FIGURE 6.

Tumor‐infiltrating and tumor‐conditioned neutrophils induce protumorigenic IL‐17A‐producing Th subset polarization through a B7‐H2‐ERK pathway. (A) CFSE‐labeled peripheral CD4⁺ T cells of GC patients or donors were co‐cultured for 4 days with autologous neutrophils from tumor tissues or autologous TTCS‐conditioned neutrophils with or without transwells. Representative data and statistical analysis of proliferated IL‐17A‐producing CD4⁺ T cells and IL‐17A production were shown (n = 3). (B) CFSE‐labeled peripheral CD4⁺ T cells of GC patients or donors pretreated with DMSO or U0126 were co‐cultured for 4 days with autologous neutrophils from tumor tissues or autologous TTCS‐conditioned neutrophils. Representative data and statistical analysis of proliferated IL‐17A‐producing CD4⁺ T cells and IL‐17A production were shown (n = 3). (C) Autologous neutrophils isolated from tumor or non‐tumor tissues, and TTCS‐ or NTCS‐conditioned neutrophils were pretreated with or without human B7‐H2 neutralizing antibody or control IgG (20 µg/ml) for 2 h. Then peripheral CD4⁺ T cells were co‐cultured with these neutrophils for 4 days. The ERK1/2 and p‐ERK1/2 proteins in CD4⁺ T cells were analyzed by western blot. (D) Representative analysis of IL‐17A‐expressing (brown) CD4⁺ cells (red) in tumor tissues of GC patients by immunohistochemical staining. Scale bars: 20 microns. *P < 0.05; **P < 0.01; ^n.s. P > 0.05 for groups connected by horizontal lines