Cross-presentation of NY-ESO-1 cytotoxic T lymphocyte epitope fused to human heat shock cognate protein 70 by dendritic cells

Abstract

The cancer-testis antigen NY-ESO-1 has been implicated as one of the most attractive candidates for a cancer vaccine. However, a protein vaccine generally meets inefficient antigen presentation to CD8(+) T cells, which could be overcome by combination with an appropriate adjuvant. Heat shock protein is a natural adjuvant and activates the antigen-presenting cells to channel exogenous antigens into the classical major histocompatibility complex class I antigen-processing pathway (cross-presentation). Therefore, we genetically fused a minigene encompassing the NY-ESO-1 cytotoxic T lymphocyte (CTL) epitope 157-165 (ESO p157-165) to the human heat shock cognate protein 70 (hsc70) and expressed the resulting fusion proteins in Escherichia coli. By using a human leukocyte antigen-A*0201-restricted NY-ESO-1-specific CTL clone, the cross-presentation of ESO p157-165 by monocyte-derived dendritic cells (mo-DC) pulsed with the fusion protein was evaluated. The fusion protein-pulsed mo-DC activates the CTL clone much more efficiently than the free NY-ESO-1 protein-pulsed mo-DC. Moreover, the magnitude of the CTL activity was comparable between ESO p157-165 and the fusion protein of hsc70 and ESO p157-165 (hsc70-ESO p157-165 fusion protein). In addition, the CTL activation induced by the fusion protein, but not by the epitope, was inhibited by paraformaldehyde fixation of the mo-DC and by treatment with lactacystin, a specific inhibitor for the proteasome. Finally, the hsc70-ESO p157-165 fusion protein-pulsed DC was able to induce an antigen-specific T-cell response. These results suggest that the hsc70-ESO p157-165 fusion protein is therefore considered to be a promising candidate as a cancer vaccine.

Figure 1

Construction of plasmids encoding human heat shock cognate protein 70 (hsc70) and NY‐ESO‐1 cytotoxic T lymphocyte (CTL) epitope fusion genes. A minigene encoding the NY‐ESO‐1 CTL epitope 157‐165 was fused to the N‐ or C‐terminus of the hsc70 gene. These fusion genes were inserted into the pQE31 expression vector via 5′BamHI and 3′KpnI restriction sites.

Figure 2

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE) of the fusion proteins. NY‐ESO‐1 cytotoxic T lymphocyte epitope 157‐165 (ESO p157‐165) was conjugated with human heat shock cognate protein 70 (hsc70) at either the C‐ or N‐terminus. Both of these fusion proteins were detected on the Coomassie‐stained SDS‐PAGE. Lane 1, hsc70–ESO p157‐165; lane 2, ESO p157‐165/hsc70; lane 3, hsc70; M, marker.

Figure 3

Removal of endotoxin from lysates of Escherichia coli. After purification, these fusion proteins were treated with Kurimover I and II to remove contaminated lipopolysaccharide (LPS). The concentration of LPS, determined by the Limulus ES‐II test, was less than 50 pg/mL (less than 2.0 endotoxin unit [EU]/mg fusion proteins).

Figure 4

Phenotype of monocytes and monocyte‐derived dendritic cells. The monocytes isolated by magnetic‐bead selection from the peripheral blood mononuclear cells were harvested immediately (day 0) and cultured with granulocyte/macrophage colony‐stimulating factor (GM‐CSF) and interleukin (IL)‐4 for 5 days. The monocyte‐derived dendritic cells were used in the following assays.

Figure 5

Maturation of monocyte‐derived dendritic cells (mo‐DC). On day 6, mo‐DC were stimulated with lipopolysaccharide (LPS) (250 ng/mL or 20 pg/mL at final concentration) or the fusion protein of human heat shock cognate protein 70 (hsc70) and ESO p157‐165 (hsc70–ESO p157‐165 fusion protein) (20 pg/mL LPS and 350 µg/mL protein at final concentration), and the cells were harvested 24 h later. The cells were stained using the antibodies indicated, and were analyzed by flow cytometry.

Figure 6

Presentation of NY‐ESO‐1 cytotoxic T lymphocyte (CTL) epitope to NY‐ESO‐1‐specific CD8⁺ T cells by monocyte‐derived dendritic cells (mo‐DC) exposed to the fusion protein of human heat shock cognate protein 70 (hsc70) and ESO p157‐165 (hsc70–ESO p157‐165 fusion protein). The mo‐DC from a human leukocyte antigen (HLA)‐A*0201‐positive healthy donor were exposed for 2 h in plain RPMI with a NY‐ESO‐1 synthetic peptide (ESO p157‐165 at 5 µg/mL), hsc70–ESO p157‐165 fusion protein (at 350 µg/mL), recombinant hsc70 (at 350 µg/mL), or NY‐ESO‐1 protein (at 16 µg/mL). The two clones, namely clones 5 and 49, from a single donor were used as HLA‐A2‐restricted NY‐ESO‐1‐specific CD8⁺ T‐cell clones. (a) A photograph of the wells in an enzyme‐linked immunospot assay using clone 5. (b) The data in the panel represent the average number of positive spots stimulated by clone 49.

Figure 7

Paraformaldehyde inhibits cross‐presentation of a NY‐ESO‐1 cytotoxic T lymphocyte (CTL) epitope. The monocyte‐derived dendritic cells (mo‐DC) were harvested at day 5, then fixed for 15 min with 1% paraformaldehyde in phosphate‐buffered saline (pH 7.4), and used for assays. The enzyme‐linked immunospot assays using the human leukocyte antigen‐A2‐restricted NY‐ESO‐1‐specific CD8⁺ T‐cell clone were carried out as described above. Solid bars, no paraformaldehyde; gray bars, paraformaldehyde added.

Figure 8

Lactacystin inhibits cross‐presentation of a NY‐ESO‐1 cytotoxic T lymphocyte (CTL) epitope. The monocyte‐derived dendritic cells (mo‐DC) were harvested at day 5, then cultured for an additional 12 h in the presence or absence of lactacystin (10 µM) in normal growth medium, and used for assays. The enzyme‐linked immunospot assays using the human leukocyte antigen‐A2‐restricted NY‐ESO‐1‐specific CD8⁺ T‐cell clone were carried out as described above. Solid bars, no lactacystin; gray bars, lactacystin added.

Figure 9

ESO p157‐165‐specific T cells detected by tetramer. (a) The CD8⁺ cells were isolated using magnetic beads. (b) After three stimulations by monocyte‐derived dendritic cells (mo‐DC) pulsed with human heat shock cognate protein 70–ESO p157‐165 fusion protein, CD4⁺ T cells whose antigen specificity was unknown grew. The expanded CD8⁺ T cells were detected specifically by human leukocyte antigen‐A2 tetramer complexed with ESO p157‐165 (d) but not with influenza A matrix p58‐66 (c).