Figure 4.
LPS/TLR4 activates IFITM4P/PD-L1 signaling pathway to promote immune escape
(A) C57BL/6J mice were implanted with 5 × 105 B16F10 cells expressing IFITM4P or vector and received PD-1 mAb or IgG isotype control (IgG2a) treatment. The timeline of tumor induction and treatment is shown. (B) Tumor volumes. (C) Introduction of IFITM4P in B16F10 cells significantly increased tumor volume in C57BL/6J mice. PD-1 mAb significantly reduced tumor volume in C57BL/6J mice bearing B16F10 cells expressing IFITM4P. (D) No significant difference in body weight of mice was found in each group. Mouse body weight was measured every 3 days. (E) qRT-PCR showed a dose-dependent increase in LPS-induced IFITM4P transcription in Leuk-1 cells. (F) qRT-PCR showed inhibition of TLR4 shRNA on LPS-induced (100 μg/mL) IFITM4P expression in Leuk-1 cells. (G) qRT-PCR showed inhibition of TAK-242 (1 μM) on LPS-induced (100 μg/mL) IFITM4P expression in Leuk-1 cells. (H) Inhibition of TAK-242 (1 μM) on IFITM4P promoter-driven luciferase activity in response to LPS (100 μg/mL) in 293T cells. (I) Schematic timeline of modified mouse tongue leukoplakia/SCC model. (J) (a) Normal tongue (cage I) and (b–d) typical tongue leukoplakia (cages II–IV). (c and d) Tongue leukoplakia lesions were larger and rougher in texture (cages III and IV). (e–h) Histopathological diagnoses: NM (e) (cage I), OL with moderate dysplasia (f) (cage II), OL with severe dysplasia and local early invasive SCC (g and h) (cages III and IV). (i–l) IHC Staining of PD-L1. Negative staining (i) (cage I). Staining of local early invasive SCC was stronger (k and l) (cages III and IV) than that of tongue leukoplakia (j) (cage II). Staining was stronger in early invasive SCC areas than in nearby OL areas (k). (m–p) FISH staining of IFITM4P. Negative staining (m) (cage I). Staining of local early invasive SCC was stronger (o and p) (cages III and IV) than that of tongue leukoplakia (n) (cage II). Staining was stronger in early invasive SCC areas than in nearby OL areas (o) (e–p, 200×). (K) qRT-PCR and WB confirmed that in 4NQO-induced tongue leukoplakia/SCC mouse model, the expression of IFITM4P increased with progression of the disease. Compared with the ddH2O control group, LPS significantly increased the expression of IFITM4P and promoted the carcinogenesis of tongue leukoplakia. (L) Schematic timeline of the PD-1 mAb treatment in early tongue leukoplakia mouse model. (M) PD-1 mAb is effective in leukoplakia treatment, especially for leukoplakia induced by 4NQO and LPS. Macroscopic view before and after PD-1 mAb treatment. (N) Ratio of tongue lesion score before and after PD-1 mAb treatment was evaluated. Data from (E), (F), and (G) are shown as mean ± SD from three independent experiments; ∗p < 0.05. Data from (B,C,D), (H), (J,K) and (M,N) are shown as mean ± SD from six independent experiments. ∗P < 0.05.