Host-oriented Peptide Evaluation Using Whole Blood Assay for Generating Antigen-specific Cytotoxic T Lymphocytes

Abstract

A whole blood assay using antigenic peptide was established to predict host cytotoxic T lymphocyte (CTL) precursor status. Blood samples from HLA-A24 donors and colorectal cancer patients were directly diluted with RPMI-1640 medium to a 20% blood concentration, then distributed to tubes and a peptide of an HLA-A24-restricted CEA peptide panel (20 μM) was added to the tubes. Incubation was performed for 4-5 days and supernatants were subjected to ELISA specific for IFN-gamma protein. It was observed that certain CEA peptides could stimulate the diluted blood samples to produce IFN-gamma. Only the peripheral blood mononuclear cells (PBMCs) that were purified from the IFN-gamma-positive samples of the whole blood assay showed positive spots, detected with IFN-gamma ELISPOT assay, and could proliferate with the stimulation of immobilized anti-CD3 antibody plus interleukin-2 (CD3/IL-2 system). The proliferating PBMCs expressed cytotoxic activity against HLAA24+ CEA-expressing tumor cells and the TISI target cells pulsed with the CEA peptide that had been used to stimulate the PBMCs to produce IFN-gamma, but they did not kill the target cells pulsed with peptides that had failed to stimulate IFN-gamma production, nor did they kill the target cells alone. Theses findings suggest that the IFN-gamma production of the blood samples detected by the whole blood assay identifies the peptide that can induce the CEA antigen-specific CTL response. Detection of IFN-gamma gene expression using real-time-PCR analysis could identify the peptide within 6 hours, which is earlier than the protein analysis by ELISA. The whole blood assay using the CEA peptide panel for healthy donors and colorectal cancer patients revealed that IFN-gamma-inducible peptides were different among the individual samples tested, indicating that the CEA peptides that should be used for generating CTLs are different in individual patients. The whole blood assay using a CEA antigen peptide panel is simple and beneficial for identifying candidate peptides. The host-oriented peptide evaluation (HOPE) approach may provide hope for the augmentation of clinical efficacies for peptide-based cancer immunotherapy.