Fig. 7: PD-1 blockade prevents afferent lymphatic migration of CD25⁺Foxp3⁺CD4 Tregs from TILs to the dLNs and increases Treg conversion to CD25- IFNγ highTregs.

a Immunohistochemistry analysis of Treg subsets in TILs of mice bearing melanoma treated with anti-PD-1 (Rmp1-14), anti-CD80 (1G10), and isotype rat IgG. MFI of Foxp3, CD25, and CD4. Magnification ×20, scale bar 80 μm. b, c Intracellular IFNγ and T-bet, or surface PD-1 expression in CD25⁺ or CD25⁻ Treg subsets of TILs and dLNs, in mice treated as in (a) and assessed by flow cytometry. Representative gating strategy, dot plots, and histograms in TILs and dLNs (b) and summary of cell frequencies of all treated groups (c). d IFNγ, T-bet, and PD-1 expression in CD4 Teffs in TILs. e–g Transfer of anti-PD-1 treated CD25⁺Foxp3GFP⁺CD4⁺ iTregs (1 × 10⁶) into B16F10-bearing mice. Scheme of intratumoral cell transfer (e). Flow cytometry analysis of transferred Treg numbers (f) and IFNγ and T-bet expression (g) in TILs and dLNs. Representative dot blots and histograms shown. Data representative of 3 (a–d) or 2 (e–g) independent experiments (4 mice/group). Mean ± SEM. *P < 0.05 by one-way ANOVA. Source data are provided as a Source Data file.