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.     

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.     

Phylogenetic conservation of glycoprotein 96 ability to interact with CD91 and facilitate antigen cross-presentation

Although the ability of gp96 to activate APCs and generate CD8 CTLs against peptides they chaperone through interaction with the endocytic receptors CD91 is supported by solid evidence, its biological relevance in immune surveillance is debated. We have used an evolutionary approach to determine whether gp96 interacts with receptors expressed on APCs and promotes MHC class I cross-presentation of minor histocompatibility Ags (H-Ags) to CTLs in the frog Xenopus. We show that in Xenopus gp96 binds the CD91 homolog at the surface of peritoneal leukocytes, and that this binding is inhibited by molar excess of unlabeled gp96 or the CD91 ligand alpha2-macroglobulin, by anti-CD91 Ab and by the specific CD91 antagonist receptor-associated protein. Surface binding followed by internalization of gp96 was confirmed by fluorescent microscopy. Furthermore, adoptive transfer of peritoneal leukocytes pulsed with as little as 800 ng of gp96 chaperoning minor H-Ags, but not minor H-Ag-free gp96, induces potent CD8 T cell infiltration and Ag-specific accelerated rejection of minor H-locus disparate skin grafts. Inhibition of gp96-CD91 interaction by pretreatment with anti-CD91 Ab and receptor-associated protein impairs both CD8 T cell infiltration and acute skin graft rejection. These data provide evidence of the conserved ability of gp96 to facilitate cross-presentation of chaperoned Ags by interacting with CD91. The persistence of this biological process for >350 million years that separate mammals and amphibians from a common ancestor strongly supports the proposition that gp96 and CD91 are critically involved in immune surveillance.     

热激蛋白gp96免疫学功能及在主动免疫治疗肿瘤和传染病中的应用

热激蛋白gp96可特异性结合来源于肿瘤和病毒的抗原肽,与抗原呈递细胞表面CD91等受体作用进入胞内,并在内质网中将结合的抗原通过抗原呈递链呈递给MHCI类分子,激活特异性T细胞。同时,与细胞表面Toll样受体(TLR)TLR2、TLR4等相互作用,激活天然免疫。近期研究发现调节性T细胞(Treg)对gp96免疫功能有显著抑制作用,随着对影响gp96免疫功能的免疫抑制机制的深入了解,以及利用汉逊酵母表达有免疫活性的全长gp96蛋白体系的建立,gp96将在治疗肿瘤及传染性疾病中发挥更大的作用。     

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.     

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.     

Molecular and cellular requirements for enhanced antigen cross-presentation to CD8 cytotoxic T lymphocytes

MHC class I-mediated cross-priming of CD8 T cells by APCs is critical for CTL-based immunity to viral infections and tumors. We have shown previously that tumor-secreted heat shock protein gp96-chaperoned peptides cross prime CD8 CTL that are specific for genuine tumor Ags and for the surrogate Ag OVA. We now show that tumor-secreted heat shock protein gp96-chaperoned peptides enhance the efficiency of Ag cross-priming of CD8 CTL by several million-fold over the cross-priming activity of unchaperoned protein alone. Gp96 also acts as adjuvant for cross-priming by unchaperoned proteins, but in this capacity gp96 is 1000-fold less active than as a peptide chaperone. Mechanistically, the in situ secretion of gp96-Ig by transfected tumor cells recruits and activates dendritic cells and NK cells to the site of gp96 release and promotes CD8 CTL expansion locally. Gp96-mediated cross-priming of CD8 T cells requires B7.1/2 costimulation but proceeds unimpeded in lymph node-deficient mice, in the absence of NKT and CD4 cells and without CD40L. Gp96-driven MHC I cross-priming of CD8 CTL in the absence of lymph nodes provides a novel mechanism for local, tissue-based CTL generation at the site of gp96 release. This pathway may constitute a critically important, early detection, and rapid response mechanism that is operative in parenchymal tissues for effective defense against tissue damaging antigenic agents.     

