Adjuvanticity of α2-Macroglobulin, an
Independent Ligand for the Heat Shock
Protein Receptor CD91
This information is current as
of June 17, 2017.
Robert J. Binder, David Karimeddini and Pramod K.
Srivastava
J Immunol 2001; 166:4968-4972; ;
doi: 10.4049/jimmunol.166.8.4968
http://www.jimmunol.org/content/166/8/4968
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References
Adjuvanticity of ␣2-Macroglobulin, an Independent Ligand for
the Heat Shock Protein Receptor CD911
Robert J. Binder, David Karimeddini, and Pramod K. Srivastava2
H
eat shock proteins (HSPs)3 are abundant intracellular
proteins present in all cells. A subset of HSPs such as
gp96, hsp90, hsp70, and calreticulin possess unique immunological properties (1): they bind a broad array of peptides,
and immunization with the HSP-peptide complexes is highly effective at eliciting potent peptide-specific, MHC I-restricted, CTL
responses. For this reason, the HSPs, although of mammalian origin, are potent T cell adjuvants (2). Surprisingly small quantities of
peptides, e.g., tens of picograms, if complexed to HSPs are sufficient to elicit CTL responses (2), and immunization with HSPpeptide complexes is very sensitive to abrogation of APCs of the
immunized host (3). These observations had led us to propose in
1993 that HSPs interact with APCs through specific receptors,
which are responsible for their anomalous and potent immunological properties (4). It was subsequently demonstrated that gp96peptide complexes are taken up by APCs and the gp96-chaperoned
peptide, after internal processing in the APC, is re-presented on the
MHC I molecule of the APC (5). A cell surface receptor for the
HSP gp96, expressed on APCs, has indeed been identified recently
as CD91 or ␣2-macroglobulin (␣2M) receptor (6). Gp96, with its
chaperoned peptides, binds this receptor, allowing for its internalization and for the re-presentation of the chaperoned peptides.
mAbs to CD91 inhibit such re-presentation by gp96, as do other
ligands for CD91, such as ␣2M. ␣2M is a serum protein that has
some chaperone-like properties. It binds a wide array of molecules
such as cytokines and proteinases and internalizes them through its
receptor CD91 (7). Because gp96 and ␣2M appear to bind to the
Center for Immunotherapy of Cancer and Infectious Diseases, University of Connecticut School of Medicine, Farmington, CT 06030
Received for publication December 1, 2000. Accepted for publication February 6,
2001.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grant CA84479 and a
research agreement with Antigenics, Inc., in which P.K.S. has a significant financial
interest.
2
Address correspondence and reprint requests to Pramod Srivastava, University of
Connecticut School of Medicine, MC1601, Farmington, CT 06030-1920. E-mail address: [email protected]
Abbreviations used in this paper: HSP, heat shock proteins; ␣2M, ␣2-macroglobulin;
PEC, peritoneal exudate cells; SA, serum albumin.
3
Copyright © 2001 by The American Association of Immunologists
same receptor, CD91, we have asked the question whether ␣2M,
like gp96, can bind peptides, whether ␣2M-peptide complexes, like
gp96-peptide complexes, can elicit peptide-specific CTL responses, and whether ␣2M-peptide complexes, like gp96-peptide
complexes, can be re-presented by APCs.
Materials and Methods
Cell lines and reagents
RAW264.7 was a gift from C. Nicchitta (Duke University, Durham, NC),
and RAW309Cr.1 was purchased from American Type Culture Collection
(Manassas, VA). Peritoneal exudate cells (PEC) were obtained from the
peritoneum of BALB/c or C57BL/6 mice (The Jackson Laboratory, Bar
Harbor, ME) by flushing with cold PBS. Peptides AH1–20 and OVA20
were synthesized at Genemed Synthesis (San Francisco, CA). AH1–20
refers to a 20-mer extended variant (NH2-RVTYHSPSYVYHQFERRAKCOOH) of the Ld-binding epitope AH1 (NH2-SPSYVYHQF-COOH) of
the CT26 mouse colon carcinoma (8). OVA20 refers to a 20-mer extended
variant (NH2-SGLEQLESIINFEKLTEWTS-COOH) of the Kb-binding
epitope OVA8 (SIINFEKL) of OVA (9). CT26 and OVA-expressing E.G7
cells (10) were obtained from our cell culture bank.
Iodination of peptides
This was conducted by using IODO-BEADS (Pierce, Rockford, IL) as per
the manufacturers protocol. Free 125I was removed by using kwik-sep gel
filtration columns (Pierce) as per the manufacturers protocol.
