In vitro effects of CSA, RAPA, and MPA on Treg cells. In vitro effects of CSA, RAPA, and MPA on allostimulated Treg cells with respect to suppressor function (A), FoxP3 expression (B,D), and IL-2 levels in the primary coculture (C). (A) Fresh isolated C57B/6 Treg cells (CD4⁺CD25^highH-2k^b+) were incubated with Balb/c APCs (CD11c H-2k^d) with CSA (100 ng/mL), RAPA (10 ng/mL), or MPA (50 ng/mL). Treg cells were reisolated by sorting for CD4⁺CD25^highH-2k^b+ cells after 72 hours of primary coculture. Reisolated Treg cells were then incubated for 96 hours with γ-irradiated (30 Gy) APCs (CD11c⁺H-2k^d+) and CFSE-labeled T_CONV cells (CD4⁺CD25^-H-2k^b+Thy-1.2⁺), with each population 2 × 10⁵ cells. Cell division was monitored by levels of CFSE dilution. The congenic markers Thy-1.1 and Thy-1.2 were used to distinguish between Treg and T_CONV cells, respectively. Histograms show the FACS profile of CFSE⁺ T_CONV cells. Numbers of events in each cell division (n) are indicated below the respective peak. The percentage of undivided CD4⁺CFSE⁺ cells in each culture condition is indicated next to the right upper corner of the respective histogram. One representative experiment of 4 is presented. Allostimulated Treg cells suppress alloantigen-driven expansion of CFSE⁺ T_CONV (^*) significantly more strongly than autostimulated Treg cells (+; P = .027). CSA-exposed Treg cells are still suppressive, but significantly less as compared to RAPA (#P = .031) and addition of IL-2 (50 IU/mL) to the activation culture restores Treg function partially (‡; P = .037). (B) Relative FoxP3 mRNA expression level in Treg cells (H-2k^b+) exposed to allogeneic APCs (H-2k^d+) alone or in conjunction with RAPA (10 ng/mL), MPA (50 ng/mL), CSA (100 ng/mL), or CSA (100 ng/mL) plus IL-2 or exposed to autologous APCs (H-2k^d). The increase of FoxP3 expression in Treg cells during allostimulation is reduced by CSA addition and restored by IL-2. Data represent means ± SD of triplicates (^**P = .004; ^***P = .008; ^****P = .007). (C) IL-2 concentrations measured by ELISA in supernatants from the following cocultures are depicted (y-axis): Treg cells (H-2k^b) incubated with APCs (H-2k^d) and CSA (▪), RAPA (▵), or MPA (•)at different concentrations (x-axis) after 72 hours. CSA but not RAPA or MPA reduces IL-2 levels in the Treg activation culture in a dose-dependent manner. (D) Histograms depict the intracellular expression profile of FoxP3 in live-gated Treg cells exposed to medium, CSA (100 ng/mL), RAPA (10 ng/mL), or MPA (50 ng/mL) for 72 hours (filled histogram, unstained cells; open histogram, FoxP3 staining). The mean fluorescence intensity (MFI) for FoxP3 expression is significantly lower in Treg cells reisolated from CSA (213 ± 24) as compared to cultures containing RAPA (412 ± 42) or MPA (392 ± 31; P < .05).