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J Immunol 2014; 193:5765-5771; ;
doi: 10.4049/jimmunol.1401417
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References
Robert Julian Binder
Brief Reviews
The
Journal of
Immunology
Functions of Heat Shock Proteins in Pathways of the
Innate and Adaptive Immune System
Robert Julian Binder
H
eat shock proteins (HSPs) were inadvertently discovered in 1962 when Drosophila salivary glands
were grown overnight at elevated temperatures and
were found to be highly upregulated (1). Several HSPs have
been discovered since then, and they are grouped into families
based on m.w. (e.g., hsp10, hsp40, hsp60, hsp70, hsp90), although family members may be structurally unrelated (2).
The ability of HSPs to chaperone nascent polypeptides for
proper folding and expression is their best known function
(2); however, a significant role for HSPs in the immune
system has emerged in the past three decades and is the focus
of this review. The usefulness of HSP-mediated immune
responses for immunotherapy of disease is mentioned. The
immunological roles for HSPs, in situ or in the extracellular
environment, rely fully or partially on the fundamental property of a select few HSPs to act as a chaperone of peptides,
in addition to whole proteins.
Peptide-binding properties of select HSPs
The intracellular association of HSPs with peptides has been
demonstrated on structural, biochemical, and immunological
levels. The bacterial homolog of mammalian hsp70, DnaK,
was crystallized in 1996, together with its associated peptide
occupying a peptide-binding groove (3, 4). Peptide binding
sites have been identified for calreticulin (5) and hsp90 (6), as
well as its endoplasmic reticulum (ER) paralogue gp96 (7).
Peptides associate with each of these HSPs with low affinity
Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh,
PA 15261
Received for publication June 3, 2014. Accepted for publication September 7, 2014.
This work was supported by National Institutes of Health Grants CA90440, CA137133,
and AI079057.
Address correspondence and reprint requests to Dr. Robert Julian Binder, University of
Pittsburgh School of Medicine, E1058 Biomedical Science Tower, 200 Lothrop Street,
Pittsburgh, PA 15261. E-mail address: [email protected]
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401417
and no or low specificity (8–10). The peptides are generally 10–
30 aa in length and are generated during normal protein
turnover in cells (8–10). Because the peptide binding sites for
each HSP are different, the case can be made for distinct associated peptide repertoires. It is important to note that the
association of HSPs with peptides is not an artifact of cell lysis
(11). In many immunological systems tested, peptides derived
from antigenic proteins were shown to be associated with cytosolic (hsp90 and hsp70) and ER (calreticulin and gp96)
HSPs. These antigenic peptides include those derived from viral
(12–18) or bacterial (19) proteins from infected cells, tumor
Ags from malignant cells (20–22), model Ags from transfected
cells (23–27), and alloantigens from MHC-mismatched cells
(28, 29). Immunologically, peptides chaperoned by HSPs reflect the antigenic fingerprint of a cell. Finally, the association of
HSPs with peptides appears to be an evolutionarily conserved
property, because HSPs from bacteria (3), amphibians (30),
rodents (12–14, 17–19, 21–25, 31), and primates (16, 20) can
bind peptides. The HSPs and MHC are the two prominent
families of peptide-binding proteins in immunology.
In situ HSPs and the immune system
HSPs and the MHC class I Ag-presentation pathway. The classical
view of the pathway that leads to productive peptide presentation by MHC class I (MHC I) begins with protein
degradation by the cytosolic proteasome. Although the source
of proteasome substrates is under intense debate (32, 33),
extended polypeptides are cleaved to small peptides with the
correct C-termini, but extended N-termini, for occupying
MHC I. For several years these peptides were inexplicably
assumed to diffuse from the proteasome to the TAP for
transportation into the ER (34). ER peptides are loaded
onto nascent MHC H chains with the assistance of the
peptide loading complex (PLC) comprising TAP, tapasin,
ERp57, and calreticulin. During loading onto MHC I, peptides simultaneously undergo a final round of N-terminal
trimming by ER-associated protease (34). Peptide-loaded
MHC I H chains associate with b2M, and the trimeric
complex is transported through the Golgi and ultimately
expressed on the cell surface, where it can be recognized by
T cells. Studies from several laboratories argue strongly for
Abbreviations used in this article: DC, dendritic cell; ER, endoplasmic reticulum; HSP,
heat shock protein; MHC I, MHC class I; PLC, peptide-loading complex; RA, rheumatoid arthritis; Treg, regulatory T cell.
Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00
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For more than 50 years, heat shock proteins (HSPs)
have been studied for their role in protecting cells from
elevated temperature and other forms of stress. More
recently, several roles have been ascribed to HSPs in
the immune system. These include intracellular roles
in Ag presentation and expression of innate receptors,
as well as extracellular roles in tumor immunosurveillance and autoimmunity. Exogenously administered HSPs
can elicit a variety of immune responses that have been
used in immunotherapy of cancer, infectious diseases,
and autoimmune disease. The Journal of Immunology,
2014, 193: 5765–5771.
5766
resultant peptides can be presented by MHC I, a process referred to as cross-presentation (34). There is debate regarding
the cellular compartment in which Ags are processed for
cross-presentation (34, 50). Vesicular bodies into which Ag
is internalized, such as endosomes or phagosomes, may
become specialized peptide-processing and MHC I–loading
compartments through recruitment of selective ER proteins
that perform these roles. MHC I–peptide complexes in these
vesicles recycle to the cell surface (50). A prevalent second
pathway involves trans-endosomal membrane transport of
the Ag to the cytosol where it enters the classical MHC I
Ag-presentation pathway (34). In support of this, the
proteasome dependence for cross-presentation of Ags by
APCs was shown (34). hsp90 and hsp70 are intimately
involved in the extrusion of Ags from the endosomes to the
cytosol through a putative pore structure (51, 52). This
mechanism involves the unfolding and linearization of the
protein Ags by hsp90 prior to extrusion. Pharmacological
inhibition of hsp90 or its deletion in cross-presenting APCs
or Ag rendered structurally inflexible through fixation
abrogated Ag cross-presentation (51). Interestingly, hsp70
was shown to negatively regulate Ag extrusion, arguing for cooperative regulation of this process by two cytosolic HSPs.
These specific roles for hsp70 and hsp90 may account,
at least in part, for the extraordinary efficiency of extracellular HSPs at cross-presenting Ags that are chaperoned by
them (as described below). Endogenous HSPs, through functionality or expression levels, appear to be key regulators
of Ag cross-presentation. Coincidentally, hsp90 and hsp70
are induced in response to stress and IFNs, the precise situations during which cross-presentation is required.
gp96 as a chaperone of innate immune receptors. gp96 has a welldescribed function in the ER for folding nascent polypeptides
into their final conformation (2). These client proteins are
wholly dependent on gp96 for proper expression. All TLRs,
with the exception of TLR3, are client proteins of gp96 (53).
Folding of TLRs is dependent on dimerization of gp96 and its
cochaperone CNPY3 also present in the ER lumen. Following
gp96 depletion in APCs, TLRs are rapidly removed from
the cell surface and intracellular vesicles, rendering APCs
functionally unresponsive to corresponding TLR ligands, as
measured by a lack of NF-kB activation and secretion of
cytokines. Structural studies on gp96 identified the TLRbinding domains, which are distinct from ATP-binding
domains (54). Several other client proteins, such as a and b
integrins, known for their roles in influencing immune
responses, bind to the same domain. Thus, mice deficient
in gp96 expression are severely susceptible to bacterial
infection (55). TLRs are the critical sensors for recognition
of microorganisms, and their expression patterns are closely
related to function. Upstream processes that determine TLR
expression are expected to regulate immunity to pathogens;
thus, gp96 is a master chaperone that can control immune
responses with fine specificity based on selectivity and
titratability in client binding.
Extracellular HSPs and influence of adaptive immunity
Collectively, HSPs are the most abundant proteins in cells, and
their liberation into the extracellular environment is a key
immunological indicator of loss of cellular integrity. HSPs in
the extracellular environment are a product of pathologic cell
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modifications to this oversimplistic classical pathway. First,
hydrophobic amino acids present in short peptides (which
cannot assume higher-ordered structures) cannot exist in the
aqueous intracellular environment without being shielded (35),
a role provided by HSPs (Fig. 1). Second, diffusion as a
transport mechanism is too uneconomical to satisfy the
calculated efficiency of production of peptide/MHC I on
the cell surface (36, 37). This efficient process can be
satisfied by factoring in the HSPs. hsp70, hsp90, gp96, and
calreticulin were proposed, in 1994, to form a relay line for
efficient transport of peptides from the proteasome to MHC I
(38). The following evidence strongly supports this proposal.
