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. 2021 Jan 19;11(7):3392–3416. doi: 10.7150/thno.52435

Figure 4.

Figure 4

ILT4 in EGFR-activated tumor cells impaired the proliferation and cytotoxicity of T cells. (A-B) High ILT4 expression in tumor cells of NSCLC tissues was correlated with decreased infiltration and IFN-γ generation in CD3+ T cells detected by IHC analysis. (A) Images of ILT4 expression, T cell infiltrates, and IFN-γ levels, brown granules represent positive staining. (B) Average numbers of T cells in 80 patients. The cutoff scores for ILT4-high and -low were the same as in Figure 3I. Scale bar: 20 µm. (C) Patients with high ILT4 expression displayed significantly decreased IFN-γ levels in tumor-infiltrating T cells compared with ILT4-low patients by IHC analysis. The histogram shows the average proportion of IFN-γ+ T cells from 80 patients. (D-E) T cells co-cultured with ILT4-downregulated PC9 and H1975 cells showed increased proliferation ability compared with the counterpart group. PC9 and H1975 cells were first transfected with ILT4-knockdown lentivirus for 48 h, and then co-cultured with CD3+ T cells for 4 days at 1:2 ratio. The proliferation ability of T cells was assessed by the CFSE proliferation assay. (D) Flow cytometry results and (E) Average results from 3 independent experiments. (F-G) Transfection of ILT4-knockdown lentivirus in PC9 and H1975 cells inhibited T cell apoptosis compared with the control lentivirus group. Flow cytometry was performed to assess apoptotic T cells after co-culturing with different tumor cells. (F) Representative results for T cell apoptosis and (G) Results from 3 independent experiments. (H-I) ILT4 knockdown in PC9 and H1975 cells increased IFN-γ production in T cells. T cells co-cultured with tumor cells were collected and evaluated by flow cytometry to determine IFN-γ expression levels. (H) Flow cytometry results and (I) Average results from 3 independent experiments. (J) T cells co-cultured with ILT4-downregulated PC9 and H1975 cells released more IFN-γ into the supernatant than those co-cultured with control tumor cells. IFN-γ secretion in the same supernatant as in (F-G) by ELISA. (K) T cells co-cultured with ILT4-downregulated PC9 and H1975 cells displayed increased cytolytic ability compared with those co-cultured with control tumor cells. T cells were co-cultured with ILT4-downregulated or control PC9 and H1975 cells at different E: T ratios for 4 days, and the cytolysis assay was used to assess the killing ability of T cells. *, p < 0.05; **, p < 0.01; ***, p < 0.001. E:T ratio: Effector: target cell ratio; LV-shILT4: lentivirus carrying ILT4 shRNA; LV-shNC: lentivirus carrying control shRNA.