Figure 8.

ILT4 blockade enhanced the efficacy of PD-L1 inhibitor in EGFR wild-type NSCLC in vivo. (A-B) ILT4 or PD-L1 blockade markedly slowed H1299 growth in humanized NSG mice compared with the control group, and combination treatment generated the lowest tumor growth rate. EGFR-overexpressing H1299 cells (3×10⁶ cells/mouse) were first transfected with ILT4 knockdown or control lentiviruses, and then inoculated into the right flanks of 6-8-week female NSG mice. On day 7 post tumor inoculation, 2×10⁷ PBMCs from healthy volunteers were intravenously injected into each mouse. 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 for each mouse. Scale bar: 2 cm. (C) Tumors in ILT4 or PD-L1 blockade group showed significantly smaller tumor weight than the control group, while combination therapy showed synergy in tumor inhibition. The histogram shows the mean ± SD of tumor weights from each group at the experiment endpoint (n = 8 mice/group). (D-E) Spleen, blood and tumor tissues in ILT4 or PD-L1 blockade group showed significantly higher CD3⁺ T cell frequency than the control group, while the combination therapy group showed the highest CD3⁺ T cell infiltrates. (D) The CD3⁺ T cell frequency in mouse spleen and blood was detected by flow cytometry and (E) tumors were detected by IHC respectively. (F-G) ILT4 or PD-L1 blockade increased IFN-γ expression in T cells of spleen, blood and tumors relative to the control group, and combination therapy yielded the highest IFN-γ in T cells. The IFN-γ expression in mouse spleen, blood and tumors was detected by flow cytometry and IHC, respectively. (H-I) The distribution of CD4⁺ and CD8⁺ T cell subsets in spleen (H) and blood (I) was not altered by single or combination blockade of ILT4 and PD-L1 as determined by flow cytometry (J-K) ILT4 or PD-L1 blockade increased IFN-γ expression in CD4⁺ and CD8⁺ T cells of spleen (J) and blood (K) with the highest IFN-γ level in combination therapy group by flow cytometry analysis. *, p < 0.05; **, p < 0.01; ***, p < 0.001. LV-shILT4: lentivirus carrying ILT4 shRNA; LV-shNC: lentivirus carrying control shRNA; ns: no significance.