T cell proliferation assays

For proliferation assays the T cells were labeled with 1 μM carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen). Cells were plated at 5 × 10⁵ cells/well in 96-well flat-bottom plates and incubated for 5 days following addition of IL-2. The extent of T cell proliferation is proportional to the decrease in CFSE-fluorescence, with every cell division decreasing the CFSE fluorescence intensity of the daughter cells by 50%. The frequency of CFSE^low T cells was used as a measurement of total T cell proliferation, and flow cytometry gates were set using untreated control conditions as the baseline. For long-term IL-2 culture experiments IL-2 (30 U/mL) was added every 2–3 days and samples were harvested at the indicated time-points of the 31 days experimental period. Upon harvesting, cells were washed, labeled with specific mAbs (CD3, CD4, CD8, CD28 and CD151), and subjected to flow cytometric analysis.