Phosphate-modified CpG oligonucleotides induce in vitro maturation of human myeloid dendritic cells

Myeloid dendritic cells (DCs) play an important role in the immune response; therefore, the search for compounds that can effectively activate DCs is a needful goal. This study was aimed to investigate the effect of synthetic CpG oligodeoxynucleotides (CpG-ODN) on the maturation and allostimulatory activity of myeloid DCs in comparison with other PAMP and DAMP molecules. For the research, we synthesized known CpG-ODN class C (SD-101 and D-SL03) containing thiophosphate internucleotide groups, and their original phosphate-modified analogues (SD-101M and D-SL03M) with mesylphosphoramide internucleotide groups (M = μ-modification). The effects of CpG-ODN and other activators were evaluated on DCs generated from blood monocytes in the presence of GM-CSF and IFN-α (IFN-DC) or IL-4 (IL4-DC). Evaluation of the intracellular TLR-9 expression showed that both types of DCs (IFN-DC and IL4-DC) contained on average 52 and 80 % of TLR-9-positive cells, respectively. The CpG-ODNs studied enhanced the allostimulatory activity of IFN-DCs, and the effect of μ-modified CpG-ODNs was higher than that of CpG-ODNs with thiophosphate groups. The stimulating effect of CpG-ODN at a dose of 1.0 μg/ml was comparable (for D-SL03, D-SL03M, SD-101) with or exceeded (for SD-101M) the effect of LPS at a dose of 10 μg/ml. At the same time, IFN-DCs were characterized by greater sensitivity to the action of CpG-ODNs than IL4-DCs. The enhancement of DC allostimulatory activity in the presence of CpG-ODNs was associated with the induction of final DC maturation, which was confirmed by a significant decrease in the number of CD14+DC, an increase in mature CD83+DC and a trend towards an increase in CD86+DC. Interestingly, the characteristic ability of LPS to enhance the expression of the co-stimulatory molecule OX40L on DCs was revealed only for the μ-analogue SD-101M. In addition, CpG-ODNs (SD-101 and SD-101M) had a stimulatory effect on IFN-γ production comparable to the action of LPS. The data obtained indicate a stimulating effect of CpG-ODN on the maturation and allostimulatory activity of human myeloid DCs, which is more pronounced for μ-modified analogs.

1. Alyamkina E.A., Leplina O.Yu., Sakhno L.V., Chernykh E.R., Ostanin A.A., Efremov Ya.R., Shilov A.G., Orishchenko K.E., Likhacheva A.S., Dolgova E.V., Nikolin V.P., Popova N.A., Zagrebelniy S.N., Bogachev S.S., Shurdov M.A. Effect of double-stranded DNA on maturation of dendritic cells in vitro. Cell. Immunol. 2010;266(1): 46-51. DOI 10.1016/j.cellimm.2010.08.011.

2. Banchereau J., Briere F., Caux C., Davoust J., Lebecque S., Liu Y.J., Palendran B., Palucka K. Immunobiology of dendritic cells. Annu. Rev. Immunol. 2000;18:767-811. DOI 10.1146/annurev.immunol.18.1.767.

3. Bauer S., Kirschning C.J., Hacker H., Redecke V., Hausmann S., Akira S., Wagner H., Lipfordet G.B. Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition. Proc. Natl. Acad. Sci. USA. 2001;98(16):9237-9242. DOI 10.1073/pnas.161293498.

4. Behboudi S., Chao D., Klenerman P., Austyn J. The effects of DNA containing CpG motif on dendritic cells. Immunology. 2000;99(3): 361-366. DOI 10.1046/j.1365-2567.2000.00979.x.

5. Cehim G., Chies J.A.B. In vitro generation of human monocyte-derived dendritic cells methodological aspects in a comprehensive review. An. Acad. Bras. Ciênc. 2019;91:e20190310. DOI 10.1590/0001-3765201920190310.

6. Chelobanov B.P., Burakova E.A., Prokhorova D.V., Fokina A.A., Stetsenko D.A. New oligodeoxynucleotide derivatives containing N-(methanesulfonyl)-phosphoramidate (mesyl phosphoramidate) internucleotide group. Russ. J. Bioorg. Chem. 2017;43(6):664-668. DOI 10.1134/S1068162017060024.

7. Dyakonova V.A., Dambaeva S.V., Pinegin B.V., Khaitov R.M. Study of interaction between the polyoxidonium immunomodulator and the human immune system cells. Inter. Immunopharm. 2004;4(13): 1615-1623. DOI 10.1016/j.intimp.2004.07.015.

8. Hoene V., Peiser M., Wanner R. Human monocyte-derived dendritic cells express TLR9 and react directly to the CpG-A oligonucleotide D19. J. Leuk. Biol. 2006;80(6):1328-1336. DOI 10.1189/jlb.0106011.

9. Iwasaki A., Medzhitov R. Toll-like receptor control of the adaptive immune responses. Nat. Immunol. 2004;5(10):987-995. DOI 10.1038/ni1112.

10. Jounai N., Kobiyama K., Takeshita F., Ishii K.J. Recognition of damage-associated molecular patterns related to nucleic acids during inflammation and vaccination. Front. Cell. Infect. Microbiol. 2013; 2:168. DOI 10.3389/fcimb.2012.00168.

11. Kabanov V.A. From synthetic polyelectrolytes to polymer-subunit vaccines. Pure Appl. Chem. 2004;76(9):1659-1677. DOI 10.1351/pac200476091659.