1) Disruption of peptide binding to hsp70 or hsp90 with
deoxyspergualin or tanespimycin, respectively, abrogates
peptide presentation by MHC I (39, 40). Knock down of
hsp90a with small interfering RNA produces a similar
phenotype (41). 2) When peptides are eluted from highly
purified, apparently homogenous HSPs, MHC-binding
peptides and their precursors (intermediates of the processing
events) are found (8–10, 12–29). 3) Transfer of progressively
trimmed peptides between HSPs and MHC I has been observed
in the ER (42). 4) Naturally occurring free peptides cannot be
detected in cells even after the most rigorous of experimental
exploration (11). Once cells or their derived lysates are treated
with ATP or mild acid, agents known to release peptides from
chaperones, peptides can be readily detected (11). 5) Peptides
transported by TAP associate with gp96 and other ER
chaperones, including protein disulfide isomerase (43).
These HSPs adhere to certain specifications to satisfactorily
transfer peptides for MHC I presentation. The ubiquitous
expression of hsp70, hsp90, calreticulin, and gp96 in cells
essentially ensures that the MHC I–presentation pathway
remains functional in all nucleated cells. IFN-g inducibility of
a majority of the components of the MHC I–presentation
pathway also applies to HSPs involved in the pathway (44,
45). The receipt and delivery of peptides by HSPs suggest
a physical association of those HSPs with other components
of the pathway. hsp90 was shown to physically associate with
the proteasome and, in certain instances, replace the PA28
subunits (46, 47), situating hsp90 in an optimal position to
receive peptides being generated by the proteasome. hsp70
and hsp90 form heteromultimeric complexes in the cytosol
with other cochaperones (48). Although this complex is essential for client protein–refolding roles, peptide transfer
within this complex has yet to be tested. hsp70 also were
shown to be in physical proximity to TAP (49), an association
that would allow delivery of HSP-chaperoned peptides to
TAP for onward translocation into the ER. This suggests that
the proteasomes, hsp70, hsp90, and TAP could exist as
a single cytosolic multimeric unit akin to the PLC. Within the
ER, the PLC has been well defined, with calreticulin playing
a peptide-binding role. The transfer of peptides among calreticulin, gp96, and MHC I in the ER has been documented
(42). The apparent lack of association of gp96 (and ERassociated protease) with other PLC members may reflect
a weaker or transient interaction with this complex, considering the other significant roles for gp96 in ER biology. Other
unidentified chaperones may be involved in this pathway.
Cytosolic HSPs in endosome–cytosol trafficking of cross-presented Ags.
APCs capture Ags from the extracellular space and internalize
them into vesicular bodies. Following processing of the Ags,
BRIEF REVIEWS: HSPs IN THE IMMUNE SYSTEM
The Journal of Immunology
5767
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FIGURE 1. (AA) hsp90 and hsp70 are intricately involved in shuttling peptides from the proteasome to TAP in the cytosol. In the ER, gp96 and calreticulin
(CRT) sequentially transfer peptides from TAP to MHC. (AB) hsp90 and hsp70 regulate the extrusion of Ags from endosomes to the cytosol through a putative
pore during Ag cross-presentation. (AC) gp96 folds and controls the expression of TLRs and other client proteins. HSPs released from tumor cells through
a variety of mechanisms (BA) are endocytosed by CD91 expressed on APCs (BB). HSPs and the chaperoned peptides are processed within the cell (BC) before
presentation of the peptide on MHC molecules to CD4 or CD8 T cells (BD). (BE) CD91 also serves as a signaling receptor, controlling the expression of
cytokines and costimulatory molecules. (BF) Thus, HSPs can prime diverse Th cell responses.
