FIGURE 5 from Characterizing Neutrophil Subtypes in Cancer Using scRNA Sequencing Demonstrates the Importance of IL1β/CXCR2 Axis in Generation of Metastasis-specific Neutrophils
Authors: Fetit Rana , McLaren Alistair S. , White Mark , Mills Megan L. , Falconer John , Cortes-Lavaud Xabier , Gilroy Kathryn , Lannagan Tamsin R.M. , Ridgway Rachel A. , Nixon Colin , Naiker Varushka , Njunge Renee , Clarke Cassie J. , Whyte Declan , Kirschner Kristina , Jackstadt Rene , Norman Jim , Carlin Leo M. , Campbell Andrew D. , Sansom Owen J. , Steele Colin W. 
Published: February 2024

<p>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 <i>Mrp8-Cre-Cxcr2<sup>+/+</sup></i> (WT) or <i>Mrp8-Cre-Cxcr2</i><sup>fl/fl</sup> (CXCR2fl) mice were harvested 28 days following intrasplenic injection of villinCreER <i>Kras</i><sup>G12D/+</sup><i>Trp53<sup>f</sup></i><sup>l/fl</sup><i>Rosa26</i><sup>N1CD/+</sup> (KPN) cells. These were placed in coculture with T cells from WT mice, and analyzed through flow cytometry 44 hours later. <b>A,</b> 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<sup>+</sup>, CD3<sup>+</sup>/CD4<sup>+</sup>, and CD3<sup>+</sup>/CD8<sup>+</sup> 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>B,</b> Proportion of CD3<sup>+</sup> T cells that proliferated in coculture with WT and CXCR2fl neutrophils from tumor-bearing livers (<b>B</b>–<b>J</b>, four replicates from 2 WT mice and six replicates from 3 CXCR2fl mice). <b>C,</b> Expansion index of CD3<sup>+</sup> T cells in coculture with WT and CXCR2fl neutrophils from tumor-bearing livers. <b>D,</b> Division index of CD3<sup>+</sup> T cells in coculture with WT and CXCR2fl neutrophils from tumor-bearing livers. <b>E,</b> Proportion of CD4<sup>+</sup> T cells that proliferated in coculture with WT and CXCR2fl neutrophils from tumor-bearing livers. <b>F,</b> Expansion index of CD4<sup>+</sup> T cells in coculture with WT and CXCR2fl neutrophils from tumor-bearing livers. <b>G,</b> Division index of CD4<sup>+</sup> T cells in coculture with WT and CXCR2fl neutrophils from tumor-bearing livers. <b>H,</b> Proportion of CD8<sup>+</sup> T cells that proliferated in coculture with WT and CXCR2fl neutrophils from tumor-bearing livers. <b>I,</b> Expansion index of CD8<sup>+</sup> T cells in coculture with WT and CXCR2fl neutrophils from tumor-bearing livers. <b>J,</b> Division index of CD8<sup>+</sup> T cells in coculture with WT and CXCR2fl neutrophils from tumor-bearing livers. <b>K,</b> Proportion of CD3<sup>+</sup> T cells that proliferated in coculture with WT and CXCR2fl neutrophils from blood of tumor-bearing mice (<b>K</b>–<b>S,</b> three replicates from 2 WT mice and four replicates from 2 CXCR2fl mice). <b>L,</b> Expansion index of CD3<sup>+</sup> T cells that proliferated in coculture with WT and CXCR2fl neutrophils from blood of tumor-bearing mice. <b>M,</b> Division index of CD3<sup>+</sup> T cells that proliferated in coculture with WT and CXCR2fl neutrophils from blood of tumor-bearing mice. <b>N,</b> Proportion of CD4<sup>+</sup> T cells that proliferated in coculture with WT and CXCR2fl neutrophils from blood of tumor-bearing mice. <b>O,</b> Expansion index of CD4<sup>+</sup> T cells that proliferated in coculture with WT and CXCR2fl neutrophils from blood of tumor-bearing mice. <b>P,</b> Division index of CD4<sup>+</sup> T cells that proliferated in coculture with WT and CXCR2fl neutrophils from blood of tumor-bearing mice. <b>Q,</b> Proportion of CD8<sup>+</sup> T cells that proliferated in coculture with WT and CXCR2fl neutrophils from blood of tumor-bearing mice. <b>R,</b> Expansion index of CD8<sup>+</sup> T cells that proliferated in coculture with WT and CXCR2fl neutrophils from blood of tumor-bearing mice. <b>S,</b> Division index of CD8<sup>+</sup> T cells that proliferated in coculture with WT and CXCR2fl neutrophils from blood of tumor-bearing mice. *, <i>P</i> < 0.05 on unpaired <i>t</i> test. **, <i>P <</i> 0.01 on unpaired <i>t</i> test.</p>