Figure 3. A specific subset of Treg cells with the ability to reprogram is identified by labile Eos expression at rest.

(A) Thymic cells from Foxp3^GFP mice were disaggregated and incubated with cycloheximide (CHX) or vehicle control (“resting”) for 1 hr in vitro as described in Supplemental Procedures, then stained for Eos vs. cell-surface markers. Arrows indicate the Eos-labile subset. Representative of 15 similar experiments, using both Foxp3^GFP and WT B6 mice.

(B) Resting thymic Treg cells showed similar expression of Foxp3, Eos, intracellular CTLA-4 and surface CD25 between CD38⁺ (Eos-labile) and CD38^NEG (Eos-stable) subsets.

(C) Thymic cells from Foxp3^GFP-Thy1.1 donors were incubated with CHX for 1 hr to improve separation of sorting markers, then Treg cells sorted into Eos-labile (CD38⁺CD103^NEG) and Eos-stable (CD38^NEGCD103⁺) cohorts and transferred into B6 mice. Mice were challenged with OT-I + vaccine and VDLNs analyzed on day 4. Representative of 4 experiments.

(D) Splenic Treg cell subsets were sorted as in the previous panel, and transferred into Cd40lg^−/− hosts. Mice received CFSE-labeled OT-I + vaccine, and VDLNs analyzed on day 4. Representative of 5 experiments.

(E) Thymic cells from IL-6 deficient mice or WT B6 controls were tested in a 4 hr CHX assay, then stained for CD38 and CD69 as markers of the Eos-labile Treg cell subset. Examples of the Foxp3 gating for each strain are shown at left.

Each panel representative of at least 3 experiments, or as noted. See also Figure S3.