6 | See next page for caption. NATuRE MEDICINE | www.nature.com/naturemedicine Articles Nature MediciNeExtended 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+Te t+ (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 10 6 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. NATuRE MEDICINE | www.nature.com/naturemedicine Articles Nature MediciNe Extended Data Fig. 7 | See next page for caption. NATuRE MEDICINE | www.nature.com/naturemedicine Articles Nature MediciNeExtended Data Fig. 7 | (A) Representative density plots for intracellular cytokine staining (ICS) of patient G8 ex vivo and IVS-expanded leukopak PBMCs stimulated overnight with either DMSO or patient-specific CD8 Pool (Supplementary T able 2) to assess production of IFNγ, CD107α, TNFα, and IL-2 in CD8+ T cells. Cells were gated on PBMCs (FSC-A vs SSC-A), singlets (FSC-A vs FSC-H), live cells (FSC-A vs Live/Dead), CD8 T cells and cytokine+ cells. (B) Polyfunctionality was assessed via Boolean gating of CD8+cytokine+ populations (background subtracted). Pie charts show polyfunctionality of ex vivo PBMCs (left pie chart) and post-IVS PBMCs (right pie chart). Pie arcs depict individual cytokines in red (CD107α), lime green (IFNγ), turquoise (TNFα), and navy blue (IL-2). Pie sections depict frequencies of cells expressing multiple cytokines as outlined in Pie Category legend. Main functional pie slices are colored red (quadruple positive for CD107α, IFNγ, TNFα, and IL-2), orange (triple positive for CD107α, IFNγ, and TNFα), and yellow (double positive for CD107α and IFNγ). (C) Polyfunctionality was assessed via Boolean gating of CD4 +cytokine+ populations (background subtracted). Pie charts show polyfunctionality of post-IVS PBMCs for patient G2 (left pie chart) and G8 (right pie chart). Pie arcs depict individual cytokines in red (CD107α), lime green (IFNγ), turquoise (TNFα), and navy blue (IL-2). Pie sections depict frequencies of cells expressing multiple cytokines as outlined in Pie Category legend. Main functional pie slices are colored red (quadruple positive for CD107α, IFNγ, TNFα, and IL-2), orange (triple positive for CD107α, IFNγ, and TNFα), and yellow (double positive for CD107α and IFNγ). (D) T arget cell killing by IVS-expanded PBMCs from patients G8 (A*03:03, C*04:01). Graphs show relative viability based on NucRed count and normalized to effector:target (E:T) ratio of 10:1 DMSO control over time. Data are shown as mean +/- SD from duplicate wells for targets alone stimulated with DMSO (gray circles) or cognate peptide (black squares), and for 10:1 E:T co-cultures stimulated with DMSO (light blue circles) or cognate peptide (navy blue squares). NATuRE MEDICINE | www.nature.com/naturemedicine Articles Nature MediciNe Extended Data Fig.