Fig. 4: Neoantigen-reactive CD8 T cells are polyfunctional, kill targets presenting cognate peptides in multiple HLA alleles and are increased in on-treatment tumor samples.
a, Representative density plots for patient G2 ICS for CD8+ cells producing IFN-γ, CD107α, TNF-α and IL-2 in ex vivo or IVS-expanded PBMCs stimulated overnight with DMSO or patient-specific CD8 pool. Cells were gated on PBMCs (forward scatter (FSC)-A versus side scatter (SSC)-A), singlets (FSC-A versus FSC-H), live cells (FSC-A versus Live/Dead), CD8 or CD4 T cells (CD8 versus CD4) and cytokine+ cells. b, Polyfunctionality was assessed via Boolean gating of CD8+cytokine+ populations (background subtracted). Pie charts show polyfunctionality of ex vivo PBMCs and post-IVS PBMCs. Pie arcs depict individual cytokines in red (CD107α), lime green (IFN-γ), turquoise (TNF-α) 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, IFN-γ, TNF-α and IL-2), orange (triple positive for CD107α, IFN-γ and TNF-α) and yellow (double positive for CD107α and IFN-γ). c, Target cell killing by IVS-expanded PBMCs from patients G2 (B*08:01, B*57:01, A*30:04). Graphs show relative viability based on NucRed count and normalized to effector-to-target ratio of 10:1 DMSO control over time. Data are shown as mean ± s.d. from duplicate wells for targets alone stimulated with DMSO (gray circles) or cognate peptide (black squares) and for 10:1 effector/target co-cultures stimulated with DMSO (light blue circles) or cognate peptide (navy blue squares). d, DNA sequencing of TCR-β chains in tumor samples in baseline and on-treatment samples from patients G1 and G3. TCR-β chains expanded in PBMCs, in both PBMCs and tumors, and contracted are shown.
