Rsistence. Similarly, previous reports have found that IL-2 can play a role in enhancing Th1 mediated CAL-120 web responses but these were not affected by sCD25 at the concentrations used in this study [31] (Figure 2A, 3B). As Th17 responses are clearly elevated under these conditions, these data may reflect differing levels of sensitivity among pathogenic versus regulatory CD4+ T cell subsets towards the AKT inhibitor 2 effects of IL-2 signalling. Alternatively, it is possible that Tregs, which express constitutively high levels of surface CD25 (and the heterotrimeric IL-2R) in comparison to Th17 cells, may be competitively less sensitive to sequestration of circulating IL-2 by sCD25. Studies are ongoing to determine how limiting doses 
of IL-2 may differentially impact CD4+ T cell responses and how sCD25 might influence these events. It is clear from our in vivo studies that the ability of sCD25 to enhance Th17 responses on a per cell basis is only observed in the periphery and not at the site of inflammation where although percentages of both Th17 and Th1 cells are remarkably unaltered upon sCD25 treatment, both are present in significantly increased numbers (Figure 1). Although this may reflect the effects of sCD25 on T cell expansion, this seems unlikely given that we observed no such effects in vitro (Figure 3D). A further possible explanation for this is an increased plasticity or inter-conversion between both subsets at the site of inflammation. However, this is unlikely given that the IL-17A 25837696 eGFP mouse used in these studies allows the identification of cells which also have a legacy of IL-17A expression. As such, increased plasticity would be evident as an increase in the percentage of GFP+ve cells expressing IFNc which was not detected. More likely, these data indicate that enhanced antigen-specific Th17 cells in the periphery can facilitate the infiltration of both pathogenic Th1 and Th17 cells to the site of inflammation as has been previously reported [32]. In direct relevance to this study, the use of humanized antiCD25 antibodies is showing considerable promise as a potential therapeutic for Multiple Sclerosis [33]. Although efficacy for this approach has been demonstrated in early clinical trials, the exact mechanism through which these antibodies inhibit disease remains obscure. It is noteworthy that these antibodies bind sCD25 and block its ability to sequester IL-2 [34]. As levels sCD25 are elevated among MS patients [10], blockade of its immunomodulatory effects with anti-CD25 could conceivably play an important part in the mechanism of action of Anti-CD25. Together these data demonstrate the immunomodulatory activity of the soluble form of the IL-2R alpha chain in vivo for the first time and indicate that these effects are mediated by its capacity to act as a decoy receptor for secreted IL-2. Although biochemical studies indicate that CD25 in isolation has a significantly lower affinity for IL-2 when compared to the heterotrimeric IL-2R complex, it has been demonstrated to bind IL-2 efficiently and its ability to suppress IL-2 mediated responses in vitro has been extensively reported [10,13,26,30]. The association between elevated levels of sCD25 found in the sera of autoimmune patients and the presence of specific susceptibility alleles at the CD25 gene locus offer perhaps the clearest indication that sCD25 plays a role in autoimmune pathogenesis [10]. Although whether elevated levels of sCD25 are causally linked to the pathogenesis of human a.Rsistence. Similarly, previous reports have found that IL-2 can play a role in enhancing Th1 mediated responses but these were not affected by sCD25 at the concentrations used in this study [31] (Figure 2A, 3B). As Th17 responses are clearly elevated under these conditions, these data may reflect differing levels of sensitivity among pathogenic versus regulatory CD4+ T cell subsets towards the effects of IL-2 signalling. Alternatively, it is possible that Tregs, which express constitutively high levels of surface CD25 (and the heterotrimeric IL-2R) in comparison to Th17 cells, may be competitively less sensitive to sequestration of circulating IL-2 by sCD25. Studies are ongoing to determine how limiting doses of IL-2 may differentially impact CD4+ T cell responses and how sCD25 might influence these events. It is clear from our in vivo studies that the ability of sCD25 to enhance Th17 responses on a per cell basis is only observed in the periphery and not at the site of inflammation where although percentages of both Th17 and Th1 cells are remarkably unaltered upon sCD25 treatment, both are present in significantly increased numbers (Figure 1). Although this may reflect the effects of sCD25 on T cell expansion, this seems unlikely given that we observed no such effects in vitro (Figure 3D). A further possible explanation for this is an increased plasticity or inter-conversion between both subsets at the site of inflammation. However, this is unlikely given that the IL-17A 25837696 eGFP mouse used in these studies allows the identification of cells which also have a legacy of IL-17A expression. As such, increased plasticity would be evident as an increase in 
the percentage of GFP+ve cells expressing IFNc which was not detected. More likely, these data indicate that enhanced antigen-specific Th17 cells in the periphery can facilitate the infiltration of both pathogenic Th1 and Th17 cells to the site of inflammation as has been previously reported [32]. In direct relevance to this study, the use of humanized antiCD25 antibodies is showing considerable promise as a potential therapeutic for Multiple Sclerosis [33]. Although efficacy for this approach has been demonstrated in early clinical trials, the exact mechanism through which these antibodies inhibit disease remains obscure. It is noteworthy that these antibodies bind sCD25 and block its ability to sequester IL-2 [34]. As levels sCD25 are elevated among MS patients [10], blockade of its immunomodulatory effects with anti-CD25 could conceivably play an important part in the mechanism of action of Anti-CD25. Together these data demonstrate the immunomodulatory activity of the soluble form of the IL-2R alpha chain in vivo for the first time and indicate that these effects are mediated by its capacity to act as a decoy receptor for secreted IL-2. Although biochemical studies indicate that CD25 in isolation has a significantly lower affinity for IL-2 when compared to the heterotrimeric IL-2R complex, it has been demonstrated to bind IL-2 efficiently and its ability to suppress IL-2 mediated responses in vitro has been extensively reported [10,13,26,30]. The association between elevated levels of sCD25 found in the sera of autoimmune patients and the presence of specific susceptibility alleles at the CD25 gene locus offer perhaps the clearest indication that sCD25 plays a role in autoimmune pathogenesis [10]. Although whether elevated levels of sCD25 are causally linked to the pathogenesis of human a.
