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Jun 2020
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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   

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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), cGAMP delivery (cGAMP传递), Protein purification (蛋白质纯化), Ribonucleoprotein complex (核糖核蛋白复合体), Interferon stimulation in vitro (干扰素体外刺激)

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

  1. STINGΔTM protein purification

    The DNA sequences (sources: Tmem173 NM_028261 Mouse Tagged ORF Clone, Origene Catalog number: MR227544, STING TMEM173 NM_198282 Human Tagged ORF Clone, Origene Catalog number: RC208418) of STINGΔTM protein (138 to 378 amino acids for mouse STING, 139 to 379 amino acids for human STING) were synthesized as gBlock DNA fragments (Integrated DNA Technologies) and cloned into the pSH200 expression vector (linearized via Nco I and Not I restriction enzymes) with a hexa-histidine-tag at the N-terminus (Figure 1). Plasmids for STINGΔTM mutants such as S365A were then generated via site-specific mutagenesis. DE3 Escherichia coli (E. coli) was used to express the proteins (mouse STINGΔTM in BL21 DE3, human STINGΔTM in Rosetta DE3). Lysogeny Broth (LB) containing the antibiotic ampicillin (100 mg/L) was used for bacteria culture, shaker conditions were 37 °C 220 rpm for growth and 18 °C 220 rpm for induction.



    Figure 1. Plasmid maps of (A) pSH200_His6_TEV_human STING (139-379aa) and (B) pSH200_His6_TEV_mouse STING (138-378aa)


    Day 1: Bacteria culture

    1. Pick DE3 E. coli from glycerol stock and culture in 50 ml of LB with ampicillin at 37 °C overnight.


    Day 2: Induction
    1. Transfer 50 ml of overnight culture into a flask containing 1 L LB with ampicillin and culture for approximately 2 h at 37 °C until the culture’s OD600 reaches 0.4. This OD value is critical for expressing human STINGΔTM in Rosetta DE3, as we have found that higher concentration can compromise protein purity; for the expression of mouse STINGΔTM in BL21 DE3, the OD600 can be in the range of 0.4-0.8 without compromising yield and purity.

    2. Cool the 1 L culture on ice and add 0.5 ml 1 M IPTG, then shake at 18 °C for approximately 20 h.


    Day 3: Protein purification
    1. After induction, centrifuge bacteria cultures at 6,000 × g for 20 min.

    2. Collect pellets and wash once with 30 ml PBS, then lyse in 20 ml protein binding buffer (Recipe 1) with 20 mg lysozyme, 200 μl Triton X-100, 1 mM PMSF (replenished every 30 min until Cobalt binding) and one tablet of cOmplete protease inhibitor cocktail tablets at room temperature for 20 min with gentle rotation at 20 rpm. After cells are lysed, the proteins should be kept ice-cold throughout the purification to minimize degradation (using cold protein binding buffer, protein elution buffer, and protein storage buffer).

    3. Cool cell lysate on ice water and sonicate with Misonix sonicator 3000 at 18 W (with 3-s on and 5-s off intervals to prevent heat-deactivation of the protein) for a total of 5 min.

    4. After sonication, centrifuge cell lysate at 14,000 × g at 4 °C for 30 min.

    5. Wash 0.5 ml HisPur Cobalt Resin with 5 ml protein binding buffer, then add to the bacteria supernatant separated from the centrifugation, along with 20 μl Triton X-114 (to eliminate endotoxin) for Cobalt-HisTag binding (4 °C for 1 h with gentle rotation at 20 rpm).

    6. After binding, carefully aspirate the supernatant and wash the Cobalt resin twice (4 °C for 30 min each time with gentle rotation at 20 rpm) with 5 ml protein binding buffer containing 5 μl Triton X-114.

    7. Transfer washed Cobalt resin into a Poly-Prep chromatography column with 5 ml of protein binding buffer. After all the protein binding buffer has drained out under gravity, add 1.5 ml protein elution buffer (Recipe 2) and collect the elution.

