Phenotypic characterisation of CD4+CD25+ T cells from immunised IFN-γR KO and wild-type DBA/1 mice. (a, b) CD4+CD25+ T cells isolated from IFN-γR KO and wild-type mice show a similar expression pattern of activation markers, in naive (a) and immunised (b) conditions. CD4+ T cells were purified from the lymph node cells of eight IFN-γR KO and wild-type DBA/1 mice, either naive or having been immunised 21 days previously with collagen type II in complete Freund's adjuvant (purity more than 99%). CD4+ T cells were stained for CD25 in combination with CD69, CD62L, CD44 or cytolytic T lymphocyte-associated antigen-4 (CTLA-4). Dead cells were excluded by gating on propidium iodide-negative cells. The numbers represent the percentages of CD4+CD25+ cells within the indicated marker. (c) Decreased Foxp3 mRNA levels in CD4+CD25+ Treg cells from immunised mice. Lymph node cells were isolated from eight naive or immunised IFN-γR KO and wild-type DBA/1 mice. Purified CD4+ T cells were stained with anti-CD25-FITC and phycoerythrin-conjugated anti-CD4, and sorted. The purity of the sorted CD4+CD25+ population was more than 97%. cDNA samples were prepared from 2 × 105 cells of each population and were subjected to real-time quantitative PCR analyses. The relative quantity of Foxp3 in each sample was normalised to the quantity of β-actin. Error bars indicate standard error of the means of two (CD4+CD24+ cells from naive mice) or three (CD4+CD25+ cells from immunised mice) independent experiments. *P < 0.05 for comparison with Foxp3 expression of cells isolated from immunised wild-type mice (Mann–Whitney U-test).