Scavenger receptor-A mediates gp96/GRP94 and calreticulin internalization by antigen-presenting cells

gp96 (GRP94) elicits antigen-presenting cell (APC) activation and can direct peptides into the cross- presentation pathways of APC. These responses arise through interactions of gp96 with Toll-like (APC activation) and endocytic (cross-presentation) receptors of APC. Previously, CD91, the alpha2-macroglobulin receptor, was identified as the heat shock/chaperone protein receptor of APC. Recent data indicates, however, that inhibition of CD91 ligand binding does not alter gp96 recognition and uptake. Furthermore, CD91 expression is not itself sufficient for gp96 binding and internalization. We now report that scavenger receptor class-A (SR-A), a prominent scavenger receptor of macrophages and dendritic cells, serves a primary role in gp96 and calreticulin recognition and internalization. gp96 internalization and peptide re-presentation are inhibited by the SR-A inhibitory ligand fucoidin, although fucoidin was without effect on alpha2-macroglobulin binding or uptake. Ectopic expression of SR-A in HEK 293 cells yielded gp96 recognition and uptake activity. In addition, macrophages derived from SR-A-/- mice were substantially impaired in gp96 binding and uptake. These data identify new roles for SR-A in the regulation of cellular responses to heat shock proteins.     

Cell-surface Processing of the Metalloprotease Pro-ADAMTS9 Is Influenced by the Chaperone GRP94/gp96

A disintegrin-like and metalloprotease domain with thrombospondin type 1 motifs 9 (ADAMTS9) is a highly conserved metalloprotease that has been identified as a tumor suppressor gene and is required for normal mouse development. The secreted ADAMTS9 zymogen undergoes proteolytic excision of its N-terminal propeptide by the proprotein convertase furin. However, in contrast to other metalloproteases, propeptide excision occurs at the cell surface and leads to decreased activity of the zymogen. Here, we investigated the potential cellular mechanisms regulating ADAMTS9 biosynthesis and cell-surface processing by analysis of molecular complexes formed by a construct containing the propeptide and catalytic domain of pro-ADAMTS9 (Pro-Cat) in HEK293F cells. Cross-linking of cellular proteins bound to Pro-Cat followed by mass spectrometric analysis identified UDP-glucose:glycoprotein glucosyltransferase I, heat shock protein gp96 (GRP94), BiP (GRP78), and ERdj3 (Hsp40 homolog) as associated proteins. gp96 and BiP were present at the cell surface in an immunoprecipitable complex with pro-ADAMTS9 and furin. Treatment with geldanamycin, an inhibitor of the HSP90α family (including gp96), led to decreased furin processing of pro-ADAMTS9 and accumulation of the unprocessed pro-ADAMTS9 at the cell surface. gp96 siRNA down-regulated the levels of cell-surface pro-ADAMTS9 and furin, whereas the levels of cell-surface pro-ADAMTS9, but not of cell-surface furin, were decreased upon treatment with BiP siRNA. These data identify for the first time the cellular chaperones associated with secretion of an ADAMTS protease and suggest a role for gp96 in modulating pro-ADAMTS9 processing.     

GRP94/gp96 elicits ERK activation in murine macrophages. A role for endotoxin contamination in NF-kappa B activation and nitric oxide production

Vaccination of mice with GRP94/gp96, the endoplasmic reticulum Hsp90, elicits a variety of immune responses sufficient for tumor rejection and the suppression of metastatic tumor progression. Macrophages are a prominent GRP94/gp96 target, with GRP94/gp96 reported to activate macrophage NF-kappa B signaling and nitric oxide production, as well as the MAP kinase p38, JNK, and ERK signaling cascades. However, recent studies report that heat shock protein elicited macrophage activation is due, in large part, to contaminating endotoxin. To examine the generality of this finding, we have investigated the role of endotoxin in GRP94/gp96-elicited macrophage activation. We report that GRP94/gp96 binds endotoxin in a high-affinity, saturable, and specific manner. Low endotoxin calreticulin and GRP94/gp96 were purified, the latter using a novel method of depyrogenation; this resulted in GRP94/gp96 and calreticulin preparations with endotoxin levels substantially lower than those of previously reported preparations. Low endotoxin GRP94/gp96 retained its native conformation, ligand binding activity, and in vitro chaperone function, yet did not activate macrophage NF-kappa B signaling, nitric oxide production or inducible nitric-oxide synthase production. Low endotoxin GRP94/gp96 and calreticulin did, however, elicit a marked increase in ERK phosphorylation at protein concentrations as low as 2 microg/ml. These results are discussed with respect to current understanding of the contributions of endotoxin and heat shock/chaperone proteins to the stimulation of innate immune responses.     