    8. Desalt the protein elution with ZebaTM Spin Desalting Columns (40k MWCO 10 ml). Add 10 ml of protein storage buffer (Recipe 3) into the column and allow it to completely drain under gravity (until no liquid remains above the resin surface). Add the 1.5 ml protein elution dropwise onto the middle of the resin and allow it to drain completely. Finally, collect five 1 ml fractions with protein storage buffer (add 1 ml of storage buffer then collect the flow-through as “Fraction 1”, then add another 1 ml of storage buffer and collect “Fraction 2” … all the way till “Fraction 5”). Combine Fractions 3 and 4 (which contains the majority of the desalted protein) for SDS-PAGE characterization and BCA quantification of protein concentration. Fractions 1, 2 may contain minimal amounts of protein, fraction 5 may contain protein with imidazole. The concentrations of each fraction may also be quantified through the BCA assay.

    9. For storage, 1 mM DTT was added and protein solution was aliquoted then stored in -80 °C. Protein function can be maintained for years under this storage condition, but can be compromised by multiple freeze-thaw cycles.


  2. Cell lines used for in vitro STING activation

    Human embryonic kidney 293T (HEK293T) cells are deficient in cGAMP synthase (cGAS) and STING proteins, but express other essential proteins in downstream STING signaling, including TANK-binding kinase 1 (TBK1) and Interferon regulatory factor 3 (IRF3). Therefore, it provides a good model for studying the function of cGAMP-STINGΔTM without interactions with endogenous cGAMP or STING proteins (Figure 2). Additionally, it can be transfected to overexpress full-length mutant STING proteins (for example HAQ STING). To detect STING activation, we generated an interferon (IFN) reporter cell line by integrating an IFN-stimulated response element (ISRE) that drives luciferase expression through the stable transfection of pGL4.45[luc2p/ISRE/Hygro] plasmid selected in hygromycin (200 μg/ml). HEK293T cells are not capable of uptaking cGAMP-STINGΔTM complex directly, so commercial transfection reagents (we primarily used TransIT-X2, though others like Lipofectamine also work) are required in order to deliver cGAMP-STINGΔTM complex into the cells. HEK293T cells are cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin.

      Mouse macrophage RAW264.7 and dendritic cell DC2.4 both express cGAS and STING, as well as TBK1 and IRF3 (Figure 2). They are also capable of internalizing cGAMP-STINGΔTM complex without the help of a transfection reagent. To detect STING activation, we can measure the cell secreted CXCL10 concentration in the medium with enzyme-linked immunosorbent assay (ELISA) or quantify the interferon-β mRNA level with qPCR. The aforementioned media contains no cells – medium that contains cells is denoted simply as ‘cells’. In addition, a reporter cell line derived from RAW264.7: RAW-BlueTM ISG cells can be used to study the kinetics of STING activation in vitro, and since the Secreted embryonic alkaline phosphatase (SEAP) – IFN assay only requires 20 μl of media, it can be performed at multiple time points post treatment. RAW264.7 cells are cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. For SEAP-IFN assay, RAW-BlueTM ISG cells are cultured in DMEM with 10% heat-inactivated (56 °C, 30 min) FBS and 1% penicillin/streptomycin. DC2.4 cells are cultured in Roswell Park Memorial Institute (RPMI) medium with 10% FBS and 1% penicillin/streptomycin.

      All cells are cultured in a 37 °C, 5% CO2 incubator, used at low passage number and tested negative for Mycoplasma contamination.



    Figure 2. Immunoblotting of endogenous expression of cGAS, STING, TBK1, and IRF3 proteins from HEK293T, RAW264.7, and DC2.4 cells


  3. IFN-luciferase assay with HEK293T-luc2p/ISRE/Hygro

    Day 1: Preparation of Cells
    1. Seed HEK293T-luc2p/ISRE/Hygro cells into 96-well plates (clear bottom flat white plates for luciferase assay) at a density of 3 × 105 cells/ml in 100 μl media per well.


