Lung Expansion of Th1, Th2, Th17 and Tregs Figure 4. Expression of T cell transcription factors, T-bet+(Th1), GATA3+(Th2), RORct+(Th17) and Foxp3+(Tregs) cells in the CD4+CD252 cells in lung during allergic inflammation. A) Number of lung CD4+CD252 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+CD252 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+CD252 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+CD252 cells after exposure of mice to OVA compared to PBS. doi:10.1371/journal.pone.0019889.g004 cells(Figure 5B,D,H) and 50% of Th17 cells (Figure 5F) are newly produced in CD4+CD252 population, suggesting that the presence of the IL-2Ra increases the proliferative activity of these cells. When the number of newly produced cells in the S- G2/M phase is examined, more than 95% of the newly produced Th1 and Th2 cells in both CD25+ and CD252 populations were proliferating in situ, compared to only 65% of the newly produced Th17 and Treg cells in CD25+ and CD252 populations. Together, these data show that the majority of Th1, Th2, Th17 and Treg cells in CD25+ population are newly produced after allergen exposure, whereas only half of the Th1, Th2, Th17 and Treg cells in CD252 population are newly produced after exposure. This confirms the proliferative enhancement in the presence of IL-2 receptor in the lung during allergen exposure. Next we estimated the Th1, Th2, Th17 and Treg cells presence in the CD4+ cell pool. T effector cells in S and G2/M phase i.e.: Th1 (CD4+CD25+/2Tbet+BrdU+7-AAD++), Th2 (CD4+CD25+/2 GAT A-3+BrdU+7-AAD++), Th17 (CD4+CD25+/2RORct+BrdU+7-A AD++) and Treg (CD4+CD25+/2Foxp3+BrdU+7-AAD++) cells are expressed as % of total CD4+ T cells. Among T effector cells, the Th2 cells had the higher fold increase after allergen exposure in both CD25+ (Th1: 11, Th2: 31, Th17: 8, and Treg: 10) and CD252 (Th1: 10, Th2: 23, Th17: 9, and Treg: 10 respectively). The distribution of T cell transcription factors differs in the lung tissue during allergic airway inflammation As shown by FACS, all of the investigated T effector cells and Treg cells in the lung were increased in numbers, with many in a proliferative state, during allergic inflammation. It has been reported that T cells can traffic into the inflamed lung tissue during allergic inflammation, but their micro environmental distribution has so far not been investigated. Therefore, we stained lung tissue from OVA/PBS and OVA/OVA mice for T- bet (Th1), GATA-3 (Th2), Foxp3 (Treg cells) and RORct (Th17) expressing cells by immunohistochemistry (Figure 6A), and quantified their respective presence in three different areas of the lung, including lung peribronchial tissue, lung perivascular tissue and lung alveolar tissue. The immunohistochemical staining of different transcription factor positive cells is docu- mented in the nucleus in the three different lung compartments of OVA /OVA animals (Figure 6A). In addition, also MBP+ cells, representing eosinophils, were present in the lung tissue of allergen exposed mice (Figure 6A). The mice acquired a significant accumulation of T-bet+, GATA-3+ and Foxp3+ cells in the peribronchial, perivascular and alveolar lung tissue after allergen exposure, while RORct+ cells increased only in the alveolar tissue, but not in the peribronchial and perivascular tissue (Figure 6B). Focusing in T effector cell relevant distribution, PLoS ONE | www.plosone.org 7 May 2011 | Volume 6 | Issue 5 | e19889