FIGURE 5 from Characterizing Neutrophil Subtypes in Cancer Using scRNA Sequencing Demonstrates the Importance of IL1β/CXCR2 Axis in Generation of Metastasis-specific Neutrophils

Loss of neutrophil-specific CXCR2 attenuates suppression of T-cell proliferation in neutrophils from the metastatic niche in mice bearing tumors. Neutrophils from the liver and blood of littermate mice either Mrp8-Cre-Cxcr2^+/+ (WT) or Mrp8-Cre-Cxcr2^fl/fl (CXCR2fl) mice were harvested 28 days following intrasplenic injection of villinCreER Kras^G12D/+Trp53^fl/flRosa26^N1CD/+ (KPN) cells. These were placed in coculture with T cells from WT mice, and analyzed through flow cytometry 44 hours later. A, Flow cytometry gating of T cells and neutrophils from coculture. Cells were gated on FSC-A/SSC-A (not shown), FCS-A/FSC-H for singlets, Live/Dead, CD3, and CD4/CD8 to define CD3⁺, CD3⁺/CD4⁺, and CD3⁺/CD8⁺ populations for analysis. Cell trace yellow allows for tracking of T-cell proliferation. The signal for each T-cell division becomes subsequently dimmer, and allows for calculation of the number of T-cell generations. Gating obtained from CXCR2fl mouse (blue) and unstimulated T cell control (red). B, Proportion of CD3⁺ T cells that proliferated in coculture with WT and CXCR2fl neutrophils from tumor-bearing livers (B–J, four replicates from 2 WT mice and six replicates from 3 CXCR2fl mice). C, Expansion index of CD3⁺ T cells in coculture with WT and CXCR2fl neutrophils from tumor-bearing livers. D, Division index of CD3⁺ T cells in coculture with WT and CXCR2fl neutrophils from tumor-bearing livers. E, Proportion of CD4⁺ T cells that proliferated in coculture with WT and CXCR2fl neutrophils from tumor-bearing livers. F, Expansion index of CD4⁺ T cells in coculture with WT and CXCR2fl neutrophils from tumor-bearing livers. G, Division index of CD4⁺ T cells in coculture with WT and CXCR2fl neutrophils from tumor-bearing livers. H, Proportion of CD8⁺ T cells that proliferated in coculture with WT and CXCR2fl neutrophils from tumor-bearing livers. I, Expansion index of CD8⁺ T cells in coculture with WT and CXCR2fl neutrophils from tumor-bearing livers. J, Division index of CD8⁺ T cells in coculture with WT and CXCR2fl neutrophils from tumor-bearing livers. K, Proportion of CD3⁺ T cells that proliferated in coculture with WT and CXCR2fl neutrophils from blood of tumor-bearing mice (K–S, three replicates from 2 WT mice and four replicates from 2 CXCR2fl mice). L, Expansion index of CD3⁺ T cells that proliferated in coculture with WT and CXCR2fl neutrophils from blood of tumor-bearing mice. M, Division index of CD3⁺ T cells that proliferated in coculture with WT and CXCR2fl neutrophils from blood of tumor-bearing mice. N, Proportion of CD4⁺ T cells that proliferated in coculture with WT and CXCR2fl neutrophils from blood of tumor-bearing mice. O, Expansion index of CD4⁺ T cells that proliferated in coculture with WT and CXCR2fl neutrophils from blood of tumor-bearing mice. P, Division index of CD4⁺ T cells that proliferated in coculture with WT and CXCR2fl neutrophils from blood of tumor-bearing mice. Q, Proportion of CD8⁺ T cells that proliferated in coculture with WT and CXCR2fl neutrophils from blood of tumor-bearing mice. R, Expansion index of CD8⁺ T cells that proliferated in coculture with WT and CXCR2fl neutrophils from blood of tumor-bearing mice. S, Division index of CD8⁺ T cells that proliferated in coculture with WT and CXCR2fl neutrophils from blood of tumor-bearing mice. *, P < 0.05 on unpaired t test. **, P < 0.01 on unpaired t test.