E IL-2R was affected in these cells. IL-2 is expressed early during the first 24 hours after TCR Autophagy stimulation of CD4+ T cells and activation of Jak3-STAT5 dependent signal pathways in T cells during this time is considered to be largely driven by the autocrine effects IL-2. sCD25 significantly decreased levels of STAT5 activation in Th17 cells demonstrating its ability to inhibit signalling downstream of the IL-2R (Figure 5A). IL-2 dependent activation of STAT5 signalling is known 
to directly inhibit earlysCD25 Enhances Th17 ResponsessCD25 Enhances Th17 ResponsesFigure 3. sCD25 enhances Th17 cell responses in vitro. (A B) Purified naive CD4+ T cells were activated under either Th17 or Th1 inducing conditions (as described in methods) in the presence of a range of concentrations of sCD25 (20, 10, 5 or 1 mg/ml) or anti-IL-2 (10 mg/ml). Levels of IL17A or IFNc expression were determined after 96 hrs by (A) FACS and (B) ELISA. (C) Purified naive CD4+ T cells were activated under Treg inducing conditions, as described in methods, in the presence or absence of sCD25 (20mg/ml) and FoxP3 expression determined by FACS. (D) Naive CD4+ T cells were stained with CFSE (2.5 mM) prior to activation under Th17 conditions in presence or absence of sCD25 (20 mg/ml). After 96 hours, levels of intracellular IL-17A expression and CFSE dilution or 7AAD incorporation were determined by FACS. (E) Purified naive CD4+ T cells were activated under Th0, Th17 and Th17 sCD25 (20 mg/ml) conditions for 72 hours and levels of P-Stat3 (pY705) determined by FACS. All data are representative of 3 independent experiments. Statistical Epigenetics Significance determined by unpaired student’s t-test, p#0.05, **p#0.01, ***p#0.001. doi:10.1371/journal.pone.0047748.gprogramming events in the development of a Th17 response by blocking the induction of RORcT expression [9]. These data identify a novel mechanism whereby sCD25 enhanced the generation and development of proinflammatory Th17 responses through inhibiting the protolerogenic effects of IL-2R signalling. To determine the precise mechanism through which sCD25 was mediating this inhibition we considered a number of possibilities. First, sCD25 may inhibit the levels of IL-2 expressed upon T cell activation (although IL-2 neutralization by monoclonal antibodies has previously been found to enhance IL-2 expression by inhibiting an auto-regulatory negative feedback loop [20]). We observed no differences between the levels of IL-2 expressed on a per cell basis either in the presence or absence of sCD25 after 24 hours (Figure 5B). Second, sCD25 may exert its effects at the cell surface by acting to either inhibit appropriate assembly of the heterotrimeric receptor complex or inhibit IL-2 binding. To examine this possibility we used a His-tag on the soluble form of the receptor to discriminate between soluble and surface expressed forms of CD25. However, we were not able to detect any bindingof sCD25 to the cell surface 12926553 during the first 24 hours after activation (Figure 5C). In contrast, the presence of sCD25 did significantly inhibit the upregulation of endogenous surface CD25 expression (Figure 5D). This observation further indicated a role for sCD25 in inhibiting IL-2R signalling as IL-2 is recognised as an important mediator in driving surface CD25 expression early during T cell activation. Third, we investigated the possibility that sCD25 may act to sequester secreted IL-2 in the T cell microenvironment. Significantly, sCD25 inhibited t.E IL-2R was affected in these cells. IL-2 is expressed early during the first 
24 hours after TCR stimulation of CD4+ T cells and activation of Jak3-STAT5 dependent signal pathways in T cells during this time is considered to be largely driven by the autocrine effects IL-2. sCD25 significantly decreased levels of STAT5 activation in Th17 cells demonstrating its ability to inhibit signalling downstream of the IL-2R (Figure 5A). IL-2 dependent activation of STAT5 signalling is known to directly inhibit earlysCD25 Enhances Th17 ResponsessCD25 Enhances Th17 ResponsesFigure 3. sCD25 enhances Th17 cell responses in vitro. (A B) Purified naive CD4+ T cells were activated under either Th17 or Th1 inducing conditions (as described in methods) in the presence of a range of concentrations of sCD25 (20, 10, 5 or 1 mg/ml) or anti-IL-2 (10 mg/ml). Levels of IL17A or IFNc expression were determined after 96 hrs by (A) FACS and (B) ELISA. (C) Purified naive CD4+ T cells were activated under Treg inducing conditions, as described in methods, in the presence or absence of sCD25 (20mg/ml) and FoxP3 expression determined by FACS. (D) Naive CD4+ T cells were stained with CFSE (2.5 mM) prior to activation under Th17 conditions in presence or absence of sCD25 (20 mg/ml). After 96 hours, levels of intracellular IL-17A expression and CFSE dilution or 7AAD incorporation were determined by FACS. (E) Purified naive CD4+ T cells were activated under Th0, Th17 and Th17 sCD25 (20 mg/ml) conditions for 72 hours and levels of P-Stat3 (pY705) determined by FACS. All data are representative of 3 independent experiments. Statistical Significance determined by unpaired student’s t-test, p#0.05, **p#0.01, ***p#0.001. doi:10.1371/journal.pone.0047748.gprogramming events in the development of a Th17 response by blocking the induction of RORcT expression [9]. These data identify a novel mechanism whereby sCD25 enhanced the generation and development of proinflammatory Th17 responses through inhibiting the protolerogenic effects of IL-2R signalling. To determine the precise mechanism through which sCD25 was mediating this inhibition we considered a number of possibilities. First, sCD25 may inhibit the levels of IL-2 expressed upon T cell activation (although IL-2 neutralization by monoclonal antibodies has previously been found to enhance IL-2 expression by inhibiting an auto-regulatory negative feedback loop [20]). We observed no differences between the levels of IL-2 expressed on a per cell basis either in the presence or absence of sCD25 after 24 hours (Figure 5B). Second, sCD25 may exert its effects at the cell surface by acting to either inhibit appropriate assembly of the heterotrimeric receptor complex or inhibit IL-2 binding. To examine this possibility we used a His-tag on the soluble form of the receptor to discriminate between soluble and surface expressed forms of CD25. However, we were not able to detect any bindingof sCD25 to the cell surface 12926553 during the first 24 hours after activation (Figure 5C). In contrast, the presence of sCD25 did significantly inhibit the upregulation of endogenous surface CD25 expression (Figure 5D). This observation further indicated a role for sCD25 in inhibiting IL-2R signalling as IL-2 is recognised as an important mediator in driving surface CD25 expression early during T cell activation. Third, we investigated the possibility that sCD25 may act to sequester secreted IL-2 in the T cell microenvironment. Significantly, sCD25 inhibited t.