Flow cytometry
Flow cytometry was performed on a FACScan (Becton Dickinson, San
Jose, CA). For FACScan analysis, all cells were incubated with FcBLOCK (anti-CD16/32 Ab) purchased from BD PharMingen (San Diego,
CA). Rabbit anti-mouse CD91 polyclonal IgG was generated in our laboratory and has been described previously (6). The CD11b Ab was purchased from BD PharMingen. Isotype control rabbit IgG and isotype control rat IgG were used for the anti-CD91 and anti-CD11b Abs, respectively.
Purification of gp96 and peptide complexing
Gp96 was purified as published previously (11). ␣2M and serum albumin
(SA) were purchased from Sigma (St. Louis, MO). Peptides were incubated
with proteins at a 50:1 molar ratio and incubated at 50°C for 10 min
followed by a 30-min incubation at 25°C. Free (uncomplexed) peptides
were removed by size-exclusion filters (Millipore, Bedford, MA). Proteinpeptide complexes were measured in a scintillation counter and per molar
basis, each protein was observed to bind equivalent amounts of peptide
(⬃0.1% of the starting amount of the peptide). Protein-peptide complexes
were stored at 4°C until use.
0022-1767/01/$02.00
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We recently have identified CD91 as a receptor for the heat shock protein gp96. CD91 was identified initially as a receptor for
␣2-macroglobulin (␣2M). Gp96 and ␣2M are both ligands for CD91. Because gp96-chaperoned peptides can prime CD8ⴙ T cell
responses and are re-presented by APCs, we tested ␣2M for similar properties. Our studies show that ␣2M binds peptides in vitro
and that the peptides, chaperoned by ␣2M, efficiently prime peptide-specific CD8ⴙ T cell responses in mice immunized with
␣2M-peptide complexes. Furthermore, peptides chaperoned by ␣2M, like those chaperoned by gp96, can be re-presented by
CD91ⴙ APCs on their MHC I molecules. These studies demonstrate that ␣2M molecules, like the heat shock protein molecules,
are T cell adjuvants that can channel exogenous Ags into the endogenous pathway of Ag presentaion. The remarkable similarities
between an intracellular chaperone and an extracellular serum chaperone may have interesting physiological ramifications. The
Journal of Immunology, 2001, 166: 4968 – 4972.
The Journal of Immunology
4969
scribed in the previous section), was added to the cultures. As controls, the
extended peptides or the precise MHC I epitopes were added at 1 ␮M final
concentration. Culture supernatants were harvested after 20 h and assayed
for IFN-␥ by ELISA with kits purchased from Endogen (Woburn, MA) and
used according to the manufacturers protocol.
Immunizations and mixed lymphocyte tumor cell cultures
BALB/c or C57BL/6 mice were immunized with the peptides AH1–20 or
OVA20, respectively, alone or complexed to ␣2M, gp96, or albumin (50
␮g of each protein in 100 ␮l of PBS). Immunizations were performed i.p.
Mice were boosted with 50 ␮g of the same complex after 1 wk. Spleen
cells were removed 1 wk after the last immunization and cultured with
irradiated stimulator cells, the AH1-expressing CT26 cells line, or the
OVA-expressing cell line EG7. Cultures were tested for the presence of
CTL in a 51Cr release assay by using AH1-pulsed or SIINFEKL-pulsed
target cells, respectively.
Results
␣2M, gp96, and SA bind antigenic peptides in vitro
Re-presentation assays
Re-presentation assays were performed as described previously (6).
Briefly, 1 ⫻ 104 T cells were incubated with 1 ⫻ 104 of the indicated APC.
The indicated protein (40 ␮g/ml), with or without bound peptide (as de-
FIGURE 2. ␣2M-peptide complexes
prime a peptide-specific CD8⫹ T cell response. BALB/c (top) or C57BL/6 (bottom) mice were immunized twice, 1 wk
apart, with AH1–20 (top) or OVA20
(bottom) peptides uncomplexed (free) or
complexed to ␣2M, gp96, or SA or with
PBS as indicated. Spleen cells were
stimulated with irradiated CT26 (top) or
E.G7 (bottom) for 1 wk before testing
for cytotoxicity against AH1- (top) or
SIINFEKL- (bottom) pulsed target cells,
as indicated. Three mice per group were
immunized; data is shown for a representative mouse. The experiment was
performed in duplicate.