Molecular mechanisms of peptide loading by the tumor rejection antigen/heat shock chaperone gp96 (GRP94)

Complexes of gp96/GRP94 and peptides have been shown to elicit immunogenicity. We used fluorescence to understand peptide association with gp96. A pyrene-peptide conjugate was complexed with gp96 under a variety of conditions. At room temperature in low salt (20 mM NaCl), the peptide binds gp96 with a strong affinity (approximately 100-150 nM) and forms pyrene excimers, suggesting that the peptides were assembled as dimers. In high salt (2.2 M NaCl), although peptide binding was stronger (Ka approximately 55 nM) than in low salt, pyrene excimers were absent, implying that peptides were farther apart in the complex. Heat shock-activated peptide binding exhibited characteristics of both low salt and high salt modes of binding. Anisotropy and average lifetime of the bound pyrene suggested that peptides were probably located in a solvent-accessible hydrophobic binding pocket in low salt, whereas in high salt, the peptide may be buried in a less hydrophobic (more hydrophilic) environment. These results suggested that peptide-gp96 complexes were assembled in several different ways, depending on the assembly conditions. Resonance energy transfer between the intrinsic tryptophan(s) in gp96 and pyrene suggested that one or more tryptophan residues were within the critical Forster distance of 27-30 A from the pyrene in the bound peptide. It is proposed that peptides are assembled within higher order gp96 complexes (dimers, etc.) in a hydrophobic pocket and that there may be a conformational change in gp96 leading to an open configuration for peptide loading.     

Cutting edge: CD91-independent cross-presentation of GRP94(gp96)-associated peptides

GRP94(gp96) elicits CD8(+) T cell responses against its bound peptides, a process requiring access of its associated peptides into the MHC class I cross-presentation pathway of APCs. Entry into this pathway requires receptor-mediated endocytosis, and CD91 (low-density lipoprotein receptor-related protein) has been reported to be the receptor mediating GRP94 uptake into APC. However, a direct role for CD91 in chaperone-based peptide Ag re-presentation has not been demonstrated. We investigated the contribution of CD91 to GRP94 cell surface binding, internalization, and trafficking in APCs. Whereas a clear role for CD91 in alpha(2)-macroglobulin binding and uptake was readily obtained, the addition of excess CD91 ligand, activated alpha(2)-macroglobulin, or receptor-associated protein, an antagonist of all known CD91 ligands, did not affect GRP94 cell surface binding, receptor-mediated endocytosis, or peptide re-presentation. These data identify a CD91-independent, GRP94 internalization pathway that functions in peptide Ag re-presentation.     

Biophysical analysis of the endoplasmic reticulum-resident chaperone/heat shock protein gp96/GRP94 and its complex with peptide antigen

Animals vaccinated with heat shock protein (HSP)--peptide complexes develop specific protective immunity against cancers from which the HSPs were originally isolated. This autologous specific immunity has been demonstrated using a number of HSP--peptide antigen complexes. A prototypical HSP-based cancer vaccine is the gp96--peptide antigen complex, which is currently undergoing human clinical trials. Here, we analyzed the structure of a recombinant wild-type and a mutant gp96 protein and their peptide complexes using a number of biophysical techniques. Gel filtration chromatography, dynamic light scattering, and equilibrium analytical ultracentrifugation demonstrated that both a wild-type gp96 and a gp96 mutant lacking a dimerization domain formed higher order structures. More detailed analysis using scanning transmission electron microscopy indicated that both the wild-type and dimerization deletion mutant gp96 protein were organized, unexpectedly, into large aggregates. Size distributions ranged from dimers to octamers and higher. Circular dichroism and intrinsic Trp fluorescence suggested that the gp96 dimerization domain deletion mutant protein was more compact than the wild-type gp96. A fluorescent peptide antigen was synthesized, and the peptide-binding properties of wild-type and the dimerization domain deletion mutant gp96 were studied. Fluorescence lifetime and anisotropy decay showed that the bound antigenic peptide was located in a hydrophobic pocket, with considerable free space for the rotation of the probe. Deletion of the dimerization domain affected the peptide-binding microenvironment, although peptide-binding affinity was reduced by only a small extent. Peptide--gp96 complexes were extremely stable, persisting for many days in the cold. The extraordinary stability of peptide--gp96 complexes and the plasticity of the peptide-binding pocket support the proposed relay of diverse peptides to MHC and/or other molecules via molecular recognition.     

