Lung Expansion of Th1, Th2, Th17 and Tregs Figure 9. Gene expression in lung microenvironments during allergic inflammation. A) Fold change of genes related to allergic inflammation in peribronchial, perivascular and alveolar tissue. Fold change is calculated by comparing OVA exposed mice to PBS control. 3 fold change threshold indicated by dotted line was considered biological relevant. B) Network of differentially expressed genes in OVA exposed mice compared to the PBS exposed controls. A network of genes associated with allergic inflammation, generated by Ingenuity Pathways Analysis (3.0 fold change threshold), of OVA/OVA mice compared to OVA/PBS mice. The expression of these genes in three lung microenvironments was examined; peribronchial, perivascular and alveolar tissue. Lines indicate relationships between molecules (solid line = direct interaction; dashed line = indirect interaction). Arrows at the end of these lines indicate the direction of the interaction. Molecules that are up-regulated and down-regulated in the dataset are coloured red and green respectively. Darker colouring of the molecules indicates a higher fold change. Grey molecules did not meet the threshold of 3 fold change. Uncoloured molecules have been added from the Ingenuity Knowledge Base. The exact fold change values of each molecule are shown in Table 1. doi:10.1371/journal.pone.0019889.g009 has commonly been used as a marker for activated T cells [18]. CD25 is involved in T cell proliferation, activation induced cell death, as well as the actions of both Treg and effector T cells [19,20]. Soper et al. have shown that transgenic mice lacking IL- 2Ra have unchanged amounts of Tregs in periphery compared to wild type mice, while mice lacking IL-2Rb did not, suggesting that IL-2 can be effective even without expression of CD25[21]. These findings were confirmed in a recent human study, where the administration of a humanized anti-CD25 antibody in children undergoing liver transplantation did not affect the circulating Tregs, proposing that the needed IL-2 signal could be mediated by the IL-2 bc dimeric receptor [22]. Together, these findings argue that the CD252 cells may still use the IL-2 cytokine for their maintenance. However, it is not known whether they are as effective as the CD25+ cells in regulating immune response. Furthermore, CD252 cells may have other important functions. For example, CD4+CD252Foxp3+ cells has been suggested to be more effective than CD4+CD25+Foxp3+ cells in delivering T-cell mediated tolerance [15]. Similarly, CD4+CD252 peripheral Treg cells showed preventive functions in the induction of autoimmune responses [16]. These previous findings urged us to quantify not only the CD25+ CD4 cells, but also the CD252 fraction of CD4+ T cells. In this study, allergen exposure increase the number of CD4+ cells that express CD25 from 5% to approximately 25% of CD4+ cells in the lungs, confirming its role in allergic inflammation. Importantly, both the CD25+ and the CD252 populations are proliferating in the lung, although more prominently in the CD25+ cell population, arguing for a key role of IL-2 in this specific cellular process. An explanation of the proliferation of the CD252 cells could be that the cells have shed their IL-2 receptor after having unchanged proliferation. Indeed, CD25 is up-regulated on recently activated effector T cells, but this molecule is subsequently released into the microenvironment [23]. PLoS ONE | www.plosone.org 13 May 2011 | Volume 6 | Issue 5 | e19889