Heat shock cognate protein 70 encodes antigenic epitopes recognised by HLA-B4601-restricted cytotoxic T lymphocytes from cancer patients

Abstract

Heat shock cognate protein 70 (HSC70), a highly conserved protein and a member of the family of molecular chaperones, has the ability to induce cytotoxic T lymphocyte (CTL) responses through binding and carrying antigenic peptides. We demonstrated in this study that the HSC70 gene encodes two antigenic peptides recognised by HLA-B46-restricted and tumour-reactive CTLs established from tumour-infiltrating lymphocytes of a colon cancer. These HSC70-derived peptides, at amino-acid positions 106-114 and 233-241, had the ability to induce HLA-B46-restricted and peptide-specific CTLs, which are reactive to tumour cells, from peripheral blood mononuclear cells of the majority of epithelial cancer patients tested. These results, along with those from the previous studies, indicate the two ways of HSC70 involvement in the immune responses to tumours: chaperones and antigens, and thus may provide a new insight for the development of HSC70-directed cancer-specific immunotherapy.

Figure 1

Determination of the gene. (A) Identification of a gene coding for tumour antigen. Amounts of 100 ng of a clone 3H from SW620 cDNA libraries and 100 ng of the HLA-B4601 gene were cotransfected into COS-7 cells. The HLA-B5101 or HLA-B5201 gene was used as a negative control. After incubation for 48 h, the cells were tested for their ability to stimulate IFN-γ production by the OKB-CTLs. COS-7 cells transfected with HLA-A0207 and clone 3H were also served as a negative control. The results of three separate experiments are shown in the figure. The background IFN-γ production in response to COS-7 cells (<100 pg ml⁻¹) was subtracted. The values represent means of triplicate assays. ^*P<0.05 by a two-tailed Student's t-test. (B) The mRNA expression of HSC70. The mRNA expression of HSC70 in various tumour cells and normal cells was analysed by Northern blotting. Representative results are given in the figure. (C) The expression levels of the constitutively expressed HSC70 or the stress-inducible HSP70 in the samples used for Northern blotting were investigated at the protein level by means of Western blotting with anti-Hsc70 or -Hsp70 mAb, respectively, to further investigate the expression of HSP family in tumour cells. (D) Recognition of an HSC70-derived peptide by the OKB-CTLs. Various doses of the peptides were loaded onto HLA-B4601-transfected COS-7 cells for 2 h followed by addition of the OKB-CTLs at an E/T ratio of 10 : 1. After 18 h incubation, the culture supernatants were collected in order to measure the IFN-γ production. The values represent of means of duplicate assays.

Figure 2

Determination of CTL epitope peptides. (A) Two peptides, HSC70_106–114 and HSC70_233–241, were tested for their ability to induce HLA-B46-restricted and tumour-reactive CTLs from the PBMCs of HLA-B46⁺ 10 cancer patients and four HDs. The details of the method are described in the Materials and Methods section. The data showed amounts of IFN-γ produced by peptide-treated PBMC in response to HLA-B4601-transfected COS7 cells pulsed with the corresponding peptides (left column) and OSC20 (HLA-B46⁺) tumour cells (right column) at an E/T ratio of 20 : 1. The background IFN-γ release in response to HLA-B4601-transfected COS7 cells pulsed with a control peptide (p53_204–212) (<100 pg ml⁻¹) was subtracted. The background IFN-γ release in response to Kuma-1 (HLA-B46) tumour cells (<50 pg ml⁻¹) was also subtracted from the figure. The values represent the means of duplicate assays. (B) The cytotoxicity of the peptide-treated PBMCs from seven cancer patients against OSC20 (HLA-B46⁺), Kuma-1 (HLA-B46⁻), and PHA-blastoid T cells was confirmed by a 6-h ⁵¹Cr-release assay. Values represent the means of triplicate assays at three different E/T ratios. ^*P<0.05 by a two-tailed Student's t-test.

Figure 3

Inhibition tests and peptide specificity. (A) Inhibition test. IFN-γ production by the HSC70_106–114 and HSC70_233–241-stimulated PBMCs in response to OSC20 tumour cells was tested at an E/T ratio of 20 : 1 in the presence of 20 μg ml⁻¹ of anti-HLA class I, anti-HLA class II, anti-CD4, anti-CD8, anti-CD14, or anti-HLA-B, C mAb. Values represent the means of triplicate assays ^*P<0.05 by a two-tailed Student's t-test. (B) Inhibition test and peptide specificity. Cytotoxicity by the peptide-stimulated PBMCs against OSC20 was tested at an E/T ratio of 20 : 1 in the presence of 20 μg ml⁻¹ of the mAb shown above. The CTL activity was inhibited by anti-CD8, anti-HLA class I, and anti-HLA-B, C mAb, but not by any other mAbs tested. For measurement of peptide specificity, an excess amount (20 μg ml⁻¹) of a corresponding peptide (HSC70_106–114 or HSC70_233–241) or a control peptide (p53_204–212) was preloaded onto the ⁵¹Cr-labelled OSC20 tumour cells, which were used as target cells in a 6-h ⁵¹Cr-release assay. As cold cells, unlabelled EBV-B cells preloaded with either a corresponding peptide (HSC70_106–114 or HSC70_233–241), or a control peptide (p53_204–212) were added to wells containing the ⁵¹Cr-labelled OSC20 tumour cells, at a cold-to-hot cell ratio of 20 : 1. Values represent the means of triplicate assays. ^*P<0.05 by a two-tailed Student's t-test.