The messenger and the message: gp96 (GRP94)-peptide interactions in cellular immunity

Vaccination of mice with tumor-derived stress proteins, such as Hsp70 and gp96 (GRP94), can elicit antitumor immune responses, yielding a marked suppression of tumor growth and metastasis. The molecular basis for this response is proposed to reflect a peptide-binding function for these proteins. In this view, stress proteins bind the antigenic peptide repertoire of their parent cell, and when provided to the immune system, tumor-derived stress protein-peptide complexes are processed by antigen-presenting cells (APCs) to yield the subsequent activation of tumor-directed cytotoxic T lymphocyte activity. This model predicts that stress proteins, whose primary intracellular function concerns the proper folding and assembly of nascent polypeptides, intersect with the cellular pathways responsible for the generation, processing, or assembly (or all) of peptide antigens onto nascent major histocompatability class I molecules. Recent insights into the pathways for peptide generation now allow this hypothesis to be critically examined, which is the subject of this review.     

The molecular chaperone gp96/GRP94 interacts with Toll-like receptors and integrins via its C-terminal hydrophobic domain

The structural basis for molecular chaperones to discern misfolded proteins has long been an enigma. As the endoplasmic reticulum paralogue of the cytosolic HSP90, gp96 (GRP94, HSP90b1) is an essential molecular chaperone for Toll-like receptors (TLRs) and integrins. However, little is known about its client-binding domain (CBD). Herein, we provide genetic and biochemical evidence to definitively demonstrate that a C-terminal loop structure, formed by residues 652-678, is the critical region of CBD for both TLRs and integrins. Deletion of this region affects neither the intrinsic ATPase activity nor the overall conformation of gp96. However, without it, the chaperoning function of gp96 collapses. We also find a critical Met pair (Met(658)-Met(662)) for the folding of integrins but not TLRs. Moreover, we find that the TLR binding to gp96 is also dependent on the C-terminal dimerization domain but not the N-terminal ATP-binding pocket of gp96. Our study has unveiled surprisingly the exquisite specificity of gp96 in substrate binding and suggests a manipulation of its CBD as an alternative strategy for targeted therapy of a variety of diseases.     

Gp96/GRP94 is a putative high density lipoprotein-binding protein in liver

We have previously shown that three high density lipoproteins (HDL)-binding proteins in liver, of 90, 110 and 180 kDa, are structurally related. In this study, these proteins are identified as gp96/GRP94. This protein is known to occur as a homodimer and has a dual subcellular localization: it is both an endoplasmic reticulum resident protein, where it is supposed to act as a chaperonin, and a plasma membrane protein, whose significance is unknown. In ultrastructural studies the plasma membrane localization of the homodimeric form was verified. The 90-kDa protein was abundantly present at the membranes of the endosomal/lysosomal vesicles as well as at the apical hepatocyte membranes, comprising the bile canaliculi. The monomeric protein is scarcely present at the basolateral membrane of the hepatocytes, but could be demonstrated in coated pits, suggesting involvement in receptor-mediated endocytosis. Labeling of the endoplasmic reticulum was virtually absent. Gp96/GRP94 was transiently expressed in COS-1 cells. However, the expressed protein was exclusively localized in the endoplasmic reticulum. Transfection with constructs in which the C-terminal KDEL sequence had been deleted, resulted in plasma membrane localized expression of protein, but only in an extremely low percentage of cells. In order to evaluate the HDL-binding capacities of this protein, stably transfected cells were generated, using several cell types. It appeared to be difficult to obtain a prolonged high level expression of gp96. In these cases, however, a marked increase of HDL-binding activity compared with the control cells could be observed.     