    Day 2: Cell treatment
    1. For each well, mix 1 μg of STINGΔTM protein with 0.025 μg cGAMP, then add 1 μl of TransIT-X2 in OptiMEM media to a total volume of 20 μl and incubate at room temperature for 15 min.

    2. Add the mixture to the cell medium without pipetting up and down. At least 3 replicates should be performed for each treatment.

    3. Incubate treated cells for 24 h.


    Day 3: Luminescence measurement
    1. Remove plates from the incubator, aspirate medium and add 25 μl lysis buffer (five-fold diluted with DI water from the provided 5× firefly luciferase lysis buffer) to each well, then incubate at room temperature for 15 min with orbital shaking.

    2. Add 50 μl of luciferase assay buffer to each well (with D-luciferin freshly added at final concentration of 0.2 mg/ml), read plate for bioluminescence on a Tecan microplate reader.


  4. mCXCL10 ELISA with RAW264.7 and DC2.4

    Day 1: Preparation of Cells
    1. Seed RAW264.7 or DC2.4 cells into 96-well plates at a density of 2 × 105 cells/ml in 100 μl medium per well.


    Day 2: Cell treatment
    1. For each well, mix 1 μg of STINGΔTM protein with 0.025 μg cGAMP and 1 μl of TransIT-X2 in 20 μl OptiMEM medium, then incubate at room temperature for 15 min. Alternatively, for vehicle-free treatment, mix 5 μg of STINGΔTM protein with 0.125 μg cGAMP in 20 μl OptiMEM medium and incubate at room temperature for 15 min.

    2. Add the mixture to the cell medium without pipetting up and down. At least 3 replicates should be performed for each treatment.

    3. Incubate treated cells for 48 h.


    Day 3: Preparation of ELISA plates

    ELISA was performed according to the manufacturer’s protocol: Mouse CXCL10 ELISA kit (R&D, DY466 )

    1. Dilute CXCL-10 capture antibody in PBS to 2 μg/ml.

    2. Add 100 μl capture antibody solution to each well of the ELISA assay plates. Incubate at 4 °C overnight.


    Day 4: ELISA
    1. Aspirate each well of the ELISA assay plates, wash three times with Wash Buffer (PBS with 0.05% Tween 20).

    2. Add 300 μl Reagent Diluent (PBS with 1% BSA) to each well to block plates. Incubate at room temperature for 1 h with orbital shaking at 220 rpm.

    3. Aspirate plates and wash three times with Wash Buffer.

    4. Add 100 μl media of RAW264.7 or DC2.4 cells 48 h post-treatment without dilution. Incubate at room temperature for 2 h with orbital shaking at 220 rpm.

    5. Aspirate plates and wash three times with Wash Buffer.

    6. Add 100 μl of the Detection Antibody at 100 ng/ml (diluted in Reagent Diluent) to each well. Incubate at room temperature for 2 h with orbital shaking at 220 rpm.

    7. Aspirate plates and wash three times with Wash Buffer.

    8. Add 100 μl of the provided Streptavidin-HRP stock solution 40-fold diluted in Reagent Diluent to each well. Incubate at room temperature for 20 min avoiding light with orbital shaking at 220 rpm.

    9. Aspirate plates and wash three times with Wash Buffer.

    10. Add 100 μl of TMB Substrate Solution, freshly prepared from mixing equal volumes of TMB Substrate A with TMB Substrate B. Incubate at room temperature for 20 min with orbital shaking at 220 rpm, avoiding light.

    11. Add 50 μl of Stop Solution (2 N H2SO4) to each well. Gently tap the plate for mixing. Measure absorbance at 450 nm with a spectrophotometer/plate reader.


