Fig 2: Enrichment of ILT4 in CRC cells was correlated with decreased CD8+ T cell but increased Treg cell infiltration. (A and B) ILT4 expression in CRC cells was not associated with CD4+ T cell density by IHC analysis. (A) Density of CD4+ T cell infiltrates, based on ILT4 expression in the overall tumor. (B) CD4+ T cell density in the tumor center and invasive margin of ILT4-low and ILT4-high patients. (C–E) High ILT4 expression in CRC cells was correlated with decreased CD8+ T cell but increased FOXP3+ Treg cell density by IHC analysis. (C) Representative images for ILT4 expression and CD8+ T/Treg cell infiltration. (D and E) Summarized results of CD8+ T cell and Treg cell frequencies from 145 patients based on ILT4 level. (F) ILT4 expression was positively associated with FOXP3 level (r = 0.78) in TCGA database. The online tool GEPIA was used to analyze the correlation between ILT4 and FOXP3 levels. (G) ILT4 level in CRC tissues was positively correlated with the fraction of tumor-infiltrating Treg cells (r = 0.63). TCGA database, containing 458 colon adenocarcinoma patients, was used for this analysis. (H) High ILT4 expression was correlated with declined CD8+ T cell densities in the tumor centers and invasive margins of CRC tissues by IHC assay. The cutoff score for high and low ILT4 levels was the same as in Figure 1C. (I) ILT4 expression was positively correlated with Treg cell density in the tumor center but not the invasive margin of CRC tissues. The cutoff scores for high and low ILT4 levels were the same as in Figure 1C. *p<0.05; ***p<0.001.

Fig 3: ILT4 in CRC cells was inversely correlated with IFN-? production in TILs. (A and B) IHC analysis showed that CRC tissues with high ILT4 expression displayed lower IFN-? levels in TILs, compared with the CRC tissues with low ILT4 expression. (A) Representative imaging of ILT4 and IFN-? levels in CRC tissues and (B) the statistical results in 145 patients. Scale bar: 20µm. (C) High IFN-? levels in TILs were correlated with decreased lymph node involvement and earlier tumor stage, compared with the IFN-?-low group. The medium proportion of IFN-?+ cells in TILs was used to discriminate IFN-?-low and -high groups. (D) High IFN-? expression in TILs was correlated with superior patient OS. The cutoff value for high and low IFN-? was as described in (C). (E and F) IHC analysis showed that high ILT4 expression in CRC cells predicted declined IFN-?+ TIL frequency in the tumor center (E) but not in the invasive margin (F). The cutoff score for high and low ILT4 groups was the same as in Figure 1C. (G) The frequency of IFN-?+ TILs was positively correlated with CD8+ T but not CD4+ T cell density by IHC analysis. (H) IFN-? expression was positively correlated with CD8 levels in CRC tissues by analyzing TCGA database. The online tool GEPIA was used to analyze the correlation between IFN-? and CD8 levels. (I) Patients with high ILT4 and low IFN-? levels showed more advanced lymph node involvement, perineural invasion, and tumor stage than those in the ILT4-low IFN-?-high group. (J) High ILT4 in combination with low IFN-? levels predicted poor patient OS relative to the ILT4-low IFN-?-high counterpart. *p<0.05; **p<0.01; ***p<0.001.

Fig 4: Increased ILT4 expression in CRC cells predicted disease progression. (A and B). ILT4 expression was increased in the tumor cells of CRC tissues relative to epithelial cells in the adjacent normal tissues. (A) Representative images of ILT4 expression in CRC tissues. Brown granules were defined as positive staining. ILT4 was mainly located in the cell membrane and cytoplasm. (B) The summarized results from 145 patients. Scale bar: 50 µm. (C) Patients with high ILT4 expression in CRC cells showed increased frequencies of lymph node involvement, perineural invasion, and advanced tumor stage than those with low ILT4 levels. The cutoff scores for high and low ILT4 expression were = 6 and < 6, respectively. (D) Compared with patients in the ILT4-low group, those in the ILT4-high group showed markedly poor OS. (E) Compared with patients in the ILT4-low group, patients in the ILT4-high group displayed markedly poor PFS. A total of 89 patients from the GSE33113 dataset were included for PFS analysis. The cut-off value for high and low ILT4 expression was the median *p<0.05; **p<0.01; ***p<0.001.

Fig 5: ILT4-directed suppressive T cell subsets predicted poor clinical outcomes. (A) Patients with high CD8+ T cell infiltration displayed good OS relative to those with low CD8+ T cell infiltration. The medium density of tumor-infiltrating CD8+ T cells was defined as the cutoff value for CD8-high and -low groups. (B) High Treg cell infiltration in CRC tissues was associated with advanced lymph node metastasis and tumor stages. The medium proportion of Treg cells in TILs was defined as the cutoff value for Treg-high and -low groups. (C) Patients with high frequency of Treg infiltration showed poor OS compared with those in the Treg-low group. The cutoff value for Treg-high and -low was the same as in (B and D). High ILT4 level in combination with low CD8+ T cell infiltration predicted more advanced lymph node involvement, perineural invasion, and tumor stage than those observed in the ILT4-low CD8-high group. (E) Patients in the ILT4-high CD8-low group showed significantly shorter OS than those in the ILT4-low CD8-high group. (F) Compared with the patients in the ILT4 and Treg double low group, those in the double high group were prone to lymph node metastasis, perineural invasion, and advanced tumor stage. (G) High ILT4 expression in combination with high Treg infiltration indicated poor patient OS, compared with the double low group. *p<0.05; **p<0.01; ***p<0.001.