Figure 6. Store-operated Ca²⁺ entry controls proliferation and functional maturation of Treg cell precursors.

(A) Flow cytometric analysis of thymic CD4⁺ Foxp3⁺CD25⁺ Treg cells in mice treated with IL-2-immune complexes (left) and frequency of Foxp3⁺CD25⁺ Treg cells in CD4SP thymocytes (right). Control, Stim1^fl/flStim2^fl/fl (dark grey bar); DKO, Stim1^fl/flStim2^fl/fl Vaν-iCre (light grey bar). Error bars denote mean ± SEM. n=3. *, P<0.05. (B) Enhanced proliferation of Foxp3⁺CD25⁺ Treg cells following injection of IL-2-anti-IL-2 immune complexes (black lines) but not immune complexes of IL-2 and isotype control monoclonal antibody (grey lines), in the same experiment as that shown in (A). (C) Amounts of Foxp3 expression in control (black lines) and DKO (grey lines) thymocytes following injection of IL-2-anti-IL-2 immune complexes. (D) Impaired suppressive function of the recovered Treg cells. Carboxyfluorescein succinimidyl ester (CFSE)-labeled responder T cells were cocultured with Treg cells treated with IL-2-anti-IL-2 immune complexes. Control, Stim1^fl/flStim2^fl/fl Foxp3^hCD2 or Stim1^+/+Stim2^+/+ Foxp3^hCD2 (closed circle); DKO, Stim1^fl/flStim2^fl/fl CD4-Cre Foxp3^hCD2 (open circle). Error bars denote mean ± SEM. n=3. (E) Amounts of Foxp3 expression on the recovered control (black lines) and DKO (grey lines) Treg cells. (F) Cell numbers of each population after 3 days of coculture. Control. Open circle; DKO, closed circle. (G) Flow cytometric analysis of the expression of inhibitory molecules on thymic Foxp3⁺CD25⁺ Treg cells in control (black lines) and DKO (grey lines), in the same experiment as that shown in (D). (H) Flow cytometric analysis of thymic Foxp3⁺CD25⁺ Treg cells in mice treated with IL-15-immune complexes. Control, Stim1^fl/flStim2^fl/fl; DKO, Stim1^fl/flStim2^fl/fl Vaν-iCre. Data are representative of two (B to H) and more than three (A) independent experiments. See also Figure S4.