Figure 2. Fate mapping monocyte derived macrophages adopting a tissue resident phenotype after long-term residency in the peritoneal cavity.

(a) Tamoxifen (TAM) pulse-administration and IL-4c treatment (i.p.) in Cx3cr1^{CreERT2-IRESYFP/+}Rosa26^{floxed-tdTomato/+} mice previously injected with thioglycollate at 1 week (n=4), 4 weeks (n=4) or 8 weeks (n=4) post-tamoxifen gavage. (a–c) Representative flow cytometric analysis of CD206 (a) or PD-L2 or MHCII (b) and F4/80 expression on (singlet, live, Siglec-F⁻) CD11b⁺ tdTomato⁺ cells (a) from Cx3cr1^{CreERT2-IRESYFP/+} Rosa26^{floxed-tdTomato/+} mice injected with thioglycollate + IL- 4c and analyzed at 1, 4, and 8 weeks post-tamoxifen gavage. (a) Stacked bar graph showing the relative proportion of F4/80 and/or CD206 expression (a) in CD11b⁺ tdTomato⁺ cells, or (c–d) F4/80 and/or PDL2/MHCII expression. (c) Representative flow cytometry plots of PD-L2, CD206 and MHCII expression in CD11b⁺ tdTomato⁺ cells after IL-4c treatment in Cx3cr1^{CreERT2-IRESYFP/+}Rosa26^{floxed- tdTomato/+} mice pulsed with TAM and injected with thioglycollate 4 weeks (n=6) or 8 weeks (n=3) prior. (d) Converted macrophages (AAM^conv) gain expression of UCP1. Expression of UCP1 by RT-PCR on FACS sorted in CD11b⁺ tdTomato⁺CD206⁺F4/80^int 4 days after Thio+IL-4c treatment (AAM^mono; n=3), relative to CD11b⁺ tdTomato⁻CD206⁻F4/80^high cells from naïve Cx3cr1^{CreERT2-IRESYFP/+}Rosa26^{floxed- tdTomato/+} mice treated with IL-4c only (AAM^res; n=6) and CD11b⁺ tdTomato⁺ CD206⁻F4/80^high cells 8 weeks post thioglycollate treatment (AAM^conv; n=8).