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Analysis of cytotoxic T lymphocytes from a patient with hepatocellular carcinoma who showed a clinical response to vaccination with a glypican‑3‑derived peptide - PubMed

Analysis of cytotoxic T lymphocytes from a patient with hepatocellular carcinoma who showed a clinical response to vaccination with a glypican‑3‑derived peptide

Yoshitaka Tada et al. Int J Oncol. 2013 Oct.

Abstract

Glypican-3 (GPC3), which is a carcinoembryonic antigen, is overexpressed in human hepatocellular carcinoma (HCC). Previously, we performed a phase I clinical trial of GPC3‑derived peptide vaccination in patients with advanced HCC, and reported that GPC3 peptide vaccination is safe and has clinical efficacy. Moreover, we proposed that a peptide‑specific CTL response is a predictive marker of overall survival in patients with HCC who receive peptide vaccination. In this study, we established GPC3‑derived peptide-specific CTL clones from the PBMCs of an HLA-A*02:07-positive patient with HCC who was vaccinated with an HLA-A2-restricted GPC3 peptide vaccine and showed a clinical response in the phase I clinical trial. Established CTL clones were analyzed using the IFN-γ ELISPOT assay and a cytotoxicity assay. GPC3 peptide-specific CTL clones were established successfully from the PBMCs of the patient. One CTL clone showed cytotoxicity against cancer cell lines that expressed endogenously the GPC3 peptide. The results suggest that CTLs have high avidity, and that natural antigen-specific killing activity against tumor cells can be induced in a patient with HCC who shows a clinical response to vaccination with the GPC3144-152 peptide.

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Figures

Figure 1.
Figure 1.

GPC3 peptide-specific CTL clones established from the PBMCs of a patient following GPC3 peptide vaccination. (A) Changes in the frequencies of GPC3 144–152 peptide-specific CTLs before and after vaccination in a patient who showed a PR post-vaccination. Changes in the GPC3 peptide-specific CTLs are observed as differences in the number (left) and the area (right) of spots in an ex vivo IFN-γ ELISPOT assay. (B) Results of the IFN-γ ELISPOT assay against peptide-pulsed target. HLA-A * 02:07 + cancer cell line 1–87 was used as the target. The target was pulsed with the GPC3 144–152 peptide. A non-pulsed target was used as the negative control. The ratio of effector cells to target cells (E/T) is 1. (C) Results of the cytotoxicity assay against peptide-pulsed target. The 1–87 cells were used as the target. Non-pulsed and HIV 19–27 peptide-pulsed targets were used as negative controls. E/Ts are 10, 3 and 1, respectively. A representative of three experiments is shown.

Figure 2.
Figure 2.

GPC3 144–152 peptide-specific avidity of the established CTL clones. The established CTL clones were tested for avidity using 1–87 cells that were pulsed with various concentrations of the GPC3 144–152 peptide. The peptide concentration at which the curve crossed the 50% cytotoxicity mark was defined as the recognition efficiency of that clone. E/T is 10. A representative of three experiments is shown.

Figure 3.
Figure 3.

Recognition of GPC3 + cancer cells by the established CTL clones. (A) Expression of HLA-A2 (left panel) and GPC3 (right panel) on established GPC3 + HLA-A * 02:07 + cancer cells and control cells. (B) Results of the IFN-γ ELISPOT assay for the GPC3 + cancer cell line. The HLA-A * 02:07-overexpressing GPC3 + cancer cell line, JHH7/HLA-A * 02:07, was established and used as the target. JHH7/mock cells were used as the negative control. E/T ratio, 1. Data are presented as mean ± SD of three independent batches. (C) Results of the assay for cytotoxicity against the GPC3 + cancer cell line. JHH7/HLA-A * 02:07 cells were used as the target. JHH7/mock cells were used as the negative control. E/T is 3. A representative of three experiments is shown.

Figure 4.
Figure 4.

GPC3 specificity of CTL clone 24-4-2. (A) GPC3 expression levels on JHH7/HLA-A * 02:07 cells treated with GPC3-siRNA or negative (nega)-siRNA for 48 h, as determined by RT-PCR. (B) Results of the IFN-γ ELISPOT assay for JHH7/HLA-A * 02:07 cells treated with GPC3-siRNA or nega-siRNA. E/T is 1. Data are presented as mean ± SD of three independent batches.

Figure 5.
Figure 5.

CTL clone 24-4-2 shows HLA-A * 02:07 restriction. Results of the IFN-γ ELISPOT assay for healthy donor PBMCs with HLA-A2. The established CTL clone 24-4-2 and the HLA-A * 02:01-restricted, GPC3-specific CTL clone were used as effectors. E/T is 0.2. A representative of two experiments is shown.

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