GRP94 reduces cell death in SH-SY5Y cells perturbated calcium homeostasis

The endoplasmic reticulum (ER) resident-94 kDa glucose-regulated protein (GRP94), plays a pivotal role in cell death due to ER stress. In our study expression of GRP94 was increased in human neuroblastoma SH-SY5Y cells due to exposure to calcium ionophore A23187. A23187-mediated cell death was associated with activation of the major cysteine proteases, caspase-3 and calpain. Pretreatment with adenovirus-mediated antisense GRP94 (AdGRP94AS) reduced viability of SH-SY5Y cells subjected to A23187 treatment compared with wild type cells or cells with adenovirus-mediated overexpression of GRP94 (AdGRP94S). These results indicated that suppression of GRP94 is associated with accelerated cell death. Moreover, expression of GRP94 suppressed A23187-induced cell death and stabilized calcium homeostasis.     

Glucose-regulated protein 94/glycoprotein 96 elicits bystander activation of CD4+ T cell Th1 cytokine production in vivo

Glucose-regulated protein 94 (GRP94/gp96), the endoplasmic reticulum heat shock protein 90 paralog, elicits both innate and adaptive immune responses. Regarding the former, GRP94/gp96 stimulates APC cytokine expression and dendritic cell maturation. The adaptive component of GRP94/gp96 function reflects a proposed peptide-binding activity and, consequently, a role for native GRP94/gp96-peptide complexes in cross-presentation. It is by this mechanism that tumor-derived GRP94/gp96 is thought to suppress tumor growth and metastasis. Recent data have demonstrated that GRP94/gp96-elicited innate immune responses can be sufficient to suppress tumor growth and metastasis. However, the immunological processes activated in response to tumor Ag-negative sources of GRP94/gp96 are currently unknown. We have examined the in vivo immunological response to nontumor sources of GRP94/gp96 and report that administration of syngeneic GRP94/gp96- or GRP94/gp96-N-terminal domain-secreting KBALB fibroblasts to BALB/c mice stimulates CD11b(+) and CD11c(+) APC function and promotes bystander activation of CD4(+) T cell Th1 cytokine production. Only modest activation of CD8(+) T cell or NK cell cytolytic function was observed. The GRP94/gp96-dependent induction of CD4(+) T cell cytokine production was markedly inhibited by carrageenan, indicating an essential role for APC in this response. These results identify the bystander activation of CD4(+) T lymphocytes as a previously unappreciated immunological consequence of GRP94/gp96 administration and demonstrate that GRP94/gp96-elicited alterations in the in vivo cytokine environment influence the development of CD4(+) T cell effector functions, independently of its proposed function as a peptide chaperone.     

CD133表位联合热休克蛋白佐剂疫苗引发抗白血病的免疫应答

肿瘤干细胞具有肿瘤形成、自我更新和抗化疗的特性,因而受到广泛关注和研究。CD133作为重要的肿瘤干细胞标志物与肿瘤细胞的干性与恶性密切相关。本研究筛选并且鉴定了CD133的3个H2-Kd限制性的细胞毒性T细胞 (CTL) 的表位,分别是CD133419–428、CD133702–710和CD133760–769。将重组热休克蛋白gp96为佐剂联合CD133表位制备表位疫苗,将疫苗免疫CD133+白血病移植的小鼠后引发抗肿瘤的特异性T细胞免疫应答。转输CD133表位特异的T细胞同样可以抑制小鼠淋巴瘤的生长。该研究为设计抗CD133+的白血病和其他肿瘤的表位疫苗提供了依据。     

Inducers of immunogenic cancer cell death

Recently, cytokine-based pro-tumourigenic signalling has been found to play a major role in the immune system's pro-tumourigenic activity. On the other hand, other recent findings have shown that immunogenic cancer cell death triggered by certain anticancer modalities might reset the dysfunctional immune system towards the activation of a long-lasting protective anti-tumour response. Therefore, using inducers of immunogenic cell death (ICD) that can prevent or impede tumour-promoting cytokine signalling is one of the best ways of instigating or restoring efficient anti-tumour immunity. In this review it is discussed, how the different ICD inducers interact with the immune system and influence cytokine-based pro-tumourigenic signalling. We believe that it is crucial to discover or develop new anti-cancer therapeutic modalities that can induce ICD and impede tumour-promoting cytokine signalling.     

