Adjuvanticity of alpha 2-macroglobulin, an independent ligand for the heat shock protein receptor CD91

We recently have identified CD91 as a receptor for the heat shock protein gp96. CD91 was identified initially as a receptor for alpha(2)-macroglobulin (alpha(2)M). Gp96 and alpha(2)M are both ligands for CD91. Because gp96-chaperoned peptides can prime CD8(+) T cell responses and are re-presented by APCs, we tested alpha(2)M for similar properties. Our studies show that alpha(2)M binds peptides in vitro and that the peptides, chaperoned by alpha(2)M, efficiently prime peptide-specific CD8(+) T cell responses in mice immunized with alpha(2)M-peptide complexes. Furthermore, peptides chaperoned by alpha(2)M, like those chaperoned by gp96, can be re-presented by CD91(+) APCs on their MHC I molecules. These studies demonstrate that alpha(2)M 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 heat shock protein gp96: a receptor-targeted cross-priming carrier and activator of dendritic cells

Heat shock proteins like gp96 (grp94) are able to induce specific cytotoxic T-cell (CTL) responses against cells from which they originate and are currently studied in clinical trials for use in immunotherapy of tumors. We have recently demonstrated that gp96 binds to at least one yet unidentified receptor restricted to antigen-presenting cells (APCs) like dendritic cells (DCs) but not to T cells. Moreover we have shown, that for CTL activation by gp96-chaperoned peptides receptor-mediated uptake of gp96 by APCs is required. Lately, we have discovered a second function of gp96 when interacting with professional APCs. Gp96 is able to mediate maturation of DCs as determined by upregulation of MHC class II, CD86 and CD83 molecules, secretion of pro-inflammatory cytokines IL-12 and TNF-alpha and enhanced T-cell simulatory capacity. Furthermore, the gp96 receptor(s) are down-regulated on mature DCs, suggesting that the gp96 receptor(s) behave similar to other endocytic receptors like CD36, mannose receptor etc. Our findings now provide additional evidence for the remarkable immunogenicity of gp96: first, the existence of specific gp96 receptors on APCs and second, the capacity to activate dendritic cells which is strictly required to enable these highly sophisticated APCs to prime CTL responses.     

热休克蛋白-多肽复合物在肿瘤和传染性疾病免疫中的作用

热休克蛋白家族中的许多成员如gp96\,HSP90\,HSP70等具有排斥和治疗肿瘤及传染性疾病的免疫原性,进一步研究发现热休克蛋白作为分子伴侣可结合细胞中的肽库,它本身没有抗原性,抗原性由结合的短肽所决定。热休克蛋白将结合的短肽呈递给I类MHC分子,进而激活特异性CTL和记忆性T细胞,引发机体细胞免疫反应。据最新发现gp96还可能有与MHC一样的功能,可直接将结合的多肽抗原呈递给T细胞。近年来对哺乳动物的二种主要热休克蛋白gp96和HSP70的免疫机制和作为治疗性疫苗的优越性进行了详细研究,这为乙型肝炎和乙肝继发性肝癌的免疫治疗提供了新思路。     

Cutting edge: tumor secreted heat shock-fusion protein elicits CD8 cells for rejection.

The endoplasmic reticulum resident heat shock protein gp96 chaperons peptides, including those derived from tumor Ags, on their way to presentation by MHC class I. Replacement of the endoplasmic reticulum retention signal of gp96 with the Fc portion of murine IgG1 generated a secretory form of gp96, gp96-Ig. Tumor cells secreting gp96-Ig exhibited decreased tumorigenicity and increased immunogenicity in vivo and were rejected after initial growth. Rejection required CD8 T cells during the priming and effector phase. CD4 T cells were not required for rejection in either phase. Carrageenan, a compound known to inactivate macrophages in vivo, did not diminish CD8-mediated tumor rejection. Therefore, immunization with tumors secreting gp96-Ig generates efficient tumor-rejecting CD8 CTL without requirement for CD4 or macrophage help. In contrast, immunization with purified, tumor-derived gp96 or with irradiated tumor cells requires both.     

Minor histocompatibility antigen-specific MHC-restricted CD8 T cell responses elicited by heat shock proteins

In mammals, the heat shock proteins (HSP) gp96 and hsp70 elicit potent specific MHC class I-restricted CD8(+) T cell (CTL) response to exogenous peptides they chaperone. We show in this study that in the adult frog Xenopus, a species whose common ancestors with mammals date back 300 million years, both hsp70 and gp96 generate an adaptive specific cellular immune response against chaperoned minor histocompatibility antigenic peptides that effects an accelerated rejection of minor histocompatibility-locus disparate skin grafts in vivo and an MHC-specific CD8(+) cytotoxic T cell response in vitro. In naturally class I-deficient but immunocompetent Xenopus larvae, gp96 also generates an antitumor immune response that is independent of chaperoned peptides (i.e., gp96 purified from normal tissue also generates a significant antitumor response); this suggests a prominent contribution of an innate type of response in the absence of MHC class I Ags.     