  5. Quantification of mIFN-β expression by qPCR with RAW264.7 and DC2.4

    Day 1: Preparation of Cells
    1. Seed RAW264.7 or DC2.4 cells into 24-well plates at a density of 3 × 105 cells/ml in 400 μl media per well.


    Day 2: Cell treatment
    1. For each well, mix 5 μg of STINGΔTM protein with 0.125 μg cGAMP and 5 μl of TransIT-X2 in 50 μl OptiMEM medium. Incubate at room temperature for 15 min. Alternatively, for vehicle-free treatment, mix 25 μg of STINGΔTM protein with 0.625 μg cGAMP in 50 μl OptiMEM medium and incubate at room temperature for 15 min.

    2. Add the mixture to the cell medium without pipetting up and down.

    3. Incubate treated cells for 24 h.


    Day 3: RNA extraction, reverse transcription and qPCR

    Perform RNA extraction following protocol provided by RNeasy Micro Kit:

    1. Prepare fresh lysis buffer by adding 10 μl β-mercaptoethanol into 1 ml RLT buffer.

    2. Aspirate medium, wash cells once with PBS, then add 350 μl lysis buffer per well. Incubate at room temperature for 5 min.

    3. Transfer the cell lysate into Eppendorf tubes, vortex for 20 s, then add 350 μl 70% ethanol, pipette up and down, transfer to a spin column provided in the kit, and centrifuge for 15 s at 8,000 × g.

    4. Discard flowthrough, add 350 μl RW1 buffer, and centrifuge for 15 s at 8,000 × g.

    5. Prepare DNase buffer from DNase supplied in lyophilized form in glass vials. Use syringe to inject 500 μl water into the glass vial to dissolve the powder. Then open the vial and add 10 μl DNase solution + 70 μl RDD buffer to the column. Incubate at room temperature for 15 min. Then add 350 μl RW1 buffer to the spin column, centrifuge for 15 s at 8,000 × g and discard the collection tube.

    6. Place the spin column in a new 2 ml collection tube as supplied. Add 500 μl RPE buffer, centrifuge for 15 s at 8,000 × g, and discard the flow through.

    7. Add 500 μl 80% ethanol to the spin column, centrifuge for 2 min at 8,000 × g and discard the collection tube.

    8. Place the spin column in another new 2 ml collection tube as supplied. Centrifuge at 8,000 × g for 5 min, discard the collection tube.

    9. Place the spin tube in a new 1.5 ml collection tube as supplied, add 14 μl RNase-free water to the center of the spin column, and centrifuge for 1 min at 8,000 × g to elute the RNA.

    10. Measure RNA concentration of each cell sample with Nanodrop spectrophotometer.


    Reverse transcription
    1. Dilute 1 μg extracted RNA with water to a total volume of 10 μl.

    2. For each RNA sample, prepare below mixture in a PCR tube:

      Diluted RNA           10 μl

      10× RT buffer            2 μl

      dNTP                          0.8 μl

      Random primer        2 μl

      RNase inhibitor      1 μl

      Enzyme                      1 μl

      Water                         3.2 μl

      Total volume             20 μl

    3. Treat the sample with thermal cycler with the program below:

      25 °C  10 min

      37°C    2 h

      85 °C   5 min

      4 °C     Infinite hold


    qPCR

    1. Dilute qPCR primers to 10 μM working concentration.

    2. Add 80 μl water to 20 μl reverse transcription product from the previous step (this 5-fold dilution is denoted as ‘template’ in the following step)

    3. Add the following to the qPCR 96-well plate for each reaction (add primer first, then add master mix):

      Template                        1 μl

      2x SYBR mixture          10 μl

      Forward Primer           0.5 μl

      Reverse Primer            0.5 μl

      Water                          8 μl

      Total volume               20 μl


  6. IFN-SEAP assay with RAW-Blue ISG cells

    Day 1: Preparation of Cells
    1. Seed RAW264.7 or DC2.4 cells into 96-well plates at a density of 2 × 105 cells/ml in 100 μl media per well.


