Figure 8

Gating strategy for identification of respiratory Treg subsets. CD4-positive cells were gated over CD25 (A). Depending on CD25-expression, CD4⁺ CD25⁺ and CD4⁺ CD25^high populations were detected. Out of the CD4⁺ CD25^high fraction, Treg cells were identified based on Foxp3 expression. Out of the CD4⁺ CD25^high Foxp3⁺ regulatory T cell fraction, a CD39⁺ subset was identified (A). CD4⁺ CD25⁺ CD127^low/neg Tregs were identified after gating of CD4⁺ cells over CD127 (B). Subsequently, the CD4⁺ CD127^low/neg fraction was gated over CD25. To confirm, that most of the CD4⁺ CD25⁺ CD127^low/neg cells were Tregs, they were finally gated over Foxp3 (B). Isotype mAb controls were used in all gates to control for unspecific background staining. Fluorescence minus one (FMO) isotype controls were used to set the gates for CD25, Foxp3, CD39 and CD127 positive subpopulations.