Specific immunogenicity of heat shock protein gp96 derives from chaperoned antigenic peptides and not from contaminating proteins

The peptide-binding property of MHC is central to adaptive immunological functions. A similar property of heat shock proteins (HSPs) hsp70 and hsp90 has been implicated in Ag presentation by MHC and in cross-priming. The peptide-binding pocket of hsp70 has been characterized structurally and functionally and a peptide-binding site in gp96 (of hsp90 family) has been defined. Nonetheless, questions persist whether the specific immunogenicity of HSP preparations derives from the peptides chaperoned by the HSPs or by proteins contaminating the HSP preparations. Because absolute purity of a protein preparation is a metaphysical concept, other approaches are necessary to address the question. In this study, we demonstrate that the specific immunogenicity of gp96 preparations isolated from cells expressing beta-galactosidase derives from the MHC I epitope precursors associated with the gp96 and not from contaminating beta-galactosidase protein nor unassociated fragments derived from it. Although the observations here are limited to a single HSP and antigenic peptides chaperoned by it, they can be extended broadly.     

Efficient cross-presentation by heat shock protein 90-peptide complex-loaded dendritic cells via an endosomal pathway

It is well-established that heat shock proteins (HSPs)-peptides complexes elicit antitumor responses in prophylactic and therapeutic immunization protocols. HSPs such as gp96 and Hsp70 have been demonstrated to undergo receptor-mediated uptake by APCs with subsequent representation of the HSP-associated peptides to MHC class I molecules on APCs, facilitating efficient cross-presentation. On the contrary, despite its abundant expression among HSPs in the cytosol, the role of Hsp90 for the cross-presentation remains unknown. We show here that exogenous Hsp90-peptide complexes can gain access to the MHC class I presentation pathway and cause cross-presentation by bone marrow-derived dendritic cells. Interestingly, this presentation is TAP independent, and followed chloroquine, leupeptin-sensitive, as well as cathepsin S-dependent endosomal pathways. In addition, we show that Hsp90-chaperoned precursor peptides are processed and transferred onto MHC class I molecules in the endosomal compartment. Furthermore, we demonstrate that immunization with Hsp90-peptide complexes induce Ag-specific CD8(+) T cell responses and strong antitumor immunity in vivo. These findings have significant implications for the design of T cell-based cancer immunotherapy.     

In vivo and in vitro activation of T cells after administration of Ag-negative heat shock proteins

Heat shock proteins (HSP) Hsp70 and gp96 prime class I-restricted cytotoxic T cells against Ags present in the cells from which they were isolated. The immunization capacity of HSPs is believed to rely on their ability to bind antigenic peptides. In this study, we employed the well-established OVA and beta-galactosidase (beta-gal) antigenic model systems. We show that in vitro long-term established OVA and beta-gal-specific CTL clones release TNF-alpha and IFN-gamma when incubated with Ag-negative Hsp70 and gp96. In the absence of antigenic peptides, HSP-mediated secretion of TNF-alpha and IFN-gamma requires cell contact of the APC with the T cell but is not MHC-I restricted. Moreover, Hsp70 molecules purified from Ag-negative tissue, e.g., negative for antigenic peptide, are able to activate T cells in vivo, leading to significant higher frequencies in OVA-specific CD8+ T cells. In unprimed animals, these T cells lyse OVA-transfected cell lines and produce TNF-alpha and IFN-gamma after Ag stimulus. Taken together our data show that, besides the well-established HSP/peptide-specific CTL induction and activation, a second mechanism exists by which Hsp70 and gp96 molecules activate T cells in vivo and in vitro.     

Cell surface targeting of heat shock protein gp96 induces dendritic cell maturation and antitumor immunity.

gp96 is a residential heat shock protein of the endoplasmic reticulum that has been implicated in the activation of dendritic cells (DCs) for the initiation of adaptive immunity. By genetic targeting of gp96 onto the cell surface, we demonstrate that direct access of gp96 to DCs induces their maturation, resulting in secretion of proinflammatory cytokines IL-1beta, IL-12, and chemokine monocyte chemoattractant protein-1 and up-regulation of the expression of MHC class I, MHC class II, CD80, CD86, and CD40. Furthermore, surface expression of gp96 on tumor cells renders them regressive via a T lymphocyte-dependent mechanism. This work reinforces the notion that gp96 is an endogenous DC activator and unveils that the context in which Ag is delivered to the immune system, in this case surface expression of gp96, has profound influence on immunity. It also establishes a principle of bridging innate and adaptive immunity for cancer immunotherapy by surface targeting of an intracellular heat shock protein.     

