In vitro maturation and functional analysis of CD4 SP T cells upon IL-7 deprivation and anti-CD3/CD28 co-stimulation in the absence of stromal cell environment. A, Analysis of T cell development kinetics upon IL-7 removal. Diagram of key events and markers for precursor T cell development is illustrated at top. Adult HPCs were co-cultured with stromal cells for 21 days and continued on with IL-7 (IL-7 present, i and iii) or transferred to culture without IL-7 (IL-7 deprived, ii and iv) for an additional 17 days. The cells were then transferred to 96 well U bottom plates and stimulated with anti-CD3/CD28 beads for an additional 14 days. Representative results of analysis of T cell surface markers two weeks after stimulation are shown with percentage of cells indicated in the flow graph quadrons. The percentages of CD4+CD3+TCRαβ+ cell population of four independent T cell development experiments are shown. B, Analysis of effector functions of the in vitro-derived CD4 T cells. PBMCs of healthy donors and the in vitro-derived CD4 T cells were stimulated using anti-CD3/CD28 beads in the presence of IL-2, IL-7 and IL-15 for two weeks. Expression of intracellular effector cytokine (IFNγ) and T helper functional markers (IL-4, IL-17) was detected after Brefeldin A treatment; unstimulated (left panel) or PMA and ionomycin stimulated (right panel) cells were analyzed by antibody staining and flow cytometry. Note that the small percentage of in vitro-derived CD8+ cells were not CD3+ or viable propagating cells.