Noncovalent Association with Stress Protein Facilitates Cross-Priming of CD8+ T Cells to Tumor Cell Antigens by Dendritic Cells

Abstract

A viral oncogene carrying well-defined Kb/Db-restricted epitopes was expressed in a heat shock protein (hsp)-associated or nonassociated form in the murine tumor cells P815 and Meth-A. Wild-type SV40 large T-Ag (wtT-Ag) is expressed without stable hsp association; mutant (cytoplasmic cT-Ag) or chimeric (cT272-green fluorescent fusion protein) T-Ag is expressed in stable association with the constitutively expressed, cytosolic hsp73 (hsc70) protein. In vitro, remnants from apoptotic wtT-Ag- or cT-Ag-expressing tumor cells are taken up and processed by immature dendritic cells (DC), and the Kb/Db-binding epitopes T1, T2/3, and T4 of the T-Ag are cross-presented to CTL in a TAP-independent way. DC pulsed with remnants of transfected, apoptotic tumor cells cross-presented the three T-Ag epitopes more efficiently when they processed ATP-sensitive hsp73/cT-Ag complexes than when they processed hsp-nonassociated (native) T-Ag. In vivo, more IFN-γ-producing CD8+ T cells were elicited by a DNA vaccine that encoded hsp73-binding mutant T-Ag than by a DNA vaccine that encoded native, non-hsp-binding T-Ag. Three- to 5-fold higher numbers of T-Ag (T1-, T2/3-, or T4-) specific, Db/Kb-restricted IFN-γ-producing CD8+ T cells were primed during the growth of transfected H-2d Meth-A/cT tumors than during the growth of transfected Meth-A/T tumors in F1(b × d) hosts. Hence, the association of an oncogene with constitutively expressed, cytosolic hsp73 facilitates cross-priming in vitro and in vivo of CTL by DC that process material from apoptotic cells.

Priming Protective CD8 T Cell Immunity by DNA Vaccines Encoding Chimeric, Stress Protein-Capturing Tumor-Associated Antigen

“…We used tetramer staining as well as 4-h ex vivo-specific IFN-␥ expression to enumerate specific and functional CD8 T cells. Confirming our previously published data, high expression levels and enhanced immunogenicity correlated well with hsp capture (6,7,23,50,51). It is likely that hsp association but not the expression level of the Ag are the critical factor for its enhanced immunogenicity because efficient CD8 T cell priming by DNA vaccines was observed with Ags that were expressed at low levels and/or showed a high turn over rate (52)(53)(54).…”

“…Different factors may cooperate. Stress proteins can provide signals that bypass CD4 T cell help (4,41), mature DC (42), stimulate the innate immune system through, for example, TLRs, CD14, or CD91 (43)(44)(45)(46), efficiently chaperone Ag processing and/or presentation (47), or stimulate cross-presentation (7,48,49). It is unknown which of these factors contribute in the system we describe to enhance the immunogenicity of the codelivered epitope.…”

“…In contrast, expression of fusion products between a non-hsp-binding domain (e.g., the T 1-60 ) and Cterminal antigenic protein fragments is not (or only barely) detectable and induces no (or only weak) immune responses. We demonstrated the usefulness of the system in priming tumor-protective T cell responses in mice (6,7). This was achieved using viral reporter Ags but not the more informative but less immunogenic tumor-associated Ags (TAA).…”

EWI-2/CD316 Is an Inducible Receptor of HSPA8 on Human Dendritic Cells

“…It has been extensively demonstrated previously that HSPs of every class and from diverse species enhance antigen uptake and presentation by DCs and that they can also pro- on May 9, 2018 by guest http://mcb.asm.org/ mote cross-priming. Such properties were also demonstrated for the hsp70 proteins and also explicitly for HSPA8/hsc73 (15). However, an established role at the beginning of the DC activation phase does not exclude the possibility that HSPA8 exerts an additional regulatory function(s) during the ensuing subacute activation/postactivation phase.…”

Tumor-Derived Heat Shock Protein 70 Peptide Complexes Are Cross-Presented by Human Dendritic Cells

“…For Ab-blocking experiments, before adding the T cells, DCs were incubated for 1 h at 37°C with the mAb HB54 (20 g/ml) recognizing an antigenic determinant shared by HLA-A*02 and B17 (29). In some experiments, immature DCs were fixed with PFA (1%) for 10 min at room temperature, then they were washed three times with excess of VLE medium before being used in cross-presentation assays (32).…”