Af1619

GR1⁺ and CD8⁺ T cells were isolated from the spleens of 4T1 tumor bearing mice using EasySep Mouse PE, and MojoSort™ Mouse CD8⁺ T Cell Isolation Kit (BioLegend, Cat# 480008), respectively. Ly6E^hi neutrophils were generated in-vitro as described above. The GR1⁺ cells and Ly6E^hi neutrophils (1x10⁶/ml) were cultured in serum-free medium for 24 hours to generate conditioned medium (CM). CD8⁺ T cells (0.5x10⁶/ml) were cultured with CM of GR1⁺ or Ly6E^hi neutrophils cells for 24 hours, after which the cells were washed and analyzed by flow cytometry for the evaluation of activated CD8⁺ T cells (CD8⁺/CD25⁺), effector CD8⁺ T cells (CD8⁺/CD44⁺/CD62L^-), Granzyme B⁺ and Ki67⁺ cells. In some experiments, CD8⁺ T cells (0.5x10⁶/ml) were cultured with conditioned medium of Ly6E^hi neutrophils together with neutralizing antibodies anti-IL12p40 (1μg/ml) or anti-IL23p19 (2μg/ml) (R&D systems, Cat# MAB4991 and Cat# AF1619, respectively). After 24 hours, the cells were collected, washed and analyzed by flow cytometry for the evaluation of activated CD8⁺ T cells (CD8⁺/CD25⁺). The experiments were performed using five biological repeats.