Viral subversion of immunogenic cell death

While physiological cell death is non-immunogenic, pathogen induced cell death can be immunogenic and hence stimulate an immune response against antigens that derive from dying cells and are presented by dendritic cells (DCs). The obligate immunogenic “eat-me” signal generated by dying cells consists in the exposure of calreticulin (CRT) at the cell surface. This particular “eat-me” signal, which facilitates engulfment by DCs, can only be found on cells that succumb to immunogenic apoptosis, while it is not present on cells dying in an immunologically silent fashion. CRT normally resides in the lumen of the endoplasmic reticulum (ER), yet can translocate to the plasma membrane surface through a complex pathway that involves elements of the ER stress response (e.g., the eIF2α-phosphorylating kinase PERK), the apoptotic machinery (e.g., caspase-8 and its substrate BAP31, Bax, Bak), the anterograde transport from the ER to the Golgi apparatus, and SNARE-dependent exocytosis. A large panoply of viruses encodes proteins that inhibit eIF2α kinases, catalyze the dephosphorylation of eIF2α, bind to caspase-8, Bap31, Bax or Bak, or perturb exocytosis. We therefore postulate that obligate intracellular pathogens have developed a variety of strategies to subvert CRT exposure, thereby avoiding immunogenic cell death.     

Identification of the peptide-binding site in the heat shock chaperone/tumor rejection antigen gp96 (Grp94)

Heat shock protein (HSP)-peptide complexes from tumor cells elicit specific protective immunity when injected into inbred mice bearing the same specific type of tumor. The HSP-mediated specific immunogenicity also occurs with virus-infected cells. The immune response is solely due to endogenous peptides noncovalently bound to HSP. A vesicular stomatitis virus capsid-derived peptide ligand bearing a photoreactive azido group was specifically bound by and cross-linked to murine HSP glycoprotein (gp) 96. The peptide-binding site was mapped by specific proteolysis of the cross-links followed by analysis of the cross-linked peptides using a judicious combination of SDS-gel electrophoresis, mass spectrometry, and amino acid sequencing. The minimal peptide-binding site was mapped to amino acid residues 624-630 in a highly conserved region of gp96. A model of the peptide binding pocket of gp96 was constructed based on the known crystallographic structure of major histocompatibility complex class I molecule bound to a similar peptide. The gp96-peptide model predicts that the peptide ligand is held in a groove formed by alpha-helices and lies on a surface consisting of antiparallel beta-sheets. Interestingly, in this model, the peptide binding pocket abuts the dimerization domain of gp96, which may have implications for the extraordinary stability of peptide-gp96 complexes, and for the faithful relay of peptides to major histocompatibility complex class I molecule for antigen presentation.     

Molecular characteristics of immunogenic cancer cell death

Apoptotic cell death is initiated by a morphologically homogenous entity that was considered to be non-immunogenic and non-inflammatory in nature. However, recent advances suggest that apoptosis, under certain circumstances, can be immunogenic. In particular, some characteristics of the plasma membrane, acquired at preapoptotic stage, can cause immune effectors to recognize and attack preapoptotic tumor cells. The signals that mediate the immunogenicity of tumor cells involve elements of the DNA damage response (such as ataxia telangiectasia mutated and p53 activation), elements of the endoplasmic reticulum stress response (such as eukaryotic initiation factor 2alpha phosphorylation), as well as elements of the apoptotic response (such as caspase activation). Depending on the signal-transduction pathway, tumor cells responding to chemotherapy or radiotherapy can express 'danger' and 'eat me' signals on the cell surface (such as NKG2D ligands, heat-shock proteins and calreticulin) or can secrete/release immunostimulatory factors (such as cytokines and high-mobility group box 1) to stimulate innate immune effectors. Likewise, the precise sequence of such events influences the 'decision' of the immune system to mount a cognate response or not. We therefore anticipate that the comprehension of the mechanisms governing the immunogenicity of cell death will have a profound impact on the design of anticancer therapies.     

Transfer of GRP94(Gp96)-associated peptides onto endosomal MHC class I molecules

GRP94 (gp96)-associated peptides can elicit cellular immune responses, an activity thought to reflect the presence of a cell surface receptor (CD91) on antigen-presenting cells that mediates GRP94 internalization and trafficking to an amenable site for peptide transfer to major histocompatibility complex class I molecules. We report that GRP94 internalized by receptor-mediated endocytosis is trafficked to a Rab5a, CD1 and transferrin-negative, Fc receptor and major histocompatibility complex class I-positive endocytic compartment. Receptor-internalized GRP94 did not access the endoplasmic reticulum of antigen-presenting cells. To identify the site of re-presentation of GRP94-associated peptides, kinetic analyses were performed utilizing GRP94-OVA (SIINFEKL) peptide complexes, with peptide re-presentation assayed with the K^b-SIINFEKL-specific MAb, 25-D1.16. Analyses of the kinetics of re-presentation of GRP94-associated peptides, under conditions in which de novo synthesis of major histocompatibility complex class I molecules was inhibited, identified a post-endoplasmic reticulum compartment, accessed by mature major histocompatibility complex class I, as the predominant site of GRP94-associated peptide exchange onto major histocompatibility complex class I.     

