Fig 2: High-dose VEGFR2-TKI therapy induce ?dT cells producing IL17A via VEGFR1-PI3K-AKT pathway. (A) Flow cytometry analysis of VEGFR1 expression of ?dT cells (CD45+CD3+TCR?d+) in the tumor. (B) Flow cytometry analysis of VEGFR1 expression of ?dT cells derived from naive spleen treated with different doses of VEGFR2-TKI in vitro. (C) Western blot analysis of the PI3K and AKT pathways in ?dT cells from tumors with different dose of VEGFR2-TKI therapy (?dT cells were sorted from 4 tumors as one donor). (D) Flow cytometry analysis of intracellular IFN-? and IL17A expression of ?dT cells derived from naive spleens treated with different doses of VEGFR2-TKI in vitro. (E) Western blot analysis of PI3K and AKT pathways in ?dT cells derived from naive spleens treated with different doses of VEGFR2-TKI in vitro (?dT cells were sorted from 9 naive spleens as one donor). (F) Representative size of primary tumor after different dose VEGFR2-TKI therapy combined with PI3K agonist or inhibitor in vivo (n=5). (G) Intracellular IL17A expression of ?dT cells from tumors after different dose VEGFR2-TKI therapy combined with PI3K agonist or inhibitor in vivo (n=5). Data are presented as the means ± SD from one representative experiment. Similar results were obtained from three independent experiments, n=4 mice each group, unless indicated otherwise. Statistical analysis was performed by one-way ANOVA. ns, not significant, *p < 0.05, **p < 0.01, and ***p < 0.001. See also Supplementary Figure S3 .

Fig 3: An overview schematic demonstrating that high-dose anti-VEGFR2 treatment modifies the tumor immunosuppression microenvironment via “?dT cell-IL17A-neutrophil” axis.

Fig 4: Combination with immunotherapeutic monoclonal antibody can rescue high-dose VEGFR2-TKI therapy. (A) Representative weight of primary tumor and spleen after high-dose VEGFR2-TKI therapy and(or) IL17A mAb, PD-1 mAb or Ly6G mAb. (B) Frequency of CD4+ T cells and CD8+ T cells in the tumor after high-dose VEGFR2-TKI therapy and(or) IL17A mAb or Ly6G mAb. (C) Frequency of PD-1 expression on CD8+ T cells in the tumor after high-dose VEGFR2-TKI therapy and(or) IL17A mAb or Ly6G mAb. (D) The expression of ROS in neutrophils after high-dose VEGFR2-TKI therapy and (or) IL17A mAb. Data are presented as the means ± SD from one representative experiment. Similar results were obtained from three independent experiments, n=5 mice each group, unless indicated otherwise. Statistical analysis was performed by two-tailed unpaired Student’s t test (D) and one-way ANOVA (A–C). ns, not significant, *p < 0.05, **p < 0.01, and ***p < 0.001. See also Supplementary Figure S7 .

Fig 5: High-dose VEGFR2-TKI therapy facilitates N2-like neutrophils by IL17A-producing ?dT cells. (A) Flow cytometry analysis of the percentage of neutrophils (CD45+CD11b+Ly-6G+) in the tumor after VEGFR2-TKI therapy. (B) Giemsa-stained neutrophils from the tumor after VEGFR2-TKI therapy in vivo. Bar=5 µm. (C) Reactive oxygen species (ROS) production of neutrophils in the tumor after VEGFR2-TKI therapy in vivo. (D, E) Representative images of Giemsa nuclear morphology analysis (D) and Frequency of ROS expression (E) of naive bone marrow (BM)-derived neutrophils, which were co-cultured with ?dT cells sorted from tumors after different dose of VEGFR2-TKI therapy. Bar=20 µm. (F, G) Representative images of Giemsa nuclear morphology analysis (F) and MFI of ROS expression by flow cytometry analysis (G) in BM-derived neutrophils co-cultured with different tumor culture supernatants after VEGFR2-TKI therapy and (or) IL17 mAb or IFN-? mAb added in vitro. Bar=10 µm. Data are presented as the means ± SD from one representative experiment. Similar results were obtained from three independent experiments, n=4 mice each group, unless indicated otherwise. Statistical analysis was performed by two-tailed unpaired Student’s t test (A–E) and one-way ANOVA (F, G). ns, not significant, *p < 0.05, **p < 0.01, and ***p < 0.001. See also Supplementary Figure S4 .