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In vitro STING Activation with the cGAMP-STINGΔTM Signaling Complex

cGAMP-STINGΔTM信号复合物体外激活STING

YH Yanpu He
CH Celestine Hong
DI Darrell J. Irvine
JL Jiahe Li
PH Paula T. Hammond

发布: 2021年02月05日第11卷第3期 DOI: 10.21769/BioProtoc.3905 浏览次数: 7073

评审: Shyam SrivatsAnand Ravindran

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Abstract

Activating the STING (stimulator of interferon genes) signaling pathway via administration of STING agonist cyclic GMP-AMP (cGAMP) has shown great promise in cancer immunotherapy. While state-of-the-art approaches have predominantly focused on the encapsulation of cGAMP into liposomes or polymersomes for cellular delivery, we discovered that the recombinant STING protein lacking the transmembrane domain (STINGΔTM) could be used as a functional carrier for cGAMP delivery and elicit type I IFN expression in STING-deficient cell lines. Using this approach, we generated anti-tumoral immunity in mouse melanoma and colon cancer models, providing a potential translatable platform for STING agonist-based immunotherapy. Here, we report the detailed in vitro STING activation protocols with cGAMP-STINGΔTM complex to assist researchers in further development of this approach. This protocol can also be easily expanded to other applications related to STING activation, such as control of various types of infections.

Keywords: STING pathway (干扰素基因的刺激因子通路) search cGAMP delivery (cGAMP传递) search Protein purification (蛋白质纯化) search Ribonucleoprotein complex (核糖核蛋白复合体) search Interferon stimulation in vitro (干扰素体外刺激) search

Background

Over the past two decades, the STING (stimulator of interferon genes) signaling pathway has emerged as a crucial feature of the immune system and a promising therapeutic target against viral and bacterial infections, autoimmune disorders, and cancers. As such, the delivery of STING agonists to boost the immune response has become an area of great interest in both academic institutions and pharmaceutical companies (Ohkuri et al., 2017). While existing efforts have focused mostly on developing synthetic delivery vehicles (Shae et al., 2019), this assumes the presence of fully functional STING in cells. STING signaling has not only been shown to be frequently impaired in cancer cells due to epigenetic silencing of the protein (Ahn et al., 2015; Xia et al., 2016); there is also an ongoing debate on whether the general population is responsive to agonist-only therapies, since 19% of humans carry a mutated STING variant (R71H-G230A-R293Q, HAQ STING) reported to exhibit impaired function (Jin et al., 2011; Fu et al., 2015; Patel et al., 2017; Sivick et al., 2017).


To address these concerns, we engineered a truncated portion of the original STING protein to pre-assemble with STING agonists, acting as a functional carrier that can effectively trigger STING signaling even in the absence of STING proteins in mammalian cells. Our in vivo vaccination studies with this platform has shown efficient activation of B cells, cytotoxic T cells and memory precursor T cells, as well as robust anti-tumoral immunity against melanoma and colon cancer mouse models (He et al., 2020).


Here, we report the detailed protocols of our in vitro STING activation assays (Table 1) with cGAMP-STINGΔTM ribonucleoprotein complex in three cell lines: human embryonic kidney (HEK293T) cell, mouse macrophage (RAW264.7) and mouse dendritic cell (DC2.4). The purification protocol of STINGΔTM is also included to ensure the reproducibility of our work. We believe this protocol may assist further mechanistic discoveries in the signaling pathway and more engineering applications of this platform in vaccinology and cancer immunotherapy.


Table 1. Summary of in vitro STING activation assays


Materials and Reagents

Protein Purification

  1. Poly-Prep chromatography column (Bio-Rad, catalog number: 7311550 )

  2. ZebaTM Spin Desalting Columns 40k MWCO 10 ml (Thermo Fisher Scientific, catalog number: 87772 )

  3. BL21 (DE3) competent E. coli (NEB, catalog number: C2527I )

  4. RosettaTM (DE3) competent E. coli (Millipore Sigma, catalog number: 70954 )

  5. Isopropyl-β-D-thiogalactopyranoside (IPTG, Millipore Sigma, catalog number: I6758-10G )

  6. Phosphate Buffered Saline (PBS, Lonza, catalog number: 17-516F )

  7. Imidazole (Millipore Sigma, catalog number: I5513 )

  8. Lysozyme from chicken egg white (Millipore Sigma, catalog number: 62971-10G-F )

  9. TritonTM X-100 (Millipore Sigma, catalog number: T8787-250ml )

  10. TritonTM X-114 (Millipore Sigma, catalog number: 93422-250ml )

  11. Phenylmethylsulfonyl fluoride (PMSF, Millipore Sigma, catalog number: P7626-5G )

  12. cOmpleteTM Mini, EDTA free (Roche, catalog number: 11836170001 )