    Day 2: Cell treatment
    1. 2 h prior to treatment, remove the medium and replenish it with 100 μl DMEM with 10% heat-inactivated FBS and 1% penicillin/streptomycin pre-warmed to 37 °C in order to reduce the noise level in subsequent QUANTI-Blue assay.

    2. For each well, mix 1 μg of STINGΔTM protein with 0.025 μg cGAMP then 1 μl of TransIT-X2 in 20 μl OptiMEM medium, incubated at room temperature for 15 min. Alternatively, for vehicle-free treatment, mix 5 μg of STINGΔTM protein with 0.125 μg cGAMP in 20 μl OptiMEM medium and incubate at room temperature for 15 min.

    3. Add the mixture to the cell medium without pipetting up and down. At least 3 replicates should be performed for each treatment.

    4. Incubate treated cells in 37 °C incubator.


    Days 3-4: IFN-SEAP assay
    1. Take 20 μl of medium from treated cell wells and mix with 180 μl of QUANTI-Blue assay buffer in a separate 96-well plate. (Multiple time points between 12 to 48 h can be taken to study the kinetics.)

    2. Incubate the plate at 37 °C for 6 to 10 h until a visible color difference is observed (Figure 3).

    3. Determine the IFN-SEAP activity by measuring the absorbance at 635 nm with a spectrophotometer/plate reader.



      Figure 3. Example IFN-SEAP assay of mixing medium of treated RAW-Blue ISG cells with QUANTI-Blue assay buffer showing color differences due to STING signaling

Data analysis

Data can be analyzed with GraphPad Prism and statistical analyses performed with one-way analysis of variance (ANOVA) followed by Student’s t-test for statistical significance.

Recipes

  1. Protein Binding Buffer

    50 mM sodium phosphate

    0.5 M NaCl

    10 mM imidazole

    pH 7.4

  2. Protein Elution Buffer

    50 mM sodium phosphate

    0.5 M NaCl

    150 mM imidazole

    pH 7.4

  3. Protein Storage Buffer

    20 mM HEPES

    150 mM NaCl

    10% glycerol

    pH 7.4

Acknowledgments

This work was supported by the Department of Defense Congressionally Directed Medical Research Program’s (CDMRP) Ovarian Cancer Research Program, Cancer Center Support Grant (CCSG) Pilot Awards at the David H. Koch Institute for Integrative Cancer Research at MIT, the Institute for Soldier Nanotechnologies (ISN) at MIT, the Marble Center for Cancer Nanomedicine, Northeastern University Faculty start-up funding, and the Peer Reviewed Medical Research Program from the Department of Defense’s Congressionally Directed Medical Research Programs (W81XWH18PRMRPDA).

Competing interests

The authors declare that they have no competing interests.

References

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  2. Fu, J., Kanne, D. B., Leong, M., Glickman, L. H., McWhirter, S. M., Lemmens, E., Mechette, K., Leong, J. J., Lauer, P. and Liu, W. (2015). STING agonist formulated cancer vaccines can cure established tumors resistant to PD-1 blockade. Sci Transl Med 7(283): 283ra252-283ra252.
  3. 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.
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  7. Shae, D., Becker, K. W., Christov, P., Yun, D. S., Lytton-Jean, A. K., Sevimli, S., Ascano, M., Kelley, M., Johnson, D. B. and Balko, J. M. (2019). Endosomolytic polymersomes increase the activity of cyclic dinucleotide STING agonists to enhance cancer immunotherapy. Nat Nanotechnol 1.
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  9. Xia, T., Konno, H. and Barber, G. N. (2016). Recurrent loss of STING signaling in melanoma correlates with susceptibility to viral oncolysis. Cancer Res 76(22): 6747-6759.

