Preferential STAT-5 pathway usage in Tregs predicted resistance to mTOR inhibition. (A) The amount of phosphorylated p70S6, 4E-BP1, and STAT-5 was quantified by phospho-flow analysis within Tconv cells (CD4CD25⁻, left panel) and Tregs (CD4CD25^high, right panel). T cells were isolated after 48 hours of IL-2 (100 IU/mL) stimulation. The amount of phosphorylated protein as measured in MFI increased significantly for p70S6, 4E-BP1 in Tconv (4 vs 172, and 11 vs 94, P < .001), but not in Tregs. MFI for phospho-STAT5 increases in Tregs in response to IL-2 (9 vs 103, P < .001). (B) Total protein expression in naive CD4⁺CD25^high or CD4⁺CD25⁻ T cells isolated from C57BL/6 mice. Expression of p70S6 and mTOR qA increased in Tregs compared with CD4⁺CD25⁻ T cells. (C) The amount of phosphorylated p70S6, 4E-BP1, and STAT-5 is quantified by phospho-flow analysis within Tconv cells (CD4CD25⁻, filled gray histogram) and Treg cells (CD4CD25^high, black solid line) both on C57BL/6 background. T cells were isolated after 48 hours of IL-2 (100 IU/mL) and irradiated (30 Gy, γ-irradiation) allogeneic CD11c⁺ APC (BALB/c) stimulation. The amount of phosphorylated protein as measured in MFI increases significantly more in Tconv compared with Treg for p70S6, 4E-BP1 (343 vs 16, and 1035 vs 10, P < .001) in response to alloantigen and IL-2. Addition of RAPA (10 ng/mL) abrogates phosphorylation of p70S6 and 4E-BP1 (right panel). MFI for phospho-STAT5 remains constant in Tregs when RAPA is added (29 vs 32, NS).