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 þ /IgD þ /CD27 À ). 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.

T cell immunity using transgenic B lymphocytes

“…2Bd). We conclude that B cells susceptible to spontaneous transgenesis are mature B cells with the IgM ϩ ͞IgD ϩ phenotype consistent with what we observed in human B lymphocytes (26). Because T cell activation in vitro requires two signals, a specific signal by the peptide͞MHC complex and a second signal through costimulatory molecules (3, 30), we also analyzed the expression of MHC class II and class I, CD40 and CD86, on spontaneously transfected cells.…”

“…For instance, nuclear DNA released from dying B lymphocytes in germinal centers during a primary response may be internalized by neighboring B lymphocytes that are escaping death or terminal differentiation. Similarly, viral DNA from dying infected cells could also be internalized in B cells and transcribed as the type of promoter is not a limiting factor (26). Because a very low number of transgenic B lymphocytes is sufficient for T cell priming, the mechanism described here could play a role in idiotype regulation of B cells or the amplification of antigen presentation during a primary response with a contribution to the establishment of immunological memory.…”

“…Typically, murine spleen lymphocytes were rendered transgenic for plasmid DNA coding for transgenes under the control of an Ig-specific promoter by incubation in PBS without Ca 2ϩ ͞Mg 2ϩ for 60 min (26). To induce antigen-specific CD4 and CD8 T cell responses, we used heavy chain genes engineered to express heterologous T cell determinants in the complementarity-determining regions (CDRs) (27).…”

KDEL-Retained Antigen in B Lymphocytes Induces a Proinflammatory Response: A Possible Role for Endoplasmic Reticulum Stress in Adaptive T Cell Immunity

“…To this end, we used a model system, developed in this laboratory, whereby adaptive T cell responses can be induced using B lymphocytes programmed as APCs by spontaneous transgenesis of plasmid (p)DNA (19,20). In this model of immunization, pDNA codes for the H chain of a secretory Ig (IgH) molecule.…”

Protection against renal disease in (NZB × NZW)F1 lupus‐prone mice after somatic B cell gene vaccination with anti‐DNA immunoglobulin consensus peptide

“…The somatic B cell gene transfer technique has been described in detail elsewhere (11,12). Briefly, single spleen-cell suspensions were prepared from mice under aseptic conditions, and B cells were sorted for enrichment (Ͼ96%) using anti-CD19 magnetic beads (Miltenyi Biotec, Auburn, CA) on a VarioMACS separator (Miltenyi Biotec).…”