Figure 2.

Effects of nasal heat shock protein (HSP)60 on CD4⁺CD25⁺GARP⁺ regulatory T cells (T_regs) in spleens and cervical lymph nodes (CLNs). Apolipoprotein E (ApoE)^−/− mice were nasally administered HSP60 for 5 days and were killed 4 and 14 days after the final nasal treatment. Untreated mice (t = 0) were considered controls. For glycoprotein A repetitions predominant (GARP) activation, the cells (2 × 10⁶/sample) were stimulated with soluble anti‐CD3 monoclonal antibody (mAb) (1·5 μg/ml) and anti‐CD28 mAb (2 μg/ml) at 37°C with 5% CO₂ for 24 h. (a) CD4⁺ T cell subsets were gated. (b–e) Representative results for isotype controls in spleens and CLNs estimated by fluorescence activated cell sorter (FACS) analysis. (f–i) Representative results for unstimulated and stimulated CD4⁺CD25⁺GARP⁺ T_regs in spleens and CLNs estimated by FACS analysis. (j,k) The graphs represent the percentage of unstimulated and stimulated CD4⁺CD25⁺GARP⁺ T_regs in spleens and CLNs. (l,m) The graphs represent the absolute number of unstimulated and stimulated CD4⁺CD25⁺GARP⁺ T_regs in spleens and CLNs. *P < 0·05, **P < 0·01. The data are expressed as the mean ± standard error of the mean (s.e.m.) and are representative of at least three independent experiments.