Figure 3. M1p specific T cell culture.

A T cell culture was generated against the influenza M1p antigen, and the percentage of M1p-specific CD8⁺ T cells was enumerated using M1p/HLA-A*02:01 tetramers and a negative control HLA-A*02:01 tetramer, along with an anti-CD8 antibody and a lineage marker mix of CD4, CD14, CD16 and CD19 antibodies. Tetramer staining was analyzed by gating on live cells (DAPI negative), lymphocytes (FSC vs SSC), and CD8⁺Lineage⁻ cells. Only the CD8⁺Lineage⁻ cells are shown on the plots. Over 99% of the cells that fell in the live lymphocyte gates were CD8⁺Lineage⁻. Non-specific binding to the negative control tetramer was low (0.05% of CD8⁺ T-cells), while 48.4% of CD8⁺ T-cells had high avidity interactions with the M1p/HLA-A*02:01 tetramer (Figure 3A). The culture also demonstrated antigen specific cytotoxicity against M1p pulsed autologous DCs compared to PR1 pulsed autologous DCs (Figure 3B). For the cytotoxicity assay, the FSC vs SSC plot was gated to remove debris in the lower left hand corner, and DDAO-SE positive target cells (DCs) were selected on a DDAO-SE vs CMFDA plot. The percentage of dead target cells shown is the percentage of DDAO-SE positive DCs that also stained positive with the Fixable Violet Dead Cell Stain.