CD4⁺ T cells proliferating in presence of regMΦ are T-cell immunosuppressive in vitro and in vivo. (A) CD4⁺ T cells proliferating in the presence of regMΦ gain T-cell immunosuppressive capacity. CD45.1⁺CD4⁺ T cells (2 × 10⁵) were stimulated with anti-CD3/CD28 dynabeads in the presence of 4 × 10⁴ regMΦ. Five days later, CD4⁺ T cells were again sorted by MACS to obtain CD45.1⁺CD4⁺ T cells. Together with anti-CD3/CD28 dynabeads these cells were cocultured with MACS-purified, CFSE-labeled, CD45.2⁺CD4⁺ T cells at the ratios indicated. Five days later, proliferation of CD45.2⁺CD4⁺ T cells was analyzed by flow cytometry. Cells were gated on the DAPI⁻CD45.²⁺CD4⁺ population. Data are representative of at least 3 independent experiments. (B) Expression of Foxp3 by CD4⁺ T cells cultivated in the presence of regMΦ. CD4⁺ T cells (2 × 10⁵ C57BL/6) were stimulated with anti-CD3/CD28 dynabeads in the presence of 8000 or 4 × 10⁴ regMΦ as indicated. Five days later, cells were stained intracellularly with anti-Foxp3 mAb. Gates were set on CD4⁺ cells. Numbers adjacent to gated areas indicate the percentage of Foxp3⁺CD4⁺ Treg. Number in circles indicate experimental conditions described in panel C. (C) Statistical analysis of (B; mean ± SEM, n = 3). Similar results were obtained in another 2 experiments. (D-E) CD4⁺ T cells from mice immunized with OVA-maDCs in the presence regMΦ transmit antigen-specific immune suppression in vivo. (D) Experimental setup of in vivo immune tolerance induction. At day 0 syngeneic C57BL/6 mice intravenously received, OVA-maDCs, OVA-regMΦ, or OVA-maDCs together with regMΦ. At day 7, CD4⁺ T cells were isolated from LNs and spleen of different groups and 5 × 10⁶ cells/mouse were adoptively transferred into another cohort of naive C57BL/6 recipients. At day 8 these mice were immunized with OVA or KLH in CFA, and at day 15 mice were bled. Serum IgG1 levels against OVA (E) or KLH (F) were measured by ELISA. Serum was diluted serially as indicated (values represent mean ± SEM; 3 mice per group were analyzed). Similar data were obtained in a second experiment.