Thymic CD45^– radioresistant cells are sufficient for positive selection of HA-specific Tregs following lentiviral vector injection. (A, left) Shown is a representative CD45 versus forward side scatter (FSC) staining of total cells of a thymus from a TCR-HA mouse after collagenase and Dnase I digestion. (A, right) Evaluation of eGFP expression in CD45⁺ and CD45^– cells after injection of PBS (CTRL) or LvGFP vector. Similar result was obtained in another mouse. (B) Direct visualization of radioresistant transduced cells in the thymus of 9 Gy–irradiated TCR-HA mice irradiated 4 weeks after injection of PBS (CTRL) or of the eGFP-expressing vector (LvGFP). Whole thymuses were examined for eGFP fluorescence directly under a UV microscope in PBS 1 × 6 weeks after irradiation and T-cell–depleted bone marrow transfer from TCR-HA mice. Original magnification: ×40. (C) Frequencies of HA-specific cells within CD4SP CD25⁺ thymocytes or CD4⁺CD25⁺ splenocytes in TCR-HA mice injected with the control eGFP-expressing vector (LvGFP) or the HA-expressing vector (LvHA) 4 weeks prior to irradiation. Analysis was performed either 4 or 6 weeks after T-cell–depleted bone marrow transfer in a single experiment.