Lung Expansion of Th1, Th2, Th17 and Tregs Figure 3. Expression of T cell transcription factors, T-bet+(Th1), GATA3+(Th2), RORct+ (Th17) and Foxp3+(Tregs) cells in the CD4 + CD25+ cells in lung during allergic inflammation. A) Number of lung CD4+CD25+ cells (per gram tissue) expressing T-bet, GATA-3, RORct and Foxp3 after exposure of mice to PBS or OVA. Data is shown as mean 6 SEM, n = 5–8 mice/group (*** p,0.001, ** p,0.01). B) Fold change of the total number of lung cells expressing the four different transcription factors (T-bet, GATA-3, RORc and Foxp3) in CD4+CD25+ cells after exposure of mice to OVA compared to PBS. C) Pie chart showing the relative presence of Th1 (T-bet+) ,Th2 (GATA-3+),Th17 (RORct+ ) and Treg (Foxp3+) T cells expressed as percentage of CD4+CD25+ cells in OVA and PBS exposed animals. TF = transcription Factor. D) The fold change of the relative presence of Th1 (T-bet+) ,Th2 (GATA-3+),Th17 (RORct+ ) and Treg (Foxp3+) T cells expressed as percentage of CD4+CD25+ cells after exposure of mice to OVA compared to PBS. doi:10.1371/journal.pone.0019889.g003 cells, revealed substantial differences between PBS and OVA exposed animals (Figure 3C). For example, a considerable percentage of CD4+ cells in OVA/PBS exposed animals did not express any of above transcription factors, which was lower in OVA exposed animals. Among the studied transcription factors, GATA-3, changes drastically in response to OVA exposure, with a 6 fold increase compared to PBS exposure, whereas Foxp3 was decreased (Figure 3D). The expression of T cell transcription factors, T-bet+(Th1), GATA-3+(Th2), RORct+(Th17) and Foxp3+(Treg) cells in CD4+CD252 cells in the lung tissue increased after allergen exposure Expression of transcription factors for Th1, Th2, Th17 and Treg cells was observed also in CD4+ cells lacking the IL-2Ra (the CD4+CD252 population; Figure 4). The number of Th1 and Th2 cells in the CD4+CD252 cells was significantly increased in the lung tissue after allergen exposure, while the number of Th17 and Treg cells also numerically increased, but not significantly. In the CD4+CD252 cell population, the majority of the cells express transcription factors for Th17 cells, followed by Th2, Tregs and Th1 cells (Figure 4A). Evaluation of the fold increase of the cell numbers revealed that in CD4+CD252 cells, the Th2 cells dominate, however to a smaller magnitude vs CD4+CD25+ (Figure 4B). When the relevant expression of all transcription factors in CD4+ cells was calculated, relatively fewer cells express the transcription factors for Th1, Th2, Th17 and Treg cells in OVA/PBS animal compared to OVA/OVA animals (Figure 4C). Calculation of the fold increase also showed that even in the CD4+CD252 population, GATA-3 was more increased compared to the changes observed for T-bet, RORct and Foxp3( Figure 4D). Lung Th1, Th2, Th17 and Treg cells proliferate during allergic airway inflammation in both the CD4+CD25+ and CD4+CD252 cell populations As all effector T cells and Treg cells investigated increased during allergic inflammation, the degree of ongoing proliferation was also assessed in each cell type, by determining the percentage of newly produced cells and their cell cycle phase. Allergen exposure significantly increased the production of new Th1, Th2, Th17 and Treg cells in both CD25+ and CD252 populations (Figure 5A–H). However, when the percentage of newly produced cells in each population is compared, more than 90% of the Th1, Th2 and Th17 cells (Figure 5A,C,E) and about 80% of the Treg cells (Figure 5G) are newly produced in the CD4+CD25+ population, while only about 60% of the Th1, Th2 and Treg PLoS ONE | www.plosone.org 6 May 2011 | Volume 6 | Issue 5 | e19889