Constitutive expression of TNF-related activation-induced cytokine (TRANCE)/receptor activating NF-κB ligand (RANK)-L by rat plasmacytoid dendritic cells

Plasmacytoid dendritic cells (pDCs) are a subset of DCs whose major function relies on their capacity to produce large amount of type I IFN upon stimulation via TLR 7 and 9. This function is evolutionary conserved and place pDC in critical position in the innate immune response to virus. Here we show that rat pDC constitutively express TNF-related activation-induced cytokine (TRANCE) also known as Receptor-activating NF-κB ligand (RANKL). TRANCE/RANKL is a member of the TNF superfamily which plays a central role in osteoclastogenesis through its interaction with its receptor RANK. TRANCE/RANK interaction are also involved in lymphoid organogenesis as well as T cell/DC cross talk. Unlike conventional DC, rat CD4(high) pDC were shown to constitutively express TRANCE/RANKL both at the mRNA and the surface protein level. TRANCE/RANKL was also induced on the CD4(low) subsets of pDC following activation by CpG. The secreted form of TRANCE/RANKL was also produced by rat pDC. Of note, levels of mRNA, surface and secreted TRANCE/RANKL expression were similar to that observed for activated T cells. TRANCE/RANKL expression was found on pDC in all lymphoid organs as well blood and BM with a maximum expression in mesenteric lymph nodes. Despite this TRANCE/RANKL expression, we were unable to demonstrate in vitro osteoclastogenesis activity for rat pDC. Taken together, these data identifies pDC as novel source of TRANCE/RANKL in the immune system.     

Differential CD91 dependence for calreticulin and Pseudomonas exotoxin-A endocytosis

Calreticulin (CRT) interaction with cell-surface receptors is integral to its function in escorting associated peptides into the antigen-presenting cell (APC) antigen presentation pathway. Additionally, extracellular CRT is proposed to be required for lung APC interaction with collectins. In both cases, CD91 has been proposed to act as the APC cell-surface receptor requisite for mediating these processes. However, the evidence for a CRT interaction with CD91 is indirect, predicated on partial competition of cellular binding by gp96, of which CD91 has been proposed as the unique endocytic receptor, and by the CD91 ligand alpha2-macroglobulin. Here, we directly investigate the function of CD91 in binding and trafficking CRT. We find that the ability of CRT to interact with APC does not correlate with cellular CD91 expression or function. Additionally, in the first genetic test of CD91 function regarding CRT, CD91 expression neither conferred CRT association nor did CD91-deficient (CD91-/-) and CD91-expressing cells differ in their ability to traffic CRT. Finally, cellular CRT trafficking did not parallel that of Pseudomonas exotoxin-A, an obligate CD91 ligand, by the criteria of CD91 dependence, cell-type specificity and endocytic itinerary. These data identify that CRT trafficking is not, as previously hypothesized, CD91 dependent and indicate usage of alternative cellular trafficking pathways.     

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.     

Evidence that glycoprotein 96 (B2), a stress protein,functions as a Th2-specific costimulatory molecule

After the engagement of Ag receptor, most of the Th cells for their optimal activation require a second (costimulatory) signal provided by the APCs. We demonstrate the isolation and characterization of a 99- to 105-kDa protein (B2), from LPS-activated B cell surface, and its function as a Th2-specific costimulatory molecule. Appearance of B2 as a single entity on two-dimensional gel electrophoresis and as a distinct peak in reverse-phase HPLC ascertains the fact that B2 is homogeneous in preparation. Electron microscopy as well as competitive binding studies reveal that (125)I-labeled B2 specifically binds anti-CD3-activated T cell surface and also competes with its unlabeled form. Internal amino acid sequences of B2 are found to be identical with stress protein gp96. The identity of B2 as gp96 is also revealed by immunological characterization and by confocal microscopic colocalization studies of B2 and gp96 on LPS-activated B cells. Confocal imaging studies also demonstrate that gp96 can be induced on B cell surface without association of MHC molecules. Furthermore, the novel role of gp96 in Th cell proliferation skewing its differentiation toward Th2 phenotype has also been established. Ab-mediated blocking of gp96-induced signaling not only abrogates in vitro proliferation of CD4(+) T cells, but also diminishes the secretion of Th2-specific cytokines. Notably, the expression of CD91 (receptor of gp96/B2) is up-regulated on anti-CD3-activated Th cells and also found to be present on Th1 and Th2 subsets.     

