Fig 2: CD177 mRNA expression occurs in immature neutrophils. (a) Absolute transcript numbers of CD177 and established neutrophil markers CXCR2, CXCR4, and CXCL8 normalized to GAPDH, in CD177 sorted neutrophils. Values given as mean±SD, including individual values; (*: p ≤ 0.05), n = 9. (b) Gene signatures (mRNA expression) from different clusters of bone marrow neutrophils, following the patterns of maturation state, calculated from single‐cell RNA sequencing data as described in Montaldo et al. 2022 (18). (c) Single‐cell RNA sequencing of bone marrow neutrophils and their precursors, UMAP and clustering were calculated via unbiased highly variable genes and kmers, respectively (d) Corresponding expression of CD177 mRNA in individual cells within identified developmental neutrophil stages, n = 12 924. (e) Quantification of the amount of CD177+ expressing vs. CD177− cells in the identified developmental stages shown in b and c. (f) Flow cytometric analysis of the CD177 expression pattern on neutrophils of two patients with acute lymphoblastic leukemia 7 days before (dpt ‐7) (middle panel) and 28 days after (dpt 28) (right panel) hematopoietic stem cell transplantation. The CD177 expression pattern of the respective donor is shown in the left panel for comparison. The patients showed a clinically confirmed donor chimerism of >99% on dpt 28.

Fig 3: CD177 marks a stable and durable neutrophil subpopulation. (a) Exemplary flow cytometry plots showing differential CD177 expression on neutrophils (CD66b+) in three individuals. (b) Percentage of CD177‐expressing neutrophils out of total neutrophil count in 11 healthy donors measured consecutively over multiple days, up to 160 days apart, values represented as mean±SD, with individual values color‐coded for the day of measurement. (c) Percentage of CD177+ neutrophils out of total neutrophil count in 4 healthy donors, measured over the course of 24 h at different timepoints. (d) Proportion of CD177+ neutrophils found in blood or sputum of 5 healthy individuals 2 h after irritation of the oral cavity using a 10% Tabasco‐saline solution. (e) Proportion of circulating CD177+ neutrophils in 33 ischemic stroke patients at different timepoints post hospital admission, measured via flow cytometry of peripheral blood. Grey bars depict patients with changes ≥18% over time; data presented as mean±SD for individual values at indicated time points.

Fig 4: CD177 delineates an immunoregulatory neutrophil subtype in head‐and‐neck cancer. (a) Proportion of CD177 expressing tumor‐associated neutrophils (TAN) in head‐and‐neck cancer (HNC) tissue separated by tumor progression stage (early T1/2, late T3/4), data given as mean+SD (* = p ≤ 0.05), n(early) = 9; n(late) = 17. (b) Kaplan‐Meier curves, showing long‐term survival of HNC patients with >10% CD177+ TANs found within the tumor stroma (magenta) against patients with <10% CD177+ (blue) separated by early stages (T1/T2; top) and late stages (T3/T4; bottom). Significant differences according to the Log‐rank test were found for early‐stage tumors. (c) Microscopy images of UT‐SCC‐50 cells cocultured with the indicated neutrophil subpopulation for 48 h and quantification of cell‐free area. (d) Microscopy images of UT‐SCC‐50 cells cocultured with T cells and indicated autologous CD177‐FACS‐sorted neutrophils for 48 h and quantification of cell free area.

Fig 5: CD177 protein expression is developmentally fixed and absent from mature CD177− neutrophils. (a) Confocal imaging of neutrophils isolated from peripheral blood of a healthy donor, stained for CD66b (cyan), CD177 (magenta), and DAPI (grey) as nuclear counterstain; scale bar is 30 µm. (b) May‐Grünwald‐staining of FACS‐sorted and cytospun CD177− and CD177+ neutrophils used as input data for multidimensional feature comparison; scale bar is 10 µm. (c) UMAP dimension reduction of accessed shape features derived from >4000 sorted CD177− and CD177+ neutrophils, including representative images of resulting clusters.