Cutting edge: receptor-mediated endocytosis of heat shock proteins by professional antigen-presenting cells

Immunization with heat shock proteins (HSPs) induces Ag-specific CTL responses. The specificity of the immune response is based on peptides associated with HSPs. To investigate how exogenous HSP/peptide complexes gain access to the MHC class I-restricted Ag presentation pathway, we incubated the monocytic cell line P388D1 and the dendritic cell line D2SC/1 with gold-labeled HSPs gp96 and HSC70. We show that HSPs bind specifically to the surface of these APCs and are internalized spontaneously by receptor-mediated endocytosis, demonstrating the existence of specific receptors for HSPs on these cells. In addition, we observe colocalization of internalized HSPs and surface MHC class I molecules in early and late endosomal structures. These findings provide possible explanations for the immunogenicity of HSP/peptide complexes and for the transfer of HSP-associated peptides onto MHC class I molecules.     

Direct priming and cross-priming contribute differentially to the induction of CD8+ CTL following exposure to vaccinia virus via different routes

To explore the relative importance of direct presentation vs cross-priming in the induction of CTL responses to viruses and viral vectors, we generated a recombinant vaccinia vector, vUS11, expressing the human CMV (HCMV) protein US11. US11 dislocates most allelic forms of human and murine MHC class I heavy chains from the lumen of the endoplasmic reticulum into the cytosol, where they are degraded by proteasomes. Expression of US11 dramatically decreased the presentation of viral Ag and CTL recognition of infected cells in vitro without significantly reducing total cell surface MHC class I levels. However, because US11 is an endoplasmic reticulum resident membrane protein, it cannot block presentation by non-infected cells that take up Ag through the cross-priming pathway. We show that the expression of US11 strongly inhibits the induction of primary CD8(+) CTLs when the infection occurs via the i.p. or i.v. route, demonstrating that direct priming is critical for the induction of CTL responses to viral infections introduced via these routes. This effect is less dramatic following i.m. infection and is minimal after s.c. or intradermal infection. Thus, classic MHC class I Ag presentation and cross-priming contribute differentially to the induction of CD8(+) CTLs following exposure to vaccinia virus via different routes.     

Dendritic cells cross-dressed with peptide MHC class I complexes prime CD8+ T cells

The activation of naive CD8+ T cells has been attributed to two mechanisms: cross-priming and direct priming. Cross-priming and direct priming differ in the source of Ag and in the cell that presents the Ag to the responding CD8+ T cells. In cross-priming, exogenous Ag is acquired by professional APCs, such as dendritic cells (DC), which process the Ag into peptides that are subsequently presented. In direct priming, the APCs, which may or may not be DC, synthesize and process the Ag and present it themselves to CD8+ T cells. In this study, we demonstrate that naive CD8+ T cells are activated by a third mechanism, called cross-dressing. In cross-dressing, DC directly acquire MHC class I-peptide complexes from dead, but not live, donor cells by a cell contact-mediated mechanism, and present the intact complexes to naive CD8+ T cells. Such DC are cross-dressed because they are wearing peptide-MHC complexes generated by other cells. CD8+ T cells activated by cross-dressing are restricted to the MHC class I genotype of the donor cells and are specific for peptides generated by the donor cells. In vivo studies demonstrate that optimal priming of CD8+ T cells requires both cross-priming and cross-dressing. Thus, cross-dressing may be an important mechanism by which DC prime naive CD8+ T cells and may explain how CD8+ T cells are primed to Ags that are inefficiently cross-presented.     