5768
1) Tumor Ags have been grouped into neoantigens resulting from mutated, overexpressed, or abnormally
expressed proteins (67), although the former are ubiquitous and appear to be the most immunogenic (67).
Priming of tumor-specific T cells requires that these
tumor Ags are transferred from the Ag-expressing (tumor) cell to APCs for cross-presentation (68). Mutated
self proteins, expressed by a few abnormal cells, set the
limitations for the amount of material available for
cross-presentation during tumorigenesis. These limits
have been quantified and are in the picogram range
(69, 70). However, cross-presentation of soluble protein
Ags, as measured in several model Ag and pathogen
experimental systems, requires ∼10 to hundreds of
micrograms [e.g., (66, 71)]. The five orders of disparity
between these two quantities are satisfied when HSP–
peptide complexes are considered as the mediators of Ag
transfer (69). Peptides chaperoned by HSPs are efficiently cross-presented by APCs, and the efficiency is
largely attributed to the presence of an endocytic receptor CD91 (60–62, 72). The mechanisms associated
with cross-presentation of HSP-chaperoned peptides
have been quantified and detailed in a large body of
work (60–63, 73). Importantly, femtogram to picogram
levels of a specific peptide complexed to 10 mg of gp96,
hsp70, or calreticulin are sufficient for cross-presentation
(74). My group proposed that the HSP–peptide–CD91
mode of Ag transfer must be used during tumorigenesis given limiting Ag levels. Mice deficient in CD91
expression on APCs failed to mount immune responses
to growing tumors (75), a phenotype previously seen
in IFN-g– or rag-deficient mice (64). CD91-deficient
mice or tumors expressing endogenous inhibitors of
CD91 fail to adequately cross-present Ag and stimulate T cell responses. These observations point to an
indispensable role for endogenous tumor-derived
HSPs and their receptor CD91 in establishing immune responses to a developing tumor. In other
pathological situations in which Ag load is considerably higher, other modes of Ag transfer may apply
(76).
2) Tumors, being of self origin, lack expression of pathogenassociated molecular patterns, which typically elicit costimulation. We do not know the origin of costimulation during tumorigenesis. Purified gp96, hsp70, and
calreticulin were shown to stimulate and mature APCs,
which are associated with cytokine release and upregulation of costimulatory molecules (77, 78). HSPstimulated dendritic cells (DCs) are perfectly suited
for priming T cells. Incidentally, CD91 was shown to
be a signaling receptor for immunogenic HSPs activating p38 MAPK and NF-kB (78). The repertoire of
cytokines released and costimulatory molecules expressed are specific to the CD91+ APC and the HSP
used, but routinely includes IL-1b, TNF-a, IL-6, IL12, and GM-CSF (78). HSP-stimulated APCs upregulate CD80, CD86, CD40, and MHC class II (77).
In a tumor microenvironment, with the release of
multiple HSPs and with APCs in this locale, the response is of the Th1 type, capable of rejecting the
tumor.
As a single entity, the HSP–peptide complexes are capable
of priming T cell responses. These responses are considered in
juxtaposition with other intracellular molecules capable of
stimulating APCs, such as HMGB1, dsDNA, and uric acid
(79).
Cellular necrosis, which is a near-universal occurrence in
cancer, appears to be the most rational mechanism for extracellular HSP release (56). However, there are other
mechanisms through which HSPs are accessible to their
receptor(s) on immune cells (57–59, 80). Therefore, immunologically dangerous situations can be classified quantitatively and qualitatively according to the potential for
release of HSPs, or more generally, intracellular content,
which provide signals that may complement, supersede, or
antagonize those originating from other pathways (e.g.,
pathogen-associated molecular pattern–pattern recognition
receptor) or, in the absence of other apparent signals, elicit
exclusive responses.
Extracellular HSPs and etiology of autoimmunity. During homeostasis, it is necessary that HSPs are largely inaccessible to
their receptors. Sustained release of HSPs into the extracellular
environment would be expected to trigger autoimmunity based
on the observed proinflammatory conditions elicited by some
HSPs. Although there is no formal experimental demonstration of this expectation, there is some suggestion of it in various
pathological conditions.
