Induction of HO-1 results in reduced T-cell proliferation and expression of MHC class II by APCs. (A) Expression of MHC class II by splenic APCs. Mice were treated twice with PBS (□, 11 mice) or hemin (750μM, ■, 7 mice) at a 7-day interval. Spleens were recovered 72 hours after the second treatment. The expression of MHC class II was measured using an LSRII flow cytometer (BD Biosciences) on dendritic cells (identified as CD11c⁺ and F4/80⁻ cells), macrophages (F4/80⁺ and CD11c⁻ cells), and B lymphocytes (B220⁺ and F4/80⁻ and CD11c⁻ cells). Geometric mean fluorescence intensities (MFI) were measured for each mouse on each individual cell population with FACSDiva software Version 5.0.1 (BD Biosciences) and are expressed for each mouse relative to the mean MFI calculated for all PBS-treated mice. Data represent mean ± SD. Statistical significance in the differences were assessed using the Mann-Whitney test. The expression of MHC class II by APCs after the first and third series of treatments is shown in supplemental Figure 2. (B) Proliferation of splenic T cells. Spleens from PBS- (□) and hemin-treated mice (■) were recovered 72 hours after the second treatment. Splenocytes (1.25 × 10⁶ cells/mL) were incubated for 72 hours alone or with 2 μg/mL ConA. Cell proliferation was measured by incorporation of tritiated thymidine (0.5 μCi/well) for an additional 16 hours, and is expressed as counts per minute (cpm; left panels). Stimulation indexes were calculated for the splenocytes of each mouse by dividing the cpm obtained in the presence of ConA by that obtained in medium alone (right panel). Horizontal bars represent mean ± SD. Significance of differences was statistically assessed using the Mann-Whitney test. (C) Levels of regulatory T cells on HO-1 induction in FVIII-deficient mice. Levels of T cells positive for CD4, CD25, and FoxP3 expression were analyzed by flow cytometry in spleens from mice treated twice with PBS (□) or hemin (■) at a 7-day interval, 72 hours after the second treatment. Horizontal bars represent the arithmetic means. All data are from 2 independent experiments.