Binding of the viral immunogenic octapeptide VSV8 to native glucose-regulated protein Grp94 (gp96) and its inhibition by the physiological ligands ATP and Ca2+

The molecular chaperone Grp94 (gp96) of the endoplasmic reticulum (ER) lumen plays an essential role in the structural maturation and/or secretion of proteins destined for transport to the cell surface. Its proposed role in binding and transferring peptides for immune recognition is, however, controversial. Using SPR spectroscopy, we studied the interaction of native glycosylated Grp94 at neutral pH and 25 and 37 degrees C with the viral immunogenic octapeptide RGYVYQGL (VSV8), derived from vesicular stomatitis virus nucleoprotein (52-59). The peptide binds reversibly with low affinity ([A]0.5 approximately 640 microM) and a hyperbolic binding isotherm, and the binding is partially inhibited by ATP and Ca2+ at concentrations that are present in the ER lumen, and the effects are explained by conformational changes in the native chaperone induced by these ligands. Our data present experimental support for the recent proposal that, under native conditions, VSV8 binds to Grp94 by an adsorptive, rather than a bioselective, mechanism, and thus further challenge the proposed in vivo peptide acceptor-donor function of the chaperone in the context of antigen-presenting cell activation.     

Binding of antigenic peptide to the endoplasmic reticulum-resident protein gp96/GRP94 heat shock chaperone occurs in higher order complexes. Essential role of some aromatic amino acid residues in the peptide-binding site

Vaccination with heat shock protein gp96-antigenic peptide complexes produces a powerful specific immune response against cancers and infectious diseases in some experimental animal models, and gp96-peptide complexes are now being tested in human clinical trials. gp96 appears to serve as a natural adjuvant for chaperoning antigenic peptides into the immune surveillance pathways. A fundamental issue that needs to be addressed is the mechanism of binding of antigenic peptide to gp96. Here, we show using scanning transmission electron microscopy that recombinant gp96 binds peptide in stable multimeric complexes, which may have biological significance. To open the possibility for genetically engineering gp96 for improved immunogenicity and to understand if molecular recognition plays a role in the binding of antigenic peptide, we mutagenized some specific aromatic amino acids in the presumed peptide-binding pocket. Replacement of Tyr-667 or Tyr-678 to Ala reduced affinity for peptide whereas conversion of Trp-654 to Tyr increased peptide binding. Similarly, changing Trp-621 to Phe or Leu or Ala or Ile negatively affected peptide binding whereas changing Trp-621 to Tyr or Val positively affected peptide binding. Probing the peptide microenvironment in gp96-peptide complexes, suggested that hydrophobic interactions (and perhaps hydrogen bonding/stacking interactions) may play a role in peptide loading by gp96.     

Reinforcing the immunogenic cell death to enhance cancer immunotherapy efficacy

Immunogenic cell death (ICD) has been a revolutionary modality in cancer treatment since it kills primary tumors and prevents recurrent malignancy simultaneously. ICD represents a particular form of cancer cell death accompanied by production of damage-associated molecular patterns (DAMPs) that can be recognized by pattern recognition receptors (PRRs), which enhances infiltration of effector T cells and potentiates antitumor immune responses. Various treatment methods can elicit ICD involving chemo- and radio-therapy, phototherapy and nanotechnology to efficiently convert dead cancer cells into vaccines and trigger the antigen-specific immune responses. Nevertheless, the efficacy of ICD-induced therapies is restrained due to low accumulation in the tumor sites and damage of normal tissues. Thus, researchers have been devoted to overcoming these problems with novel materials and strategies. In this review, current knowledge on different ICD modalities, various ICD inducers, development and application of novel ICD-inducing strategies are summarized. Moreover, the prospects and challenges are briefly outlined to provide reference for future design of novel immunotherapy based on ICD effect.