HIV-1 gp120 mannoses induce immunosuppressive responses from dendritic cells

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 is a vaccine immunogen that can signal via several cell surface receptors. To investigate whether receptor biology could influence immune responses to gp120, we studied its interaction with human, monocyte-derived dendritic cells (MDDCs) in vitro. Gp120 from the HIV-1 strain JR-FL induced IL-10 expression in MDDCs from 62% of donors, via a mannose C-type lectin receptor(s) (MCLR). Gp120 from the strain LAI was also an IL-10 inducer, but gp120 from the strain KNH1144 was not. The mannose-binding protein cyanovirin-N, the 2G12 mAb to a mannose-dependent gp120 epitope, and MCLR-specific mAbs inhibited IL-10 expression, as did enzymatic removal of gp120 mannose moieties, whereas inhibitors of signaling via CD4, CCR5, or CXCR4 were ineffective. Gp120-stimulated IL-10 production correlated with DC-SIGN expression on the cells, and involved the ERK signaling pathway. Gp120-treated MDDCs also responded poorly to maturation stimuli by up-regulating activation markers inefficiently and stimulating allogeneic T cell proliferation only weakly. These adverse reactions to gp120 were MCLR-dependent but independent of IL-10 production. Since such mechanisms might suppress immune responses to Env-containing vaccines, demannosylation may be a way to improve the immunogenicity of gp120 or gp140 proteins.     

Re-examination of CD91 function in GRP94 (glycoprotein 96) surface binding, uptake, and peptide cross-presentation

GRP94 (gp96)-peptide complexes can be internalized by APCs and their associated peptides cross-presented to yield activation of CD8(+) T cells. Investigations into the identity (or identities) of GRP94 surface receptors have yielded conflicting results, particularly with respect to CD91 (LRP1), which has been proposed to be essential for GRP94 recognition and uptake. To assess CD91 function in GRP94 surface binding and endocytosis, these parameters were examined in mouse embryonic fibroblast (MEF) cell lines whose expression of CD91 was either reduced via RNA interference or eliminated by genetic disruption of the CD91 locus. Reduction or loss of CD91 expression abrogated the binding and uptake of receptor-associated protein, an established CD91 ligand. Surface binding and uptake of an N-terminal domain of GRP94 (GRP94.NTD) was unaffected. GRP94.NTD surface binding was markedly suppressed after treatment of MEF cell lines with heparin, sodium chlorate, or heparinase II, demonstrating that heparin sulfate proteoglycans can function in GRP94.NTD surface binding. The role of CD91 in the cross-presentation of GRP94-associated peptides was examined in the DC2.4 dendritic cell line. In DC2.4 cells, which express CD91, GRP94.NTD-peptide cross-presentation was insensitive to the CD91 ligands receptor-associated protein or activated α(2)-macroglobulin and occurred primarily via a fluid-phase, rather than receptor-mediated, uptake pathway. These data clarify conflicting data on CD91 function in GRP94 surface binding, endocytosis, and peptide cross-presentation and identify a role for heparin sulfate proteoglycans in GRP94 surface binding.     

