Regulatory T cells suppress autoreactive CD4+ T cell response to bladder epithelial antigen

Figure 7 Bladder infiltrating CD4⁺ Treg cells are functionally active and express inhibitory effector molecules in urothelium-ovalbumin^GFP-Foxp3/OT-II mice. Bladder single-cell suspensions were prepared from URO-OVA^GFP-Foxp3/OT-II mice (8 wk) and analyzed by flow cytometry or sorted for GFP-positive (Foxp3⁺) and GFP-negative (Foxp3^-) CD4⁺ T cells. A: Bladder infiltrating CD4⁺ T cells consist of both GFP-positive (Foxp3⁺) and GFP-negative (Foxp3^-) populations by flow cytometry. Gate was set on lymphocytes according to scatter criteria; B: Flow cytometric analysis of surface CD44, CD45RB, CD62L and CD69 on bladder infiltrating GFP-positive (Foxp3⁺) and GFP-negative (Foxp3^-) CD4⁺ T cells. Gate was set on CD4⁺ T cells; C: RT-PCR analysis of TGF-β, IL-10, FGL2 and GITR mRNAs in bladder infiltrating GFP-positive (Foxp3⁺) and GFP-negative (Foxp3^-) CD4⁺ T cells. GAPDH was used as an internal control. M: Marker; N: GFP-negative (Foxp3^-) CD4⁺ T cells; P: GFP-positive (Foxp3⁺) CD4⁺ T cells; URO^GFP-Foxp3/OT-II: Urothelium-ovalbumin^GFP-Foxp3/OT-II mice; RT-PCR: Reverse transcriptase-polymerase chain reaction; TGF: Transforming growth factor; GITR: Glucocorticoid-induced tumor necrosis factor receptor.