Ex vivo characterization of circulating CD4⁺CRTH2⁺ T cells. (A) CRTH2⁺ cells were enriched to more than 70% by positive selection from peripheral-blood CD4⁺CD45RO⁺ T cells (see “Materials and methods”). Shown is their frequency in the parental PBMC preparation and in the enriched fraction in a typical experiment. (B) CRTH2⁺ cell–enriched preparations consistently produced higher levels of IL-4 and lower levels of IFN-γ than CD4⁺CD45RO⁺CRTH2⁻ cells in response to stimulation with PMA and ionomycin. Mean ± SEM cytokine production from 6 (IL-4) or 5 donors (IFN-γ). **P < .005. (C) GATA-3 expression in CRTH2-sorted peripheral-blood CD4⁺CD45RO⁺ T cells. Real-time RT-PCR and flow cytometry were used for quantitative analysis of GATA-3 expression at the RNA and protein level. The top panels show the mean (± SEM) GATA-3 and T-bet RNA levels in sorted preparations from 6 donors. The bottom panel shows GATA-3 flow cytometric determination in CRTH2⁺ (solid line) and CRTH2⁻ cells (dotted line) from an individual donor (filled histogram: isotype control) and, inset, the mean ± SEM percent GATA-3⁺ cells in CRTH2⁺ (filled bar) and CRTH2⁻ preparations (empty bar) in 3 donors. *P < .05.