Factors affecting the efficiency of CD8+ T cell cross-priming with exogenous antigens

Processing of exogenous protein Ags by APC leads predominantly to presentation of peptides on class II MHC and, thus, stimulation of CD4+ T cell responses. However, "cross-priming" can also occur, whereby peptides derived from exogenous Ags become displayed on class I MHC molecules and stimulate CD8+ T cell responses. We compared the efficiency of cross-priming with exogenous proteins to use of peptide Ags in human whole blood using a flow cytometry assay to detect T cell intracellular cytokine production. CD8+ T cell responses to whole CMV proteins were poorly detected (compared with peptide responses) in most CMV-seropositive donors. Such responses could be increased by using higher doses of Ag than were required to achieve maximal CD4+ T cell responses. A minority of donors displayed significantly more efficient CD8+ T cell responses to whole protein, even at low Ag doses. These responses were MHC class I-restricted and dependent upon proteosomal processing, indicating that they were indeed due to cross-priming. The ability to efficiently cross-prime was not a function of the number of dendritic cells in the donor's blood. Neither supplementation of freshly isolated dendritic cells nor use of cultured, Ag-pulsed dendritic cells could significantly boost CD8 responses to whole-protein Ags in poorly cross-priming donors. Interestingly, freshly isolated monocytes performed almost as well as dendritic cells in inducing CD8 responses via cross-priming. In conclusion, the efficiency of cross-priming appears to be poor in most donors and is dependent upon properties of the individual's APC and/or T cell repertoire. It remains unknown whether cross-priming ability translates into any clinical advantage in ability to induce CD8+ T cell responses to foreign Ags.     

Immunization with gp96 from Listeria monocytogenes-infected mice is due to N-formylated listerial peptides.

N-Formylated (N-f-met) peptides derived from proteins of the intracellular bacterium Listeria monocytogenes generate a protective, H2-M3-restricted CD8 T cell response in C57BL/6 mice. N-f-met peptide-specific CTL were generated in vitro when mice previously immunized with gp96 isolated from donor mice infected with L. monocytogenes were stimulated with these peptides. No significant peptide-specific CTL activity was observed in mice immunized with gp96 from uninfected animals. Masses corresponding to one N-f-met peptide were found by matrix-assisted laser desorption/ionization-mass spectrometry on gp96 isolated from C57BL/6 mice infected with L. monocytogenes, but not on gp96 from noninfected mice. Therefore, bacterial N-f-met peptides from intracellular bacteria can bind to gp96 in the infected host, and gp96 loaded with these peptides can generate N-f-met-peptide-specific CTL. We assume a unique role of gp96 in Ag processing through the H2-M3 pathway.     

Calreticulin displays in vivo peptide-binding activity and can elicit CTL responses against bound peptides.

Calreticulin is an endoplasmic reticulum (ER) chaperone that displays lectin activity and contributes to the folding pathways for nascent glycoproteins. Calreticulin also participates in the reactions yielding assembly of peptides onto nascent MHC class I molecules. By chemical and immunological criteria, we identify calreticulin as a peptide-binding protein and provide data indicating that calreticulin can elicit CTL responses to components of its bound peptide pool. In an adoptive immunotherapy protocol, dendritic cells pulsed with calreticulin isolated from B16/F10.9 murine melanoma, E.G7-OVA, or EL4 thymoma tumors elicited a CTL response to as yet unknown tumor-derived Ags or the known OVA Ag. To evaluate the relative efficacy of calreticulin in eliciting CTL responses, the ER chaperones GRP94/gp96, BiP, ERp72, and protein disulfide isomerase were purified in parallel from B16/F10.9, EL4, and E.G7-OVA tumors, and the capacity of the proteins to elicit CTL responses was compared. In both the B16/F10.9 and E.G7-OVA models, calreticulin was as effective as or more effective than GRP94/gp96 in eliciting CTL responses. Little to no activity was observed for BiP, ERp72, and protein disulfide isomerase. The observed antigenic activity of calreticulin was recapitulated in in vitro experiments, in which it was observed that pulsing of bone marrow dendritic cells with E.G7-OVA-derived calreticulin elicited sensitivity to lysis by OVA-specific CD8+ T cells. These data identify calreticulin as a peptide-binding protein and indicate that calreticulin-bound peptides can be re-presented on dendritic cell class I molecules for recognition by CD8+ T cells.     

CpG-DNA activates in vivo T cell epitope presenting dendritic cells to trigger protective antiviral cytotoxic T cell responses

MHC class I-restricted T cell epitopes lack immunogenicity unless aided by IFA or CFA. In an attempt to circumvent the known inflammatory side effects of IFA and CFA, we analyzed the ability of immunostimulatory CpG-DNA to act as an adjuvant for MHC class I-restricted peptide epitopes. Using the immunodominant CD8 T cell epitopes, SIINFEKL from OVA or KAVYNFATM (gp33) from lymphocytic choriomeningitis virus glycoprotein, we observed that CpG-DNA conveyed immunogenicity to these epitopes leading to primary induction of peptide-specific CTL. Furthermore, vaccination with the lymphocytic choriomeningitis virus gp33 peptide triggered not only CTL but also protective antiviral defense. We also showed that MHC class I-restricted peptides are constitutively presented by immature dendritic cells (DC) within the draining lymph nodes but failed to induce CTL responses. The use of CpG-DNA as an adjuvant, however, initiated peptide presenting immature DC progression to professional licensed APC. Activated DC induced cytolytic CD8 T cells in wild-type mice and also mice deficient of Th cells or CD40 ligand. CpG-DNA thus incites CTL responses toward MHC class I-restricted T cell epitopes in a Th cell-independent manner. Overall, these results provide new insights into CpG-DNA-mediated adjuvanticity and may influence future vaccination strategies for infectious and perhaps tumor diseases.     

