Figure 1. Direct inhibitory effect of TLR7 ligands on Th17 differentiation from naïve T cells.

(A) TLR7 ligands inhibited human Th17 cell differentiation from naïve CD4⁺ T cells in vitro. Human naïve CD4⁺ T cells were cultured with Th17 polarization condition medium in the presence of TLR7 ligands (Imiq and Lox) or TLR2 ligand (Pam3) for 6 days. Results shown in left panel are representative intracellular staining of IL-17-producing T cells in differentiated Th17 cells with different TLR ligand treatments using flow cytometry analyses after stimulation with PMA and ionomycin for 5 hours. Results shown in right panel are the summary of IL-17-producing cells in differentiated Th17 cells with indicated TLR ligand treatments in human naïve CD4⁺ T cells isolated from different donors. Result of each dot shown is derived from an individual donor and data shown are mean ± SD. *p<0.05, compared with the medium only group determined by paired t-test. (B) Imiq and Lox, but not Pam3 inhibited mouse Th17 cell differentiation from naïve CD4⁺ T cells in vitro. Intracellular staining of IL-17-producing cells was performed using flow cytometry analyses following Th17 differentiation from mouse naïve CD4⁺ T cells in the presence of imiq, lox or Pam3 for 6 days. (C) Imiq and Lox, but not Pam3 suppressed IL-17 mRNA expression in mouse CD4⁺ T cells during Th17 polarization. Mouse CD4⁺ T cells were cultured in Th17 polarization condition medium in the presence of Imiq and Lox and Pam3 for 6 days. IL-17 and INF-γ mRNA expression levels were determined by real-time PCR. The expression level of each gene was normalized to GAPDH expression and adjusted to the levels in naïve CD4⁺ T cells. Data are means ± SD from 3 representative naïve CD4⁺ T cells. ** p< 0.01, compared with the medium only group determined by paired t-test. (D) Imiq, but not Lox and Pam3 inhibited mouse Th1 polarization from naïve CD4⁺ T cells. Mouse CD4⁺ T cells were cultured in Th1 differentiation medium in the presence of indicated TLR ligands for 6 days. INF-γ-producing T cells were analyzed using flow cytometry after stimulation with PMA and ionomycin for 5 hours. (E) No inhibitory effect of TLR7 ligands on mouse Th2 polarization from naive CD4⁺ T cells. Mouse CD4⁺ T cells were cultured in Th2 differentiation medium in the presence of indicated TLR ligands for 6 days. IL-4-producing T cells were analyzed using flow cytometry after stimulation with PMA and ionomycin for 5 hours. (F) No inhibitory effect of TLR7 ligands on mouse Treg polarization from naive CD4⁺ T cells. Naïve CD4⁺ T cells from FoxP3-GFP transgenic mice were cultured in Treg differentiation medium in the presence of indicated TLR ligands for 6 days. GFP⁺ T cells were analyzed using flow cytometry.