Monitoring antigen-specific cellular immune response using an RT-ddPCR-based assay for IFN-γ gene expression

This study evaluated the effectiveness of the Reverse Transcription-droplet digital PCR (RT-ddPCR) method in measuring T-cell-mediated immunity by quantifying IFN-γ mRNA expression. The results demonstrated that peak IFN-γ expression occurred approximately 4 h after stimulation of whole blood and peripheral blood mononuclear cells (PBMCs) by stimulants. The fold activation of IFN-γ mRNA expression in 100 µL of blood challenged with CEF peptides was lower than that observed in PBMCs. Our findings highlighted the effectiveness of the RT-ddPCR assay in detecting IFN-γ mRNA expression with the least number of cells and at the lowest stimulant concentration for varying numbers of PBMCs. Additionally, there was a strong and significant positive correlation between the number of SARS-CoV-2-specific spot-forming units (SFUs) and the fold activation of IFN-γ gene expression (r = 0.78, p < 0.02) in PBMC from 10 donors vaccinated with SARS-CoV-2, further supporting the usefulness of the RT-ddPCR method. Importantly, even with just 10,000 PBMCs, we detected SARS-CoV-2-specific IFN-γ mRNA induction. The RT-ddPCR and ELISpot assays demonstrated similar sensitivities and measured IFN-γ activation by low concentrations of stimulants. This study suggests that the RT-ddPCR method effectively assesses T-cell mediated immunity through IFN-γ expression, offering a feasible alternative to the ELISpot assay.