Fig 2: The expression of 4 CRIGs and FDX1 in GC. (A) The IHC scores of NRP1, CXCR4, LGR6, CTLA4, and FDX1 in GC tissues and adjacent normal tissues. (B) The representative IHC images of NRP1, CXCR4, LGR6, CTLA4, and FDX1 in GC tissues and adjacent normal tissues (scale bar: 40 μm). (C) The representative IHC images of NRP1, CXCR4, LGR6, CTLA4, and FDX1 in the same GC tissues (scale bar: 100 μm). (D) The correlation between NRP1, CXCR4, LGR6, CTLA4 and FDX1 in the GC tissues. Data represent the mean ± SD. Student’s t-test was used to determine statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 3: Expression of CRIGs ANOS1, CTLA4, ITGAV, CXCR4, NRP1, FABP3 and LGR6 in GC single cells. (A) Cell t-SNE distribution among different samples. (B) T-SNE distribution of 25 independent cell clusters. (C) T-SNE distribution of cell types after SingleR annotation. (D–J) Violin plots of ANOS1, CTLA4, ITGAV, CXCR4, NRP1, FABP3 and LGR6 abundance in GC at Single Cell Level. (K–Q) The t-SNE distribution of ANOS1, CTLA4, ITGAV, CXCR4, NRP1, FABP3 and LGR6 abundance in GC at Single Cell Level.

Fig 4: Verification of the levels of immunocytes infiltration and inhibitory immune checkpoints expression in collected CSCC and ADC samples. (A) Representative images of CD8A (T cells) and CD20 (B cells) in collected CSCC patients. (B) Representative images of CD8A (T cells) and CD20 (B cells) in collected ADC patients. (C, D) Statical charts displayed that CD8A (T cells) and CD20 (B cells) were more abundant in CSCC compared to ADC patients in our collected samples. (E, F) Representative staining of T cells (CD3/CD4/CD8) and inhibitory immune checkpoints (PD1/PD-L1/CTLA-4) in CSCC and ADC patients, respectively. (G, H) Statical charts displayed that T cells (CD3/CD4) and some immune inhibitory checkpoints (PD1/PD-L1/CTLA4) were more abundant in CSCC compared to ADC patients in our collected samples. * P <0.05; ** < P <0.01, *** P <0.001; ns: not significant.

Fig 5: Chemokines and immune‐negative regulators in T cells and macrophages associated with liver injury. (A) Left: Heatmap of gene expression of immune regulation, chemokines, complements, cytokines, and interferons (rows) for all 82 patients (columns) in the four phases of CHB. Right: 95% credible intervals of ALT and AST slopes versus gene expression in Left. Colored bubbles indicate positive (red) or negative slopes (blue) (B) Left: Radar charts of the PSEA coefficient value (βP) for genes of different reference cell types in chronic hepatitis patients. Right: Changes in gene coefficients of different reference cell types comparing chronic hepatitis to HBeAg‐positive chronic infection. (C, D) Partial residual plot with fitted posterior lines and violin plot of slope distributions from the fitted lines in chronic hepatitis (red) and HBeAg‐positive chronic infection (blue) for gene expression contribution by T cells and iMac cells. Using the established PSEA model, we analyzed upregulated immune checkpoint genes and chemokine genes to discern their predominant expression cell types and expression variations across different clinical stages. (E) Our findings indicated that during the immune‐active phase, immune checkpoint genes (e.g., CTLA4, EOMES, TIGIT, LGALS3) were predominantly expressed by infiltrating T lymphocytes, implying that T cell exhaustion drives inflammation. Moreover, chemokine genes strongly correlated with ALT levels, such as CCL20 and CXCL8, were primarily expressed by macrophages, suggesting that macrophages secrete chemokines to recruit immune‐exhausted T lymphocytes into liver tissues. (F) Workflow for clinical histopathology validation: Liver slides from 12 CHB patients in the hepatitis stage and five CHB patients in the infection phase were analyzed. Multiplex immunostaining was performed on eight sequential slides using the parameters outlined in the blue box. These images were created using Biorender (https://biorender.com/). ALT, alanine aminotransferase; CHB, chronic hepatitis B; HBeAg, hepatitis B e‐antigen; PSEA, population‐specific expression analysis.