Background: Synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs are potent immunomodulators and are of great interest as potential adjuvants in HIV vaccine development. ODN have been shown to activate cells carrying Toll-like receptor 9 (TLR-9) found on plasmacytoid dendritic cells (PDC), B-cells, and, to a lesser degree, monocytes. Isolated PDC stimulated with ODN are able to upregulate IFN-g production by isolated T-cells. We investigated whether B-cells would influence the production of cytokines in response to stimulation with phytohemaglutinin (PHA) in combination with various ODN in vitro.

Method: PBMC from random blood donors were isolated by Ficoll separation. Magnetic cell sorting with anti-CD19 conjugated beads was used to obtain PBMC depleted of B-cells. Both PBMC and B-cell depleted samples were incubated at 37°C with ODN at a concentration of 5 µg/ml with or without stimulation with PHA at 0.2 µg/ml for 12–24 hrs, and culture supernatants were analyzed by ELISA for production of multiple cytokines, including IFN-g and IL-10. Student’s T-test used to compare mean concentrations of cytokine production.

Results: IFN-g levels were increased by ODN co-stimulation 2–3 fold above control levels (either lacking ODN or using a non-stimulatory ODN) (controls: 72 ±3 pg/ml vs ODN: 288 ±13 pg/ml; p = 0.002). However, IL10 levels were also increased 2–5 fold above controls. Depletion of B-cells resulted in a 2-fold augmentation of IFN-g production (PBMC: 288 ±13 pg/ml vs B-cell depleted PBMC: 728 ±33 pg/ml; p = 0.003) and a decrease in IL10 production (PBMC: 491 ±27 pg/ml vs B-cell depleted PBMC: 138 ±11 pg/ml; p = 0.003).

Conclusion: ODN significantly enhance the production of IFN-g in PBMC in vitro; however, the presence of B cells inhibits IFN-g production. This attenuation is likely related to stimulation of IL-10 by B-cells in response to ODN. While studies utilizing isolated PDC and T-cells have shown that ODN stimulate IFN-g from T-cells, the presence of B-cells in vivo may significantly effect HIV-specific responses to vaccines using ODN as adjuvants. Further understanding of the factors affecting differential stimulation of B-cells as opposed to dendritic cells and other APCs by ODN is required.