Volume 12, Issue 3 (9-2024)                   JoMMID 2024, 12(3): 201-207 | Back to browse issues page

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Moradi Pordanjani P, Bolhassani A, Agi E. Differential Immunostimulatory Effects of EGFP and +36 GFP on Immune Cells. JoMMID 2024; 12 (3) :201-207
URL: http://jommid.pasteur.ac.ir/article-1-698-en.html
Differential Immunostimulatory Effects of EGFP and +36 GFP on Immune Cells
Parisa Moradi Pordanjani , Azam Bolhassani * , Elnaz Agi
Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran
Abstract:   (165 Views)
Introduction: Green fluorescent protein (GFP) and its variants are pivotal in tracking gene expression across various gene delivery systems. While GFP is typically employed for intracellular reporting, it can be modified to display on cell surfaces for labeling. Previous research indicates GFP might have immunogenic effects, notably enhancing tumor-specific T cell responses. This study explores the immunostimulatory differences between enhanced GFP (EGFP) and the supercharged variant, +36 GFP. Methods: Recombinant EGFP and +36 GFP proteins were generated using an Escherichia coli expression system. Murine bone marrow-derived dendritic cells (BMDCs) were generated using established protocols. Splenocytes were isolated from murine spleens via mechanical disruption and red blood cell lysis. The RAW 264.7 macrophage cell line was cultured in complete DMEM medium. Immune cells were then incubated with varying concentrations of EGFP and +36 GFP, separately, for 48 h. Cytokine levels (IFN-γ, TNF-α, IL-10) were quantified using sandwich ELISA.
Keywords: Green Fluorescent Protein, Enhanced GFP, Supercharged +36 GFP, Immunogenicity, Cytokines
Full-Text [PDF 1033 kb]   (94 Downloads)    
Type of Study: Original article | Subject: Immune responses, deficiencies and vaccine candidates
Received: 2024/12/9 | Accepted: 2024/09/11 | Published: 2024/12/22
References
1. Arpino JAJ, Rizkallah PJ, Jones DD. Crystal structure of enhanced green fluorescent protein to 1.35 Å resolution reveals alternative conformations for Glu222. PLoS One. 2012; 7 (10): e47132. [DOI:10.1371/journal.pone.0047132] [PMID] [PMCID]
2. Stripecke R, del Carmen Villacres M, Skelton DC, Satake N, Halene S, Kohn DB. Immune response to green fluorescent protein: implications for gene therapy. Gene Ther. 1999; 6 (7): 1305-12. [DOI:10.1038/sj.gt.3300951] [PMID]
3. Koelsch KA, Wang Y, Maier-Moore JS, Sawalha AH, Wren JD. GFP affects human T cell activation and cytokine production following in vitro stimulation. PLoS One. 2013; 8 (4): e50068. [DOI:10.1371/journal.pone.0050068] [PMID] [PMCID]
4. Gambotto A, Dworacki G, Cicinnati V, Kenniston T, Steitz J, Tuting T, et al. Immunogenicity of enhanced green fluorescent protein (EGFP) in BALB/c mice: identification of an H2-Kd-restricted CTL epitope. Gene Ther. 2000; 7 (23): 2036-40. [DOI:10.1038/sj.gt.3301335] [PMID]
5. Liu HS, Jan MS, Chou CK, Chen PH, Ke NJ. Is green fluorescent protein toxic to the living cells? Biochem Biophys Res Commun. 1999; 260 (3): 712-7. [DOI:10.1006/bbrc.1999.0954] [PMID]
6. Taghizadeh RRSJ. CFP and YFP, but not GFP, provide stable fluorescent marking of rat hepatic adult stem cells. J Biomed Biotechnol. 2008; 2008: 453590. [DOI:10.1155/2008/453590] [PMID] [PMCID]
7. Sherley JL. Asymmetric cell kinetics genes: the key to expansion of adult stem cells in culture. Stem Cells. 2002; 20 (6): 561-72. [DOI:10.1634/stemcells.20-6-561] [PMID]
8. Inoue H, Ohsawa I, Murakami T, Kimura A, Hakamata Y, Sato Y, et al. Development of new inbred transgenic strains of rats with LacZ or GFP. Biochem Biophys Res Commun. 2005; 329 (1): 288-95. [DOI:10.1016/j.bbrc.2005.01.132] [PMID]
9. Pletnev S, Gurskaya NG, Pletneva NV, Lukyanov KA, Chudakov DM, Martynov VI, et al. Structural basis for phototoxicity of the genetically encoded photosensitizer Killer Red. J Biol Chem. 2009; 284 (46): 32028-39. [DOI:10.1074/jbc.M109.054973] [PMID] [PMCID]
10. Ma C, Malessa A, Boersma AJ, Liu K, Herrmann A. Supercharged proteins and polypeptides. Adv Mater. 2020; 32 (20): 1905309. [DOI:10.1002/adma.201905309] [PMID]
11. Wang L, Geng J, Chen L, Guo X, Wang T, Fang Y, et al. Improved transfer efficiency of supercharged 36+GFP protein mediate nucleic acid delivery. Drug Deliv. 2022; 29 (1): 386-98. [DOI:10.1080/10717544.2022.2030430] [PMID] [PMCID]
12. Thompson DB, Cronican JJ, Liu DR. Engineering and identifying supercharged proteins for macromolecule delivery into mammalian cells. Methods Enzymol. 2012; 503: 293-319. [DOI:10.1016/B978-0-12-396962-0.00012-4] [PMID] [PMCID]
