The concentration (in pg/ml) was determined using a standard curve with known amounts of IL-2 added to the ELISA plate. While sustained Foxp3 Pexidartinib datasheet gene expression is required for the suppressive function of natural Tregs,29 its expression is also up-regulated in activated human Teffs.4–6 Thus, a challenge in the study of Tregs in humans is the difficulty in discriminating between recently activated CD25+ FoxP3+ Teffs and the

subset of resting Tregs in which FoxP3 can be expressed at similar levels. In this regard, other markers that help to discriminate Tregs from Teffs can be used in combination with FoxP3 expression for the study of freshly isolated and ex vivo activated T cells.4,30 We used unfractionated PBMC rather than purified Tregs/Teffs in order to study them within the context of a broader population of immune cells.

To study the relationship between human natural Tregs and Teffs upon polyclonal activation, total PBMC were stimulated with anti-CD3 (5, 100 or 1000 ng/ml) and the expression of FoxP3, IFN-γ and IL-2 was determined on CD4+ cells by flow cytometry at days 3, 7 and 10, as previously reported.4 This system relies on ‘presentation’ of anti-CD3 antibody to T cells by Fc receptors on antigen-presenting cells, a situation that resembles T-cell receptor (TCR) activation in response to its natural MI-503 research buy ligand [i.e. peptide/major histocompatibility complex (MHC) complexes] in vivo.4 In addition, as the assay is performed on total PBMC, it avoids the requirement of T-cell purification, a condition that may affect the activation state of the cells. In the absence of TCR stimulation, rTregs (defined as CD4+ FoxP3low IFN-γNeg IL-2Neg) remained fairly stable at day 3 of culture (compare Figs 1a and 1d). In contrast, as previously described,4 anti-CD3 activation of PBMC induced a dramatic increase in the percentage of FoxP3-positive cells, peaking at day 3 post-stimulation (compare Figs 1d and g, and data not shown). Furthermore, among these cells, two novel cell

populations were distinguished based on the expression levels of FoxP3 and the effector cytokines IFN-γ and IL-2. These cells were PD184352 (CI-1040) identified as CD4+ FoxP3HI IFN-γNeg IL-2Neg and CD4+ FoxP3Low IFN-γPos IL-2Pos (Fig. 1g,h), representing activated Tregs and Teffs, respectively.4,6 From these experiments, the highest expression of FoxP3 was observed at day 3 using 100 ng/ml of anti-CD3 (Fig. 1g and data not shown); this concentration was used in the subsequent assays. In addition, aTeffs were further defined as IFN-γPos, which include both FoxP3Neg and FoxP3Low cells. In order to address the mechanism of CD4+ FoxP3HI cell generation, we determined the expression of Ki-67, a marker of cell proliferation.31 At day 3 post-TCR stimulation, 20% of CD4+ FoxP3HI cells were Ki-67 positive (Fig. 1i), supporting the conclusion that this cell population is expanded through proliferation.