MDPs educated by nnLNSCs (regMΦ) are poorly immunogenic but T-cell immunosuppressive in vitro. (A) regMΦ control T-cell proliferation. Cells were incubated as indicated at 37°C for 3 days before the total numbers of DAPI⁻CD45.1⁺CD4⁺Vα2⁺ OTII cells was determined (mean ± SD of pooled data from 2 independent experiments). (B) Same as panel A; CSFE dilution of OTII cells labeled with the dye prior incubation (circles with numbers indicate conditions depicted in panel A). (C-D) regMΦ do not dampen early activation of OTII cells. Expression of the activation-related markers (C) CD25 and (D) CD69 on OTII cells after 24 hours incubation under experimental conditions shown in panel A were assessed by flow cytometry. Numbers adjacent to gated areas indicate percentage of gated cells. DAPI⁻CD45.1⁺CD4⁺Vα2+ T cells are shown. Data are representative of at least 3 independent experiments. (E-F) The influence of regMΦ on IFNγ and IL-2 levels in the coculture supernatants. After 3 days coculture, IFNγ and IL-2 present in the supernatant were analyzed by cytometric bead array. (G) nnLNSCs are not directly T-cell immunosuppressive. gp38⁺CD45⁻ and gp38⁻CD45⁻ nnLNSCs were sorted from peripheral or meseneric LNs by flow cytometry and 8 × 10³ stromal cells were seeded per well. One day later, 2 × 10⁵ MACS-isolated CD4⁺ T cells were added and stimulated with anti-CD3/CD28 dynabeads. After 3 days of culture [³H]-thymidine was added. The incorporation of [³H]-thymidine for 15 hours is shown. (E-G) Values represent mean ± SEM of 3 wells from 1 representative of 2 independent experiments.