Fig. 2: LysM^Cre+ Clec4a2^flox/DTR mice allow selective ablation of vascular CLEC4A2⁺ macrophages.

a Representative images of CD64 (red)-stained thoracic aorta sections from LysM^Cre+ Clec4a2^flox/DTR and Clec4a2^flox/DTR mice 24 h after a saline or diphtheria toxin (DT) injection (n = 4 mice each; Scale bar: 100 µm). b–e Cell numbers of CLEC4A2⁺ macrophages, Ly6C⁺ monocytes, CD11c⁺ macrophages, and neutrophils in whole aortas of LysM^Cre+ Clec4a2^flox/DTR mice at 1, 2, 3, 5, 7, 14, and 70 days post administration of DT. Day 0 represents the saline-treated control group (b–e: n = 3–9 mice in each group, pooled from two to three independent experiments). Ordinary one-way ANOVA with Holm–Sidak’s multiple-comparison test. b **P = 0.0011 (Day 1), ****P < 0.0001 (Day 2), **P = 0.0033 (Day 3). c *P = 0.0167. d and e not significant (n.s.). f The mean fluorescent intensity (MFI) of CLEC4A2 expression in vascular macrophages post depletion. FMO stands for fluorescence minus one. g–l LysM^Cre+ Clec4a2^flox/DTR and Clec4a2^flox/DTR mice received three intraperitoneal injections of saline or DT in a week. Aortic cells were analysed by flow and mass cytometry. g–j Representative flow cytometry plots depict macrophages, Ly6C⁺ monocytes, cDC2 and neutrophils from aortas of saline or DT-treated LysM^Cre+ Clec4a2^flox/DTR mice and DT-treated Clec4a2^flox/DTR mice. Cell numbers of each population in the three groups (n = 4 mice; the data are representative of three independent experiments). Ordinary one-way ANOVA with Holm-Sidak’s multiple comparisons test. g ***P = 0.0006 (saline vs. DT and DT vs. negative DT control). h *P = 0.0166 (saline vs. DT), *P = 0.0402 (DT vs. negative DT control). i and j: n.s. k Overlaid tSNE plots of vascular leucocytes from saline or DT-treated LysM^Cre+ Clec4a2^flox/DTR mice by mass cytometry. Leucocyte populations were identified based on expression of 32 markers. l Cell numbers in 9 aortic leucocyte populations across the 2 treatment groups by mass cytometry (n = 4 mice; the data are representative of two independent experiments). Two-tailed Student’s t-test. CLEC4A2⁺ Mac: **P = 0.0025, Ly6C⁺ Mon: **P = 0.0011. All data are presented as mean ± SEM. Source data are provided as a Source Data file.