Deoxycytidyl-deoxyguanosine oligonucleotide classes A, B, and C induce distinct cytokine gene expression patterns in rhesus monkey peripheral blood mononuclear cells and distinct alpha interferon responses in TLR9-expressing rhesus monkey plasmacytoid dendritic cells

Abstract

To determine if deoxycytidyl-deoxyguanosine oligonucleotides (CpG ODN) can be used effectively as nonspecific inducers of innate immune defenses for preventative or therapeutic interventions in infectious disease models for nonhuman primates, the present study evaluated the response of rhesus monkey peripheral blood mononuclear cells to three different synthetic CpG ODN classes by defining the cytokine gene expression patterns and by characterizing IFN-alpha/beta responses. Depending on the type and dose of CpG ODN used for stimulation, distinct gene expression patterns were induced. CpG ODN class A (CpG-A ODN) and CpG-C ODN, but not CpG-B ODN, were potent inducers of alpha interferon (IFN-alpha), and this response was due to IFN-alpha production by TLR9-positive plasmacytoid dendritic cells. Importantly, there was a dose-dependent increase in IFN-alpha responses to CpG-A ODN but a dose-dependent decrease in IFN-alpha responses by CpG-B ODN. The most sustained IFN-alpha response was induced by CpG-A ODN and was associated with a stronger induction of interferon regulatory factor 7 and the induction of several interferon-stimulated genes. In contrast, and independent of the dose, CpG-B ODN were the weakest inducers of IFN-alpha but the most potent inducers of proinflammatory cytokines. CpG-C ODN induced cytokine gene expression patterns that were intermediate between those of CpG-A and CpG-B ODN. Thus, the different types of CpG ODN induce different post-TLR9 signaling pathways that result in distinct cytokine gene expression patterns. Based on these findings, A and C class CpG ODN, but not B class CpG ODN, may be particularly suited for use as therapeutic or prophylactic antiviral interventions.

FIG. 1.

Dose-dependent changes in IFN-α mRNA levels for CpG ODN-stimulated rhesus monkey PBMC. The average increases (± standard errors of the means [SEM]) in IFN-α mRNA levels for three donors at 6 h after stimulation with 5 or 50 μg CpG-A (black bars), CpG-B (open bars), and CpG-C ODN (striped bars) are shown relative to IFN-α mRNA levels in medium-only control cultures of the same PBMC. Statistically significant, dose-dependent changes in IFN-α mRNA levels for a specific type of CpG ODN are indicated by P values below the x axis. Statistically significant differences in IFN-α mRNA levels dependent on the type of CpG ODN used after stimulation with 50 μg CpG ODN are indicated by P values above the bars.

FIG. 2.

IFN-α/β mRNA levels in CpG ODN-stimulated rhesus monkey PBMC. The average increase (±SEM) in IFN-α and IFN-β mRNA levels of 10 donors at various time points after CpG ODN stimulation (5 μg) is shown relative to IFN-α and IFN-β mRNA levels in medium-only control cultures of the same PBMC. (A) Increase in IFN-α mRNA levels. The inset shows the kinetics of IFN-α mRNA induction in rhesus monkey PBMC in the first 12 h after CpG-B ODN stimulation. Average mRNA levels for three different donors are shown. (B) Increase in IFN-β mRNA levels. The inset shows the kinetics of IFN-β mRNA induction in rhesus monkey PBMC in the first 12 h after CpG-B ODN stimulation. Average mRNA levels for three different donors are shown. IFN-α/β mRNA levels in class A, B, and C CpG ODN-stimulated PBMC are represented by open triangles, open circles, and open diamonds, respectively. The asterisks denote statistically significant differences (P < 0.05) between groups.

FIG. 3.

IFN-α protein levels in supernatants of PBMC stimulated by CpG-A, CpG-B, and CpG-C ODN (5 μg) as measured by ELISA. Average IFN-α protein levels for the same donors as in Fig. 2 are shown. Medium, medium-only control cultures.

