Fig 2: ILT4 expression in NSCLC cells was induced by EGFR activation. (A-B) ILT4 expression in tumor cells of NSCLC tissues was positively correlated with pEGFR levels by IHC analysis. Tumor samples were sequentially sectioned and stained with ILT4 and pEGFR primary antibodies (A) Representative images of ILT4 and pEGFR co-localization, brown granules define positive staining. (B) Statistical results from 80 patients. Scale bar: 20 µm. (C-D) Inhibition of EGFR activation using gefitinib significantly decreased mRNA (C) and protein (D) expression of ILT4 in a concentration-dependent manner in PC9 and HCC827 cells harboring activating EGFR mutation. PC9 and HCC827 cells were treated with a gradient concentration of gefitinib for 24 h before real-time PCR and Western blot analyses. (E-F) Inhibition of EGFR activation using osimertinib but not gefitinib markedly reduced mRNA (E) and protein (F) expression of ILT4 in EGFR T790M-mutant H1975 cells. The cells were treated with osimertinib (0.1 µM) or gefitinib (0.1 µM) for 24 h, and mRNA and protein expression of ILT4 were analyzed by real-time PCR and Western blotting. (G-H) Knockdown of EGFR in PC9 and H1975 cells decreased ILT4 mRNA (G) and protein (H) levels. PC9 and H1975 cells were transfected with specific EGFR siRNA, and the mRNA or protein expression of ILT4 and EGFR was analyzed 72 h after transfection using real-time PCR and Western blotting. (I-J) EGF stimulation of EGFR wild-type H1299 cells elevated ILT4 expression in both concentration- (I) and time-dependent (J) manners. (I-J) Average results from 3 independent experiments. H1299 cells were treated with different concentrations of EGF for 24 h or with 100 ng/mL EGF for different durations and Western blotting was performed to determine ILT4, EGFR, and pEGFR levels. (K-L) Overexpression of EGFR in H1299 cells increased pEGFR, ILT4 (L) and EGF (K) levels determined by Western blotting or real-time PCR. ILT4 and pEGFR expression were detected 48 h after transfection of EGFR overexpression plasmid using Western blotting, while EGF level was detected by real-time PCR. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Gef: gefitinib; NC: normal control; OE: overexpression plasmid; Osi: osimertinib; si: small interfering RNA.

Fig 3: Combined ILT4 and PD-L1 blockade in EGFR-activated NSCLC cells synergistically improved TAM- and dysfunctional T cell-mediated immunosuppression. (A-B) ILT4 or PD-L1 blockade in PC9-GR and H1299 cells inhibited the migration ability of TAMs, while combined ILT4 or PD-L1 blockade showed a stronger suppressive effect compared with either antibody alone. PC9-GR and EGF-preactivated H1299 cells were first pretreated with anti-ILT4 (500 ng/mL)/anti-PD-L1 (500 ng/mL)/combined anti-ILT4 (250 ng/mL) and anti-PD-L1 (250 ng/mL) for 8 h. CM from these cells was collected for macrophage culture and TAM induction. The migration ability of TAMs was evaluated by the Transwell migration assay. (A) Images of TAM migration, and (B) Average results from 3 independent repetitions. Scale bar: 50 µm. (C-F) ILT4 or PD-L1 blockade in PC9-GR and EGF-preactivated H1299 cells decreased the expression and secretion of CCL2 and CCL5, while combined blockade showed a synergistic effect. (C-D) Decreased CCL2 and CCL5 mRNA expression detected by real-time PCR, (E-F) Decreased CCL2 and CCL5 secretion detected by ELISA. Tumor cells were treated and conditioned media were collected as described in (A). (G-I) ILT4 or PD-L1 blockade in PC9-GR and EGF-preactivated H1299 cells reversed tumor-induced M2-like markers in TAMs, while combined ILT4 and PD-L1 blockade displayed more significant reversion compared with either blockade alone. TAMs were induced as described in (A), and M2-like markers in TAMs were determined by flow cytometry. (G) Flow cytometry results and (H-I) Average results of CD163 and CD206 from 3 independent experiments. (J-K) ILT4 or PD-L1 blockade in PC9-GR and EGF-preactivated H1299 cells increased T cell proliferation, while combined ILT4 or PD-L1 blockade showed a synergistic effect on T cell proliferation. PC9-GR and H1299 cells were treated as mentioned in (A), and co-cultured with anti-CD3-preactivated CD3+ T cells for 4 days at the ratio of 1:2. CFSE proliferation assay was performed to determine the proliferation of T cells. (J) Images of flow cytometry and (K Average results from 3 independent experiments. (L-M) ILT4 or PD-L1 blockade in PC9-GR and EGF-preactivated H1299 cells suppressed T cell apoptosis, while combined ILT4 and PD-L1 blockade showed a more significant suppression than either antibody alone. T cells co-cultured with tumor cells for 2 days were evaluated for apoptotic T cells by flow cytometry. (L) Flow cytometry results and (M) Average results from 3 independent experiments. (N) ILT4 or PD-L1 blockade in PC9-GR and EGF-preactivated H1299 cells promoted IFN-? secretion in co-cultured T cells, while combined blockade released more abundant IFN-? level than either antibody alone. T cells as in (J) were evaluated for IFN-? level by ELISA. (O) T cells co-cultured with PC9-GR and H1299 cells pretreated with anti-ILT4 or anti-PD-L1 had stronger tumoricidal ability compared with those co-cultured with IgG-pretreated tumor cells, and T cells co-cultured with combination antibody-pretreated tumor cells showed the strongest tumor cytolytic activity. Tumor cells were pretreated with different antibodies as mentioned in (A) and then co-cultured with tumor cells at an E:T ratio of 5:1. Cytolysis assays were employed to determine the killing ability of T cells. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Anti: neutralization antibody; CM: conditioned medium.

