c-Myb contributes Th2 cell development and maintenance through regulation of GATA-3 expression. (A) Normal human peripheral blood CD4⁺ cells were transduced with lentivirus constructs expressing c-myb shRNA and GFP. Cells expressing GFP were sorted from nonexpressing cells and then further selected using MACS MicroBeads for CD4⁺CD45RO⁻ cells. These were then cultured under Th1- or Th2-promoting conditions for 7 days. c-myb, GATA-3, T-bet, IL-4, and IFN-γ expression was measured by quantitative RT-PCR before and after stimulation. (B) Intracellular IL-4 levels in the cells analyzed in panel A. Before flow cytometric analysis, the cells were cultured for an additional 6 hours with phorbol myristate acetate (50 ng/mL), calcium ionomycin (500 ng/mL), and a protein transport inhibitor (BD GolgiStop). The histogram shows intracellular IL-4 in cells stimulated under Th2 cell-promoting conditions. Dotted line represents cells transfected with control shRNA; black line, cells transfected with c-myb shRNA; and gray line, control cells stained with mouse IgG-phycoerythrin (antibody control). (C) CD4⁺CD45RO⁺ (effector/memory) cells were transduced with control, or c-myb shRNA expression lentivirus and then stimulated with CD3/CD28 beads and IL-2 for 5 days. Poststimulation mRNA expression levels were measured by quantitative RT-PCR. Data are representative of at least 2 independent experiments. *P < .05.