小鼠热休克蛋白gp96羧基端片段的克隆、表达及纯化

热休克蛋白(Heat shock protein)gp96(Grp94)是近年来新发现的一类糖蛋白,除了分子伴侣的功能外,现有越来越多的献报道了它在先天性免疫和获得性免疫中的重要作用。gp96可以促进抗原呈递细胞的成熟以及细胞因子的分泌。热休克蛋白抗原肽复合体可以引起特异性的细胞毒T淋巴细胞效应,应用这个特点可以设计抗病毒及抗肿瘤药物。但是gp96全长分子量大,蛋白在大肠杆菌中表达量低,不稳定,难纯化。组织提取的gp96又受组织来源和样品量的限制。对gp96的结构和功能的研究带来困难。克隆并表达了小鼠热休克蛋白gp96的羧基端560．751aa约四分之一长的功能片段,该段包含gp96的一个肽结合区和二聚化位点。将该功能片段在大肠杆菌中进行融合表达,纯化后将融合的片段切掉,并对目的片段进行了分析,结果表明该段可能是形成二聚体密切相关的片段,为进一步研究其结构和功能打下基础。     

Glycoprotein 96-activated dendritic cells induce a CD8-biased T cell response

Heat shock proteins (Hsps) are able to induce protective immune responses against pathogens and tumors after injection into immunocompetent hosts. The activation of components of the adaptive immune system, including cytotoxic T lymphocytes specific for pathogen- or tumor-derived peptides, is crucial for the establishment of immunoprotection. Hsps acquire these peptides during intracellular protein degradation and when released during necrotic cell death, facilitate their uptake and Minor Histocompatibility Complex (MHC)-restricted representation by professional antigen-presenting cells (APCs). In addition, the interaction of Hsps with APCs, including the Endoplasmatic Reticulum (ER)-resident chaperone glycoprotein 96 (Gp96), induces the maturation of these cells by Toll-like receptor (TLR)-mediated signaling events. We now provide evidence that in contrast to lipopolysaccharides (LPS)-mediated dendritic cell (DC) maturation, the interaction of Gp96 with DCs leads to the preferential expansion of antigen-specific CD8-positive T cells in vitro and in vivo. This CD8 preference induced by mouse and human DCs did not correlate with enhanced levels of interleukin-12 secretion. Thus, despite the fact that both LPS and Gp96 activate DCs in a TLR4-dependent manner, the experiments of this study clearly demonstrate qualitative differences in the outcome of this maturation process, which preferentially favors the expansion of CD8-positive T cells.     

Evolution of the immunomodulatory role of the heat shock protein gp96.

In mammals, certain heat shock proteins (hsps) participate in specialized responses to stressors associated with pathogens or tumors, and as such, act as agents of immune surveillance interacting with both innate and adaptive immunity. We are investigating the conservation of this role throughout the class of vertebrates. We have shown that in Xenopus as in mammals, gp96, the major resident of the endoplasmic reticulum, generates MHC-restricted thymus-dependent immunity in vivo and CR in vitro against minor histocompatibility (H) antigens. By as yet unclear mechanisms that may involve classical MHC-unrestricted cytotoxic CD8+ T cells, gp96 also elicits peptide-specific responses against MHC-class I-negative tumors in adult frogs that may involve cytotoxic NK, MHC-unrestricted CD8+ T and NK/T cells. In naturally MHC class I-deficient but immunocompetent Xenopus larvae, gp96 also generates an innate type of anti-tumor response that is independent of chaperoned peptides. Finally, in a subset of Xenopus sIgM+ B cells, a substantial fraction of gp96 is directed to the cell surface by an active process that is upregulated by bacterial stimulation. This may allow gp96 to access the extracellular compartment without necrosis. Given the dual abilities of gp96 to chaperone antigenic peptides and to modulate innate immune responses, we propose that stimulated B cells that are up-regulating surface gp96 can directly interact with antigen presenting cells (APC) and/or T helper (Th) cells to trigger or amplify immune responses.