Calreticulin in the immune system: ins and outs.

Calreticulin is a calcium-binding chaperone that has several functions in the immune response. In the endoplasmic reticulum (ER), calreticulin facilitates the folding of major histocompatibility complex (MHC) class I molecules and their assembly factor tapasin, thereby influencing antigen presentation to cytotoxic T cells. Although calreticulin is normally ER-resident, it is found at the cell surface of living cancer cells and dying cells. Here, calreticulin promotes cellular phagocytic uptake. In tumor vaccine models, drugs that induce cell surface calreticulin confer enhanced tumor protection in an extracellular calreticulin-dependent manner. Much remains to be understood about the roles of calreticulin in these distinct functions. Further investigations are important towards advancing basic knowledge of glycoprotein-folding pathways, and towards developing new cancer therapeutic strategies.

Endoplasmic reticulum calcium depletion impacts chaperone secretion, innate immunity, and phagocytic uptake of cells.

A number of immunological functions are ascribed to cell surface-expressed forms of the endoplasmic reticulum (ER) chaperone calreticulin (CRT). In this study, we examined the impact of ER stress-inducing drugs upon cell surface CRT induction and the resulting immunological consequences. We showed that cell surface expression of CRT and secretion of CRT, BiP, gp96, and PDI were induced by thapsigargin (THP) treatment, which depletes ER calcium, but not by tunicamycin treatment, which inhibits protein glycosylation. Surface expression of CRT in viable, THP-treated fibroblasts correlated with their enhanced phagocytic uptake by bone marrow-derived dendritic cells. Incubation of bone marrow-derived dendritic cells with THP-treated fibroblasts enhanced sterile IL-6 production and LPS-induced generation of IL-1ß, IL-12, IL-23, and TNF-α. However, extracellular CRT is not required for enhanced proinflammatory responses. Furthermore, the pattern of proinflammatory cytokine induction by THP-treated cells and cell supernatants resembled that induced by THP itself and indicated that other ER chaperones present in supernatants of THP-treated cells also do not contribute to induction of the innate immune response. Thus, secretion of various ER chaperones, including CRT, is induced by ER calcium depletion. CRT, previously suggested as an eat-me signal in dead and dying cellular contexts, can also promote phagocytic uptake of cells subject to ER calcium depletion. Finally, there is a strong synergy between calcium depletion in the ER and sterile IL-6, as well as LPS-dependent IL-1ß, IL-12, IL-23, and TNF-α innate responses, findings that have implications for understanding inflammatory diseases that originate in the ER.

The polypeptide binding conformation of calreticulin facilitates its cell-surface expression under conditions of endoplasmic reticulum stress.

We define two classes of calreticulin mutants that retain glycan binding activity; those that display enhanced or reduced polypeptide-specific chaperone activity, due to conformational effects. Under normal conditions, neither set of mutants significantly impacts the ability of calreticulin to mediate assembly and trafficking of major histocompatibility complex class I molecules, which are calreticulin substrates. However, in cells treated with thapsigargin, which depletes endoplasmic reticulum calcium, major histocompatibility complex class I trafficking rates are accelerated coincident with calreticulin secretion, and detection of cell-surface calreticulin is dependent on its polypeptide binding conformations. Together, these findings identify a site on calreticulin that is an important determinant of the induction of its polypeptide binding conformation and demonstrate the relevance of the polypeptide binding conformations of calreticulin to endoplasmic reticulum stress-induced interactions.

Modes of calreticulin recruitment to the major histocompatibility complex class I assembly pathway.

Major histocompatibility complex (MHC) class I molecules are ligands for T-cell receptors of CD8(+) T cells and inhibitory receptors of natural killer cells. Assembly of the heavy chain, light chain, and peptide components of MHC class I molecules occurs in the endoplasmic reticulum (ER). Specific assembly factors and generic ER chaperones, collectively called the MHC class I peptide loading complex (PLC), are required for MHC class I assembly. Calreticulin has an important role within the PLC and induces MHC class I cell surface expression, but the interactions and mechanisms involved are incompletely understood. We show that interactions with the thiol oxidoreductase ERp57 and substrate glycans are important for the recruitment of calreticulin into the PLC and for its functional activities in MHC class I assembly. The glycan and ERp57 binding sites of calreticulin contribute directly or indirectly to complexes between calreticulin and the MHC class I assembly factor tapasin and are important for maintaining steady-state levels of both tapasin and MHC class I heavy chains. A number of destabilizing conditions and mutations induce generic polypeptide binding sites on calreticulin and contribute to calreticulin-mediated suppression of misfolded protein aggregation in vitro. We show that generic polypeptide binding sites per se are insufficient for stable recruitment of calreticulin to PLC substrates in cells. However, such binding sites could contribute to substrate stabilization in a step that follows the glycan and ERp57-dependent recruitment of calreticulin to the PLC.

MHC class I assembly: out and about.

The assembly of major histocompatibility complex (MHC) class I molecules with peptides is orchestrated by several assembly factors including the transporter associated with antigen processing (TAP) and tapasin, the endoplasmic reticulum (ER) oxido-reductases ERp57 and protein disulfide isomerase (PDI), the lectin chaperones calnexin and calreticulin, and the ER aminopeptidase (ERAAP). Typically, MHC class I molecules present endogenous antigens to cytotoxic T lymphocytes (CTLs). However, the initiation of CD8(+) T-cell responses against many pathogens and tumors also requires the presentation of exogenous antigens by MHC class I molecules. We discuss recent developments relating to interactions and mechanisms of function of the various assembly factors and pathways by which exogenous antigens access MHC class I molecules.