We postulate that the affinity of this mAb for the canine epitope is low, a view supported by a recent study in which a specific anti-canine CD25 MI-503 in vivo mAb was developed in mice.55 A proportion of the ACT-1-negative cells may therefore be CD25+, which would reconcile this apparent anomaly with the observation that the majority of Foxp3/FOXP3+ T cells in both rodents and humans are CD25+. Stimulation of mononuclear cells derived from peripheral LNs with Con A for up to 120 hr elicited a significant increase in percentage and MFI of FOXP3 expression by both CD4+

and CD8+ T cells (Fig. 2). This phenomenon occurred in the absence of exogenous IL-2 or TGF-β, though the addition of low concentrations of IL-2 augmented CD25 and FOXP3 expression (Fig. 3a). Robust increases in CD25 expression were also observed in a recent study of CD4+ T cells derived from PB stimulated with Con A, yielding parallel increases in FOXP3 expression.64 However, similar experiments performed in an earlier study failed to elicit significant increases in the proportions of FOXP3+ CD4+ T cells without the addition of IL-2 and TGF-β,49 ABT-888 nmr presumably reflecting differences in experimental conditions. Interestingly, in our study removal of the stimulus and continued culture disclosed a FOXP3high

population of lymphocytes that was IFN-γ− and predominantly CD4+ (Fig. 2d). Both the high level of FOXP383,84 and the lack of IFN-γ expression – Foxp3 directly represses the Ifng gene85,86 – suggested that this population was regulatory in nature, supported by our subsequent functional studies in vitro (Fig. 3d). Two alternative, non-mutually exclusive explanations for the increased proportion and absolute numbers of FOXP3+ T cells with polyclonal stimulation were considered – namely, up-regulation of FOXP3 in cells

that were originally either FOXP3intermediate or FOXP3−, or proliferation of pre-existing FOXP3+ T cells. The impressive increase in MFI of FOXP3 suggested that up-regulation of this molecule had occurred in individual cells, but parallel proliferation of pre-existing Treg cells could not be excluded. Reasoning that in both mice and humans Helios expression is restricted to nTreg cells and is not Galeterone induced by stimulation, even in the presence of TGF-β, we explored the expression of Helios in cells that had been stimulated in an identical manner to those for the functional studies. We observed an impressive increase in the number of FOXP3+ Helios+ cells with Con A stimulation, arguing for the proliferation of pre-existing nTreg cells. However, Helios expression was not limited to the FOXP3high population, which we speculated were Treg cells on the basis of their IFN-γ− phenotype in earlier studies (Fig. 2d).