Antigenic peptides chaperoned by ␣2M elicit a CD8⫹ T cell
response
Gp96-peptide and ␣2M-peptide complexes were generated with
AH1–20 and OVA20, and the preparations were filtered through
size exclusion filters to remove free peptides. Mice were immunized i.p. twice, 1 wk apart, with 50 ␮g of each complex in PBS,
per immunization. Control mice were immunized with 1 ␮g of
peptide alone (uncomplexed). Spleen cells were removed 1 wk
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FIGURE 1. ␣2M, gp96, and SA bind peptides in vitro. A, ␣2M, gp96, or
SA were incubated with 125I-labeled AH1–20 peptide at 50°C for 10 min
followed by a 30-min incubation at 25°C. Free peptide was removed as
described in Materials and Methods. Protein-peptide complexes were analyzed on a 5–15% gradient SDS-PAGE (top). Gels were exposed to film
and autoradiographed (bottom). B, ␣2M or SA (1 ␮g) were incubated with
increasing quantities of 125I-labeled AH1–20 peptide at 37°C. For SA, only
the reaction with the highest quantity of peptide is shown. All other experimental conditions are the same as in A. The binding was measured as
in A. Top panels are the autoradiographs of the silver-stained gels (bottom).
Gp96 and SA have been shown previously to bind peptides in vitro
by coincubation of proteins and peptides at 50°C (2). We tested
whether ␣2M could bind peptides under similar conditions. Gp96,
␣2M, and SA were mixed with iodinated preparations of AH1–20
peptide (described in Materials and Methods) for 30 min at 50°C
and the mixtures were analyzed by SDS-PAGE (to detect the proteins) and autoradiography (to detect bound peptides). All three
proteins were seen to bind peptides (Fig. 1A) and the proteinpeptide interaction was observed to be stable under denaturing
SDS-PAGE. This has been observed previously (2) for gp96 and
SA and was now observed for ␣2M as well. Similar data was
obtained with the OVA20 peptide (data not shown).
Binding at 50°C is obviously an experimentally created artifactual condition. We tested the binding of peptides to ␣2M and SA at
37°C as well. The experimental conditions and assays described
above were used except that the binding reaction was conducted at
37°C. Titrated quantities of the peptide AH1–20 were used in binding to ␣2M or to SA. It was observed that AH1–20 bound to ␣2M
but not SA in a dose-dependent and saturable manner (Fig. 1B).
4970
␣2M-PEPTIDE COMPLEXES PRIME PEPTIDE-SPECIFIC CD8⫹ T CELL RESPONSES
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FIGURE 3. CD91-expressing APC can re-present peptides chaperoned by ␣2M or gp96. A, RAW264.7 and CD11b⫹ PEC express significant levels of
CD91 but RAW309Cr.1 does not. RAW264.7, RAW309Cr.1, and PEC were stained with anti-CD91 and anti-CD11b Abs. RAW264.7 cells also were
stained with isotype control Abs (first panel on left); rabbit IgG and rat IgG, respectively. B, RAW264.7 cells (top) or RAW309Cr.1 cells (bottom) were
used as APCs for re-presentation of complexes of ␣2M, gp96, or SA with AH1–20, to AH1-specific T cells. Release of IFN ␥ was monitored as a marker
of T cell stimulation. The contents of each well are indicated. Each well was tested in duplicates and the experiment was repeated three times. C, CD11b⫹
PECs from C57BL/6 mice were used as APCs for re-presentation of complexes of ␣2M, gp96, or SA with OVA20 to SIINFEKL-specific T cells. Release
of IFN-␥ was monitored as a marker of T cell stimulation. The contents of each well are indicated. Each well was tested in duplicates and the experiment
was repeated three times.
The Journal of Immunology
after the last immunization and incubated in vitro with irradiated
stimulator cells (CT26 or E.G7, respectively) for 1 wk. Cytotoxic
assays were performed on the cultures by using P815 (H-2d) cells
pulsed with AH1 or EL4 cells (H-2b) pulsed with SIINFEKL as
indicated in Fig. 2. Complexes of AH1–20 or OVA20 with ␣2M or
gp96 were able to prime an AH1-specific CTL response in
BALB/c immunized mice or an OVA8-specific CTL response in
C57BL/6 mice, respectively (Fig. 2). Either peptide complexed
with SA did not elicit a CTL response, as observed previously (2),
nor did mice immunized with peptide alone, protein alone (uncomplexed to peptide), or with PBS.
Peptides chaperoned by ␣2M are re-presented by CD91expressing APCs
␣2M-peptide complexes, and as controls, gp96-peptide complexes
and SA-peptide complexes, were tested in a re-presentation assay.
We chose three types of APCs for the re-presentation: the macrophage cell line RAW264.7 and CD11b⫹ PEC, both of which express
high levels of the gp96-receptor CD91; and the macrophage cell line
RAW309Cr.1, which expresses little or no CD91 (Fig. 3A).
