(A) Control or apoptotic MPs were stained with FITC-labeled CD44-Fc (30 μg/ml; blue line) and analyzed by FACS. Control staining was with FITC-labeled human IgG-Fc (red line). (B) MP preparations were pretreated with 50 μg/ml CD44-Fc chimeric protein or human IgG-Fc protein (hIgG-Fc). DCs were treated overnight with control or apoptotic MPs and subsequently poly I:C stimulated, and cytokine production was analyzed the following day. Data are representative of at least 3 independent experiments. The P value (unpaired Student’s t test) for indicated comparisons is shown. (C) DCs were pretreated with 50 μg/ml anti-CD44 blocking monoclonal antibody (BD Biosciences; clone 515) or mouse IgG isotype control. An alternative method involved pretreating plasma-derived MPs with CD44-Fc or hIgG-Fc control. DCs were then treated with MPs derived from uninfected or AHIV donors and subsequently stimulated with poly I:C. IL-12p70 secretion by DCs was analyzed the next day, and levels of IL-12p70 from AHIV MP-treated DCs were normalized to levels from DCs treated with MPs derived from uninfected donor plasma. Results are representative of at least 5 separate experiments with different control and AHIV donors. The P values (paired Student’s t test) for indicated comparisons are shown. (D) DCs were treated with microbead-bound anti-CD44, mouse IgG1 control, or microbeads only. DCs were subsequently poly I:C or LPS stimulated, and cytokine levels were assessed (IL-12p70 for poly I:C; IL-6 for LPS; LPS-stimulated DCs do not produce substantial IL-12p70). The P values (unpaired Student’s t test) for indicated comparisons are shown.