简介

[摘要]通过给予STING激动剂环状GMP-AMP(cGAMP)激活STING(干扰素基因的刺激物)信号通路已显示出在癌症免疫治疗中的广阔前景。尽管目前最先进的方法主要集中在将cGAMP封装进脂质体或聚合物小体中以进行细胞递送,但我们发现缺少跨膜结构域(STINGΔTM)的重组STING蛋白可以用作cGAMP递送的功能载体。在STING缺陷型细胞系中诱导I型IFN表达。使用这种方法,我们在小鼠黑素瘤和结肠癌模型中产生了抗肿瘤免疫力,为基于STING激动剂的免疫疗法提供了潜在的可翻译平台。在这里,我们报告与cGAMP-STINGΔTM复合物的详细体外STING激活方案,以帮助研究人员进一步开发这种方法。该协议还可以轻松扩展到与STING激活相关的其他应用程序,例如控制各种类型的感染。


[背景]在过去的二十年中,STING(干扰素基因的刺激物)信号传导途径已成为免疫系统的关键特征,并有望成为针对病毒和细菌感染,自身免疫性疾病和癌症的治疗靶标。因此,递送STING激动剂以增强免疫应答已经成为学术机构和制药公司的极大兴趣领域(Ohkuri等人,2017)。尽管现有的努力主要集中在开发合成运载工具上(Shae et al。,2019),但这是假设细胞中存在功能齐全的STING。由于该蛋白的表观遗传沉默,不仅表明STING信号转导在癌细胞中经常受损(Ahn等,2015; Xia等,2016)。关于普通人群是否仅对激动剂疗法有反应,也有一个持续的争论,因为19%的人携带突变的STING变异体(R71H-G230A-R293Q,HAQ STING),据报道其功能受损(Jin等, 2011; Fu等,2015; Patel等,2017; Sivick等,2017)。

为了解决这些问题,我们设计了原始STING蛋白的截短部分,以与STING激动剂进行预组装,以作为功能性载体,即使在哺乳动物细胞中不存在STING蛋白的情况下,也可以有效触发STING信号传导。我们使用该平台进行的体内疫苗接种研究显示,B细胞,细胞毒性T细胞和记忆前体T细胞能够有效激活,并且具有针对黑素瘤和结肠癌小鼠模型的强大抗肿瘤免疫力(He等,2020)。

在这里,我们报告了在三种细胞系中用cGAMP-STINGΔTM核糖核蛋白复合物进行的体外STING激活测定的详细方案(表1):人胚肾(HEK293T)细胞,小鼠巨噬细胞(RAW264.7)和小鼠树突状细胞(DC2) .4)。还包括STINGΔTM的纯化方案,以确保我们工作的可重复性。我们认为,该协议可能有助于进一步的机制发现,以及该平台在疫苗学和癌症免疫疗法中的更多工程应用。

