A. Tregs were enriched by CD25 selection and stained for Foxp3 pre- and post-expansion with irradiated, anti-CD3 Ab loaded KT64 CD86 aAPCs. B. Overall relative expansion of T cells from day 0 to day 20 in the presence or absence of rapamycin (100ng/ml). A. and B. are from the same experiment and are representative of data collected using 9 different donors. C. Enriched Tregs were expanded with rapamycin (100 ng/ml) and the percentage of Foxp3 expressing cells at the end of culture was determined by flow cytometry. Data are compiled from 15 independent experiments. D. Enriched Tregs were expanded without rapamycin and the percentage of Foxp3 expressing cells at the end of culture was determined by flow cytometry. Data are compiled from 9 independent experiments. Correlation between Foxp3 expression and relative cell expansion was determined by nonlinear regression analysis.

A. Diagram of Foxp3 expression vectorsFreshly purified CD4⁺CD25⁺ Tregs (left panel) and CD4⁺CD25⁻ T effector cells (right panel) were stained with anti-Foxp3 Ab (shaded histograms) or an isotype control Ab (open histograms) and analyzed by flow cytometry. B. 28S RNA-normalized mRNA levels of pim 2 were measured from freshly isolated CD4⁺CD25⁺ and CD4⁺CD25⁻ T lymphocyte populations as determined by quantitative RT-PCR analysis. The data plotted as the mean ± SD of triplicate determinations from an individual donor. C. Western blot detection of pim 2 and actin (loading control) from freshly isolated CD4⁺CD25⁺ and CD4⁺CD25⁻ T cell populations shown in A. Lysates correspond to 5 × 10⁵ cell equivalents. Data is representative of 3 independent experiments.

A. Diagram of constructs used in this study. B. CD4⁺CD25⁻ T cells were activated with CD3/CD28 Ab coated beads and transduced with lentiviral vectors expressing either YFP-2A-Foxp3 or YFP-2A-Foxp3Δ2. The correlation between Foxp3 and YFP expression was determined by flow cytometry. C. YFP-2A-Foxp3, YFP-2A-Foxp3Δ2 and untransduced CD4⁺CD25⁻ T cells were re-stimulated with PMA (50 ng/ml) and ionomycin (500 ng/ml) for 4 hours and intracellular IL-2 expression was detected by flow cytometry. D. 28S-normalized mRNA levels of pim 2 from either YFP-2A-Foxp3, YFP-2A-Foxp3Δ2, or GFP transduced CD4⁺CD25⁻ T cells shown in A. was determined by quantitative RT-PCR analysis. The cells were harvested 17 days after transduction. The data are plotted as the mean ± SD of triplicate determinations from one donor. E. CD4⁺CD25⁻ T cells were activated with CD3/CD28 Ab coated beads and transduced with lentiviral vectors expressing YFP-2A-Foxp3, YFP-2A-Foxp3Δ2 or GFP. 3 days post-transduction the percentage of transduced cells was determined and half the culture was placed in rapamycin (100 ng/ml) containing medium. After an additional 7 (middle panels) or 14 (bottom panels) days of culture, the percentage of transduced cells was measured by flow cytometry. Data is representative of 3 independent experiments.