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death, abnormal active secretory mechanisms, or nonconventional cellular expression on the membrane (56–59). A
select group of HSPs, when extracellular, is recognized by
cellular immune sentinels. Such recognition allows HSPs to
be critical mediators of immune responses, initiating specific
immune responses against cancers or pathogen-infected cells,
exacerbating pre-existing inflammatory conditions, or suppressing ongoing immunity. The surprising discovery of HSP
receptors on APCs afforded a molecular description of these
immunological mechanisms (60–63) (Fig. 1B).
Extracellular HSPs are necessary for immunosurveillance of cancer.
The ability of the immune system to recognize aberrant,
premalignant cells and eliminate them before they become
pathological is the hallmark of tumor immunosurveillance
(64). Immunosurveillance and resulting immunoediting include an elimination, equilibrium, and escape phase (64).
T cell responses, initiated during the elimination phase, are
primarily responsible for rejection of the tumorigenic cells
(65). T cells receive signal 1 through TCR recognition of
Ag presented by MHC. Recognition of costimulatory
ligands on APCs by corresponding receptors on T cells
provides signal 2. These signals are provided in the context
of a cytokine milieu. In a majority of cases, these signals are
sufficient for T cell priming, and all abnormal premalignant
cells are eliminated. A failure of the immune system to
completely eliminate the abnormal cells sets the stage for an
equilibrium phase, a state that can persist for many years.
Cancers then develop when they escape immune control.
The conventional mechanism of priming T cells has been
largely elucidated using experimental systems with model
Ags or pathogens (66). These mechanisms are not wholly
applicable during tumorigenesis and have been revised to
include the immunological effects of HSPs for two reasons.
BRIEF REVIEWS: HSPs IN THE IMMUNE SYSTEM
The Journal of Immunology
hsp70 (see Ref. 102)], in patients with autoimmune diabetes
[with hsp60 (95, 103)], and in virally infected patients [with
hsp70 (104)].
The plasticity of HSP-mediated immunity (i.e., priming
Th1, Th2, Th17, and/or Tregs) depends on the ability of HSPs
to engage local APCs through cell surface receptors, thereby
stimulating diverse costimulatory signals (77, 78, 95). CD91
is one such prominent receptor. When HSPs are introduced
at higher doses, we speculate that additional APCs and/or
(lower-affinity) cell surface receptors (63) could be targeted.
The 24mer peptide of hsp60 offers a clear example: it was
shown to bind TLR2 and TLR4 mediating anti- and proinflammatory conditions, respectively (95). The repertoires of
cytokines released by bone marrow–derived dendritic cells,
peritoneal exudate cells, and lymph node DCs stimulated
with HSPs are overlapping, but distinct, and dependent on
receptor usage (78). The overall signal received by the cell
from engagement of multiple signaling receptors, as well as
the resulting costimulation, determines the Th cell response
that is primed. The recent demonstration that blocking Treg
generation allowed gp96 to mediate stronger peptide-specific
CTL responses in BALB/c mice provides further evidence for
this dynamic interplay, as do the studies on hsp60 (95). Although many of the experimental systems are necessarily
minimalistic, it is important to consider the sum of these
signals during pathology in conjunction with signals emanating from other engaged (pattern recognition) receptors.
Conclusions
It has been 30 years since Srivastava and colleagues first described the immunogenicity of HSPs. The HSPs (and the
receptor CD91) are evolutionarily ancient; thus, it is unsurprising that the immune system has evolved to use HSPs in
conserved pathways. As we continue to better understand their
functions, other roles for HSPs in innate and adaptive immunity may be uncovered, not only in the diseased state but
also during homeostasis. With the recent appreciation of
various commensals in regulating immune responses, for example, we are yet to appreciate the role of commensal-derived
HSPs in generating signals that may affect those emanating
from PRRs.
Acknowledgments
I thank Drs. Abigail Sedlacek and Yu Jerry Zhou for advice and discussions.
Disclosures
R.J.B. is a named inventor of intellectual property that is being evaluated under
his National Institutes of Health grants, and the technology has been licensed
to a company in which R.J.B. has no ownership interest or consulting contract
and from which he has no sponsored research agreements. His significant financial interest consists only of a share of license fees and possible future royalties from the commercialization of his invention.
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