CD4⁺Foxp3⁺LAP⁺ Tregs exhibit potent suppression ability in a TGF-β–dependent manner.

<p>(A) The expression of TGF-β in CD4⁺Foxp3⁺LAP⁺, CD4⁺Foxp3⁺LAP⁻, and CD4⁺Foxp3⁺ Tregs was analyzed by FACS. T cells were stimulated in vitro as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108554#s2" target="_blank">Materials and methods</a>, and TGF-β expression was compared between these three groups by paired <i>t</i>-test (**P<0.01). (B) CD4⁺CD25⁺LAP⁺ T cells and CD4⁺CD25⁺LAP⁻ T cells were separated by cell sorting using FACSAria and stimulated with anti-CD3 and anti-CD28 antibodies for 96 h. T cell proliferation was assessed by thymidine [³H] incorporation and expressed as the mean ± SD of three independent experiments; the data were analyzed by paired <i>t</i>-test (*P<0.05). (C) Anti-TGF-β antibody (10 µg/mL) was added to CD4⁺CD25⁺LAP⁺ Tregs co-cultured with CD4⁺CD25⁻LAP⁺ T cells (ratio of 1∶1) and activated by anti-CD3 and anti-CD28 mAbs. The percentage of suppression in the presence or absence of anti-TGF-β mAb is expressed as the mean ± SD of three independent experiments. *P<0.05 by paired <i>t</i>-test.