Impaired suppressive function of regulatory T cells from transgenic mice. (A) Splenocytes from C57BL/6 mice were stained with anti-CD4 and anti-CD25 antibodies, which was followed by Foxp3 intracellular staining, as described in "Materials and methods." The dot plots of CD25+ and Foxp3+ proportions after gating on CD3+CD4+ T cells are shown. The results shown are from one of three representative experiments. (B) The absolute number of regulatory T cells (Tregs) (CD4+CD25+Foxp3+ cells) from the spleens of C57BL/6 mice were calculated and are shown here (n = 3 mice for each group; P > 0.05). (C) CD4+ T cells from the spleens of B6 wild-type (Wt) or transgenic (Tg) mice were enriched and sorted into a CD4+CD25+ subset as the regulatory cells and a CD4+CD25- subset from Wt C57BL/6 as the responder cells. The number of responder cells was fixed at 5 × 104. The inhibition function of Tregs was measured by in vitro assay as described in "Materials and methods." The percentage inhibition by different populations of Tregs was calculated by using the formula described in "Materials and methods." cpm, counts per minute. (D) The results shown are from one of three representative experiments. (E) B6 Wt and Tg mice were crossed with Foxp3-RFP mice. CD4+ T cells from the spleens of Wt or Tg red fluorescent protein (RFP) mice were enriched and sorted into a CD4+RFP+ subset as the regulatory cells and a CD4+RFP- subset from Wt C57BL/6 as the responder cells. The responder cells (5 × 104/well) were cocultured with an equal number of regulatory cells for the inhibition assay described above. The results from one of three representative repeated experiments are shown.
Tao et al. Arthritis Research & Therapy 2011 13:R212 doi:10.1186/ar3545