Figure 1.

SiglecF^high CD11b⁺ Ly-6G⁺ Cells Resemble Neutrophils

(A) Cells obtained from lung tissue of KP1.9 tumor-bearing mice (day 29 after intravenous tumor cell injection) were stained by flow cytometry to identify eosinophils (CD11b⁺ Ly-6G⁻ SiglecF⁺), SiglecF^low (CD11b⁺ Ly-6G⁺ SiglecF^low), and SiglecF^high neutrophils (CD11b⁺ Ly-6G⁺ SiglecF^high). Representative dot plots are shown (pre-gated on live cells).

(B) Suspensions of CD45⁺ cells for single-cell RNA sequencing were prepared from murine KP1.9 lung tumors (T) (n = 2) and lung tissue of healthy mice (H) (n = 2; Engblom et al., 2017; Zilionis et al., 2019). Major cell types (neutrophils highlighted in red) were identified by a Bayesian cell classifier as reported in Zilionis et al. (2019), and neutrophils were defined as tumor (T)-Siglecf^high, tumor (T)-Siglecf^low, and healthy (H)-Siglecf^low based on the expression of genes correlated to SiglecF (Engblom et al., 2017). The heatmap shows a comparison of the 3 neutrophil subsets and of alveolar macrophages (Mø₄), monocytes, dendritic cells (DCs), and basophils (rows) to immune profiles defined by the Immgen consortium (columns).

(C) Representative histogram (left) and quantification of geometric mean fluorescence intensity (gMFI) followed by fluorescence-minus one (FMO) signal subtraction (right) of CCR3 expression measured by flow cytometry in eosinophils and SiglecF^low and SiglecF^high neutrophils (day 29 after intravenous tumor cell injection; n = 4 mice/group).

(D) Representative histogram (left) and quantification of delta gMFI (right) of SiglecE expression measured by flow cytometry in eosinophils and SiglecF^low and SiglecF^high neutrophils (day 29 after intravenous tumor cell injection; n = 4 mice/group).

Data are represented as mean ± SEM. For comparisons between two groups, Student’s two-tailed t test was used. ^∗∗∗∗p < 0.0001. See also Figure S1.