Stable Foxp3 Expression in CD25⁺CD4⁺ Tregs

<p>CD25⁺CD4⁺ Tregs were isolated ex vivo from pooled spleen and LN single-cell suspensions, labeled with CFSE, and then transferred into syngenic 8-wk-old recipients (2 × 10⁶/mouse). Before transfer, sorted cells were analyzed for intracellular Foxp3 expression by FACS. Fourteen days after transfer, splenocytes of recipient mice were stained for CD4, CD25, and Foxp3, and analyzed by FACS. About 0.1% of total splenocytes and, accordingly, 3% of total Foxp3⁺ splenocytes were donor derived (CFSE⁺). Representative dot and histogram plots from four independently analyzed mice were selected. Numbers display frequency of cells within indicated populations. Comparable results were obtained for cells isolated from peripheral or mesenteric LNs (unpublished data). The bars in the left and right graphs indicate the marker gate for Foxp3⁺ cells. The box in the middle graph indicates the region that was used to gate for CD4⁺CFSE⁺ cells. These gated cells (CFSE⁺) are depicted in the right graph.