Extended Data Fig. 8. Cloning and dose response of antigen-specific T cells.

a–c, Cloning and screening of TCRs corresponding to CD8 T cells with highly differential gene expression relative to influenza-specific T cells. Seven TCRs were selected from the refined CD8 sc data based on highly differential gene expression relative to influenza-specific T cells. These TCRs were cloned into the Jurkat/NFAT luciferase reporter system and first screened against autologous LCL pre-loaded with pools of putative MANA peptides (10μg/ml) based on the respective patient’s WES and MANA predictions. Three TCRs recognized a MANA peptide pool, one each from patients MD01-005 (a), MD01-004 (b), and MD043-011 (c). The reactive MANA was then mapped from the reactive peptide pool by stimulating the TCR-transfected Jurkat cell with autologous LCL pre-loaded with 10μg/ml of each individual MANA within the reactive pool (centre). Dose–response curves were then generated for each MANA-specific TCR (right). Data are shown as relative luminescence units. A (+) sign indicates the positive response. d, Functional characterization of MANAFEST-identified and screening-identified TCRs. 2D projection of clones identified from the MANAFEST assay (red) and clones identified via cloning of TCRs corresponding to T cells with differential gene expression relative to influenza-specific T cells (green) is shown for patients MD01-004, MD01-005, and MD043-011. CD8 T cell clusters are marked with the same colour code as Fig. 2b. e, Viral-specific TCRs and MANA-specific TCRs from one patient with MPR and two patients without MPR were cloned into the Jurkat reporter system and tested against titrating concentrations of relevant peptide. The average log₁₀ relative luminescence of viral-specific TCRs (blue, 3 clonotypes from 3 different patients), MANA-specific MPR TCRs (green, 3 clonotypes from 1 patient with MPR), and MANA-specific non-MPR TCRs (red, 7 clonotypes from 2 patients without MPR) was compared at each peptide titration. Data are shown as a Box and Whiskers plot. The middle bar shows the median, with the lower and upper hinges corresponding to the 25^th and 75^th percentiles, respectively (interquartile range, IQR). The upper whisker extends from the hinge to the largest value no further than 1.5 * IQR from the hinge. The lower whisker extends from the hinge to the smallest value at most 1.5 * IQR of the hinge. Comparisons of relative luminescence units for viral-specific vs MANA-specific T cell clonotypes at different titrations were performed using two-sided Wilcoxon rank sum test. ns: P > 0.05; *, 0.01 < P < 0.05.