CD4⁺ RTEs are characterized by distinct transcription factor and cytokine-receptor expression patterns. (A) CD4⁺ RTEs and MN T cells were differentiated under Th0 or Th1 conditions for 5 days and then stained for intracellular T-bet. Representative histograms show Th0 and Th1 CD4⁺ RTEs (shaded) or MN T cells (open) and MN T cells stained directly ex vivo (dashed line). MFI values were normalized by expressing RTE values as a percentage of the MN value, which was set to 1, and these compiled data are presented as mean ± SEM for 4 independent experiments with cells pooled from 3-4 mice per experiment and anti-CD3 concentrations ranging from 30-500 ng/mL. *P < .05, **P < .005. qRT-PCR for (B) Tbx21, (C) Il12rb1, and (D) Cxcr3 expression on ex vivo and Th0- or Th1-differentiated CD4⁺ RTEs and MN T cells. Data are presented as mean ± SD for 3 independent experiments with cells pooled from 2-4 mice per experiment for each target. *P < .05, **P < .005, ***P < .0005. (E) CD4⁺ RTEs and MN T cells were stained for IL-4Rα directly ex vivo or 5 days after stimulation under Th0 and Th2 conditions. Representative histograms show RTEs (shaded), MN T cells (solid line), and isotype control (dotted line). MFI of IL-4Rα for Th0 RTEs was 220 ± 77, and for Th0 MN T cells, it was 532 ± 109, and this difference was significant (P = .005). MFI of IL-4Rα for Th2 RTEs was 863 ± 69, and for Th2 MN T cells, it was 948 ± 92, and this difference was not significant (n.s.; P > .05). MFI data were compiled from 3 independent experiments with cells pooled from 2-3 mice per experiment. (F) qRT-PCR for GATA3 expression on purified ex vivo and 5 day Th2-differentiated CD4⁺ RTEs and MN T cells. Data are presented as mean ± SD for 3 independent experiments with cells pooled from 2-3 mice per experiment. *P < .05.