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

<p>(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. *; <i>P</i><0.001.</p

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

<p>(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.</p

Candidate peptides derived from HIG2 restricted with HLA-A*02:01.

<p>The binding score was obtained from the BIMAS website (<a href="http://www-bimas.cit.nih.gov/molbio/hla_bind" target="_blank">http://www-bimas.cit.nih.gov/molbio/hla_bind</a>).</p

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

<p>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.</p

Image_1_Immunological analysis of hybrid neoantigen peptide encompassing class I/II neoepitope-pulsed dendritic cell vaccine.jpeg

Neoantigens/ are tumor-specific antigens that evade central immune tolerance mechanisms in the thymus. Long-term tumor-specific cytotoxic T lymphocyte activity maintenance requires class II antigen-reactive CD4+ T cells. We had previously shown that intranodal vaccination with class I neoantigen peptide-pulsed dendritic cells (DCs) induced a robust immune response in a subset of patients with metastatic cancer. The present study aimed to perform a detailed ex vivo analysis of immune responses in four patients receiving an intranodal hybrid human leukocyte antigen class II neoantigen peptide encompassing a class I neoantigen epitope (hybrid neoantigen)-pulsed DC vaccine. After vaccination, strong T-cell reactions against the hybrid class II peptide and the class I-binding neoantigen peptide were observed in all four patients. We found that hybrid class II neoantigen peptide-pulsed DCs stimulated CD4+ T cells via direct antigen presentation and CD8+ T cells via cross-presentation. Further, we demonstrated that hybrid class II peptides encompassing multiple class I neoantigen epitope-pulsed DCs could present multiple class I peptides to CD8+ T cells via cross-presentation. Our findings provide insight into the mechanisms underlying hybrid neoantigen-pulsed DC vaccine therapy and suggest future neoantigen vaccine design.</p