Lung Expansion of Th1, Th2, Th17 and Tregs Figure 2. Lung CD4+ cells proliferate de novo and in situ during allergic airway inflammation. A) Total lung CD4+ and CD4+CD25+ cells (per gram tissue) in sensitised mice exposed either to PBS or OVA on five consecutive days. B) Representative FACS plots showing the CD4+ CD252 and CD4+CD25+ the lung cells that are also positive for BrdU (newly produced) and stained with 7-AAD (DNA) to determine cell-cycle status. Cells (%) are shown for the indicated gates and quadrants. The two upper plots represent CD4+CD252 cells, and the two lower plots represent CD4+CD25+ cells. The left plots represent data from mice exposed to PBS and the right plots represent data from mice exposed to OVA. Within each plot, the G1 phase is represented by the upper left quadrant, the G0 phase in the lower left quadrant, and the S, G2/M phase in the upper right quadrant. C) Number of CD4+CD25+ lung cells (per gram tissue) (white column) demonstrating signs of proliferation, as assessed by the incorporation of BrdU (filled column) and BrdU and 7-AAD (hatched columns) after exposure of mice to PBS or OVA. D) Number of CD4+CD252 lung cells (per gram tissue) (white column) demonstrating signs of proliferation, as assessed by the incorporation of BrdU (filled column) and BrdU and 7-AAD (hatched columns) after exposure of mice to PBS or OVA. All data are shown as mean 6 SEM, n = 5–8 mice/group (*** p,0.001, ** p,0.01). doi:10.1371/journal.pone.0019889.g002 in both the CD25+ and CD252 CD4 populations are proliferating in the lung during repeated OVA exposure (S and G2/M phase) (Figure 2B). Importantly, comparison of the proliferation rate de novo and in situ revealed a greater number of proliferating CD4+CD252 T cells (64%) vs. CD4+CD25+ T cells (37%) in the lung during allergen exposure (Figure 2B). The expression of T cell transcription factors, T-bet+(Th1), GATA-3+( Th2), RORct+(Th17) and Foxp3+(Treg) cells in CD4 + CD25+ cells in the lung tissue increased after allergen exposure The presence of different effector T cells and Treg cells within the CD4+CD25+ cell compartment was determined by FACS evaluation of the expression of specific transcription factors. The number of Th1 (CD4+CD25+T-bet+), Th2 (CD4+CD25+GATA- 3 + ), Th17 (CD4+CD25+RORct+) and Treg (CD4+CD25+Foxp3+) cells were all significantly expanded in the lung tissue during allergen exposure (Figure 3A). Interestingly, the majority of the expanded cells are Th17 cells, followed by Treg, Th2 and Th1 cells. However, when determining the fold increase of Th1, Th2, Th17 and Treg cell numbers (OVA-OVA/OVA-PBS) we discovered that the GATA-3 expressing Th2 cells were increased up to 22 times more compared to any of the other effector T cells (Figure 3B). As all of the T effector cells are generated from common na¨ıve cells we were interested to evaluate the relevance between them. The relative change of cells, expressed as a percentage of all CD4+ PLoS ONE | www.plosone.org 5 May 2011 | Volume 6 | Issue 5 | e19889