Evaluation of DCs that have been ligated by hybrid ApoS targeting both CR3 and αvβ5. (A) To evaluate the ability of hybrid αvβ5/CD11b-ApoS to bind individual receptors to the same extent as “single” ApoS, we cocultured HEK293 cells that express αvβ5, but not CD11b (as assessed by FACS staining [left]), with single or hybrid ApoS for 2 hour at 4°C at different HEK293/ApoS ratios. Importantly, the hybrid αvβ5/CD11b-ApoS bound HEK293 cells to same extent as the “single” αvβ5-ApoS (right). (B,C) DCs were exposed to either αvβ5-, CD11b-, Ig-, or αvβ5/CD11b-ApoS for 2 hours and then cultured either in media alone or in the presence of LPS for 40 hours. (B) Maturation of DCs was assessed by determining the expression of CD83, CD80, and CD86 by FACS. (C) Supernatants of DC cultures were analyzed by ELISA for the presence of TNFα, IL1β, and IL6. A representative experiment is shown. Error bars represent SD of duplicate test wells. (D) ApoS-pretreated DCs were cocultured with naive CFSE-labeled CD4⁺ T cells at a 1:30 ratio. After 6 days the proliferation of T cells was assessed by analyzing the dilution of CFSE dye in T-cell membranes by FACS. (Left) Immature ApoS-exposed DCs. (Right) MCM-mimic–treated ApoS-exposed DCs. Averages and SDs of 3 experiments are shown. Stars represent P < .05 in a paired Student t test.