Identification of an HLA-A2-restricted epitope peptide derived from hypoxia-inducible protein 2 (HIG2)

Abstract

We herein report the identification of an HLA-A2 supertype-restricted epitope peptide derived from hypoxia-inducible protein 2 (HIG2), which is known to be a diagnostic marker and a potential therapeutic target for renal cell carcinoma. Among several candidate peptides predicted by the HLA-binding prediction algorithm, HIG2-9-4 peptide (VLNLYLLGV) was able to effectively induce peptide-specific cytotoxic T lymphocytes (CTLs). The established HIG2-9-4 peptide-specific CTL clone produced interferon-γ (IFN-γ) in response to HIG2-9-4 peptide-pulsed HLA-A*02:01-positive cells, as well as to cells in which HLA-A*02:01 and HIG2 were exogenously introduced. Moreover, the HIG2-9-4 peptide-specific CTL clone exerted cytotoxic activity against HIG2-expressing HLA-A*02:01-positive renal cancer cells, thus suggesting that the HIG2-9-4 peptide is naturally presented on HLA-A*02:01 of HIG-2-expressing cancer cells and is recognized by CTLs. Furthermore, we found that the HIG2-9-4 peptide could also induce CTLs under HLA-A*02:06 restriction. Taken together, these findings indicate that the HIG2-9-4 peptide is a novel HLA-A2 supertype-restricted epitope peptide that could be useful for peptide-based immunotherapy against cancer cells with HIG2 expression.

Figure 1. The IFN-γ production in response to the HIG2-9-8, HIG2-9-15, HIG2-9-4 or HIG2-10-8 peptide.

(a) The IFN-γ production from cells induced by the indicated peptide-pulsed DCs was examined by an ELISPOT assay using T2 cells. “+” indicates the wells in which cells were stimulated with T2 cells pulsed with the indicated peptide and “−” indicates the wells in which cells were stimulated with HIV peptide-pulsed T2 cells. The IFN-γ production from cells induced with HIG2-9-8 (b), HIG2-9-15 (c), HIG2-9-4 (d) or HIG2-10-8 (e) peptide stimulation after CTL expanding culture was examined by ELISA. Cells were stimulated with T2 cells pulsed with the corresponding peptide (closed diamonds) or HIV peptide (open squares) at the indicated responder/stimulator ratio (R/S ratio). Similar results were obtained from three independent experiments.

Figure 2. The expression of a HIG2-9-4 peptide-specific T cell receptor on CD8+ T cells.

The expression of the HIG2-9-4 peptide-specific T cell receptor was examined on CD3⁺CD4⁻ cells following CTL expansion culture of HIG2-9-4 peptide-induced CTLs. (a) A quadrant gate was set based on the staining results with the HIV peptide/HLA-A*02: 01 tetramer. (b) CD8⁺ T cells expressing the HIG2-9-4 peptide/HLA-A*02: 01-specific T cell receptor were detected. Similar results were obtained from three independent experiments.

Figure 3. The IFN-γ production and cytotoxic activity of a HIG2-9-4 peptide-specific CTL clone.

(a) An established CTL clone was stimulated with T2 cells pulsed with the HIG2-9-4 peptide (closed diamonds) or HIV peptide (open squares). The IFN-γ production in the culture supernatant was examined by ELISA. R/S ratio; responder/stimulator ratio. (b) The cytotoxic activity of the HIG2-9-4 peptide-specific CTL clone was examined against peptide-pulsed T2 cells (close diamond) or T2 cells pulsed with the HIV peptide (open square). E/T ratio; effector/target ratio. All experiments were performed in triplicate. The representative results from three independent experiments are shown. *P<0.001

Figure 4. The recognition of HIG2 and HLA-A*02:01-expressing cells by a HIG2-9-4 peptide-specific CTL clone.

(a) A HIG2-9-4 peptide-specific CTL clone was stimulated with COS7 cells expressing both HIG2 and HLA-A*02:01 (close diamond), or either HIG2 alone (open circle) or HLA-A*02:01 alone (open triangle), then the IFN-γ production was examined by ELISA. R/S ratio; responder/stimulator ratio. (b) The cytotoxic activity of the HIG2-9-4 peptide-specific CTL clone was examined against HLA-A*02:01-positive HIG2-expressing A498 cells (closed diamond) or HLA-A*02:01-negative HIG2-expressing Caki-1 cells (open circle). E/T ratio; effector/target ratio. All experiments were performed in triplicate. Representative results from three independent experiments are shown. *; P<0.001.

Figure 5. The HLA-A*02:06-restricted response of a HIG2-9-4 peptide-specific CTL clone.

(a) A HIG2-9-4 peptide-specific CTL clone was induced from HLA-A*02:06-positive PBMCs, and stimulated with HLA-A*02:06-positive PSCCA0922 cells pulsed with the HIG2-9-4 peptide (close diamond) or HIV peptide (open square). (b) The HIG2-9-4 peptide-specific CTL clone was stimulated with COS7 cells expressing both HIG2 and HLA-A*02:06 (close diamond), or either HIG2 alone (open circle) or HLA-A*02:06 alone (open triangle). The IFN-γ production in the culture supernatant was examined by ELISA. R/S ratio; responder/stimulator ratio. The representative results from three independent experiments are shown.