Extended Data Fig. 6
(A) Density plots showing recognition of pHLA and memory phenotypes in PBMCs from posttreatment timepoints (G1, W48, HLA-A*02:01-APC and HLA-B*44:05-PE; G8, W25, A*03:01). Pie charts showing frequencies of naïve (CD45RA+CCR7+; purple), central memory (CM; CD45RA-CCR7+; blue), effector memory (EM; CD45RA-CCR7-; green), and T effector memory RA+ (TEMRA; CD45RA+CCR7-; orange) cells in bulk CD8 and CD8+tetramer+ populations from corresponding dot plots. (B) Density plots showing recognition of peptide-HLA (HLA-A*02:01-APC) pre- and post- ChAd68 boost for patient G1. Pie charts showing frequencies of naïve (CD45RA+CCR7+; purple), central memory (CM; CD45RA-CCR7+; blue), effector memory (EM; CD45RA-CCR7-; green), and T effector memory RA+ (TEMRA; CD45RA+CCR7-; orange) cells in CD8+tetramer+ populations from corresponding dot plots. Cells were gated on lymphocytes (SSC-A vs FSC-A), single cells (FSC-H vs FSC-A), viable cells (FSC-A vs LD-AF488), CD3+ cells (FSC-A vs CD3-BV421), CD8+Tet+ (CD8-PerCP-Cy5.5 vs tetramer-PE or tetramer-APC), memory phenotypes (CD45RA-SB702 vs CCR7-PE-Cy7). (C) Supernatants from ex vivo IFNγ ELISpot (G1 W5, G2 W12, G3 W37, G7 W5, G8 W12, G10 W16, G11 W2, G12 W8) were analysed by MSD U-plex assay for levels of IL-2, TNFα, and Granzyme B (GRZB) following stimulation with patient-specific CD8 Pools or DMSO. Stacked bar graphs show mean cytokine levels (triplicate wells) in pg/ml (background subtracted) +/- SD for replicate wells for IL-2 (light blue), GRZB (orange), and TNFα (yellow). (D) PBMCs from optional leukopak donations (G2 W19, G8 W25) were analysed by ex vivo FluoroSpot assay to measure IFNγ, GRZB and IL-2 expression following overnight stimulation with DMSO or patient-specific CD8 Pools. Graphs show mean SFU per 106 PBMCs +/- SD for triplicate FluoroSpot wells stacked for IFNγ (navy blue), GRZB (orange), and IL-2 (light blue). E) MSD U-plex analysis of Granzyme B (GRZB) levels in ELISpot supernatants pre- and post-ChAd68 boost for patients G1, G3, G8, G11, G13 and G14 are shown in pg/ml (background subtracted, mean +/- SD for triplicate wells). No post ChAd68-boost samples were available for patients G9 and G12.
