Fig. 5: αTIGIT-IL2 activated CD4⁺ T cells enhance the antigen-presentation function in neutrophils and facilitate cross-talk between neutrophils and CD8⁺ T cells.

a, b Inferred cell-cell communication networks from T_reg (a) or CD4⁺ T_conv (b) cells as senders of ligands to neutrophils as receivers expressing cognate receptors in αTIGIT-IL2-treated tumors. c Gene Ontology enrichment analysis of scRNA-seq dataset in neutrophils for αTIGIT-IL2 vs. Control treatment. P values were computed with a one-sided hypergeometric test and adjusted for multiple comparisons using the Benjamini–Hochberg false discovery rate (FDR). d 6–8 weeks old female C57BL/6 J mice were inoculated with 1.4 × 10⁶ MC38-OVA-GFP cells. The tumor-bearing mice were administered the specified treatment intraperitoneally according to their respective groups (n = 5 individual animals/group) on day 8. Tumor volume was measured as indicated. e, f, 6–-8 weeks old female C57BL/6 J mice were inoculated with 1.4 × 10⁶ MC38-OVA-GFP cells. The tumor-bearing mice were administered the specified treatment intraperitoneally according to their respective groups (n = 5 individual animals/group) on day 9. Mice were sacrificed on day 16, and tumors were analyzed by flow cytometry. e Representative frequency pseudocolor of H-2Kb SIINFEKL⁺ neutrophils. f Frequency of H-2Kb SIINFEKL⁺ neutrophils in total intratumoral CD45⁺ cells. g, h 6–8 weeks old female C57BL/6 J mice were inoculated with 5 × 10⁵ MC38 cells. The tumor-bearing mice were administered the specified treatment intraperitoneally according to their respective groups (n = 3 individual animals/group) on day 7. Mice were sacrificed on day 11, and tumors were analyzed by multiplex immunohistochemistry. g Multiplex immunohistochemical analysis of tumor tissue from MC38 tumor-bearing mice treated with Control or αTIGIT-IL2. h Spatial distance from Ly6G⁺ neutrophils to CD8⁺ T cells. Data represent mean ± SEM from n  ≥  3 mice per group, and 5 fields were chosen per mouse. i 6–8 weeks old female C57BL/6 J mice were inoculated with 5 × 10⁵ MC38 cells. Tumor-bearing mice were intraperitoneally treated with PBS (n = 3 individual animals) or 10 μg αTIGIT-IL2 (n = 5 individual animals) on day 7. Mice were harvested 4 days after treatment. Tumor-infiltrating neutrophils were isolated and co-cultured with OT-1 T cells at a 1:1 ratio. The proliferation of OT-1 T cells was detected by flow cytometry. j 6–8 weeks old female C57BL/6 J mice were inoculated with 1.4 × 10⁶ MC38-OVA-GFP cells. The tumor-bearing mice were administered the specified treatment intraperitoneally according to their respective groups (n = 5 individual animals/group) on day 9. Mice were harvested on day 16, and tumors were processed and analyzed by flow cytometry. The proportion of antigen-specific CD8⁺ T cells and Ki67⁺ antigen-specific CD8⁺ T cells in tumor tissues was detected by flow cytometry. k–n 6–8 weeks old female C57BL/6 J mice were inoculated with 5 × 10⁵ MC38 cells. The tumor-bearing mice were administered the specified treatment intraperitoneally according to their respective groups (n = 5 individual animals/group) on day 7. Mice were sacrificed 4 days after treatment, and tumors were processed and analyzed by flow cytometry. l Representative frequency pseudocolor of neutrophils in CD45⁺ immune cells. m Quantification of neutrophils in MC38 tumor tissue. n Flow cytometry analysis of functional marker gene expression in intratumoral neutrophils. Data represent mean ± SEM, and each point represents an individual animal in the statistical graphics. The P value was determined by 2-tailed unpaired t-test (f, h, i, j) or one-way ANOVA followed by Tukey’s multiple-comparison test (m, n). Source data are provided as a Source Data file.