PyNTTTTGT and CpG immunostimulatory oligonucleotides: effect on granulocyte/monocyte colony-stimulating factor (GM-CSF) secretion by human CD56+ (NK and NKT) cells

CD56+ cells have been recognized as being involved in bridging the innate and acquired immune systems. Herein, we assessed the effect of two major classes of immunostimulatory oligonucleotides (ODNs), PyNTTTTGT and CpG, on CD56+ cells. Incubation of human peripheral blood mononuclear cells (hPBMC) with some of these ODNs led to secretion of significant amounts of interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α) and granulocyte/monocyte colony-stimulating factor (GM-CSF), but only if interleukin 2 (IL2) was present. IMT504, the prototype of the PyNTTTTGT ODN class, was the most active. GM-CSF secretion was very efficient when non-CpG ODNs with high T content and PyNTTTTGT motifs lacking CpGs were used. On the other hand, CpG ODNs and IFNα inhibited this GM-CSF secretion. Selective cell type removal from hPBMC indicated that CD56+ cells were responsible for GM-CSF secretion and that plasmacytoid dendritic cells (PDCs) regulate this process. In addition, PyNTTTTGT ODNs inhibited the IFNα secretion induced by CpG ODNs in PDCs by interference with the TLR9 signaling pathway. Since IFNα is essential for CD56+ stimulation by CpG ODNs, there is a reciprocal interference of CpG and PyNTTTTGT ODNs when acting on this cell population. This suggests that these synthetic ODNs mimic different natural alarm signals for activation of the immune system.Fil: Rodriguez, Juan M.. Fundacion Pablo Cassara; ArgentinaFil: Marchicio, José. Immunotech S.a.. Departamento de Investigacion y Desarrollo; ArgentinaFil: López, Mariela. Immunotech S.a.. Departamento de Investigacion y Desarrollo; ArgentinaFil: Ziblat, Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina. Immunotech S.a.; ArgentinaFil: Elias, Fernanda. Immunotech S.a.. Departamento de Investigacion y Desarrollo; ArgentinaFil: Fló, Juan. Immunotech S.a.. Departamento de Investigacion y Desarrollo; ArgentinaFil: López, Ricardo A.. Immunotech S.a.. Departamento de Investigacion y Desarrollo; ArgentinaFil: Horn, David. David Horn LLC; Estados UnidosFil: Zorzopulos, Jorge. Immunotech S.a.. Departamento de Investigacion y Desarrollo; ArgentinaFil: Montaner, Alejandro Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencias y Tecnología "Dr. Cesar Milstein"; Argentin

Effect of IL2 and PDC on the ability of immunostimulatory ODNs to stimulate GM-CSF.

<p>GM-CSF secretion on purified CD56⁺ cells from hPBMC. CD56⁺ cells and PDCs were purified using the MACS MicroBeads system (Miltenyi Biotec, Bergish Gladbach, Germany) and confirmed by flow cytometry. PDCs were added to purified CD56⁺ cells in a 1 to 10 ratio. ODNs and IL2 were added to the medium to reach a final concentration of 6 μg/ml and 400 IU/ml respectively. Data are shown as mean +SD of duplicates from three independent experiments. Two-way ANOVA interaction, p<0.001. Newman—Keuls test: *p<0.05, **p<0.005, ***p<0.0005.</p

Effect of IFNα and CpG ODNs on GM-CSF secretion induced by IMT504 in hPBMC.

<p>Addition of (A) IFNα or (B) CpG ODNs to the hPBMC culture in the presence of IL2. ODNs and IL2 were added to the medium to reach a final concentration of 6 μg/ml and 400 IU/ml, respectively. IFNα was added to the medium to reach the concentration indicated for each point in the figure. Data are shown as mean +SD of duplicates from three independent experiments. Two-way ANOVA interaction, p<0.001. Newman—Keuls test: *p<0.05, **p<0.005, ***p<0.0005.</p

Intracellular GM-CSF staining on CD56⁺ cells from hPBMC.

<p>(A) In the presence of the indicated ODN plus IL2: GM-CSF content in the cell culture without ODN stimulation was used as a control. Data are shown as mean +SD and are representative of three similar experiments. A specific response was defined as a response where the percentage of GM-CSF in CD56⁺ lymphocytes was at least 2-fold below the background observed in the medium control. The asterisk indicates statistically significant differences (p<0.05). (B) In the presence of IL2 in both CD3⁺ (NKT) and CD3^- (NK) CD56⁺ dim or bright populations: ODNs and/or IL2 were added to the medium to reach a final concentration of 6 μg/ml and 400 IU/ml respectively. Dot plots are representative of three similar experiments.</p

IL8 secretion induced by heated and cooled immunostimulatory ODNs on HEK293 cells stably transfected with the hTLR9 gene.

<p>Differences in IL8 secretion after incubation with immunostimulatory ODNs with or without previous heating (95°C, 10 min), rapid freezing in ethanol (-80°C) and thawing on ice. Data are shown as mean +SD of duplicates from three independent experiments. Two-way ANOVA interaction, p<0.001. Newman—Keuls test: *p<0.05, **p<0.005, ***p<0.0005.</p

Immunostimulatory ODN sequences and GM-CSF secretion.

<p>Induction of GM-CSF by ODNs on hPBMC in the presence of IL2. (A) ODN sequence vs. activity and (B) T content (%) vs. activity. Empty circles represent ODNs with unusual high activity. All these ODNs contain high T content and at least one PyNTTTTGT group (IMT700, IMT703, IMT702, IMT706, IMT701, IMT105, IMT104). Empty squares represent all the CpG ODNs assayed which, in addition to the CpG motif, have a high T content (IMT172, IMT560, IMT563, IMT565, IMT561, IMT710, CpG 2006). ODNs and IL2 were added to the medium to reach a final concentration of 6 μg/ml and 400 IU/ml respectively. Data are shown as mean +SD of duplicates from five independent experiments. Two-way ANOVA interaction, p<0.001. Newman—Keuls test: *p<0.05, **p<0.005, ***p<0.0005.</p

Cell populations from hPBMC and GM-CSF secretion induced by immunostimulatory ODNs.

<p>(A) hPBMC depleted of certain cell populations and (B) cell populations isolated from PBMC. Cell depletion/isolation was performed using the MACS MicroBeads (Miltenyi Biotec, Bergish Gladbach, Germany) system and confirmed by flow cytometry. ODNs and IL2 were added to the medium to reach a final concentration of 6 μg/ml and 400 IU/ml, respectively. Data are shown as mean +SD of duplicates from the same PBMC and are representative of three independent experiments. Two-way ANOVA interaction, p<0.001. Newman—Keuls test: *p<0.05, **p<0.005, ***p<0.0005.</p

Schematic interpretation of the biological action of PyNTTTTGT ODNs.

<p>Schematic interpretation of the biological action of PyNTTTTGT ODNs.</p