Figure 5. Differentiation into Id3^lo Treg cells is promoted by IL2 signaling.

(a) Id3^hi Treg cells (CD4⁺CD25⁺CD45RB^lo) purified from naive Id3^GFP/+ or Ifnar1^-/-Id3^GFP/+ mice (Thy1.2) were transferred into wt mice (Thy1.1). One day after transfer, mice were left untreated or infected with 200 pfu LCMV WE. The percentage of Id3^lo Treg cells among transferred cells (Thy1.2⁺) 7 days post-transfer in spleens of naive or LCMV WE infected host mice. Results from 1-3 mice per experiment were pooled and the mean value from each of 3-5 independent experiments is presented by the symbols. The horizontal lines are the mean of all experiments. ns = not significant (unpaired Student’s t test). (b) Id3 and CD44 expression in splenic Treg cells (CD4⁺CD25⁺) of PBS or IL2/IL2mAb treated Id3^GFP/+ mice at day 5 after treatment. (c) Total cell number of CD44^loId3^hi, CD44^hiId3^hi and CD44^hiId3^lo Treg cells (CD4⁺CD25⁺) in spleens of PBS or IL2/IL2mAB treated mice. Mean ± SEM from at least 4 independent experiments are shown. ^*p < 0.05, ^**p < 0.01 (unpaired Student’s t test). (d) Fold increase in CD44^loId3^hi, CD44^hiId3^hi and CD44^hiId3^lo Treg cells in IL2/IL2mAb treated mice compared to PBS treated mice. Mean ± SEM from at least 4 independent experiments are shown. ^**p < 0.01; ns = not significant (unpaired Student’s t test). (e-h) CD44^hiId3^hi and CD44^hiId3^lo Treg cells (CD4⁺CD25⁺) from IL2/IL2mAb treated Id3^GFP/+ mice (Thy1.2) were labeled with a proliferation dye and transferred into wt mice (Thy1.1). Transferred cells (Thy1.2⁺) were analyzed 7 days post-transfer in the spleens after PBS or IL2/IL2mAB treatment. (e) Absolute numbers of recovered Thy1.2⁺ cells. (f) Representative analysis of Id3 and CD44 expression (small insets) and proliferation of Thy1.2⁺ cells recovered after transferring CD44^hiId3^hi (blue) or CD44^hiId3^lo (orange) Treg cell populations. (g) Id3 and CD44 expression of recovered Thy1.2⁺ cells in spleens of PBS or IL2/IL2mAB treated mice that had received CD44^hiId3^hi Treg cells (left). Representative proliferation analysis of recovered phenotypically stable Thy1.2⁺CD44^hiId3^hi (blue) or newly generated Thy1.2⁺CD44^hiId3^lo (orange) cells in spleens of PBS or IL2/IL2mAB treated mice that had received CD44^hiId3^hi Treg cells (right). (h) Representative analysis of Id3 expression versus proliferation of recovered Thy1.2⁺ cells in spleens of PBS or IL2/IL2mAB treated mice that had received CD44^hiId3^hi Treg cells.