13. Mangla B, Javed S, Kohli K, Ahsan A, Ahsan W. Reassessment of therapeutic applications of carbon nanotubes: a majestic and futuristic drug carrier. Crit Rev Ther Drug Carrier Syst. 2020; 37 (4): 331-73. [DOI:10.1615/CritRevTherDrugCarrierSyst.2020032570] [PMID]
14. Krishnan Y, Rees HA, Rossitto CP, Kim SE, Hung HHK, Frank EH, et al. Green fluorescent proteins engineered for cartilage-targeted drug delivery: Insights for transport into highly charged avascular tissues. Biomater. 2018; 183: 218-33. [DOI:10.1016/j.biomaterials.2018.08.050] [PMID] [PMCID]
15. Lawrence MS, Phillips KJ, Liu DR. Supercharging proteins can impart unusual resilience. J Am Chem Soc. 2007; 129 (33): 10110-2. [DOI:10.1021/ja071641y] [PMID] [PMCID]
16. Bajpayee AG, Quadir MA, Hammond PT, Grodzinsky A. Charge based intracartilage delivery of single dose dexamethasone using Avidin nano-carriers suppresses cytokine-induced catabolism long term. Osteoarthritis Cartilage. 2016; 24 (1): 71-81. [DOI:10.1016/j.joca.2015.07.010] [PMID] [PMCID]
17. Motevalli F, Bolhassani A, Hesami S, Shahbazi S. Supercharged green fluorescent protein delivers HPV16E7 DNA and protein into mammalian cells in vitro and in vivo. Immunol Lett. 2018; 194: 29-39. [DOI:10.1016/j.imlet.2017.12.005] [PMID]
18. Kadkhodayan S, Sadat SM, Irani S, Fotouhi F, Bolhassani A. Generation of GFP native protein for detection of its intracellular uptake by cell penetrating peptides. Folia Biol. 2016; 62 (3): 103-9. [DOI:10.14712/fb2016062030103] [PMID]
19. Milani A, Agi E, Pouriayevali MH, Motamedi-Rad M, Motevalli F, Bolhassani A. Different dendritic cells-based vaccine constructs influence HIV-1 antigen-specific immunological responses and cytokine generation in virion-exposed splenocytes. Int Immunopharmacol. 2022; 113 (Pt A):109406. [DOI:10.1016/j.intimp.2022.109406] [PMID]
20. Skelton D, Satake N, Kohn DB. The enhanced green fluorescent protein (eGFP) is minimally immunogenic in C57BL/6 mice. Gene Ther. 2001; 8 (23): 1813-4. [DOI:10.1038/sj.gt.3301586] [PMID]
21. Baens M, Noels H, Broeckx V, Hagens S, Fevery S, Billiau AD, et al. The dark side of EGFP: defective polyubiquitination. PLoS One. 2006; 1 (1): e54. [DOI:10.1371/journal.pone.0000054] [PMID] [PMCID]
22. Lai W, Chang CH, Farber DL. Gene transfection and expression in resting and activated murine CD4 T cell subsets. J Immunol Methods. 2003; 282 (1-2): 93-102. [DOI:10.1016/j.jim.2003.07.015] [PMID]
23. Zhang F, Hackett NR, Lam G, Cheng J, Pergolizzi R, Luo L, et al. Green fluorescent protein selectively induces HSP70-mediated up-regulation of COX-2 expression in endothelial cells. Blood. 2003; 102 (6): 2115-21. [DOI:10.1182/blood-2003-01-0049] [PMID]
24. Re F, Srinivasan R, Igarashi T, Marincola F, Childs R. Green fluorescent protein expression in dendritic cells enhances their immunogenicity and elicits specific cytotoxic T-cell responses in humans. Exp Hematol. 2004; 32 (2): 210-7. [DOI:10.1016/j.exphem.2003.10.014] [PMID]
25. Beagles KE, Peterson L, Zhang X, Morris J, Kiem HP. Cyclosporine inhibits the development of green fluorescent protein (GFP)-specific immune responses after transplantation of GFP-expressing hematopoietic repopulating cells in dogs. Hum Gene Ther. 2005; 16 (6): 725-33. [DOI:10.1089/hum.2005.16.725] [PMID]
26. Granelli-Piperno A, Keane M, Steinman R. Evidence that cyclosporine inhibits cell-mediated immunity primarily at the level of the T lymphocyte rather than the accessory cell. Transplant. 1988; 46 (2 Suppl): 53S-60S. [DOI:10.1097/00007890-198808001-00011] [PMID]
27. Rosenzweig M, Connole M, Glickman R, Yue SP, Noren B, DeMaria M, et al. Induction of cytotoxic T lymphocyte and antibody responses to enhanced green fluorescent protein following transplantation of transduced CD34 (+) hematopoietic cells. Blood. 2001; 97 (7): 1951-9. [DOI:10.1182/blood.V97.7.1951] [PMID]
28. Jiang X, Sung YK, Tian W, Qian J, Semenza GL, Nicolls MR. Graft microvascular disease in solid organ transplantation. J Mol Med (Berlin). 2014; 92 (8): 797-810. [DOI:10.1007/s00109-014-1173-y] [PMID] [PMCID]
29. Choy JC. Granzymes and perforin in solid organ transplant rejection. Cell Death Differ. 2010; 17 (4): 567-76. [DOI:10.1038/cdd.2009.161] [PMID]
30. Vahabpour R, Basimi P, Roohvand F, Asadi H, Irani GM, Zabihollahi R, et al. Anti-viral effects of superpositively charged mutant of green fluorescent protein. Protein Pept Lett. 2019; 26 (12): 930-9. [DOI:10.2174/0929866526666190823145916] [PMID]
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Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.