Increased Treg and reduced Th17 cells in Ccr2−/− mice that received CAWS. (A) Representative FACS of circulating Tregs gated on CD4+ cells (CD4+, CD25+, Foxp3+) from Ccr2+/+ mice (upper panel) and Ccr2−/− mice (lower panel), after two cycles of CAWS. (B) Percentage of Treg in circulation in Ccr2+/+ mice and Ccr2−/− mice, 1 week prior (day −7), day 19 and day 97 post-CAWS administration. (C) Quantification of Tregs in freshly isolated splenocytes derived from Ccr2+/+ mice and Ccr2−/− mice that received a full cycle of CAWS. (D-E) Levels of IL-10 and TGF-β in cell culture supernatants of splenocytes stimulated with anti-CD3/CD28 for 60 hours in Ccr2+/+ and Ccr2−/− mice, 10 days after the first cycle of CAWS administration. (F) Percentage of Th17 cells (CD4+, IL-17A+) in spleen detected by FACS in Ccr2+/+ and Ccr2−/− mice, after full cycle of CAWS. (G) Negative correlation between circulating Tregs and Th17 cells in splenocytes cultures. (H) Treg suppression assay showing the percentage of proliferation of CD4+ responder cells from Ccr2−/− mice cultured with Treg from Ccr2+/+ mice (grey bar) and CD4+ responder cells from Ccr2+/+ mice cultured with Treg from Ccr2−/− mice (open bar) at different ratios (CD4+ responder : Treg; 1:0, 1:1 and 2:1); analyzed, after a short cycle of CAWS injections. (I) FACS analysis in blood showing the percentage of Treg in Ccr2+/+, Ccr2−/− and PPGM treated Ccr2+/+ mice for 19 days, after one cycle of CAWS. Each bar represents the mean ± SE from a representative experiment with 6–8 mice per group.
Martinez et al. BMC Immunology 2012 13:56 doi:10.1186/1471-2172-13-56