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Naturally occurring polyreactive anti-DNA mAbs derived from a nonimmunized (NZB ؋ NZW)F 1 mouse with spontaneous lupus erythematosus penetrated and accumulated in the nuclei of a variety of cultured cells. These mAbs and their F(ab)2 and Fab fragments, covalently coupled to f luorescein, peroxidase, or a 15-mer polynucleotide, also translocated to the cell nuclei. A 30-amino acid peptide corresponding to the combined sequences of the complementary-determining regions 2 and 3 of the heavy chain variable region of one mAb was able to penetrate into the cytoplasm and nucleus of cells of several lines. This peptide recognized DNA and was strongly polyreactive. Streptavidin-peroxidase conjugates complexed with the N-biotinylated peptide were rapidly translocated into cells. Similarly, peroxidase or antiperoxidase polyclonal antibodies covalently coupled to the N-cysteinylated peptide through an heterobifunctional maleimide cross-linker were also rapidly internalized and frequently accumulated in nuclei. The peptide carrying 19 lysine residues at its N-terminal was highly effective in transfecting 3T3 cells with a plasmid containing the luciferase gene. Thus, penetrating mAbs and derived peptides are versatile vectors for the intracellular delivery of proteins and genes.A long time ago, it was reported that human IgG from systemic lupus erythematosus patients with high titers directed against nuclear ribonucleoproteins and/or DNA were able to penetrate into living cells and to reach the nucleus (1). More recent studies of murine anti-DNA autoantibodies confirmed these observations and disclosed that different penetrating antibodies exhibited diverse behaviors and characteristics (2-7). In this study, we prepared several penetrating IgG anti-DNA mAbs from the spleen of a (NZB ϫ NZW)F 1 lupus mouse and examined their specificities and their abilities to act as vectors of haptens, proteins, polynucleotides, and plasmids. MATERIALS AND METHODSMice and Cell Lines. (NZB ϫ NZW)F 1 hybrids and BALB/c mice were bred in the Institut Pasteur animal facilities. Cells used were from different species and from various tissues as follows: PtK2 (Potoroo kidney fibroblasts) or CCL-39 (hamster lung), 3T3 (mouse embryo fibroblasts), and HEp-2 (human larynx carcinoma). All cells were from the American Type Culture Collection and were cultured in RPMI 1640 medium (or in DMEM for CCL-39) containing 10% heat-inactivated calf serum and supplemented with L-glutamine, sodium pyruvate, nonessential amino acids, and antibiotics (complete culture medium) at 37°C in a humidified atmosphere of 5% CO 2 /95% air.mAbs. Spleen cells from a 9-month-old nonimmunized (NZB ϫ NZW)F 1 mouse were fused with P3.X63Ag8 myeloma cells by the method of Köhler and Milstein (8), and hybridomas were selected in hypoxanthine/azaserine medium. Supernatants were tested by ELISA on double-stranded (ds) DNAcoated plates with ␤-galactosidase-labeled anti-Fc␥ conjugate prepared from sheep antiserum (9). Isotypes were determined by using anti-IgG1-, -IgG2a-, -IgG2b-,...

Expression of a mannoselfucose membrane lectin on human dendritic cellsDendritic cells (DC) are the most efficient antigen presenting cells for T lymphocytes. CDla' CD14-DC with high antigen-presenting capacities can now be obtained easily from adherent peripheral blood monocytes by culture in the presence of granulocyte/macrophage colony-stimulating factor and interleukin-4 (Sallusto et al., J . Exp. Med. 1994. 179: 1109). Human macrophages express a membrane lectin, or sugar-specific receptor, which specifically mediates the binding and endocytosis of mannose-and fucose-terminated glycoproteins and is involved in the phagocytosis of pathogens. Asimilar lectin activity was sought on cultured human DC using flow cytometry and confocal microscopy to detect binding and internalization of fluoresceinated neoglycoproteins [bovine serum albumin (BSA) substituted with sugar residues]. Several neoglycoproteins, especially a-L-fucosyl-, a-D-mannosyl-, N,N'-di-acetyl-P-chitobiosyl-and 0-Dglucosyl-BSA, were endocytosed by cultured human CDla' DC as well as by CDla-CD14-cells which were also obtained in the culture. Fuc-BSA and Man-BSA had the same number of binding sites (1.7 x 10h/cell) on CDla' DC, and bound with an affinity constant close to lo7 Vmol. Inhibition experiments indicated that these two neoglycoproteins bound to the same membrane lectin. CDla' and CDla-cells were both labeled by an antiserum specific for the human macrophage mannose receptor. The membrane lectin specific for mannose and fucose that is evidenced in these experiments on cultured DC may be similar to the macrophage membrane lectin or may share functional and structural properties with it. IntroductionDendritic cells (DC) are particularly efficient in presenting antigens to naive as well as to mature T lymphocytes [ 1-31. They initiate primary T cell responses in vivo and in vitro. They can stimulate an allogeneic mixed lymphocyte reaction with a much lower presenting celYeffector cell ratio than unseparated mononuclear cells, and they can present much lower concentrations of antigens than macrophages or nonspecific B lymphocytes [l]. DC are derived from human myeloid-lineage cells and express high levels of MHC class I and MHC class I1 molecules, CD40, CD80 (B7.1) and CD86 (B7.2) co-stimulatory molecules, several