Phenotypic and functional characteristics of CD4⁺FoxP3⁺ T_reg cells expanded in HIS-SGM3 mice. Leukocytes isolated from blood, liver, and spleen of HIS and HIS-SGM3 mice were analyzed for the expression of human CD45, CD3, CD4, FoxP3, CD25, CD45RA, CD45RO, and CTLA-4 to determine the phenotype of in vivo expanded human T_reg cells. (A) Representative histograms show CD45RO, CD25, and CTLA-4 expression on CD4⁺FoxP3⁻ (shaded area) and CD4⁺FoxP3⁺ (black outline) T cells from the blood of HIS and HIS SGM3 mice compared with isotype control (light black outline). To test their suppressive capacity, CD4⁺FoxP3⁺ T_reg cells from HIS-SGM3 mice were purified according to their CD25 expression (B) and cocultured with carboxyfluorescein succinimidyl ester (CFSE)–labeled autologous CD3⁺ T cells in the presence of anti-CD3/CD28. After 4 days of culture, the proliferation of human CD3⁺ T cells alone (C), in the presence of isolated CD4⁺CD25⁻ T cells (D) (ratio 1:1), or in the presence of CD4⁺CD25⁺ T_reg cells (E, ratio 1:1; F, ratio 2:1) was determined by flow cytometry. One of 4 individual experiments is shown.