The intracellular trafficking of chemokine receptors targeted with chemokine fusions is dependent on clathrin-associated vesicles and G-protein signaling. (A). The dependency of clathrin-coated vesicles and G-protein signaling. iDCs were treated overnight with MIP3α-gp100, or MC148-gp100 in presence or absence of 0.4 M sucrose or 2.5 ng/mL pertussis toxin (PTX). Control iDCs were incubated with MC148-D-gp100 and gp100 protein alone. As effector cells, we used splenocytes from pmel-1 mice as described in “Materials and methods.” Specificity of effector cells was tested on iDCs pulsed with hgp100_25-33 peptide or control A20 peptide, or mixing with cells such as B16 melanoma, EL4, and A20. Endocytosed chemokine fusion protein is delivered to early/late endosomes and lysosomes. iDCs were treated with 0.1 μg/mL chemokine proteins fused with gp100 (C-D) or OFA-iLRP (shown in μg/mL, B) in the presence or absence of various pharmacologic inhibitors of intracellular organelle trafficking, such as (C) wortmannin (shown in μM) and NH₄Cl (nM), or (B,D) leupeptin, chloroquine, or brefeldin A (μM), to test stimulation of activated pmel-1 T cells. (D) Chemokine fusions are cross-presented and stimulate CD8⁺ T cells. iDCs were treated with 0.1 μg/mL MIP3α-gp100 in the presence or absence of titrated doses of lactacystin (shown in μM), a specific proteasomal inhibitor, to test stimulation of activated pmel-1 T cells. The experiment was repeated 3 to 6 times, and data are average of triplicate wells. Error bars represent the SD of the mean.