Fig 4: ILT4 blockade suppressed tumor progression and immune escape in TKI-resistant EGFR mutant NSCLC in vivo. (A-B) ILT4 knockdown markedly inhibited PC9-GR growth in humanized NSG mice. However, PD-L1 blockade accelerated PC9-GR growth and combined blockade of both molecules showed no tumor inhibition compared to the control group. PC9-GR cells (3×106 cells/mouse) transfected with lentiviruses carrying ILT4-knockdown or control shRNA were subcutaneously injected into 6-8-week female NSG mice. Day 7 after tumor inoculation, 2×107 PBMCs from healthy volunteers were transplanted into each mouse via the tail vein. Anti-PD-L1 or control IgG (200 µg/mouse) were intraperitoneally injected into the tumor-bearing mice every 4 days from the day of PBMC transplant. Tumor sizes were measured every 4 days and presented as mean ± SD (n = 8 mice/group). (A) Tumor growth rate in each group and (B) Tumor images from each mouse. Scale bar: 1cm. (C) Tumors in ILT4 knockdown group showed significantly smaller tumor weight than in the control group. However, PD-L1 blockade led to higher while combined blockade generated similar tumor weight compared with the control group. The histogram shows the mean ± SD of tumor weights from each group at the experiment endpoint (n = 8 mice/group). (D-E) ILT4 knockdown increased CD3+ T cells in leukocytes in both spleens (D) and blood (E) of tumor-bearing mice. However, PD-L1 blockade alone or in combination with ILT4 inhibition did not affect CD3+ T cells in these organs. The frequency of CD3+ T cells in tumor spleens and blood were determined by flow cytometry. (F-G) ILT4 knockdown promoted the infiltration of CD3+ T cells in tumors by IHC staining. However, PD-L1 blockade alone or in combination with ILT4 inhibition did not alter the number of CD3+ T cells in tumor tissues. (F) Images of T cell infiltrates and (G) Average results from 8 mice in each group. Scale bar: 20 µm. (H-I) The IFN-? levels in CD3+ T cells from blood, spleens, and tumors were remarkably increased by ILT4 knockdown compared with PD-L1 blockade or combined ILT4 and PD-L1 blockade. (H) The IFN-? expression in blood and spleens were analyzed by flow cytometry, and (I) in tumors by IHC. (J-K) The distribution of CD4+ and CD8+ T cell subsets in mouse blood (J) and spleens (K) was not altered by inhibition of ILT4 or PD-L1 or both molecules as determined by flow cytometry analysis. (L-M) The IFN-? expression in both CD4+ and CD8+ T cells was augmented by ILT4 knockdown but not by anti-PD-L1 or combination therapy. Results are shown in spleens (L) and blood (M). The level of IFN-? expression in both organs was evaluated by flow cytometry. *, p < 0.05; **, p < 0.01; ***, p < 0.001. LV-shILT4: lentivirus carrying ILT4 shRNA; LV-shNC: lentivirus carrying control shRNA; ns: no significance.

Fig 5: Activated ERK and AKT signaling mediated EGFR-induced ILT4 expression. (A) MAPK was among the most significantly altered signaling pathways upon treatment with osimertinib in H1975 cells with osimertinib (0.2 µM) for 24 h. mRNA was extracted and microarray was performed for cluster analysis of altered signaling pathways. (B) EGFR expression in NSCLC tissues was positively correlated with EKR (r = 0.44), NF-?B1 (r = 0.38), P65 (r = 0.37), and AKT1(r = 0.32) in the TCGA database. The online tool GEPIA was used to analyze the correlation of EGFR with key modulators of MAPK, NF-?B, and AKT signaling pathways. A correlation coefficient (r) of > 0.3 was considered significant. (C) pEGFR expression in lung adenocarcinoma and squamous cell carcinoma was positively correlated with activation of MAPK1/3, AKT1/2/3, and NF-?B1 in the RPPA database. The online tool cBioportal and Spearman correlation coefficient were used to evaluate the relevance. (D-E) Treatment with a gradient concentration of gefitinib or osimertinib inhibited ERK1/2, P65, and AKT phosphorylation in PC9 (D) or H1975 (E) cells. PC9 or H1975 cells were treated with different concentrations of gefitinib or osimertinib for 24 h and Western blotting was performed to determine the phosphorylation of signaling molecules. (F) EGF stimulation activated ERK1/2, P65, and AKT phosphorylation in H1299 cells treated with 100 ng/mL EGF for 24 h before Western blotting. (G-I) Treatment with ERK (U0126) or AKT (MK2206) inhibitor rather than NF-?B (PDTC) inhibitor decreased ILT4 expression in both PC9 and H1975 cells treated with different concentrations of specific inhibitors for 72 h and Western blotting was performed. The average results from 3 independent experiments are shown. Gef: gefitinib; Osi: osimertinib.