Glycoprotein 96 can chaperone both MHC class I- and class II-restricted epitopes for in vivo presentation, but selectively primes CD8+ T cell effector function

The ability of mature T lymphocytes to develop effector capacity after encounter with cognate Ag is generally dependent upon inflammatory signals associated with infection that induce dendritic cell activation/maturation. These inflammatory signals can derive directly from pathogens or can be expressed by host cells in response to infection. Heat shock proteins (HSPs) are a class of host-derived inflammatory mediators that perform the dual function of both chaperoning MHC class I-restricted epitopes into the cross-presentation pathway of DCs and inducing the activation/maturation of these DCs to allow priming of cognate CD8(+) T cell effector responses. Although the ability of HSPs to elicit effector CD8 cell responses has been well established, their potential to prime CD4 cell effector responses has been relatively unexplored. In the current study we compared the ability of the endoplasmic reticulum-resident HSP gp96 to prime CD4 vs CD8 cells using TCR transgenic adoptive transfer systems and soluble gp96-peptide complexes. As expected, gp96 facilitated the cross-presentation of a class I-restricted peptide and priming of effector function in cognate CD8 cells. Interestingly, gp96 also facilitated the in vivo presentation of a class II-restricted peptide; however, the resulting CD4 cell response did not involve the development of effector function. Taken together, these data suggest that gp96 is an inflammatory mediator that selectively primes CD8 cell effector function.     

HIV delays IFN-α production from human plasmacytoid dendritic cells and is associated with SYK phosphorylation

Plasmacytoid dendritic cells (pDC) are the major producers of type I interferons (IFNs) in humans and rapidly produce IFN-α in response to virus exposure. Although HIV infection is associated with pDC activation, it is unclear why the innate immune response is unable to effectively control viral replication. We systematically compared the effect of HIV, Influenza, Sendai, and HSV-2 at similar target cell multiplicity of infection (M.O.I.) on human pDC function. We found that Influenza, Sendai, HSV-2 and imiquimod are able to rapidly induce IFN-α production within 4 hours to maximal levels, whereas HIV had a delayed induction that was maximal only after 24 hours. In addition, maximal IFN-α induction by HIV was at least 10 fold less than that of the other viruses in the panel. HIV also induced less TNF-α and MIP-1β but similar levels of IP-10 compared to other viruses, which was also mirrored by delayed upregulation of pDC activation markers CD83 and CD86. BDCA-2 has been identified as an inhibitory receptor on pDC, signaling through a pathway that involves SYK phosphorylation. We find that compared to Influenza, HIV induces the activation of the SYK pathway. Thus, HIV delays pDC IFN-α production and pDC activation via SYK phosphorylation, allowing establishment of viral populations.     

Administration of the stress protein gp96 prolongs rat cardiac allograft survival, modifies rejection-associated inflammatory events, and induces a state of peripheral T-cell hyporesponsiveness

High-dose gp96 has been shown to inhibit experimental autoimmune disease by a mechanism that appears to involve immunoregulatory CD4+ T cells. This study tested the hypothesis that high-dose gp96 administration modifies allograft rejection and associated inflammatory events. Wistar cardiac allografts were transplanted into Lewis recipient rats and graft function was monitored daily by palpation. Intradermal administration of gp96 purified from Wistar rat livers (100 microg) at the time of transplantation and 3 days later significantly prolonged allograft survival (14 vs 8 days in phosphate-buffered saline [PBS]-treated recipients; P = 0.009). Rejected allografts from gp96-treated animals were significantly less enlarged than allografts from their PBS-treated counterparts (2.8 vs 4.3 g; P < 0.004). Gp96 was also effective when administered on days 1 and 8 (13 vs 7 days), but not if it was derived from recipient (Lewis) liver tissue or administered on days 0, 3, and 6. In parallel studies, CD3+ T cells from gp96-treated untransplanted animals secreted less interleukin (IL)-4, IL-10, and interferon (IFN)-gamma after in vitro polyclonal stimulation than CD3+ T cells from PBS-treated animals. Gp96 administration might therefore influence the induction of immunity to coencountered antigenic challenges and inflammatory events by inducing what appears to be a state of peripheral T-cell hyporesponsiveness.     

