TGFβ1 negatively correlates with IL-9 production without affecting the surface expression of TGFβR1 and TGFβR2. (A and B) ELISA measurements of plasma IL-4, TGFβ, and IL-9 levels in HDs and CRC, GC, or breast cancer (BC) patients. (C) Correlation of plasma TGFβ1 and IL-9 cytokine levels in BC patients and HDs. The results were plotted and analyzed with the linear regression t test. (D) Tumor growth of C57BL/6 mice that were s.c. injected with B16-F10 cells and then i.v. administered control IgG; with α-PD-1; with α-PD-1 plus α-TGFβ1; or with α-PD-1 and α-TGFβ1 plus α-IL-9. The α-PD-1 or α-TGFβ1 was i.v. injected on days 7, 10, and 13 after tumor inoculation, and α-IL-9 was injected on days 10, 13, and 16 after tumor inoculation. n = 5 mice/group. (E) Immunoblot analysis of NF-κB and STAT proteins or actin and lamin B (loading controls) in cytoplasmic extracts (CEs) and nuclear extracts (NEs) of human CD4⁺ T cells derived from the indicated CRC or GC patients and HDs cultured under Th9 condition for 3 d. (F and G) Flow cytometric analysis of surface TGFβR1 and TGFβR2 expression in CD4⁺ T cells derived from the indicated CRC or GC patients and HDs cultured under Th9 condition for 3 d. Data are presented as representative plots (left panels) and summary graphs (right panels). MFI, mean fluorescence intensity. (H and I) Flow cytometric analysis of the frequencies of the naive or memory CD4⁺ T cells from the peripheral blood of HDs and CRC patients. (J) qPCR analysis of TRIM56 and IL9 mRNA expression in human HD-derived CD4⁺ T cells transduced with control siRNA or TRIM56-siRNA. (K and L) Flow cytometric analysis of the frequencies of IL-9–producing Th9 cells in human HD-derived CD4⁺ T cells were transduced with control siRNA or TRIM56-siRNA under Th9 condition for 3 d. Each panel is representative of two independent experiments, and each circle represents one human individual in A–C. Student’s t test was used. Bars, mean; error bars, SEM; ***, P < 0.001; ns, not significant.