Identification of immunogenic HLA-A*02:01 epitopes associated with HCC for immunotherapy development

Abstract

Background: HCC is the most common form of primary liver cancer, and despite recent advances in cancer treatment, it remains associated with poor prognosis and a lack of response to conventional therapies. Immunotherapies have emerged as a promising approach for cancer treatment, especially through the identification of tumor-specific immunogenic epitopes that can trigger a targeted immune response. This study aimed to identify immunogenic epitopes associated with HCC for the development of specific immunotherapies.

Methods: We used high-throughput data screening and bioinformatics tools for antigens and epitope selection. The immunogenicity of the selected epitopes was studied after coculture of peripheral blood mononuclear cells obtained from healthy donors or HCC patients with a plasmacytoid dendritic cell line loaded with the selected peptides. Specific CD8+ T cell amplification and functionality were determined by labeling with tetramers and by IFN-γ and CD107a expression (flow cytometry and ELISpot).

Results: We analyzed the transcriptional gene expression landscape of HCC to screen for a set of 16 ectopically expressed genes in a majority of HCC samples. Epitopes predicted to bind to HLA-A*02:01 with high affinity were further validated for their immunogenicity using the previously described plasmacytoid dendritic cell line in ex vivo CD8+ activation assays using patient immune cells. Three out of the 30 tested epitopes, namely FLWGPRALV (MAGE-A3), FMNKFIYEI (AFP), and KMFHTLDEL (LRRC46), elicited a strong T-cell response, in activation assays, degranulation assays, and IFN-γ secretion assays.

Conclusions: These results highlight the potential of these peptides to be considered as targets for immunotherapies. The discovery of such immunogenic epitopes should improve immune-based treatments for liver cancer in combination with the current treatment approach.

Graphical abstract

FIGURE 1

Activation frequencies in HCC (%) and signal-to-noise ratio (SNR) for the 43 preselected genes. Activation frequencies and SNR were calculated from RNA-seq databases. Gray bars represent the percentage of activation frequencies for each preselected gene. Black bars represent the SNR. Genes were ordered to increase the value of activation frequencies. Arrows point to the genes that were selected for rather a high SNR (black arrows) or activation rate (gray arrows).

FIGURE 2

Evaluation of peptide binding to HLA-A2. The fluorescence index was calculated from the median fluorescence intensities of anti-HLA-A2 staining on peptide-loaded (10 µM) and unloaded T2 cells. Cytomegalovirus HLA-B7 restricted peptide was used as negative control (black bar), and MelanA peptide was used as positive control (white bar). Mean with SD of n=4 independent experiments are shown.

FIGURE 3

Evaluation of the peptide-loaded PDC*line ability to amplify peptide-specific CD8⁺ T cells in healthy donors and HCC patients. (A) Representative flow cytometry dot plots showing the amplification from day 0 to day 28 of peptide-specific CD8⁺ T cell for 1 healthy donor after coculture with the peptide-loaded PDC*line. Gates are drawn on tetramer⁺CD3⁺CD8⁺ T alive cells. (B, C) Evolution of peptide-specific CD8⁺ T-cell frequencies from D0 (white dots) to D21 (black dots) in healthy donors (panel B, n=4–6) and in HCC patients (panel C, n=3–4). Each dot represents the frequency of peptide-specific CD8⁺ T cells among total CD8⁺ T live cells for each donor or patient. The dashed line represents the limit of the detection threshold of 0.05%. Abbreviation: HD, healthy donor.

FIGURE 4

Functionality of T-cell clones. (A) Representative result of IFN-γ ELISpot with CD8⁺ T cells from FLW-specific clone. Upper wells correspond to negative control conditions (medium and unloaded T2). Lower wells correspond to spots detected in the presence of PMA+ionomycin (positive control) or of T2 cells loaded with the FLW peptide. (B) Bar plot showing the number of spots in IFN-γ ELISpot for 100,000 cells upon coculture of T-cell clones directed against FLW (black), FMN (gray), and KMF (white) without or with unloaded or peptide-loaded T2 cells, or with PMA+ionomycin (positive control). The mean and SD of duplicate wells are shown. (C) Representative flow cytometry profiles of CD107a⁺CD8⁺ T cells from FMN-specific clones after stimulation without or with unloaded or FMN peptide-loaded T2 cells or with PMA+ionomycin (positive control). (D) Bar plot showing percentages of CD107a⁺ T cells clones directed against FLW (black), FMN (gray), and KMF (white) after culture without or with unloaded or peptide-loaded T2 cells, or with PMA+ionomycin (positive control) (1 experiment).