Fig. 8. TNFR⁺ T_regs express BATF-regulated surface markers preferentially in the HNSCC TME.

(A) Relative expression of surface markers differentially expressed on TNFR⁺ T_regs and percentage of T_reg subset in the single-cell HNSCC dataset were visualized on a dot plot. (B) Frequency of CD96, CD39, GITR, CD83, and CD74 expression determined by flow cytometry from control and BATF-null human activated T_regs from cord blood (n = 5). Samples from the same donor were connected by solid lines, and each dot indicates an individual replicate. (C) Matched TILs and PBMCs from patients with HNSCC (n = 6) and HD PBMC (n = 5) were stained for flow cytometry phenotyping. Representative flow plot of BATF expression in TILs and PBMCs from patients with HNSCC and HD PBMCs, normalized to mode scales as a percentage of the maximum count. (D) Tabular summary of percentage of cells expressing BATF from patients with HNSCC (n = 6) and HD PBMCs (n = 5). (E to J) The percentage of T_regs expressing surface markers identified in TNFR⁺ T_regs from matched TILs and PBMCs from patients with HNSCC (n = 6) and HD PBMCs (n = 5) is summarized on a bar plot. Each dot indicates an individual replicate. Bars indicate the median of expression, and error bars represent 1 SD. The coexpression of BATF expression and marker genes by MFI in HNSCC TIL T_regs was shown in scatterplots. The Pearson correlation coefficient (R) between the expression BATF and marker genes was calculated by the MFI in HNSCC TIL T_regs. The significance of Pearson correlation coefficient (P) was calculated by t test. Data in (B) were analyzed by a ratio paired t test, and data in (D) to (J) were analyzed by a nonparametric Mann-Whitney U test. P values: *P ≤ 0.05; **P ≤ 0.01.