Mutant analyses of DC-HIL-Fc function. (A) Protein structures of DC-HIL-Fc wild-type (WT) and mutants are represented schematically. Extracelluar domains (ECDs) of mutants, RAA (replacement of RGD sequence with RAA; ▴) and deletion mutants lacking PRR (amino acid [aa] 301-334) and PKD^230-355 were linked to a Fc portion of hIgG and produced in COS-1 cells. (B) After purifying mutant DC-HIL-Fc proteins, a small aliquot (2 μg/lane) was run on SDS-PAGE and then stained with Coomassie Blue to visualize protein bands. (C) T-cell activation. Highly purified DC-HIL-Fc WT or mutants (5 μg/mL each) and anti-CD3 Ab (increasing doses) were coated on microwells for culture with CD4⁺ T cells for 2 days and pulsed with ³H-thymidine for 20 hours. Results are shown as mean values ± SDs. (D) Binding of DC-HIL mutants to T cells. Activated CD4⁺ T cells were incubated with WT and mutants of DC-HIL (10 μg/mL) and analyzed for binding by FACS. Histograms of T cells stained with hIgG (filled) and a mutant (open) are overlaid. Data (C-D) shown are representative of 3 experiments.