Reactive oxygen species activate human peripheral blood dendritic cells

This study investigates the effects of hydrogen peroxide, a potent oxygen free radical donor, on the phenotype and function of dendritic cells differentiated from peripheral blood precursors. We report that hydrogen peroxide induces an up-regulation of several dendritic cell surface markers involved in interaction with T cells, including MHC Class II molecules (DQ and DR) and the co-stimulatory molecules CD40 and CD86. Moreover we have observed that H₂O₂-treated dendritic cells are more efficient in promoting T cell proliferation than normal dendritic cells and that this enhancement can be blocked using the free radical scavenger agent N-acetylcysteine. Oxygen free radicals are a common by-product of inflammation, and our results suggest they may play an important role in activation of sentinel dendritic cells, linking tissue damage to the initiation of an adaptive immune response.     

Plasmacytoid Dendritic Cell Dynamics Tune Interferon-Alfa Production in SIV-Infected Cynomolgus Macaques

IFN-I production is a characteristic of HIV/SIV primary infections. However, acute IFN-I plasma concentrations rapidly decline thereafter. Plasmacytoid dendritic cells (pDC) are key players in this production but primary infection is associated with decreased responsiveness of pDC to TLR 7 and 9 triggering. IFNα production during primary SIV infection contrasts with increased pDC death, renewal and dysfunction. We investigated the contribution of pDC dynamics to both acute IFNα production and the rapid return of IFNα concentrations to pre-infection levels during acute-to-chronic transition. Nine cynomolgus macaques were infected with SIVmac251 and IFNα-producing cells were quantified and characterized. The plasma IFN-I peak was temporally associated with the presence of IFNα⁺ pDC in tissues but IFN-I production was not detectable during the acute-to-chronic transition despite persistent immune activation. No IFNα⁺ cells other than pDC were detected by intracellular staining. Blood-pDC and peripheral lymph node-pDC both lost IFNα⁻ production ability in parallel. In blood, this phenomenon correlated with an increase in the counts of Ki67⁺-pDC precursors with no IFNα production ability. In tissues, it was associated with increase of both activated pDC and KI67⁺-pDC precursors, none of these being IFNα⁺ in vivo. Our findings also indicate that activation/death-driven pDC renewal rapidly blunts acute IFNα production in vivo: pDC sub-populations with no IFNα-production ability rapidly increase and shrinkage of IFNα production thus involves both early pDC exhaustion, and increase of pDC precursors.     

Parallel loss of myeloid and plasmacytoid dendritic cells from blood and lymphoid tissue in simian AIDS

The loss of myeloid (mDC) and plasmacytoid dendritic cells (pDC) from the blood of HIV-infected individuals is associated with progressive disease. It has been proposed that DC loss is due to increased recruitment to lymph nodes, although this has not been directly tested. Similarly as in HIV-infected humans, we found that lineage-negative (Lin(-)) HLA-DR(+)CD11c(+)CD123(-) mDC and Lin(-)HLA-DR(+)CD11c(-)CD123(+) pDC were lost from the blood of SIV-infected rhesus macaques with AIDS. In the peripheral lymph nodes of SIV-naive monkeys the majority of mDC were mature cells derived from skin that expressed high levels of HLA-DR, CD83, costimulatory molecules, and the Langerhans cell marker CD1a, whereas pDC expressed low levels of HLA-DR and CD40 and lacked costimulatory molecules, similar to pDC in blood. Surprisingly, both DC subsets were depleted from peripheral and mesenteric lymph nodes and spleens in monkeys with AIDS, although the activation status of the remaining DC subsets was similar to that of DC in health. In peripheral and mesenteric lymph nodes from animals with AIDS there was an accumulation of Lin(-)HLA-DR(moderate)CD11c(-)CD123(-) cells that resembled monocytoid cells but failed to acquire a DC phenotype upon culture, suggesting they were not DC precursors. mDC and pDC from the lymphoid tissues of monkeys with AIDS were prone to spontaneous death in culture, indicating that apoptosis may be a mechanism for their loss in disease. These findings demonstrate that DC are lost from rather than recruited to lymphoid tissue in advanced SIV infection, suggesting that systemic DC depletion plays a direct role in the pathophysiology of AIDS.