Tfh cells respond to anti-PD-1 mAb treatment in tumor-draining lymph nodes. C57BL/6 mice were injected s.c. with MC38-OVA tumor cells. After 10 days, tumor-bearing mice were treated or not with anti-PD-1 mAb (250 µg, i.v.). Draining lymph nodes were analyzed 3 days later by flow cytometry or immunofluorescence. (A and B) Representative FACS dot plot (A) and quantification (B) of BCL6⁺ PD-1^high cells among CD4⁺ T cells. Compiled from three independent experiments with a total of 15 mice per group. (C) Quantification of Ki67⁺ cells among BCL6⁺ PD-1^high CD4⁺ T cells. Compiled from three independent experiments with a total of 15 mice per group. (D) Lymph node immunofluorescence showing PD-1, CD4, and FoxP3 expression. CD19 and Ki67 staining was included to locate the B cell zone and germinal centers. Region 1 (left) shows a germinal center and region 2 (right) shows an area of the B cell zone without the germinal center. White stars (*) highlight CD4⁺ PD-1⁺ FoxP3⁻ T cells located in the B cell zone but outside germinal centers. Scale bar, 100 µm. Representative of two independent experiments with a total of three mice per group. (E) Schematic representation showing CD4⁺ PD-1⁺ FoxP3⁻ T cells in the B cell zone and CD19⁺ Ki67⁺ B cells (to identify germinal centers). (F) Distance between individual CD4⁺ PD1⁺ FoxP3⁻ T cells present in the B cell zone and the closest CD19⁺ Ki67⁺ B cells was performed to estimate T cell localization. Representative of two independent experiments with a total of three mice per group. Statistical analyses were performed using t test, ***, P < 0.001.