Human Heat Shock Protein 70 Enhances Tumor Antigen Presentation through Complex Formation and Intracellular Antigen Delivery without Innate Immune Signaling

Abstract

Heat shock proteins (HSPs) have shown promise for the optimization of protein-based vaccines because they can transfer exogenous antigens to dendritic cells and at the same time induce their maturation. Great care must be exercised in interpretating HSP-driven studies, as by-products linked to the recombinant generation of these proteins have been shown to mediate immunological effects. We generated highly purified human recombinant Hsp70 and demonstrated that it strongly enhances the cross-presentation of exogenous antigens resulting in better antigen-specific T cell stimulation. Augmentation of T cell stimulation was a direct function of the degree of complex formation between Hsp70 and peptides and correlated with improved antigen delivery to endosomal compartments. The Hsp70 activity was independent of TAP proteins and was not inhibited by exotoxin A or endosomal acidification. Consequently, Hsp70 enhanced cross-presentation of various antigenic sequences, even when they required different post-uptake processing and trafficking, as exemplified by the tumor antigens tyrosinase and Melan-A/MART-1. Furthermore, Hsp70 enhanced cross-presentation by different antigen-presenting cells (APCs), including dendritic cells and B cells. Importantly, enhanced cross-presentation and antigen-specific T cell activation were observed in the absence of innate signals transmitted by Hsp70. As Hsp70 supports the cross-presentation of different antigens and APCs and is inert to APC function, it may show efficacy in various settings of immune modulation, including induction of antigen-specific immunity or tolerance.Cytotoxic CD8 T cells have an essential role in cellular immunity in that they destroy infected or malignantly transformed cells. They are activated by the recognition of complexes of major histocompatibility complex (MHC) 4 class I and antigenic peptides present on the surface of antigen-presenting cells (APC). Conventionally, the antigenic peptides presented by MHC class I are derived from endogenous cytosolic antigens. In specialized situations, MHC class I molecules additionally present peptides derived from exogenous antigens. This noncanonical MHC class I presentation, which is referred to as cross-presentation, requires that the exogenous antigen is internalized by APCs, subsequently enzymatically processed into peptides, and channeled into the MHC class I loading pathway (1, 2). Cross-presentation is crucial for the generation of CD8 T cell responses against antigens that are not endogenously produced by APCs, such as tumor antigens and pathogen-derived proteins. In an applied setting, cross-presentation is the required pathway for the generation of protein-based vaccines that are intended to stimulate antigen-specific CD8 responses. Critical parameters that define the efficacy of a vaccine are the amount of delivered antigen and the context in which the antigen is presented to the T cells. As the physiological capacity of APCs to cross-present antigen is generally low (3), there is significant interest to de...

Inducible Heat Shock Protein 70 Reduces T Cell Responses and Stimulatory Capacity of Monocyte-derived Dendritic Cells

“…Accordingly, it was shown that the endotoxin contamination in recombinant Hsp70 is common and exclusively responsible for the described proinflammatory properties, including activation of APCs (12)(13)(14)(15)(16). It was also shown that endotoxin-free Hsp70 did not induce APC activation while still effectively delivered antigenic peptides for cross-presentation (17). In a different study, Hsp70 failed to bind to null cells overexpressing either CD14, TLR2, or TLR4 which had previously been associated with the Hsp70-mediated activation of innate immunity (8).…”

Calcium Signaling in Dendritic Cells by Human or Mycobacterial Hsp70 Is Caused by Contamination and Is Not Required for Hsp70-mediated Enhancement of Cross-presentation

“…Recombinant Hsp70 Proteins-Recombinant human (rhu)-Hsp70 protein was either purchased from Stressgen, Victoria, BC, Canada (ESP-555) or was prepared in our own laboratory as described (17). After ADP affinity chromatography, buffer was exchanged using PD10 columns followed by extensive dialysis (48 h, 5000ϫ buffer volume) and diafiltration (Vivaspin 20, 30,000 MWCO PES, Sartorius Stedim Biotech).…”

“…To assess the influence of nucleotide carryover, rhuHsp70 preparations were used after PD10 column and diafiltration with a less intensive dialysis step (Hsp70/nc ϩ ). Endotoxin was depleted, as described (17). Only proteins with endotoxin below 0.5 EU/mg rhuHsp70 were used.…”

“…C-terminally His 6 -tagged recombinant protein was produced in E. coli strain Rosetta2(DE3) (Invitrogen, Karlsruhe, Germany) and purified using Ni-nitrilotriacetic acid-agarose (Qiagen, Hilden, Germany) as described (18). Endotoxin depletion was performed as described (17).…”

“…In addition, we have shown that human Hsp70 from melanoma cells can chaperone tumor-associated peptides (tyrosinase) and deliver them to DCs allowing cross-presentation and T cell stimulation (16). Recently, we used recombinant human (rhu)Hsp70 to define in more detail the critical parameters for the induction of immune effector responses (17). Highly purified and endotoxin-depleted rhuHsp70 was found to efficiently deliver exogenous peptides to DCs resulting in T cell activation, provided that peptide and Hsp70 had formed stable complexes before exposure to DCs.…”