Monocytes are required for optimum in vitro stimulation of bovine peripheral blood mononuclear cells by non-methylated CpG motifs

Bacterial DNA and synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs within certain flanking base pairs are recognized as a danger signal by the innate immune system of vertebrates. Using lymphocyte proliferative response (LPR) and IFN-γ secretion assays, a panel of 38 ODN was screened for immunostimulatory activity on bovine peripheral blood mononuclear cells. ODN composed of a nuclease resistant phosphorothioate backbone and a leading 5′-TCGTCGTT-3′ motif with two 5′-GTCGTT-3′ motifs were highly stimulatory in both assays. Flow cytometric analysis and cell-specific surface marker labeling determined that B-cells (surface IgM +) were the primary cell population responding in the LPR assay. Depletion of T cells (CD3 +) from the PBMC population did not affect IFN-γ secretion or B-cell proliferation when cultured with CpG-ODN. However, depletion of monocytes (DH59B +) completely abrogated the ability of CpG-ODN to stimulate IFN-γ secretion, and significantly reduced the B-cell proliferative response. These data establish the identity of an optimal immunostimulatory CpG motif for cattle and demonstrate that monocytes play a pivotal role in the ability of cell populations to respond to CpG-ODN. These data provide insight for future studies investigating the mechanism of CpG-ODN bioactivity and its application in novel vaccine formulations and immunotherapy.