Yonsei Med J.  2010 Jul;51(4):594-598. 10.3349/ymj.2010.51.4.594.

The Change of Immunoactivity of Dendritic Cells Induced by Mouse 4-1BBL Recombinant Adenovirus

• Youlin K
• Xiaodong W
• Xiuheng L
• Zhiyuan C
• Hengcheng
• Hui C
• Botao J

• KMID: 1805201
• DOI: http://doi.org/10.3349/ymj.2010.51.4.594

Abstract

PURPOSE
The purpose of this study is to construct a recombinant adenovirus vector carrying mouse 4-1BBL and observe its effects in dendritic cells.
MATERIALS AND METHODS
Mouse 4-1BBL cDNA was taken from the plasmid pcDNA3-m4-1BBL and subcloned into adenovirus shuttle plasmid pAdTrack-CMV, and then transformed into competent BJ5183 with plasmid pAdEasy-1. After recombination in E. coli, Ad-4-1BBL was packaged and amplified in HEK 293 cells. The expression of 4-1BBL in Ad-4-1BBL-transfected mouse prostate cancer cell line RM-1 was detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. After the co-culture of dendritic cells (DCs) with Ad-4-1BBL-transfected RM-1 cells, interleukin (IL)-6 and IL-12 production were assessed by enzyme-linked immunosorbent assay (ELISA) and co-stimulatary moleculs (CD80 and CD86) on DCs were analyzed by flow cytometry.
RESULTS
The levels of IL-6 (3,960 pg/mL) and IL-12 (249 pg/mL) production in Ad-m4-1BBL-pulsed DCs were more than those in none-pulsed DCs. The differences were statistically significant (p < 0.05). The expression of co-stimulatary molecules (CD80 and CD86) was up-regulated in Ad-m4-1BBL-pulsed DCs.
CONCLUSION
The results indicated the recombinant mouse 4-1BBL can effectively activate DCs.

Keyword

Dendritic cell; 4-1BBL; adenovirus

Figure

• 

  Fig. 1 Identification of pAdTrack-CMV-4-1BBL by restriction endonucleases digestion. M. λ-EcoT14 DNA Marker; 1, pAdTrack-CMV digested with Bgl II; 2 and 3, pAdTrack-CMV-m4-1BBL digested with Bgl II/Hind III.

• 

  Fig. 2 Analysis of pAd-m4-1BBL by PacI. 1, 2 and 3, pAd-m4-1BBL digested with PacI; M. λ/Hind III DNA Marker.

• 

  Fig. 3 Detection of 4-1BBL mRNA by RT-PCR. RM-1 cells were transfected with pAd-4-1BBL. Total RNA was extracted, and 4-1BBL mRNA was detected by RT-PCR. (A) M, DL 2000 DNA marker; 1, RM-1 cells transfected with pAd-4-1BBL; 2, RM-1 cells. (B) β-actin was used as reference. mRNA, messenger ribonucleic acid, RT-PCR, reverse transcription polgmerase chain reaction.

• 

  Fig. 4 Detection of 4-1BBL protein by Western blot. Total cell lysates were harvested and presence of 4-1BBL protein was detected by anti-4-1BBL polyclonal antibody. A specific band was identified in RM-1 cells transfected with Ad-4-1BBL but not in none-transfected RM-1 cells. β-actin was used as reference.

• 

  Fig. 5 Activation of DCs by 4-1BBL. Bone-marrow-derived DCs from C57BL/6 mice were co-cultured with non-transfected, Ad-eGFP-transfected or Ad-4-1BBL-transfected RM-1 cells. Flow cytometry showed expression of CD80, CD86 molecules was significantly increased 4-1BBL. The experiment was repeated three with representative data shown. DCs, dendritic cells.

• 

  Fig. 6 Ad-4-1BBL-transfected RM-1 cells induce IL-6/IL-12 production by DCs. DCs were co-cultured with Ad-eGFP-transfected, Ad-4-1BBL-transfected or none-transfected RM-1 cells. Cell-free supernatants were collected at 48 h and IL-6/IL-12 were measured by Elisa. (A) The level of IL-6 in DC/Ad-4-1BBL group was more than that in DC and DC/Ad-eGFP group, *p < 0.05. (B) The production of IL-12 in DC/Ad-4-1BBL group was higher than that in DC and DC/Ad-eGFP group, *p < 0.05. Data are expressed as mean ± SD. Similar results were obtained from three independent experiments. IL, interleukin; SD, standard deviation.

Reference

1. Nencioni A, Grünebach F, Schmidt SM, Müller MR, Boy D, Patrone F, et al. The use of dendritic cells in cancer immunotherapy. Crit Rev Oncol Hematol. 2008. 65:191–199.

2. Schwarz H. Biological activities of reverse signal transduction through CD137 ligand. J Leukoc Biol. 2005. 77:281–286.

3. Laderach D, Movassagh M, Johnson A, Mittler RS, Galy A. 4-1BB co-stimulation enhances human CD8(+) T cell priming by augmenting the proliferation and survival of effector CD8(+) T cells. Int Immunol. 2002. 14:1155–1167.

4. Lu ZY, Condomines M, Tarte K, Nadal L, Delteil MC, Rossi JF, et al. B7-1 and 4-1BB ligand expression on a myeloma cell line makes it possible to expand autologous tumor-specific cytotoxic T cells in vitro. Exp Hematol. 2007. 35:443–453.

5. Lee HW, Park SJ, Choi BK, Kim HH, Nam KO, Kwon BS. 4-1BB promotes the survival of CD8+ T lymphocytes by increasing expression of Bcl-xL and Bfl-1. J Immunol. 2002. 169:4882–4888.

6. Croft M. Co-stimulatory members of the TNFR family: keys to effective T-cell immunity? Nat Rev Immunol. 2003. 3:609–620.

7. Kudo-Saito C, Hodge JW, Kwak H, Kim-Schulze S, Schlom J, Kaufman HL. 4-1BB ligand enhances tumor-specific immunity of poxvirus vaccines. Vaccine. 2006. 24:4975–4986.

8. Xiao H, Huang B, Yuan Y, Li D, Han LF, Liu Y, et al. Soluble PD-1 facilitates 4-1BBL-triggered antitumor immunity against murine H22 hepatocarcinoma in vivo. Clin Cancer Res. 2007. 13:1823–1830.

9. Futagawa T, Akiba H, Kodama T, Takeda K, Hosoda Y, Yagita H, et al. Expression and function of 4-1BB and 4-1BB ligand on murine dendritic cells. Int Immunol. 2002. 14:275–286.

10. Wiethe C, Dittmar K, Doan T, Lindenmaier W, Tindle R. Provision of 4-1BB ligand enhances effector and memory CTL responses generated by immunization with dendritic cells expressing a human tumor-associated antigen. J Immunol. 2003. 170:2912–2922.

11. Nencioni A, Grünebach F, Schmidt SM, Müller MR, Boy D, Patrone F, et al. The use of dendritic cells in cancer immunotherapy. Crit Rev Oncol Hematol. 2008. 65:191–199.

12. Hu Y, He Y, Srivenugopal KS, Fan S, Jiang Y. In vitro antitumor cytotoxic T lymphocyte response induced by dendritic cells transduced with DeltaNp73alpha recombinant adenovirus. Oncol Rep. 2007. 18:1085–1091.