title: The D-6 mouse monoclonal antibody recognizes the CD74 cytoplasmic tail.
abstract: The invariant chain (Ii; CD74) is a multifunctional protein of the immune system and a major player in the presentation of exogenous antigens to T cells. In the endoplasmic reticulum (ER), Ii assists the folding and trafficking of MHC class II molecules. In the present study, we characterized the recently commercialized D-6 monoclonal antibody (MAb) made against a polypeptide spanning the entire sequence of the p33 isoform of human Ii. Using transgenic mice expressing the human p35 isoform, we showed by flow cytometry that D-6 only slightly cross-reacts with mouse Ii in permeabilized splenocytes. Analysis of the human B lymphoblastoid cell line LG2 revealed that D-6 recognizes Ii only upon membrane permeabilization. Variants of Ii bearing specific mutations or deletions were transfected in human cells to map the D-6 epitope. Our results showed that this MAb binds to the N-terminal cytoplasmic domain of Ii and that the epitope was destroyed upon mutagenesis of the two leucine-based endosomal targeting motifs. Thus, D-6 cannot be used for rapid flow cytometric assessment of CD74 cell surface expression and would be ineffective as a drug conjugate for the treatment of hematological malignancies.
authors.affiliation: Laboratoire d'Immunologie Moléculaire, Département de Microbiologie, Infectiologie et Immunologie, Faculté de Médecine, Université de Montréal , Montréal, Canada .
authors.forename: Laetitia
authors.initials: L
authors.lastname: Genève
authors.forename: Catherine
authors.initials: C
authors.lastname: Gauthier
authors.forename: Jacques
authors.initials: J
authors.lastname: Thibodeau
mesh_terms: D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000276:immunology* / Q000378:metabolism; D058846:Antibodies, Monoclonal, Murine-Derived / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D000944:Antigens, Differentiation, B-Lymphocyte / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D002460:Cell Line; D018604:Epitope Mapping; D005434:Flow Cytometry; D000949:Histocompatibility Antigens Class II / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D058846:Antibodies, Monoclonal, Murine-Derived; D000944:Antigens, Differentiation, B-Lymphocyte; D000949:Histocompatibility Antigens Class II; C045084:invariant chain