12. Kawasaki T., Kawai T. Toll-like receptor signaling pathways. Front. Immunol. 2014. DOI 10.3389/fimmu.2014.00461.

13. Krug A., Towarowski A., Britsch S., Rothenfusser S., Hornung V., Bals V., Giese T., Engelmann H., Endres S., Krieg A.M., Hartmann G. Toll-like receptor expression reveals CpG DNA as a unique microbial stimulus for plasmacytoid dendritic cells which synergizes with CD40 ligand to induce high amounts of IL-12. Eur. J. Immunol. 2001;31(10):3026-3037. DOI 10.1002/1521-4141(2001010)31:10<3026::aid-immu3026>3.0.co;2-h.

14. Levy R., Reagan P.M., Friedberg J.W., Bartlett N.L., Gordon L.I., Leung A., Peterkin J., Xing B., Coffman R., Janssen R., Candia A., Khodadoust M., Frank M.J., Long S.R., Czerwinski D.K., Chu M. SD-101, a novel class C CpG-oligodeoxynucleotide (ODN) toll-like receptor 9 (TLR9) agonist, given with low dose radiation for untreated low grade B-cell lymphoma: interim results of a phase 1/2 trial. Blood. 2016;128(22):2974. DOI 10.1182/blood.V128.22.2974.2974.

15. Li T., Wu J., Zhu S., Zang G., Li S., Lv X., Yue W., Qiao Y., Cui J., Shao Y., Zhang J., Liu Y.-J., Chen J. A novel C type CpG oligodeoxynucleotide exhibits immunostimulatory activity in vitro and enhances antitumor effect in vivo. Front. Pharmacol. 2020;11:8. DOI 10.3389/fphar.2020.00008.

16. Marshall J.D., Fearon K.L., Higgins D., Hessel E.M., Kanzler H., Abbate C., Yee P., Gregorio J., Cruz T.D., Lizcano J.O., Zolotorev A., McClure H.M., Brasky K.M., Murthy K.K., Coffman R.L., Nest G.V. Superior activity of the type C class of ISS in vitro and in vivo across multiple species. DNA Cell Biol. 2005;24(2):63-72. DOI 10.1089/dna.2005.24.63.

17. Miroshnichenko S.K., Patutina O.A., Burakova E.A., Chelobanov B.P., Fokina A.A., Vlassov V.V., Altman S., Zenkova M.A., Stetsenko D.A. Mesyl phosphoramidate antisense oligonucleotides as an alternative to phosphorothioates with improved biochemical and biological properties. Proc. Natl. Acad. Sci. USA. 2019;116(4): 1229-1234. DOI 10.1073/pnas.1813376116.

18. Orishchenko K.E., Ryzhikova S.L., Druzhinina Y.G., Ryabicheva T.G., Varaksin N.A., Alyamkina E.A., Dolgova E.V., Rogachev V.A., Proskurina A.S., Nikolin V.P., Popova N.A., Strunov A.A., Kiseleva E.V., Leplina O.Y., Ostanin A.A., Chernykh E.R., Sidorov S.V., Mayorov V.I., Bogachev S.S., Shurdov M.A. Effect of human double-stranded DNA preparation on the production of cytokines by dendritic cells and peripheral blood cells from relatively healthy donors. Cancer Ther. 2013;8:191-205.

19. Polovinkina V.S., Markov E.Yu. Structure and immune adjuvant properties of CpG-DNA. Meditsinskaya Immunologiya = Medical Immunology (Russia). 2010;12(6):469-476. DOI 10.15789/1563-0625-2010-6-469-476. (in Russian)

20. Powell B.S., Andrianov A.K., Fusco P.C. Polyionic vaccine adjuvants: another look at aluminum salts and polyelectrolytes. Clin. Exp. Vaccine Res. 2015;4(1):23-45. DOI 10.7774/cevr.2015.4.1.23. Rothenfusser S., Tuma E., Endres S., Hartmann G. Plasmacytoid dendritic cells: the key to CpG. Hum. Immunol. 2002;63(12):1111-1119. DOI 10.1016/s0198-8859(02)00749-8.

21. Scheiermann J., Klinman D.M. Clinical evaluation of CpG oligonucleotides as adjuvants for vaccines targeting infectious diseases and cancer. Vaccine. 2014;32(48):6377-6389. DOI 10.1016/j.vaccine.2014.06.065.

22. Shirota H., Klinman D.M. Recent progress concerning CpG DNA and its use as a vaccine adjuvant. Expert Rev. Vaccines. 2014;13(2):299- 312. DOI 10.1586/14760584.2014.863715.

23. Shirota H., Tross D., Klinman D.M. CpG oligonucleotides as cancer vaccine adjuvants. Vaccines (Basel). 2015;3(2):390-407. DOI 10.3390/vaccines3020390.

24. Tada H., Aiba S., Shibata K., Ohteki T., Takada H. Synergistic effect of Nod1 and Nod2 agonists with toll-like receptor agonists on human dendritic cells to generate interleukin-12 and T helper type 1 cells. Infect. Immun. 2005;73(12):7967-7976. DOI 10.1128/IAI.73.12.7967-7976.2005.

25. Ukyo N., Hori T., Yanagita S., Ishikawa T., Uchiyama T. Costimulation through OX40 is crucial for induction of an alloreactive human T-cell response. Immunology. 2003;109(2):226-231. DOI 10.1046/j.1365-2567.2003.01648.x.

26. Veglia F., Gabrilovich D.I. Dendritic cells in cancer: the role revisited. Curr. Opin. Immunol. 2017;45:43-51. DOI 10.1016/j.coi.2017.01.002.

27. Yang L., Wu X., Wan M., Yu Y., Yu Y., Wang L. CpG oligodeoxynucleotides with double stem-loops show strong immunostimulatory activity. Int. Immunopharmacol. 2013;15(1):89-96. DOI 10.1016/j.intimp.2012.10.020.