Extended Data Fig. 1: Selection criteria for the integrated single-cell analysis and gating strategies.

a, Violin plots depicting single-cell expression levels for BCL6, CXCR5 and FOXP3 transcripts (left panel) in tumor-infiltrating CD4⁺ T cells of an exemplary dataset⁶¹; dotted lines indicate threshold used for defining positive cells. The scatter plot (right panel) shows expression levels of BCL6 and CXCR5 transcripts in FOXP3-expressing CD4⁺ T cells b, Gating strategy (surface panel) to sort tumor-infiltrating T_REG (LIN^–CD45⁺CD3⁺CD4⁺CXCR5^–CD127^–CD25⁺) and T_FR (LIN^–CD45⁺CD3⁺CD4⁺CXCR5⁺GITR⁺) cells is shown in the representative FACS plots. c, Gating strategy (intracellular panel) to identify tumor-infiltrating T_REG (LIN^–CD45⁺CD3⁺CD4⁺CXCR5^–FOXP3⁺BCL-6^-) and T_FR (LIN^–CD45⁺CD3⁺CD4⁺BCL-6⁺FOXP3⁺) cells is shown in the representative FACS plots. d, Representative immunohistochemistry staining for one of the ten NSCLC patients in (Fig. 1d–i) is shown, PanCK (white), CD4 (light blue), CXCR5 (yellow), CD20 (magenta) FOXP3 (green) and BCL-6 (red), scale bars are 25 μm.