Chemokine or defensin fusion proteins are taken up, processed, and presented by APCs in vitro via chemokine receptor. Titrated amounts of protein (shown in ng/mL), 91-101 peptide, or an irrelevant peptide derived from A20 lymphoma V_L chain were incubated with BALB/c mice splenocytes (A,C-D). APCs were then washed, irradiated, and placed in culture with epitope-specific 7A10B2 T-cell line for 48 hours, and IFN-γ was assayed in culture supernatants. (B) The same assay as in panel A, except BM-derived iDCs were treated with recombinant proteins (100 ng/mL). Control treatment groups were iDCs or matured by overnight treatment with LPS (10 ng/mL). DCs were pulsed with 0.2 μg/mL 91-101 peptide or with 10 μg/mL irrelevant peptide. (C-D) Assessment of antigen presentation pathway of chemokine fusion proteins. Splenocytes were treated with 2 and 10 μg/mL 91-101 peptides or 100 ng/mL recombinant proteins alone or together with various inhibitors of intracellular trafficking and processing, such as brefeldin A (500 μM), and monensin (0.67 μg/mL), leupeptin (5 μg/mL) and chloroquine (50, 10, and 1 μM; Figure 3D), and lactocystin (50, 10, and 1 μM, Figure 3D). Representative of 5 (A), 6 (B), and 4 (C-D) consecutive experiments performed in duplicate wells. Results are presented as pg/mL IFN-γ ± SEM. ***P < .002 as compared MIP3αVL315 versus MIP3α+ sFv315 or mDF2βsFv315 versus sFv315, respectively (C).