Fig. 1

Blimp1⁺ Treg and T_FR cells are accumulated in the tumor. a-b) Blimp1-YFP reporter mice (n = 5) were inoculated with B16-OVA and immunized with NP-OVA/CFA at day 0 and NP-OVA/IFA at day 7. Cells from spleens and tumors were analyzed at day 21 by flow cytometry. Blimp1 = YFP. a) Frequencies of indicated subsets. eTreg: CD62L^loCD44^hiTreg. b) Expression of indicated molecules in Blimp1⁺Foxp3⁺Treg cells. c) Foxp3^YFP-Cre mice (n = 5) were established with B16-OVA/NP-OVA model as in a. left, expression of T_FR (PD-1⁺Bcl6⁺Foxp3⁺CD4⁺CD3⁺), T_FH (PD-1⁺Bcl6⁺Foxp3⁻CD4⁺CD3⁺) and GC B-cells (GL-7⁺Fas⁺CD19⁺); right, Ratios of T_FR:T_FH or T_FR:GC B in the spleen or tumor. Data represent one of two (a-b) or at least three (c) independent experiments. d) Representative analysis of indicated molecules in Treg cells (CD25⁺CD127⁻CD4⁺CD3⁺) from melanoma metastatic lung (M) or uninvolved lung tissue (Control, C) of the same patient. e) Frequency of Treg cells and mean fluorescence intensity (MFI) of indicated molecules as presented in d. f) T_FR (PD-1⁺CXCR5⁺CD25⁺CD127⁻CD4⁺CD3⁺) and CD27⁺CD38⁺ B-cells (gated on IgD⁻CD19⁺CD3⁻) from melanoma metastatic lung (M) or uninvolved lung tissue (C) of the same patient. g) Frequency of each subset in f. Other melanoma metastatic tissues are included in e and g: 1, lung; 2, liver; 3, lymph node. h) IF staining of T_FR (CXCR5⁺Foxp3⁺CD4⁺) and T_FH (CXCR5⁺Foxp3⁻CD4⁺) cells in two FFPE melanoma metastatic lymph node sections (160 ×). Insets with a 4-fold magnification highlight T_FR (arrowheads) and T_FH (*) cells. i-j) Relative fractions, indicated by % with matched colors, of CXCR5^hi and CXCR5^lo (based on median expression value cutoff) patients within FOXP3^hi (i) or PRDM1^hi (j) group (top 33%) in primary and metastatic patients from the TCGA-SKCM dataset. ns, no significance, * P < 0.05, ** P < 0.01 and **** P < 0.0001 (a,c, unpaired two-tailed Student’s t-test; e,g, paired two-tailed Student’s t-test). Bars, mean ± SEM