Fig 2: Galectin-9+ epithelial cells (Epi) correlates with poor neoadjuvant anti-PD-1 response and limited tumor killing capacity of CD8+ TRMs. (A) Paraffin sections from non-small cell lung cancer (NSCLC) patients (scale bars represent 50 µm) were stained with anti-human Epcam (green), anti-human galectin-9 (red), and DAPI (blue) for immunofluorescent (IF) staining. (B) Expression plots depict average Z-transformed normalized expression of common immune checkpoint ligands genes in each subgroup of Epi in NSCLC after anti-PD-1 treatment. n=6. (C) Volcano plots presenting the differentially expressed genes between Part-PR groups and Non-PR groups. P<0.05, |LogFC|>1. (D) Expression plots depict average Z-transformed normalized expression of representative genes in each subgroup of Epi in NSCLC after anti-PD-1 treatment. n=6. (E) Heatmaps showing relative genes expression of galectin-9+ Epcam, cDC2, and CD8+ TRM. n=19. Standardized by Z score. (F) The box plot shows the percentage of CD39+ CD38+ TRM, CD39− CD38− TRM, CD1c+ cDC2 and pathological residual tumor cells in galecin-9 low or galecin-9 high tumor. n=19, *p<0.05; **p<0.01; ****p<0.0001. (G) A549 were cocultured with CD8+ TRMs and CD1c+ DC2s (1:2:2) for 24 hours with or without anti-TIM-3 (10 µg/mL) treatment in vitro. The line graph shows the percentage of CD39+ in CD8+ TRMs. T (Untreated), n=6; **p<0.01. (H) A549 were cocultured with CD8+ TRMs and CD1c+ DC2s (1:2:2) for 24 hours with or without anti-TIM-3 (10 µg/mL) treatment in vitro. The line graph shows the percentage of 7-AAD+ in A549. T (Untreated), n=9; *p<0.05, ***p<0.001, ****p<0.0001. (I) A549 were cocultured with CD8+ TRMs and CD1c+ DC2s (1:2:2) for 24 hours with or without anti-PD-1 (10 µg/mL) or anti-TIM-3 (10 µg/mL) treatment in vitro. The line graph shows the percentage of 7-AAD+ in A549. T (Untreated), n=9; *p<0.05; **p<0.01, ***p<0.001.

Fig 3: Tumor cells inhibit the maturity of CD1c+ DC2 via galectin-9. (A) Heatmap of mean log-normalized expression of different proteins in dendritic cells (DCs). n=35. (B) The box plot shows the mean log-normalized expression of PD-L1, PD-L2, CTLA-4, and TIM-3 in DCs. n=35. NS p≥0.05; *p<0.05; ***p<0.001; ****p<0.0001. (C) The box plot shows the mean log-normalized expression of TIM-3 in CD141+ DC1s and CD1c+ DC2s. T (Anti-PD-1), n=23; T (Untreated), n=6; N (Untreated), n=6. NS p≥0.05; *p<0.05; ***p<0.001. (D) The box plot shows the mean log-normalized expression of TIM-3 in CD141+ DC1s and CD1c+ DC2s. Part-PR, n=10; Non-PR, n=13; T (Untreated), n=6. NS p≥0.05; *p<0.05. (E) Expression plots depict average Z-transformed normalized expression of common immune checkpoint genes in each subgroup of DCs in NSCLC after anti-PD-1 treatment. n=6. (F) The box plot shows the percentage of HLA-A/B/C+ (left) and CD86+ (right) in CD1c+ DC2s. T (Untreated), n=4; N (Untreated), n=4. *p<0.05. (G) The box plot shows the percentage of HLA-A/B/C+ in CD86− or CD86+ CD1c+ DC2s. T (Untreated), n=4; N (Untreated), n=4. **p<0.01. (H) Normalized expression of HLA-A and CD86 genes between all samples in CD1C+ CLEC10A+ DC2s. Violin plot indicates the range of normalized expression; each black dot refers to a captured cell; width indicates the number of cells at the indicated expression level. (I) CD1c+ DC2s and CD8+ TRMs are cocultured with galectin-9+ epithelial cells (Epi) with control, isotype (10 µg/mL) or anti-TIM-3 (10 µg/mL) treatment for 24 hours after anti-PD-1 (10 µg/mL) treatment in vitro (Epi:DC:T=5:1:1). Cells sorting from T (Untreated) (n=6) were analyzed by flow cytometry. The line graph shows the percentage of CD86 (left) and HLA-A/B/C (right) in CD1c+ DC2s. *p<0.05; **p<0.01; ***p<0.001.

Fig 4: Association between intratumoural STING signature and ICs expression. (A–H) Representative IHC image and quantification of the expression level of STING and ICs including PD-L1/PD-1, GAL-9/TIM-3 and GAL-3/LAG-3 in lung cancer patients. (I) Experimental timeline and indicated treatment groups (three intravenous injections, diABZI: 0.75 mg per kg (body weight), n = 6 mice). (J) Statistical analysis tumor volume on day 13 of mice with CT-26 tumors receiving the indicated treatments. (K–O) Representative flow cytometric densities of total immune cells (CD45+), T lymphocytes (CD3+), DCs (CD11c+MHC-II+), Macrophages (CD11b+F4/80+) and NK cells (CD3−CD49b+) in the tumors receiving diABZI or PBS at three days after the last injections (n = 6 mice). (P–R) Representative flow cytometric quantification of the expression level of PD-1, TIM-3 and LAG-3 in tumor-infiltrating CD3+ T cells of CT-26-bearing mice receiving diABZI or PBS at three days after the last injections (n = 6 mice). All data are presented as the mean ± S.D. The p values were determined by two-tailed Student's t-test for (B–H) and (J–R).