Fig 2: Differentiation and apoptosis of HL-60 cells in the presence of ATRA and DMSO. HL-60 cells were differentiated into a neutrophil-like state by stimulation with ATRA (1 µmol/l) or DMSO (1.25%) for three to seven days in serum-containing medium (RPMI-1640 supplemented with 10% FBS) or serum replacement medium (X-VIVO™ 15 Medium). (A-D) CD11b, (E-H) CD16 and (I-L) CD66b expression was used as a neutrophil surface marker to measure the differentiation state in (A, E, I) ATRA-dHL-60 (FBS), (B, F and J) DMSO-dHL-60 (FBS), (C, G and K) ATRA-dHL-60 (X-VIVO) and (D, H and L) DMSO-dHL-60 (X-VIVO). (M-O) CD11b, CD66b and CD16 expression was compared between the four protocols during three to seven days of differentiation; one-way ANOVA with Dunnett's test for multiple comparisons was used for comparing the expression of surface markers on the fifth differentiation day. (M) CD11b expression in was significantly higher in the DMSO-dHL-60 (FBS) group than in the ATRA-dHL-60 (X-VIVO) group, *P<0.05. (N) The expression of CD66b was also significantly higher in the DMSO-dHL-60 (FBS) group compared with that in the ATRA-dHL-60 (FBS) group, *P<0.05. (O) The DMSO-dHL-60 (FBS) group had the highest expression of CD16 among the groups; **P<0.01. (P) Cell viability was similar from the third day to the fifth day and decreased significantly from the sixth day. Samples were measured using flow cytometry and data were analyzed using FlowJo software. The experiments were performed three times and a representative experiment is presented. ATRA, all-trans retinoic acid; DMSO, dimethyl sulfoxide; dHL-60, differentiated HL-60 cells; d, days.

Fig 3: ILC1-like cells have a unique gene signature distinct from NK cells.(a) CB mononuclear cells (MNCs) were enriched prior to sorting via biotin-labelled antibodies (anti-CD3/CD19/CD14/CD66b) and sorted for ILC1-like cells, CD56dim, and CD56bright NK cells (see Figure 1—figure supplement 1 for sorting strategy). RNA sequencing was done on the Illumina platform. The heat map indicates the top 100 differentially expressed genes between ILC1-like cells and CD56bright NK cells including CD56dim NK cells. (b) A two-dimensional principle component analyses based on the top 2000 differentially transcribed genes of CD56bright NK cells, CD56dim NK cells, and ILC1-like cells is shown. (c) A four-way plot with a cut off at a log2 fold change ±1 (dotted lines) and adjusted p-values of 0.05 showing differently expressed genes of CB CD56bright NK cells compared to CD56dim NK cells and ILC1-like cells. Blue dots represent genes with an adjusted p-value<0.05 with a fold change >1. Green dots represent genes with an adjusted p-value <0.05 with a fold change between >1 (x-axis) and <1 (y-axis). Grey dots represent genes with an adjusted p-value >0.05. Red dots represent genes with an adjusted p-value<0.05 with fold rates < 1 (x-axis) and >1 (y-axis). Selected genes differentially expressed between NK cells subsets and ILC1-like cells are highlighted. (d) CB MNCs (n = 3) gated on ILC1-like cells, CD56bright NK cells, and CD56dim NK cells, respectively were analyzed by flow cytometry for selected NK, T, and ILC markers. Representative histograms for NK cell-specific factors containing CD94, NKG2A, CD56, and EOMES (upper panel), ILC1-specific factors containing CD117, CD127, CD7, CD161, and TBET (middle panel), and T cell-specific factors CD6, CD5, CD28, as well as a Mix of CD3/TCRaß/TCR?d (bottom panel). (e) Representative dot plot of the expression of CD5 and CD161 within CB ILC1-like cells with representative quantification of CD5+ (green bar and box) and CD161+ (yellow bar and box), (n = 5). The height of the bar represents the mean ± SEM. Levels of significance were calculated with a non-parametric t test (Mann-Whitney), ** p-value <0.01. Data represent at least three different donors and experiments. Figure 1—source data 1.R code for RNAseq data analyses used in Figure 1.This file contains the R code to generate the heatmap, PCA, and Four- Way plot displayed in Figure 1.

Fig 4: Neutrophils co-localise with apoptotic tumour cells and tumour-associated macrophages in CRC pseudolumina.A Flow cytometric analysis of dissociated CRC tissues and matched normal mucosae of treatment-naïve CRC patients. For gating strategy, see Fig. S7A. Graphs present median and 95% CI (%CD66b+ cells, n = 15) or mean ± SD (surface marker MFIs, n = 10–12). *P < 0.05, **P < 0.01, ns not significant, as calculated by Wilcoxon signed-rank test (%CD66b+ cells) or two-tailed paired t-tests (surface marker MFIs). B Representative CD66b immunohistochemistry (IHC) of a CRC tissue. Red and black arrows indicate stromal and intraluminal neutrophils, respectively, while dotted lines illustrate CRC pseudolumina. Scale bars, 100 µm. C, D Consecutive IHC sections of representative CRC specimens stained for M30, cleaved caspase-3 and CD66b (C upper panel), CD3, CD68 and CD66b (C lower panel) and IL-8 and CD66b (D). Arrows indicate positive staining in CRC pseudolumina. Scale bars, 100 µm. E Quantification of cleaved caspase-3-expressing, M30-expressing, IL-8-expressing, CD66b-expressing, CD68-expressing and CD3-expressing CRC pseudolumina in IHC sections of 35 CRC patients. Patients with at least 20 IHC-positive pseudolumina/cm2 tissue area were considered positive in the presented Venn diagrams. F CD66b and M30 immunofluorescence staining of a CRC pseudolumen. Arrows indicate close contact between CD66b-positive neutrophils and M30-positive apoptotic tumour cells. Images are representative of three CRC patients. G CD66b and CD68 immunofluorescence staining of CRC pseudolumina. Arrows indicate close contact between CD66b-positive neutrophils and CD68-positive macrophages. Images are representative of three CRC patients.

Fig 5: Overall study design and neutrophil enrichment. (A) Polymorphonuclear neutrophils (PMN) were enriched from the peripheral whole blood of participants. The isolated PMN were assessed for neutrophil enrichment by CD16 surface staining and analyzed by flow cytometry. RNA were extracted from enriched neutrophils and subjected to microarray analysis followed by quantitative RT-PCR validation of top differentially expressed genes. (B, C) Flow cytometry based evaluation of the enriched PMN population based on CD16 and CD66b surface staining of representative ACLF (B) and CLD (C) samples.