Fig. 6: CD39⁺CD9⁺ IMs directly inhibit NETosis of neutrophils derived from NDA mice in CD39 and CD9-dependent manner.

a Representative confocal microscopy images of immunostaining for citH3 (red), MPO (green), and DAPI (blue) in co-cultured slides. Merged citH3⁺MPO⁺ NETs are shown in yellow. Ly6G⁺ neutrophils were co-cultured with CD39⁺CD9⁺ IMs or CD39^-CD9^- monocytes on chamber slides. b Colocalization area (µm²) of citH3⁺MPO⁺ NETs in each FOV quantified using Zen software (Neutrophils (n = 16), Neutrophils+CD39⁺CD9⁺ IMs (n = 16), Neutrophils+CD39^-CD9^- monocytes (n = 9), Neutrophils+CD39⁺CD9⁺ IMs + POM1 (n = 16), Neutrophils + CD39⁺CD9⁺ IMs + 100 μM ATP (n = 6), Neutrophils+CD39⁺CD9⁺ IMs + 1 μM ATP (n = 16), Neutrophils+CD39⁺CD9⁺ IMs + 0.1 μM ATP (n = 9), Neutrophils+CD39⁺CD9⁺ IMs + 0.01 μM ATP (n = 9), and Neutrophils + CD39⁺CD9⁺ IMs + αCD9 (n = 16). c Representative photomultiplier tube (PMT) images showing immunostaining for MHCII (red) taken after 2 hr in the co-culture system. d Number of cell aggregates per FOV. Aggregates were defined as cells that adhered to at least five other cells (Netrophils (n = 22), Neutrophils + CD39⁺CD9⁺ IMs (n = 22), Neutrophils + CD39⁺CD9⁺ IMs + αCD9 (n = 22), Neutrophils+CD39⁺CD9⁺ IMs + POM1 (n = 17), Neutrophils+CD39⁺CD9⁺ IMs + 100 μM ATP (n = 17), and Neutrophils + CD39^-CD9^- monocytes (n = 17)). e Representative confocal microscopy images of immunostaining for citH3 (red), MPO (green), and MHCII (blue) in co-cultured slides. f Colocalization area (µm²) of citH3⁺MPO⁺ NETs in each FOV. Scale bar = 20 μm. (Netrophils (n = 22), Neutrophils + CD39⁺CD9⁺ IMs (n = 22), Neutrophils + CD39⁺CD9⁺ IMs + αCD9 (n = 22), Neutrophils + CD39⁺CD9⁺ IMs + POM1 (n = 7), Neutrophils+CD39⁺CD9⁺ IMs + 100 μM ATP (n = 18), and Neutrophils + CD39^-CD9^- monocytes (n = 18)). Data are shown as mean ± s.e.m. Significance was determined by one-way ANOVA with Tukey’s post hoc correction (b, d, f).