The Ld-restricted epitope AH1 (derived from the gp70 Ag of
murine colon carcinoma CT26, as described in Materials and
Methods) was used as the first model system. Complexes of ␣2M,
gp96, or SA with an AH1–20 peptide (designed to inhibit direct
charging of surface Ld of the APC by AH1) were pulsed onto
RAW264.7 cells that were used to stimulate a Ld/AH-1-specific
CD8⫹ T cell clone. As shown in Fig. 3B, T cells were stimulated
by the re-presenting APC and secreted IFN-␥. Control APCs
pulsed with ␣2M or gp96 without AH1–20 did not stimulate T
cells. The CD91-nonbinding protein SA, which binds effectively to
peptides (Fig. 1), did not stimulate T cells when pulsed onto APC
at any concentration tested. When the same experiment was repeated with the CD91-negative APC, RAW309Cr.1, no re-presentation of either gp96-chaperoned peptides or of ␣2M-chaperoned
peptides was detected.
In a second system, complexes of ␣2M, gp96 or SA with SIINFEKL
(OVA8)-precursor OVA20 were pulsed onto CD11b⫹ cells (purified
from PEC). These pulsed APCs were used to stimulate the
Kb/SIINFEKL-specific CD8⫹ T cell clone, 4G3. As shown in Fig. 3C,
4G3 T cells secreted IFN-␥ in response to APC pulsed with gp96OVA20 or ␣2M-OVA20 complexes but not with SA-OVA20 com-
plexes. The control wells that lacked OVA20 did not show re-presentation (Fig. 3C).
We next compared the efficiency of re-presentation of peptides
chaperoned by gp96 and ␣2M. Equivalent molar amounts of gp96
and ␣2M, complexed to AH1–20, were pulsed onto RAW264.7
cells. Pulsed APCs were used to stimulate AH1-specific T cells,
and the amount of T cell stimulation (as monitored by IFN-␥ release) was measured. Fig. 4 shows that on a molar basis, the gp96peptide complexes are re-presented with a slightly higher efficiency than the ␣2M-peptide complexes, even though both
molecules chaperone approximately equal amount of peptides. The
differences in efficiency is more clearly seen at the lower concentrations, where gp96-chaperoned peptides are re-presented 1.8
times more efficiently than peptides chaperoned by ␣2M. As noted
in the legend to Fig. 4, peptides chaperoned by SA were not represented, even at very high molar concentrations.
Discussion
Our studies show that ␣2M can bind peptides in vitro. In its SDS
stability, quantity, and temperature sensitivity, this binding appears
to be qualitatively similar to that observed for gp96 as reported
previously (2). ␣2M is known to bind a wide array of cytokines and
proteases by an unusual mechanism, and it is conceivable that the
peptide-binding observed here recapitulates that mechanism (7).
␣2M-Ag complexes have been demonstrated previously to elicit a
strong Ab response (12, 13) and a CD4⫹ T cell response (14) after
sequestration by APCs. These responses are to be expected of an
exogenous Ag. The novelty of our studies lies in the demonstration
that despite exogenous administration, ␣2M-Ag complexes, like
HSP-peptide complexes, elicit, a peptide-specific, CD8⫹ T cell
response.
The re-presentation of ␣2M-chaperoned peptides, as observed
here, provides mechanistic insight into the adjuvanticity of ␣2M. It
appears that CD91 is central to this adjuvanticity: peptide-binding
proteins like gp96 and ␣2M that bind CD91 can have their peptides
re-presented on MHC I, whereas those like SA that do not bind
CD91 do not. Conversely, APCs that express CD91 can re-present
gp96-chaperoned and ␣2M-chaperoned peptides, whereas CD91negative APCs cannot. CD91 seems to be a unique portal into the
APC that allows entry of the materials through it, a special opportunity of entering the endosomal-cytosolic compartment. This
should be a promising area of cell biological investigation.
During identification of CD91 as the receptor for gp96, we proposed a model (6) in which ␣2M samples the extracellular milieu
and the HSPs the intracellular milieu: altogether, the CD91 molecule becomes a mechanism for the APC to sample the entire
antigenic milieu of an organism. The new and remarkable similarities between intracellular chaperones (HSPs) and an extracellular serum chaperone (␣2M) observed by us here lead us to
strengthen our faith in that model and to ask whether the unusual
protein-trapping mechanism, so unique for ␣2M, is indeed primarily an Ag sampling device par excellence. These ideas may have
some bearing on immune response to cancers and infectious diseases and on mechanisms of peripheral tolerance.
Acknowledgments
We thank Sreyashi Basu, Toyoshi Matsutake, and Thirumalai Ramalingham of our laboratory for critically reading the manuscript. We also thank
Maria Bausero for technical support in some of the experiments.
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FIGURE 4. Gp96-chaperoned peptides are re-presented arguably more
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