title: Spontaneous transgenesis of human B lymphocytes.
abstract: DNA can cross the cell membrane by natural means, but the functional relevance of this phenomenon has not been fully elucidated. Here, we analyzed spontaneous transgenesis of human B cells using plasmid DNA coding for a functional immunoglobulin (Ig) heavy chain gene under the control of a B-cell-specific promoter. Using polymerase chain reaction (PCR), reverse transcriptase-PCR, and flow cytometry in combination, spontaneous transgenesis was documented in Burkitt's lymphoma cell lines, Epstein-Barr virus-transformed cell lines, and peripheral blood B lymphocytes of the mature naïve phenotype (IgM(+)/IgD(+)/CD27(-)). By immunoelectron microscopy, the internalized DNA was seen in the lysosomes/late endosomes and in the cytosol proximal to the nucleus. Importantly, spontaneously transgenic B cells processed and presented to major histocompatibility complex (MHC)-restricted T lymphocytes a peptide expressed in the transgenic product. This is the first demonstration that primary B lymphocytes possess a program for the spontaneous internalization of DNA, which in turn imparts the cell with new immunological functions. As spontaneous transgenesis is obtained using a nonviral vector, does not require prior cell activation, and is not associated with chromosomal integration, the findings reported here open new possibilities for genetic manipulations of mature naïve B lymphocytes for therapy and vaccination.
authors.affiliation: The Department of Medicine and Cancer Center, University of California, San Diego, La Jolla, CA 92093-0837, USA.
authors.forename: G
authors.initials: G
authors.lastname: Filaci
authors.forename: M
authors.initials: M
authors.lastname: Gerloni
authors.forename: M
authors.initials: M
authors.lastname: Rizzi
authors.forename: P
authors.initials: P
authors.lastname: Castiglioni
authors.forename: H-D
authors.initials: HD
authors.lastname: Chang
authors.forename: M C
authors.initials: MC
authors.lastname: Wheeler
authors.forename: R
authors.initials: R
authors.lastname: Fiocca
authors.forename: M
authors.initials: M
authors.lastname: Zanetti
mesh_terms: D001402:B-Lymphocytes / Q000276:immunology* / Q000648:ultrastructure; D045744:Cell Line, Tumor; D004247:DNA / Q000008:administration & dosage*; D005434:Flow Cytometry; D015870:Gene Expression; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007167:Immunotherapy / Q000379:methods; D016253:Microscopy, Immunoelectron; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D014162:Transfection / Q000379:methods; D019076:Transgenes
chemical_list: D007143:Immunoglobulin Heavy Chains; D004247:DNA