Fig. 3

The influence of the leukemia microenvironment on the neutrophil phenotype. a Scheme of the experiment. Bone marrow Ly6G⁺ cells isolated from C57BL6/J WT donors were cocultured ex vivo alone (CTR) with splenocytes isolated from healthy donors (SPL) or from TCL1 leukemia-bearing mice (TCL1) at a 1:2 ratio for 19 h. Neutrophils and splenocytes were separated by Transwells to avoid direct cell‒cell interactions. b The percentage of bone marrow Ly6G⁺ cells with low (CD62L^lo) and high (CD62L^hi) expression of the CD62L surface marker upon incubation with no other cells (CTR), with splenocytes isolated from the healthy donors (SPL) or from TCL1 leukemia-bearing mice (TCL1) assessed by flow cytometry (upper panels); the expression of the IL4R surface marker on the bone marrow Ly6G⁺ upon incubation with no other cells (CTR), with splenocytes isolated from healthy donors (SPL) or from TCL1 leukemia-bearing mice (TCL1) assessed by flow cytometry (lower left panel, MFI – mean fluorescence intensity); the percentage of propidium iodide (PI)-positive bone marrow Ly6G⁺ cells upon incubation with no other cells (CTR), with splenocytes isolated from healthy donors (SPL) or from TCL1 leukemia-bearing mice (TCL1) assessed by flow cytometry (lower right panel). The graphs present both the value for each individual sample (points) and the mean value (bars), n = 5–8, Mann‒Whitney U test, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. c The bone marrow Ly6G⁺ cells were incubated with splenocytes isolated from healthy donors (SPL) or from TCL1 leukemia-bearing mice (TCL1) with the presence of isotype antibody (ISO) or anti-IL-10 antibody (αIL-10) added to the culture medium in a final concentration of 10 µg/ml for 19 h. The graphs show the expression of the CD62L and IL4R surface markers assessed by flow cytometry. The results are presented as both the value for each individual sample (points) and the mean value (bars), n = 4, Mann‒Whitney U test, ns – not significant, *p ≤ 0.05