e. CD25+ cells) were depleted before activation (Fig. 2a; compare whole versus CD25-depleted populations on day 0). In contrast to control PBMC, depletion of CD25+ cells resulted in loss of CD4+ FoxP3HI cells at day 3 post-activation (Fig. 2a; DAPT solubility dmso compare whole versus CD25-depleted populations on day 3). Moreover, if carboxyfluorescein succinimidyl ester (CFSE)-labelled CD25Neg cells were reintroduced

into these polyclonally activated PBMC, there was significantly greater Teff proliferation in PBMC depleted of Tregs (Fig. 2b). Together, these data provide evidence to support the conclusion that aTregs derive from a starting pool of rTregs within PBMC. To study the effect of IFN-I on the generation of aTregs, freshly isolated PBMC were stimulated with anti-CD3 in the absence or presence of human leucocyte IFN (predominantly IFN-α) at 100 or 1000 U/ml or purified recombinant human IFN-β. Then, the total number of CD4 T cells and the generation of aTregs (CD4+ FoxP3HI IFN-γNeg)

and aTeffs (CD4+ FoxP3Low/Neg IFN-γPos) were analysed for separate normal donors after 3 days of polyclonal activation without or with added IFN-α (Fig. 3) or IFN-β (Fig. S1). While there was no consistent inhibitory p38 MAPK inhibitor review or stimulatory effect of IFN-α on total CD4 cell numbers (Fig. 3a,b), there was an average of 42% (P = 0·03) and 50% (P = 0·005) inhibition of aTreg generation in the presence of 100 and 1000 U/ml of IFN-α, respectively (Fig. 3c,d). In contrast, the presence of IFN-α tended

to increased the number of aTeff cells with an average of 53% increase in the number of aTeff cells using 1000 Units IFN-α (P = 0·06) (Fig. 3e,f). In contrast, although IFN-β significantly suppressed Treg activation, this cytokine also tended to decrease Teff activation at the higher concentration (Fig. S1). Although the number of donor PBMC tested with IFN-β was limited, the results may suggest that IFNs α and β may exert distinct effects on lymphocyte homeostasis during cell activation. As a result of the opposite effects of IFN-α on aTreg and aTeff, there was an alteration in the balance Flavopiridol (Alvocidib) between regulatory and effector cells as represented by the aTreg:aTeff ratio. Across all seven donors, this balance tended to favour aTregs in the absence of IFN-α (average aTreg:aTeff ratio = 1·4). However, the substantial suppression of aTreg generation induced by IFN-α caused a statistically significant shift in the mean aTreg:aTeff ratio for all seven donors [ratio = 0·7 for 100 U IFN-α (P = 0·05) and 0·5 for 1000 U IFN-α (P = 0·01)] such that aTeffs outnumbered aTregs on average by 2:1. Together, these data suggest that IFN-α significantly suppresses generation of activated Tregs in polyclonally activated PBMC, and at the same time promotes an increase in IFNγ-producing aTeffs.