Induced ER chaperones regulate a receptor-like kinase to mediate antiviral innate immune response in plants.

Mounting an effective innate immune response against pathogens requires the rapid and global reprogramming of host cellular processes. Here we employed complementary proteomic methods to identify differentially regulated proteins early during a plant's defense response. Besides defense-related proteins, constituents of the largest category of upregulated proteins were cytoplasmic- and ER-residing molecular chaperones. Investigating the significance of upregulated ER chaperones, we find that silencing of ER-resident protein disulfide isomerases NbERp57 and NbP5 and the calreticulins NbCRT2 and NbCRT3 led to partial loss of N immune receptor-mediated defense against Tobacco mosaic virus (TMV). Furthermore, NbCRT2 and NbCRT3 were required for the expression of a previously uncharacterized induced receptor-like kinase (IRK). IRK is a plasma membrane-localized protein required for N-mediated hypersensitive response, programmed cell death, and resistance to TMV. These data support a model in which ER-resident chaperones are required for the accumulation of membrane-bound or secreted proteins during plant innate immunity.

Calreticulin requires an ancillary adjuvant for the induction of efficient cytotoxic T cell responses.

Molecular chaperones stimulate the immune system to induce both protective immune responses and therapeutic tumor rejection. However, the underlying basis for this immunogenic activity is not well understood. A variety of chaperones, including calreticulin, hsp70 and grp94, function as vehicles to efficiently traffic associated peptides into professional antigen presenting cells. Importantly, these chaperones have also been proposed to function as adjuvants by stimulating the dendritic cell activation and co-stimulatory responses required to elicit peptide-specific CD8(+) T cell cytolytic activity. The efficacy of chaperone-mediated tumor rejection has been attributed to the ability of chaperones to function in both of these capacities. However, purified calreticulin has not previously been assessed for its ability to elicit DC maturation and, moreover, recent data indicates that it is not efficient at inducing Nf-kappaB activity which often accompanies or stimulates DC maturation. Here we use two complementary methods to produce endotoxin-free calreticulin and demonstrate that it does not measurably mature or activate dendritic cells both in vitro and in vivo. Additionally, a calreticulin/peptide complex required the addition of an exogenous adjuvant to elicit in vivo cytotoxic CD8(+) T cell responses. These data are discussed with respect to current models for chaperone-derived immune responses and in regard to rational vaccine design.

Development of an in vitro system for the study of allergens and allergen-specific immunoglobulin E and immunoglobulin G: Fcepsilon receptor I supercross-linking is a possible new mechanism of immunoglobulin G-dependent enhancement of type I allergic reactions.

BACKGROUND: IgE-dependent activation of mast cells (MCs) is a key pathomechanism of type I allergies. In contrast, allergen-specific IgG Abs are thought to attenuate immediate allergic reactions by blocking IgE binding and by cross-linking the inhibitory Fcgamma receptor IIB on MCs. OBJECTIVES: To establish a defined in vitro system using human MCs to study the biological activity of allergens and to investigate the role of allergen-specific IgE and IgG. METHODS: Purified human intestinal MCs sensitized with different forms of specific IgE Abs were triggered by monomeric and oligomeric forms of recombinant Bet v 1, the major birch pollen allergen, in the presence or absence of allergen-specific IgG Abs. Results MCs sensitized with an anti-Bet v 1 IgE mAb or sera obtained from birch pollen allergic patients released histamine and sulphidoleukotrienes after exposure to oligomeric Bet v 1. Monomeric Bet v 1 provoked mediator release only in MCs sensitized with patients sera but not in MCs sensitized with anti-Bet v 1 IgE mAb. Interestingly, MC activation could be induced by supercross-linking of monomeric Bet v 1 bound to monovalent IgE on MCs with a secondary allergen-specific IgG pAb. By using IgG F(ab')2 fragments we provide evidence that this effect is not a result of IgG binding to Fcgamma receptors. CONCLUSION: This assay represents a new tool for the in vitro study of MC activation in response to natural and genetically modified allergens. Fcepsilon receptor I supercross-linking by allergen-specific IgG Abs provides a possible new mechanism of IgG-dependent enhancement of type I allergic reactions.

Exogenous stress proteins enhance the immunogenicity of apoptotic tumor cells and stimulate antitumor immunity.

We have previously reported that apoptotic tumor cells can be either immunogenic or nonimmunogenic in vivo, depending on whether or not these cells are heat stressed before induction of apoptosis. Stressed apoptotic cells express heat shock proteins on their plasma membranes and dendritic cells are capable of distinguishing them from nonstressed apoptotic cells. Here we provide evidence that when purified heat shock protein 70 or chaperone-rich cell lysate (CRCL) from syngeneic normal tissue is used as an adjuvant with nonimmunogenic apoptotic tumor cells in vaccination, potent antitumor immunity can be generated. This antitumor immunity is mediated by T cells because antitumor effects are not observed in either severe combined immunodeficiency or T cell-depleted mice. We further demonstrate that vaccination of mice with apoptotic tumor cells mixed with liver-derived CRCL as adjuvant were capable of enhancing the production of T(H)1 cytokines, inducing specific cytotoxic T lymphocytes and eliciting long-lasting antitumor immunity. Stress proteins from autologous normal tissue components therefore can serve as danger signals to enhance the immunogenicity of apoptotic tumor cells and stimulate tumor-specific immunity