Modulatory role of calreticulin as chaperokine for dendritic cell-based immunotherapy

Abstract

SummaryHeat shock proteins (HSPs) play a regulatory role for maturation of antigenpresenting cells (APCs) such as dendritic cells (DCs) and macrophages. Whereas HSP70 has been shown to enhance the maturation of human DCs via a nuclear factor kappa-B (NF-kB)-dependent pathway, the regulatory role of calreticulin (CRT), which is a HSP with similar functions to HSP70, is not well studied. To investigate the role of CRT as adjuvant in cell activation and co-stimulatory responses we determined the effects of CRT on human APC maturation in comparison to that of HSP70. To facilitate eukaryotic endotoxin-free CRT protein expression, three different methods were compared. We demonstrate that CRT induces the maturation of human DCs and increases the production of proinflammatory cytokines via the NF-kB pathway. CRT-mediated maturation was qualitatively similar to that induced by HSP70. Interestingly, priming of monocytes with HSPs showed an even more prominent effect on maturation than exposure of immature DCs to these compounds. A higher expression of CD86, CD83 and CCR7 on mature DCs were found in response to CRT. Our data provide novel insights into the role of extracellular HSPs as chaperokines in the processes of APC generation and may thus be useful to improve adoptive immunotherapy.

Calreticulin Release at an Early Stage of Death Modulates the Clearance by Macrophages of Apoptotic Cells

“…Altogether our results suggest that CRT mainly intracellular on viable cells, rapidly exposed on apoptotic cell surface and efficiently released is a significant “inducing signal” for macrophages, able to modulate macrophage polarization. In support to this proposition, Bajor and collaborators ( 22 ) have previously demonstrated that CRT induced the maturation of human DCs and increases the production of pro-inflammatory cytokines. Of note, this DC maturation was found particularly clear when the conditioned medium obtained by monocytes exposed to CRT was used.…”

“…The resulting phenotype of macrophages is associated with the secretion of the chemokine IL-8, an increase in the migration speed, and a decrease in the apoptotic cells phagocytosis ability, together with a decrease in the expression of the major histocompatibility complex of class II. These results highlight the profound consequences of the release of CRT by early dying cells, acting as a “chaperokine” ( 22 ), on the immune system.…”

A novel pathway combining calreticulin exposure and ATP secretion in immunogenic cancer cell death

“…The depletion of PERK, PI3K p110α, and LRP1 (to a relatively lesser extent) reduced the immunogenicity of phox‐ER‐stressed cancer cells in vivo . The milder effect of LRP1 depletion on immunogenicity could probably be explained by the fact that even extracellularly secreted, non‐surface tethered form of CRT can mediate DC maturation (Bajor et al , 2011) and antitumour immunity (Wang et al , 2011a). However, in contrast to previous observations for anthracyclines (Panaretakis et al , 2009), depletion of caspase‐8 had no effect on phox‐ER stress‐induced immunogenicity.…”

HSP70 Enhances Immunosuppressive Function of CD4+CD25+FoxP3+ T Regulatory Cells and Cytotoxicity in CD4+CD25− T Cells

“…The Limulus amebocyte lysate assay (LAL) was used to exclude endotoxin contamination of the purified protein (Rapid Endo-Test, Lonza, Verviers, Belgium, sensitivity 0.005 EU/ml). Endotoxin levels in the HSP70 preparation was determined by LAL assay and were found to be below 0.2 EU/ml (HSP70: 0.15 EU/ml) which has been referred to as endotoxin-free [25], [26]. Recently we were able to demonstrate the T-cell response as specific for recombinant sHSP70 by performing control experiments with proteins which were expressed and isolated under the same conditions (recombinant Sema7A, CMVpp65) [24].…”