FIG. 4.

Frequency of IFN-α-positive cells after 6 h of stimulation with different concentrations of the CpG ODN. The top row shows the gating strategy based on a forward and side scatter gate that included lymphocytes, monocytes, and dendritic cells (left panel) and the frequency of IFN-α-positive cells in medium-only negative control cultures (middle panel) and in HSV-stimulated positive control cultures (right panel). Note that frequencies of IFN-α-positive cells in medium-only control cultures never exceed 0.009% of PBMC. HSV-stimulated PBMC served as positive controls. The relative frequencies of IFN-α-positive cells after stimulation of rhesus monkey PBMC with various doses of CpG-A (second row), CpG-B (third row), and CpG-C ODN (fourth row) as determined by FACS analysis are shown. Data for one out of three representative donors are shown. APC, allophycocyanin.

FIG. 5.

Intracellular TLR9 staining in rhesus monkey cells. (A) The histograms indicate the levels of TLR9 staining in CD3⁺ T cells, CD20⁺ B cells and CD3⁻CD20⁻CD123⁺ PDC from a rhesus monkey spleen cell suspensions using TLR9 antibody at a concentration of 0.05 μg per 1 × 10⁶ cells. The gating hierarchy is indicated by arrows. In each histogram, the percentage of TLR9-positive cells in each cell population based on the cutoff established by gating on the cells with the highest TLR9 staining shown in the top panel is shown. (B) Graphic overlays of mouse IgG1 control antibody histograms and TLR9 histograms are shown for CD3⁺ T cells (green lines), CD20⁺ B cells (blue lines) and CD3⁻CD20⁻CD123⁺ PDC (red lines) of rhesus monkey PBMC. Antibodies were used at 2 μg per 1 × 10⁶ cells. Values for TLR9 staining indicate the MFI for each cell population.

FIG. 6.

The phenotype of IFN-α-producing cells after CpG ODN stimulation. Representative examples of IFN-α-producing cell frequencies at 6 h after stimulation with CpG-A, CpG-B, or CpG-C ODN. Data for one out of three representative donors after CpG-A, CpG-B, CpG-C ODN, and HSV stimulation are shown. The phenotype was determined by gating on IFN-α-positive cells (see Fig. 4) and then gating on Lin-CD123⁺ (x axis) and CD123⁺ (y axis) cells. The Lin-CD123⁺ cells were then gated for analysis of HLA-DR expression. (Note that medium control cultures are not included in this figure, as frequencies of IFN-α-secreting cells did not exceed 0.009% of PBMC).

FIG. 7.

Gene expression levels for IRF-7 and ISG. (A) Average IRF-7 mRNA levels (± SEM) for eight donors are shown in response to 5 μg CpG ODN stimulation. (B and C) Average mRNA levels (± SEM) for OAS and IP-10/CXCL10, respectively, are shown. Increases in mRNA levels are relative to medium control cultures. (D) IP-10/CXCL10 protein levels as measured by ELISA of the supernatants of the same cultures used for gene expression analysis. Medium, medium-only control cultures.

FIG. 8.

Gene expression levels of proinflammatory cytokines after stimulation of rhesus monkey PBMC with 5 μg of CpG ODN. (A) TNF-α mRNA levels. (B) IL-12p40 mRNA levels. (C) IL-6 mRNA levels. (D) IFN-γ mRNA levels. The average increase in mRNA levels (± SEM) for 10 donors are shown compared to mRNA levels for medium-only control cultures.

FIG. 9.

Dose-dependent changes in mRNA levels of proinflammatory cytokines. The average increases (± SEM) in proinflammatory mRNA levels for three donors at 6 h after stimulation with 5 or 50 μg CpG-A, CpG-B, and CpG-C ODN are shown relative to the same cytokine mRNA levels in medium-only control cultures of the same PBMC. (A) TNF-α mRNA levels. (B) IL-12p40 mRNA levels. (C) IL-6 mRNA levels. (D) IFN-γ mRNA levels.