Bone marrow dendritic cells deficient for CD40 and IL-23p19 are tolerogenic in vitro

Document Type : Original Article


1 Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran

2 Department of Laboratory Sciences, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran

3 Department of Neurology, Thomas Jefferson University, Philadelphia, PA 19107, USA

4 Immunology Department, Shiraz University of Medical Sciences, Shiraz, Iran


Objective(s): In addition to pro-inflammatory role, dendritic cells (DCs) can also be anti-inflammatory when they acquire tolerogenic phenotype. In this study we tested the role of CD40 and IL-23p19 in antigen presenting function of bone marrow-derived DCs (BMDCs) by comparing BMDCs derived from CD40 knockout (CD40KO-DCs) and IL-23p19 (IL-23p19KO-DCs) knockout mice with those from C57BL/6 mice (Cont-DCs). We have focused on CD40 and IL-23, as these molecules have well established pro-inflammatory roles in a number of autoimmune and inflammatory diseases.
Materials and Methods: The expression of maturation markers MHC II and co-stimulatory molecules CD40, CD80, and CD86 was analyzed by flow cytometry, while the expression of CD40 and IL-23p19 was measured by RT-PCR. The capacity of BMDCs to activate CD4+ T cells was evaluated by 3H-thymidine incorporation, and the capacity of BMDCs to uptake antigen was evaluated using fluorescent OVA and flow cytometry.
Results: The lack of CD40 or IL-23p19 had no effect on uptake of FITC-OVA by the DCs, confirming their immature phenotype. Moreover, CD40KO-DCs had significantly reduced capacity to stimulate proliferation of CD4+ T cells. CD4+ T cells activated by CD40KO-DCs and IL-23p19KO-DCs produced significantly less IFN-γ (P-value ≤0.05), while CD4+ T cells stimulated by IL-23p19KO-DCs produced less GM-CSF and more IL-10 than Cont-DCs.
Conclusion: This study shows that CD40KO-DCs and IL-23p19KO-DCs have a marked tolerogenic potency in vitro. Future in vivo studies should determine if and to what extent DCs lacking CD40 or IL-23 have a potential to be useful in therapy of autoimmune inflammation.