  13. Bovine Serum Albumin (BSA, Millipore Sigma, catalog number: A3608 )

  14. HisPurTM Cobalt Resin (Thermo Fisher Scientific, catalog number: 89964 )

  15. 1,4-Dithiothreitol (DTT, Millipore Sigma, catalog number: 10197777001 )

  16. Sodium Phosphate Monobasic (Millipore Sigma, catalog number: S3139 )

  17. Sodium Phosphate Dibasic (Millipore Sigma, catalog number: S3264 )

  18. Sodium Chloride (Millipore Sigma, catalog number: S9888 )

  19. HEPES (Millipore Sigma, catalog number: H3375 )

  20. Glycerol (Millipore Sigma, catalog number: G5516 )

  21. Protein Binding Buffer (see Recipes)

  22. Protein Elution Buffer (see Recipes)

  23. Protein Storage Buffer (see Recipes)


Cells and cell culture media

  1. HEK293T and RAW264.7 cells were obtained from the American Type Culture Collection (ATCC)

  2. DC2.4 cells were obtained from Rock lab, University of Massachusetts Medical School, MA, USA

  3. RAW-Blue ISG cells were obtained from Invivogen

  4. Dulbecco’s modified Eagle’s medium (DMEM, Corning, catalog number: 10-041-CV )

  5. Roswell Park Memorial Institute (RPMI) medium (Corning, catalog number: 10-013-CV )

  6. 0.25% Trypsin-EDTA (Gibco, catalog number: 25200-056 )

  7. Fetal bovine serum (FBS, Gibco, catalog number: 10437-028 )

  8. Penicillin-Streptomycin Solution, 100x (Corning, catalog number: 30-002-CI )


IFN-luciferase assay

  1. 96-well clear bottom white plate (Millipore Sigma, catalog number: CLS3610 )

  2. pGL4.45[luc2p/ISRE/Hygro] Vector (Promega)

  3. Firefly Luciferase Assay Kit (Biotium, catalog number: 30075-2 )

  4. TransIT-X2® Transfection Reagent (Mirus, catalog number: MIR 6004 )


RNA extraction, Reverse Transcription, and qPCR

  1. LightCyclerTM 480 Multiwell Plate 96 clear with Sealing Foils (Roche, catalog number: 05102413001)

  2. RNeasyTM micro kit (Qiagen, catalog number: 74004 )

  3. Beta-Mercaptoethanol (βME, Millipore Sigma, catalog number: M6250-10ML )

  4. Reverse transcription kit (Thermo Fisher Scientific, catalog number: 4374966 )

  5. SYBRTM Green PCR Master Mix (Thermo Fisher Scientific, catalog number: 4309155 )

  6. qPCR primers used for detection: mIFN-β-F: 5’-GCCTTTGCCATCCAAGAGATGC-3’, mIFN-β-R: 5’-ACACTGTCTGCTGGTGGAGTTC-3’, mActin-F: 5’-CATTGCTGACAGGATGCAGAAGG-3’, and mActin-R: 5’-TGCTGGAAGGTGGACAGTGAGG-3’ (ordered from IDT as custom oligo DNA)


mCXCL10 ELISA

  1. Greiner Bio-One MICROLONTM 600 High Binding 96-Well ELISA Assay Microplates (Fisher Scientific, catalog number: 07-000-627 )

  2. Mouse CXCL10 ELISA kit (R&D, catalog number: DY466 )

  3. TMB Substrate Set (BioLegend, catalog number: 421101 )

  4. TWEEN 20 (Millipore Sigma, catalog number: P9416-100ml )


SEAP-IFN assay

  1. QUANTI-BlueTM Solution (Invivogen, catalog code: rep-qbs )

Equipment

  1. Misonix sonicator 3000

  2. Real-time PCR system (Roche, model: LightCycler 480 )

  3. Nanodrop spectrophotometer (Thermo Fisher, model: ND-1000 )

  4. Thermal cycler (Bio-Rad, model: T100 )

  5. Plate reader (Tecan, model: Infinite M200 )

Software

  1. GraphPad Prism

Procedure

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文章信息

版权信息

© 2021 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

  1. He, Y., Hong, C., Irvine, D. J., Li, J. and Hammond, P. T. (2021). In vitro STING Activation with the cGAMP-STINGΔTM Signaling Complex. Bio-protocol 11(3): e3905. DOI: 10.21769/BioProtoc.3905.
  2. He, Y., Hong, C., Yan, E. Z., Fletcher, S. J., Zhu, G., Yang, M., Li, Y., Sun, X., Irvine, D. J. and Li, J. (2020). Self-assembled cGAMP-STINGΔTM signaling complex as a bioinspired platform for cGAMP delivery. Sci Adv 6(24): eaba7589.

    3. ris ris Download Citation in RIS Format

分类

免疫学 > 免疫细胞功能 > 细胞因子

细胞生物学 > 细胞信号传导

癌症生物学 > 肿瘤免疫学 > 细胞生物学试验

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