Extended Data Fig. 5: TCR-seq analysis of tumor-infiltrating T_REG and T_FR cells.

a, the pie chart illustrates the mean percentage of T_FR clonotypes that were shared with T_REG cells (light blue) and non-T_REG cells (gray) respectively, from 4 patients with the highest numbers of clonally expanded FOXP3-expressing cells from a published single-cell RNA-seq dataset²⁰. The lower panel plot displays the percentage of T_FR clonotypes that overlap with 4-1BB^– or 4-1BB⁺ tumor-infiltrating T_REG cells. b, Euler diagram depicting the degree of clonal overlap between T_REG, T_FH and T_FR cells. c, Representative TraCer plot of patient 1010²⁰ depicting all clonally expanded cells, color indicates the type of tumor-infiltrating CD4⁺ T cells: non-T_REG (gray, FOXP3^–), 4-1BB^– T_REG (green), 4-1BB⁺ T_REG (red) and T_FR (yellow) cells. d, Single-cell pseudotime trajectory of 4-1BB^–, 4-1BB⁺ T_REG, clonally expanded, TCR-sharing T_REG and T_FR cells (indicated with colored circles) constructed using the Monocle3 algorithm. e, Correlation of Monocle component 1 (x-axis) with the genes commonly unregulated in 4-1BB⁺ T_REG, clonally expanded, TCR-sharing T_REG and T_FR cells compared to 4-1BB^– T_REG cells (y-axis). The solid line represents LOESS fitting between the shared signature and Monocle component 1. f, flow cytometric analysis of the frequency (left panel, P = 0.002 for indicated comparison), MFI (middle panel, P = 0.002 for indicated comparison) for 4-1BB expression in tumor-infiltrating CD8⁺ T cells, T_REG and T_FR cells (n = 10 treatment naïve NSCLC patients as in Fig. 2a–d). Data are mean + /− s.e.m. Significance for comparisons were computed using two-tailed Wilcoxon matched-pairs signed-rank test between T_REG and T_FR cells.

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