Figure 2. Cx3cr1 deficiency does not affect resorption and immune function in Cx3cr1⁺ i-OCLs.

(A) Schematic representation of the differentiation of i-OCLs from BM-derived DCs of WT Cx3cr1^GFP/+ and Cx3cr-deficient Cx3cr1^GFP/GFP mice. (B) RNA-seq analysis on sorted GFP⁺ i-OCLs (representing Cx3cr1⁺ i-OCLs) differentiated from BM-derived DCs of WT Cx3cr1^GFP/+ and Cx3cr-1deficient Cx3cr1^GFP/GFP mice (gated as in Figure 2—figure supplement 1). Volcano-plot indicates –Log₁₀ adjusted p value versus log₂ fold-change comparing gene expression in i-OCL subsets. (C) Matrix dissolution activity of sorted GFP⁺ i-OCLs from WT Cx3cr1^GFP/+ and Cx3cr1-deficient Cx3cr1^GFP/GFP mice seeded at the same cell density on a calcified matrix was evidence by red alizarin staining of the mineralized matrix. Unstained areas correspond to the resorbed areas. Left panel: representative images of resorbed area. Scale bar = 100 µm. Right panel: quantification of resorbed areas presented as mean ± SD percentage of three independent biological replicates each in triplicates. (D) FACS analysis of fluorescent-OVA uptake among GFP⁺ i-OCLs from Cx3cr1^GFP/+ and Cx3cr1^GFP/GFP mice. Left panel: representative density plots and right panel: percentage of OVA⁺ cells from five independent experiments. (E) FACS analysis of GFP⁺ i-OCLs from Cx3cr1^GFP/+ and Cx3cr1^GFP/GFP mice in at least four independent experiments. (F) T cell proliferation assay on CD4⁺ T cells labelled with CFSE and cultured in the presence of OVA-challenged GFP⁺ i-OCLs differentiated from Cx3cr1^GFP/+ and Cx3cr1^GFP/GFP mice and analyzed by FACS after 4 days of coculture. (G) CD4⁺ T cells activated by OVA-challenged GFP⁺ i-OCLs from Cx3cr1^GFP/+ and Cx3cr1^GFP/GFP mice were analyzed for their expression of TNFα and IFNγ by FACS after intracytoplasmic staining of these cytokines. n.s., no significant difference.

Figure 2—figure supplement 1. Gating strategy for mature Cx3cr1⁺ and Cx3cr1^neg i-OCLs.

(A) Gating strategy for the analysis and sorting of mature GFP⁺ (Cx3cr1⁺) and GFP^neg (Cx3cr1^neg) i-OCLs using flow cytometry. After gating on total cells, doublets were excluded and cells with ≥3 nuclei (≥3N) cells were considered as OCLs and distinguished from 1 to 2 nucleated (1–2N) cells based on their Hoechst 33342 staining level. OCLs were analyzed based on their GFP expression and GFP⁺ (Cx3cr1⁺) and GFP^neg (Cx3cr1^neg) i-OCLs were sorted.

Figure 2—figure supplement 2. Comparative transcriptomic analysis of Cx3cr1⁺ i-OCLs from Cx3cr1^GFP/GFP and Cx3cr1^GFP/+ mice.

(A) Comparative RNA-seq analysis between GFP⁺ (Cx3cr1⁺) i-OCLs from KO Cx3cr1^GFP/GFP and WT Cx3cr1^GFP/+ mice. Heatmap visualization of the z-scored expression for selected genes involved in OCL differentiation, T cell stimulation and antigen presentation. (B) Relative mRNA expression of GFP⁺ (Cx3cr1⁺) i-OCLs from KO Cx3cr1^GFP/GFP and WT Cx3cr1^GFP/+ mice. Results are represented as the mean with 95% confidence interval of three independent biological replicates conducted in triplicates. n.s., no significant difference.