Generation of a panel of monoclonal antibodies against atypical chemokine receptor CCX-CKR by DNA immunization

IntroductionChemokines are regulated by a family of ‘atypical’ chemokine receptors, D6, DARC and CCX-CKR, each of which efficiently internalizes its cognate chemokine ligands. Development of monoclonal antibodies (MAbs) that would recognize CCX-CKR on the cell surface will be helpful to identify primary CCX-CKR-expressing cell types and analyze the fate of CCX-CKR after ligand binding to the receptor.MethodsWe generated IgG MAbs recognizing the cell-surface CCX-CKR by DNA immunization using a molecular adjuvant, and analyzed the epitope recognized by the MAbs. Then, the reactivities of the MAbs with CCX-CKR-transfected cells, and also hepatocytes and hepatic tumor lines were evaluated. Finally, we also tested the ligand-like activities of the MAbs, namely, induction of internalization of CCX-CKR by the MAbs.ResultsA panel of MAbs reacting with CCX-CKR expressed on the cell surface was prepared. The panel was a small one, consisting of only ten MAbs, but was rich in terms of diversity of the Ig isotypes and of the epitopes. Epitope analyses revealed that all the 10 MAbs recognized at least three different, although very close, peptide structures of the N-terminal domain. Three MAbs, namely, 2F11, 13E11 and 14F10, were selected to represent the panel. All of the MAbs were applicable for flow cytometry and immunoflurescent assays and immunoprecipitation. The reactivity of the 2F11 MAb was also confirmed by western blotting. Endogenous expression of CCX-CKR on human hepatocytes and hepatic tumor cell lines was demonstrated using the 13E11 MAb. Interestingly, binding of the 13E11 MAb with B300-19 cells expressing CCX-CKR resulted in induction of CCX-CKR internalization.DiscussionThis panel of MAbs may be expected to prove valuable for further study of the functions of this silent chemokine receptor, including those related to the homeostasis of lymphoid cells, and to the growth and metastasis of hepatic cancer.