Figure 78.

Gating strategy to identify Ag specific human CD4⁺ T cells. Human lymphocytes were gated based on physical parameters (FSC-SSC), then doublets were excluded using FSC-A and FSC-A parameters. Dead cells were excluded using viability stain 780. T cells were identified as CD3⁺ and among these CD4⁺ T cells were selected. Antigen-specific CD4⁺T cells were identified as cells expressing CD154; CD4⁺CD154⁺ T cells were then evaluated for IFN-γ, TNF-α, and IL-2 expression. Representative plots of PBMCs unstimulated (medium, negative control) stimulated with SEB (positive control) or Spike peptide pools, from a COVID-19 recovered subject evaluated at 7 days from SARS-CoV-2 vaccination, are shown.