1. Wittamer V, Franssen JD, Vulcano M, Mirjolet JF, Le Poul E, Migeotte I, et al. Specific recruitment of antigen-presenting cells by chemerin, a novel processed ligand from human inflammatory fluids. J Exp Med 2003;198:977-985.
2. Blanco P, Palucka AK, Pascual V, Banchereau J. Dendritic cells and cytokines in human inflammatory and autoimmune diseases. Cytokine & growth factor reviews 2008;19:41-52.
3. Steinman RM, Hawiger D, Nussenzweig MC. Tolerogenic dendritic cells. Annu Rev Immunol 2003;21:685-711.
4. Kapsenberg ML. Dendritic-cell control of pathogen-driven T-cell polarization. Nat Rev Immunol 2003;3:984-993.
5. Švajger U, Obermajer N, Jeras M. Dendritic cells treated with resveratrol during differentiation from monocytes gain substantial tolerogenic properties upon activation. Immunol 2010;129:525-535.
6. Torres-Aguilar H, Blank M, Jara LJ, Shoenfeld Y. Tolerogenic dendritic cells in autoimmune diseases: crucial players in induction and prevention of autoimmunity. Autoimmun Rev 2010;10:8-17.
7. Chamorro S, García-Vallejo JJ, Unger WWJ, Fernandes RJ, Bruijns SCM, Laban S, et al. TLR triggering on tolerogenic dendritic cells results in TLR2 up-regulation and a reduced proinflammatory immune program. J Immunol 2009;183:2984-2994.
8. Popov I, Li M, Zheng X, San H, Zhang X, Ichim TE, et al. Preventing autoimmune arthritis using antigen-specific immature dendritic cells: a novel tolerogenic vaccine. Arthritis Res Ther 2006;8:R141.
9. Steinman L. A brief history of TH17, the first major revision in the TH1/TH2 hypothesis of T cell–mediated tissue damage. Nat Med 2007;13:139-145.
10. Thomson A, Robbins P. Tolerogenic dendritic cells for autoimmune disease and transplantation. Ann Rheum Dis 2008;67(Suppl 3):iii90-iii6.
11. Rea D, van Kooten C, van Meijgaarden KE, Ottenhoff THM, Melief CJM, Offringa R. Glucocorticoids transform CD40-triggering of dendritic cells into an alternative activation pathway resulting in antigen-presenting cells that secrete IL-10. Blood 2000;95:3162-3167.
12. Mackey MF, Gunn JR, Maliszewski C, Kikutani H, Noelle RJ, Barth Jr RJ. Cutting edge: Dendritic cells require maturation via CD40 to generate protective antitumor immunity. J Immunol 1998;161:2094-2098.
13. Zheng X, Suzuki M, Zhang X, Ichim TE, Zhu F, Ling H, et al. RNAi-mediated CD40-CD154 interruption promotes tolerance in autoimmune arthritis. Arthritis Res Ther 2010;12:R13.
14. Vaknin-Dembinsky A, Balashov K, Weiner HL. IL-23 is increased in dendritic cells in multiple sclerosis and down-regulation of IL-23 by antisense oligos increases dendritic cell IL-10 production. J Immunol 2006;176:7768-7774.
15. Liu Z, Yadav PK, Xu X, Su J, Chen C, Tang M, et al. The increased expression of IL-23 in inflammatory bowel disease promotes intraepithelial and lamina propria lymphocyte inflammatory responses and cytotoxicity. J Leukoc Biol 2011;89:597-606.
16. Sun Mei LJ-d, JIANG Rui, JIN Cheng-yan, GAO Nan, LUO Shu-li, WANG Chun-guang WB, WANG Rong-you, ZHANG Xing-yi. Construction and Characterization of Lentiviral shRNA Expression Vector Targeting Rat CD40 Gene in Dendritic Cells. Chem Res Chinese Universities 2009;25: 666-672.
17. Blazar BR, Taylor PA, Panoskaltsis-Mortari A, Buhlman J, Xu J, Flavell RA, et al. Blockade of CD40 ligand-CD40 interaction impairs CD4+ T cell-mediated alloreactivity by inhibiting mature donor T cell expansion and function after bone marrow transplantation. J Immunol 1997;158:29-39.
18. Kalantari T, Karimi MH, Ciric B, Yan Y, Rostami A, Kamali‐Sarvestani E. Tolerogenic dendritic cells produced by lentiviral‐mediated CD40‐and interleukin‐23p19‐specific shRNA can ameliorate experimental autoimmune encephalomyelitis by suppressing T helper type 17 cells. Clin Exp Immunol 2014;176:180-189.
19. Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature 1998;392:245-252.
20. Inaba K, Inaba M, Romani N, Aya H, Deguchi M, Ikehara S, et al. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J Exp Med 1992;176:1693-1702.
21. Gordon JR. A practical guide to cellular & molecular research methods in immunology. . Fifth edition ed. University of Saskatchewan, Saskatoon, Canada: Immunology Research Group; 2006.
22. Hilkens CM, Isaacs JD, Thomson AW. Development of dendritic cell-based immunotherapy for autoimmunity. Int Rev Immunol 2010;29:156-183.
23. Taams L, Vukmanovic-Stejic M, Salmon M, Akbar A. Immune regulation by CD4+ CD25+ regulatory T cells: implications for transplantation tolerance. Transpl immunol 2003;11:277-285.
24.    Groux H, Fournier N, Cottrez F, editors. Role of dendritic cells in the generation of regulatory T cells. Semin Immunol 2004: 16:99-106.
25.Grewal IS, Flavell RA. The Role of CD40 Ligand in Costimulation and T‐Cell Activation. Immunol. Rev 1996;153:85-106.
26. Inaba K, Witmer-Pack M, Inaba M, Hathcock K, Sakuta H, Azuma M, et al. The tissue distribution of the B7-2 costimulator in mice: abundant expression on dendritic cells in situ and during maturation in vitro. J Exp Med 1994;180:1849-1860.
27. Vremec D, Shortman K. Dendritic cell subtypes in mouse lymphoid organs: cross-correlation of surface markers, changes with incubation, and differences among thymus, spleen, and lymph nodes. J Immunol1997;159:565-573.
28. Steinman RM, Turley S, Mellman I, Inaba K. The induction of tolerance by dendritic cells that have captured apoptotic cells. J Exp Med 2000;191:411-416.
29. Dhodapkar MV, Steinman RM, Krasovsky J, Munz C, Bhardwaj N. Antigen-specific inhibition of effector T cell function in humans after injection of immature dendritic cells. J Exp Med 2001;193:233-238.
30. Shortman K, Heath WR. Immunity or tolerance? That is the question for dendritic cells. Nat Immunol. 2001;2:988-989.
31. Xia CQ, Peng R, Beato F, Clare‐Salzler M. Dexamethasone induces IL‐10‐producing monocyte‐derived dendritic cells with durable immaturity. Scand J Immunol 2005;62:45-54.
32. Yanagawa Y, Onoe K. Distinct regulation of CD40-mediated interleukin-6 and interleukin-12 productions via mitogen-activated protein kinase and nuclear factor kappaB-inducing kinase in mature dendritic cells. Immunology 2006;117:526-35.
33. Kobayashi T, Walsh PT, Walsh MC, Speirs KM, Chiffoleau E, King CG, et al. TRAF6 is a critical factor for dendritic cell maturation and development. Immunity 2003;19:353-363.
34. Griffin MD, Lutz W, Phan VA, Bachman LA, McKean DJ, Kumar R. Dendritic cell modulation by 1α, 25 dihydroxyvitamin D3 and its analogs: a vitamin D receptor-dependent pathway that promotes a persistent state of immaturity in vitro and in vivo. Proc Natl Acad Sci 2001;98:6800-6805.
35. Garcia-Gonzalez P, Morales R, Hoyos L, Maggi J, Campos J, Pesce B, et al. A short protocol using dexamethasone and monophosphoryl lipid A generates tolerogenic dendritic cells that display a potent migratory capacity to lymphoid chemokines. J Transl Med 2013;11:128-150.