the action of DSG is more likely on association of HEL peptides with
HSC73, rather than on processing by the dendritic cells. To ensure that
the action of DSG was HSC73-specific, 97.2 transfectants overexpressing
HSP60 were also given Ag in presence or absence of DSG, followed by
lysis and immunoprecipitation of HSP60. HSP60-overexpressing
transfectants exhibit enhancement of Ag processing and presentation
similar to HSC73 transfectants (our unpublished data). HSP60
immunoprecipitates, however, were antigenic in DC presentation assays,
regardless of the presence of DSG before lysis and immunoprecipitation
(Fig. 5C). This implies that DSG acted specifically on HSC73
and was capable of disrupting Ag/HSC73 interaction.
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Since DSG blocked the interaction of HSC73 with internalized
antigenic material, we next investigated whether the presence of DSG in
the Ag presentation assays would reduce or abolish presentation of
exogenous HEL to T cells. Increasing amounts of DSG were given together
with Ag, either native HEL protein or HEL[1–18] peptide in
cocultures of HSC73 transfectants or untransfected controls and T
cells. DSG inhibited presentation of HEL protein in HSC73 transfectants
and virtually abolished presentation in control 97.2 cells (Fig. 6). Presentation of peptide remained
unaffected up to a concentration of DSG of 10 µg/ml; higher amounts
of DSG appear to be toxic. Notably, a 6-fold higher concentration of
DSG was required to inhibit processing and presentation of HEL protein
by HSC73-overexpressing APC than was required for untransfected
controls. This indicates that availability of HSC73 is a critical
factor in MHC-II Ag processing.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An alternative and contiguous mechanism to peptide stabilization could involve the rapid transfer of bound, unfolded antigenic peptides by HSC73 to class II molecules whose peptide binding groove has been rendered vacant by the catalytic action of H2-M. Such a rapid transfer would be assisted by the ability of HSC73 to eject bound peptides using the energy of ATP hydrolysis.
We have shown that overexpression of HSC73 in an APC line enhances processing and presentation of protein Ag. Presentation of peptide was unaffected by HSP73 overexpression, and the levels of surface MHC-II were unchanged. We have further shown that HSC73 immunoprecipitated from Ag-fed cells could act as a source of Ag in Ag presentations assays, indicating that HSC73 is able to interact with and bind internalized Ag in APCs. The strategy used to test the coprecipitation of antigenic material did not impose any constraints on the form of Ag bound to HSC73; since the complex was subjected to processing by dendritic cells, the Ag could have been just partially unfolded, rather than fully processed. ATP treatments of these immunoprecipitates caused loss of antigenic activity, suggesting that disruption of the HSC73-Ag interaction is ATP-coupled in the same way as interactions of HSP70 family chaperones with unfolded or misfolded proteins in the endoplasmic reticulum and mitochondria have been shown to be (39). We have extended the findings (19, 21) that HSC73 is present in lysosomal compartments to show that the chaperone also colocalizes with MHC-II molecules, and is therefore correctly positioned to participate in Ag processing within the MHC-II pathway. A three-way complex between MHC-II, HSC73, and peptide, such as that suggested by Auger et al. (25), could serve to create a localized concentration of HSC73-bound antigenic peptide, which in turn would accelerate the formation of peptide/MHC-II complexes. In this scenario, MHC-II molecules could act as partner proteins to HSC73 and serve to dictate a specific function for the ubiquitous chaperone, as other partner proteins have been shown to do for HSP70s in other cellular contexts (40).
Studies of Ag processing compartments have left unclear whether antigenic epitopes are invariably generated in compartments where association with class II can occur. Harding et al. (41) have shown that certain epitopes from the same Ag require processing in different parts of the endocytic chain. This suggests that transport of antigenic peptides from degradative compartments to compartments where association with MHC-II molecules can occur is necessary for these epitopes to be displayed. HSC73 has been shown to be able to carry proteins to endolysosomal compartments from other subcellular locations (23, 24), and could conceivably fulfill this function. Also, the nonclassical entry of cytosolic proteins (42) into class II Ag processing compartments could be achieved this way. It is interesting to note that the chaperone activities of HSP70 family members are already established in the loading of MHC class I molecules and in the transport and protection of their corresponding peptides (43, 44, 45).
Our studies also probed the function of HSC73 in MHC-II Ag processing using the HSC73-specific reagent methyl 15-DSG. We showed that DSG blocked the interaction of HSC73 with internalized Ag and that it had an inhibitory effect on Ag presentation that could be diminished by overexpressing HSC73. Taken together, these results confirm and extend the observations of Hoeger et al. (37), who showed that DSG was capable of arresting presentation of processing dependent Ags, but not peptide, by human monocytes to specific T cell clones. Our results suggest that interaction of HSC73 with Ag is a crucial component of the MHC-II Ag processing pathway. The disruption of Ag/HSC73 interaction by DSG also offers an explanation of the results of Wang et al. (46), who showed that positive selection of thymocytes is affected by in vivo administration of DSG, since a lack of MHC-II molecules loaded with the appropriate ligands on thymic APCs could suspend the presentation of positively selecting stimuli to thymocytes, resulting in a phenotype analogous to the absence of crucial proteolytic enzymes (47).
Further studies are required to clarify a number of issues raised by the experiments detailed in this paper. The possibility exists that certain epitopes would not survive complete proteolytic degradation in the absence of stabilization by HSC73. In addition, the postulated function of HSC73 would render the ATPase activity of this chaperone essential, and this could be addressed by studying the functional effects of ATPase disabled HSC73. The ability of HSC73 to accelerate the formation of SDS-stable (peptide-loaded) MHC-II dimers could be investigated biochemically using in vitro systems, as well as immunological reagents, recognizing peptide-loaded MHC-II molecules.
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Footnotes |
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2 Address correspondence and reprint requests to Dr.Brigitta Stockinger, Division of Molecular Immunology, National Institute for Medical Research, Mill Hill, London NW7 1AA, U.K. E-mail address:
3 Abbreviations used in this paper: MHC-II, MHC class II; HSP, heat shock protein; HSC, heat shock cognate protein; DSG, desoxyspergualin; HEL, hen egg lysozyme.
Received for publication February 11, 1999. Accepted for publication May 27, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß dimers. J. Immunol. 152:1546.[Abstract]
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, H73.5; 
, H73.14; 
, H73.18; 
, H73.23)
are shown together with vector-only transfected controls (
).
A and E, HEL protein as Ag, given in
2-fold titration from a maximum concentration of 100 µg/ml.
HEL[1–18] (B) and HEL[46–61] (F)
synthetic peptide, given in 5-fold titration from a maximum
concentration of 5 µM. C, C5 protein as Ag, given in
2-fold titration from a maximum concentration of 10 µg/ml.
D, C5[106–121] synthetic peptide, given in 5-fold
titration from a maximum concentration of 5 µM.
