Fig 2: Representative fluorescence-based multiplex immunohistochemistry images. (A–D) CD4+ T cells (CD3, CD4), nTregs (CD4, FOXP3), CD4+ naïve cells (CD4, CD45RA), exhausted cells (CD3, PD-1), and central memory T cells (CD45RO). Cytokeratin (CK) was used to identify epithelial cells in tumor samples and define the tumor and stroma. All images were obtained using 20× zoom and were scaled digitally.

Fig 3: (A, B) Tumour immune microenvironment (TIME) analysis indicates that TILs including CD3+, CD4+, CD8+ cells and TAMs including CD68+, CD163+ cells are enriched in TIME. Granzyme B also was detected foci positive in TIME (immunohistochemical staining, Scar bar 50 μM, 400×); (C) High expression of PD-L1 as assessed by 30% (immunohistochemical staining, Scar bar 50 μM, 400×); (D), PCR-based microsatellite instability testing using five microsatellite markers (BAT25, BAT26, NR-21, NR-24, NR-27), revealed microsatellite status of stable.

Fig 4: (A) A forest plot visualizing the impact of the immune microenvironment (TME)-associated signatures and GARP on overall survival (OS) in the GSE84437, as evaluated using Cox univariate tests. (B) Kaplan–Meier curve for the high and low expression groups in the GSE84437. (C) A forest plot visualizing the impact of the immune microenvironment (TME)-associated signatures and GARP on overall survival (OS) in our cohort, as evaluated using Cox univariate tests. (D) Kaplan–Meier curve for the high and low expression groups in our cohort. (E) Kaplan-Meier plot visualizing survival associations of GARP+CD4+ T cell. The optimal cutoff point was obtained from X-tile 3.6.1 software. (F) Representative fluorescence-based multiplex immunohistochemistry images of the rate of positivity for GARP+CD4+ T cell in tissue microarray sections. (G) A plot shows the rate of positivity for CD3+ T cell, CD4+ T cell, and GARP+ CD4+ T cell. White arrows in the picture that point to the GARP+CD4+ cells.

Fig 5: Relationship between PCDHGA10 protein expression and tumor-infiltrating immune cells and immune checkpoints. (A-C) Multispectral composite of CD3, CD4, CD8, CD11b, CD66b, CD68, CK and DAPI, magnification ×200. (D) Four-color multispectral composite of LAG3, PD1, CK and DAPI, magnification ×200. Green: CK, blue: DAPI. (E) Correlation analysis of PCDHGA10 protein expression and tumor-infiltrating immune cells. (F) Correlation analysis of PCDHGA10 protein expression and immune checkpoints. DAPI: 4, 6-diamino-2-phenyl indole; CK, cytokeratin. * p < 0.05.