Lack of immune stimulation by immobilized CpG-oligodeoxynucleotide

Selected phosphorothioate oligodeoxynucleotides containing CpG (CpG-ODN) activate immune responses, including B-cell proliferation and cytokine production. The mechanism by which cells detect CpG-motifs is not known. There are conflicting reports in the literature concerning the ability of CpG-ODN linked to solid supports to stimulate immunity. We prepared a fluorescent, biotinylated CpG-ODN, a reagent that will support the growth of 7TD1 cells, a murine B-cell hybridoma line that requires CpG-ODN or interleukin-6 (IL-6) for survival. Stimulation of 7TD1 cell growth was not reduced by complexing biotinylated CpG-ODN to streptavidin, but cell growth was not supported by CpG-ODN coupled to streptavidin-coated latex, magnetic, gold, or agarose beads. A fluorescent CpG-ODN was also covalently attached to cyanogen bromide-activated Sepharose beads via a 5'-amine group. These derivatized Sepharose beads did support 7TD1 cell growth, but incubation of the beads with 7TD1 cells resulted in the appearance of fluorescence within the cells, suggesting that growth stimulation may be due to CpG-ODN leached from the beads. Our results are consistent with the need for CpG-ODN to be internalized into cells to be immunostimulatory.