Fig. 1

ExTreg cells are increased during atherogenesis. a Schematic diagram with a representative flow cytometry plot of the Treg lineage tracker-ApoE^−/− (LT-ApoE^−/−) mouse model used to track Treg development during atherosclerosis. The diagram shows two population of Treg; current Treg (RFP⁺YFP⁺) cells, which express Foxp3, and exTreg (RFP⁺YFP⁻) cells, which previously expressed Foxp3. b–d LT-ApoE^−/− mice were fed a western diet for 15 weeks. Bar graphs compare the numbers of total CD4 T cells and effector CD62L^lo cells (b), the percentages and numbers of exTreg and current Treg (c) in the aorta, and the ratio of current Treg to exTreg in the aorta and PaLN (d) of western fed-diet to chow controls. c Representative flow cytometry plots and graphs showing the percentages of exTreg and current Treg in the aorta of the above mice. (e) Current and exTreg cells were sorted from the PaLN of LT-ApoE^−/− mice and mRNA levels for Foxp3 were examined in the extracted RNA and normalized to β-actin. f Foxp3 protein expression levels in current Treg, exTreg cells and TCRβ negative cells. Representative flow cytometry histograms and bar graphs show the MFI of Foxp3 in the two cell populations. g Treg suppression assay using sorted current and exTreg from spleens and peripheral LNs from LT-ApoE^−/− mice. Sorted current and exTreg cells were cultured with CD4 depleted feeder cells from spleens of B6 mice and increasing numbers of naive effector CD45.1 CD4 T cells (CD25⁻CD44^loCD62L^hi) that were labeled with Cell Trace Violet (CTV). Cells were stimulated with soluble αCD3 for 4 days, harvested and effector cells (CD4⁺CD45.1⁺) were examined for the dilution of CTV by flow cytometry. h Flow cytometry histograms showing expression pattern of CD25 (IL-2Rα), CD62L, Nrp1, CD44 and PD1 on current Treg and exTreg cells. Results are expressed as mean ± s.e.m. from two independent experiments (n = 14) (b–d), a representative of two independent experiments (n = 3) (e, g), and a representative of two independent experiments (n = 11) (f). Statistical significant differences were at *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 (unpaired Student’s t-test)