Journal: Theranostics

Article Title: ILT4 inhibition prevents TAM- and dysfunctional T cell-mediated immunosuppression and enhances the efficacy of anti-PD-L1 therapy in NSCLC with EGFR activation.

doi: 10.7150/thno.52435

Figure Lengend Snippet: Figure 3. ILT4 in EGFR-activated tumor cells promoted TAM recruitment and M2-like polarization. (A-B) Patients with high ILT4 expression in tumor cells of NSCLC tissues showed markedly elevated infiltration of CD68+ TAMs by IHC analysis. (A) Representative images of ILT4 expression and TAM infiltration, brown granules represent positive staining. (B) Average results from 80 patients. ILT4-low and -high were defined by IHC score < 6 (median) and ≥ 6. Scale bar: 20 µm. (C-D) TAMs induced by PC9 and H1975 cells displayed increased migration ability compared with parental macrophages, whereas ILT4 knockdown in PC9 and H1975 cells restricted tumor-induced TAM migration. Tumor cells were first transfected with lentivirus carrying ILT4 shRNA for 48 h, and CM was collected for macrophage culture and TAM induction. The migration ability of TAMs was evaluated by the Transwell migration assay. (C) Images of migrated cells and (D) Average results from 3 independent experiments. Scale bar: 50 µm. *, p < 0.05; **, p < 0.01 compared with the medium group; ***, p < 0.001. ###, p < 0.001 compared with the LV-shNC group. (E-F) ILT4 knockdown in PC9 and H1975 cells decreased the secretion of CCL2 and CCL5. (E) showed ELISA results of CCL2, (F) showed CCL5. (G-H) Recombinant human CCL2 or CCL5 reversed ILT4 knockdown and decreased macrophage migration. 100 ng/mL recombinant human CCL2 or 200 ng/mL CCL5 were added to the CM of ILT4 knockdown PC9 and H1975 cells to induce TAM migration. The migration ability of TAMs was evaluated by the Transwell migration assay. (G) Images of migrated cells. (H) Average results from 3 independent experiments. Scale bar: 50 µm. *, p < 0.05; **, p < 0.01 compared with the medium group; ***, p < 0.001. compared with LV-shNC group. (I-J) Patients with high ILT4 expression in tumor cells displayed more frequent CD206+ but less CD86+ TAM accumulation in tumor tissues by IHC analysis. (I) Representative images of CD68+ TAMs, CD206+ M2-like TAMs and CD86+ M1-like TAMs frequencies; brown granules represent positive staining. (J) Average results from 80 patients. ILT4-low and -high were defined by IHC score < 6 and ≥ 6 respectively. Scale bar: 20 µm. (K-L) ILT4 knockdown in PC9 and H1975 cells decreased M2-like markers (CD163, CD206, IL-10 and Arg1) and increased M1-like markers (CD80, CD86, IL-12 and TNFα) in TAMs by real-time PCR (K) and flow cytometry (L). TAMs were induced by CM as in (D) for 24 h. (M-N) TAMs induced by ILT4-downregulated PC9 and H1975 cells promoted the proliferation (M) and IFN-γ expression (N) of T cells. For T cell proliferation assay, CD3+ T cells separated from fresh PBMCs were first pre-activated by anti-CD3 for 24 h and stained with CFSE (1:1000), and then co-cultured for 4 days with TAMs induced by CM of ILT4-downregulated PC9 and H1975 cells. Flow cytometry was performed to determine CFSE strength. For IFN-γ expression, pre-activated CD3+ T cells were co-cultured with TAMs as described above for 48 h and IFN-γ levels were analyzed by flow cytometry. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Arg1: arginase 1; CM, conditioned medium; LV-shILT4: lentivirus carrying ILT4 shRNA; LV-shNC: lentivirus carrying control shRNA.

Article Snippet: Following antigen retrieval, the sections were incubated with 3% hydrogen peroxide for 20 min. Tissue slides were then incubated overnight at 4 °C with following primary antibodies: anti-ILT4 antibody (1:50; Affinity; Cat No. DF9604), anti-pEGFR antibody (1:200; Abcam; Cat No. ab40815), anti-PD-L1 antibody (1:100; CST; Cat No.13684), anti-CD68 antibody (1:100; Abcam; Cat No. ab125212), anti-CD206 antibody (1:100; Abcam; Cat No. ab64693), anti-CD163 antibody (1:100; Abcam; Cat No. ab182422), anti-CD3 antibody (1:200; Abcam; Cat No. ab5690; 1:100; CST; Cat No. 85061), anti-IFN-γ antibody (1:50; Affinity; Cat No. DF6045), and anti-F4/80 antibody (1:200; Abcam; Cat No. ab111101), anti-CD80 antibody (1:500; Boster; Cat No. BM4121), anti-CD86 antibody (1:500; Proteintech; Cat No. 66406-1-Ig), anti-PIRB antibody (1:100; Immunoway; Cat No. YN1914).

Techniques: Expressing, Staining, Migration, Knockdown, Transfection, shRNA, Transwell Migration Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Real-time Polymerase Chain Reaction, Flow Cytometry, Proliferation Assay, Cell Culture, Control