CR3 ligation impairs T-cell priming capacity of DCs. DCs were treated with the indicated ApoS. (A) DCs were further cultured in media alone (○) or exposed to MCM-mimic (▴, ▪). After 40 hours, DCs were loaded with titrated concentrations of MP peptide, washed, fixed, and added to an MP-specific CD8⁺ T-cell clone. The number of IFNγ spot-forming cells (SFCs) detected by ELISPOT assay is shown. (B) KLH and MCM-mimic were added to DC cultures after treatment with the indicated ApoS. After 40 hours, DCs were washed, irradiated, and added to naive CD4⁺ T cells. At 10-day intervals, T cells were restimulated with the same DC subsets for a second and third time. Ten days after each restimulation (ie, days 20 and 30, respectively), T cells were added to KLH-pulsed, MCM-mimic–matured DCs, and IFNγ production was measured by ELISPOT assay. Number of responding cells per 30 000 viable seeded T cells is shown. In panels A and B, error bars represent standard deviation (SD) of triplicate wells in ELISPOT plate. (C) After exposure to ApoS, DCs were stimulated for 40 hours with MCM-mimic or LPS and then cultured with allogeneic CFSE-labeled naive CD4⁺T cells. Proliferation of T cells was measured after 6 days by dilution of CFSE dye by FACS. Numbers denote percent of proliferating cells. (D) In similar cultures as in panel C, supernatants were collected 6 days after the first or second stimulation with immature (/) or mimic-exposed DCs (mimic), and IFNγ content was measured by ELISA. The left panel shows the results of an individual experiment and the right the average inhibition with SD of multiple experiments. The production of IFNγ by control (Ig-ApoS–ligated) DCs was considered the 100% value, and percent inhibition in the CD11b-ligated DC groups (with or without mimic) was calculated. (E) Similar cultures as in panel D were monitored for the binding of annexin V to T cells by FACS. The fold increase over the control Ig-ApoS group is shown. Representative experiments from at least 2 or 3 donors are shown in all panels.