IL-7–induced survival and cell-cycle entry of RTEs can be distinguished by the dose and duration of rIL-7 stimulation. (A) CD4⁺ RTEs were isolated from UC blood by negative selection and cultured in the presence of recombinant IL-7 at doses ranging from 0.01 to 100 ng/mL. After 5 days of culture, cell viability was monitored by propidium iodide (PI) labeling and the percentages of dead cells are indicated in each histogram. Cell-cycle progression was monitored by expression of the Ki67 proliferation marker and DNA content, and the direct proliferation of daughter cells was monitored by dilution of the CFSE fluorochrome. In these latter assays, analyses were performed after gating on live cells. The percentages of cells in the indicated gates are noted. FSC indicates forward scatter. (B) CD4⁺ RTEs from UC blood were cultured for 120 hours as described above, but the time of exposure to rIL-7 was varied. Following either a 1-hour, 24-hour, or 72-hour culture in the presence of rIL-7 (10 ng/mL), cells were washed and incubations were then continued in the absence of rIL-7 for the remainder of the 120-hour period. Cell viability, cell-cycle progression, and division were assessed at 120 hours as described above. Results are representative of data obtained in 8 independent experiments.