关键字:干扰素基因的刺激因子通路, cGAMP传递, 蛋白质纯化, 核糖核蛋白复合体, 干扰素体外刺激

材料和试剂
蛋白质纯化

Poly-Prep色谱柱(Bio-Rad,目录号:7311550)
Zeba TM旋转脱盐柱40k MWCO 10 ml(Thermo Fisher Scientific,目录号:87772)
BL21(DE3)感受态大肠杆菌(NEB,目录号:C2527I)
Rosetta TM (DE3)感受态大肠杆菌(Millipore Sigma,目录号:70954)
异丙基-β-D-硫代吡喃半乳糖苷(IPTG,Millipore Sigma,目录号:I6758-10G)
磷酸盐缓冲盐水(PBS,Lonza,目录号:17-516F)
咪唑(Millipore Sigma,目录号:I5513)
鸡蛋白的溶菌酶(Millipore Sigma,目录号:62971-10G-F)
Triton TM X-100(Millipore Sigma,目录号:T8787-250ml)
Triton TM X-114(Millipore Sigma,目录号:93422-250ml)
苯甲基磺酰氟(PMSF,Millipore Sigma,目录号:P7626-5G)
cOmplete TM Mini,无EDTA(罗氏,目录号:11836170001)
牛血清白蛋白(BSA,Millipore Sigma,目录号:A3608)
HisPur TM钴树脂(Thermo Fisher Scientific,目录号:89964)
1,4-二硫苏糖醇(DTT,Millipore Sigma,目录号:10197777001)
钠P hosphate一元(Millipore公司Sigma,目录号:S3139)
磷酸氢二钠(Millipore Sigma,目录号:S3264)
氯化钠(Millipore Sigma,目录号:S9888)
HEPES (Millipore Sigma,目录号:H3375)
甘油(Millipore Sigma,目录号:G5516)
蛋白乙inding乙uffer(见食谱)
蛋白质è lution乙uffer(见食谱)
蛋白质小号torage乙uffer(见食谱)

细胞和细胞培养基

HEK293T和RAW264.7细胞获自美国典型培养物保藏中心(ATCC)
DC2.4细胞获自美国马萨诸塞州大学医学院岩石实验室
RAW-Blue ISG细胞获自Invivogen
Dulbecco改良的Eagle培养基(DMEM,Corning,目录号:10-041-CV)
罗斯威尔公园纪念学院(RPMI)介质(Corning,目录号:10-013-CV)
0.25%胰蛋白酶-EDTA(Gibco,目录号:25200-056)
胎牛血清(FBS,Gibco,目录号:10437-028)
青霉素-链霉素溶液,100x(Corning,目录号:30-002-CI)

干扰素荧光素酶测定

96孔透明底部白色板(Millipore Sigma,目录号:CLS3610)
pGL4.45 [luc2p / ISRE / Hygro]载体(Promega)
萤火虫荧光素酶检测试剂盒(生物素,目录号:30075-2)
的TransIT-X2 ®转染试剂(Mirus公司,目录号:MIR 6004)

RNA提取,逆转录和qPCR

LightCycler TM 480多孔板96透明,带密封箔(Roche,目录号:05102413001)
RNeasy TM微型试剂盒(Qiagen,目录号:74004)
β-巯基乙醇(βME,Millipore Sigma,目录号:M6250-10ML)
逆转录试剂盒(Thermo Fisher Scientific,目录号:4374966)
SYBR TM Green PCR预混液(Thermo Fisher Scientific,目录号:4309155)
用于检测的qPCR引物:mIFN-β-F:5'-GCCTTTGCCATCCAAGAGATGC-3',mIFN-β-R:5'-ACACTGTCTGCTGGTGTGGAGTTC-3',mActin-F:5'-CATTGCTGACAGGATGCAGAAGG-3'和mActin-R :5'-TGCTGGAAGGTGGACAGTGAGG-3'(从IDT订购为定制的寡核苷酸DNA)
mCXCL10酶联免疫吸附测定

Greiner Bio-One MICROLON TM 600高结合96孔ELISA分析微孔板(Fisher Scientific,目录号:07-000-627)
小鼠CXCL10 ELISA试剂盒(R&D,目录号:DY466)
TMB底物组(BioLegend,目录号:421101)
TWEEN 20(Millipore Sigma,目录号:P9416-100ml)

SEAP-IFN测定

QUANTI-Blue TM解决方案(Invivogen,目录代码:rep-qbs)

设备


Misonix超声波仪3000
ř EAL-时间PCR体系(Roche公司,型号:的LightCycler 480)
Nanodrop分光光度计(Thermo Fisher,型号:ND-1000 )
牛逼有源冰箱,循环仪(Bio-Rad公司,型号:T100)
P晚读者(Tecan公司,型号:M200无限)

软洁具


GraphPad棱镜

程序


STING ΔTM蛋白纯化
的DNA sequenc ES(来源:Tmem173 NM_028261小鼠标记ORF克隆,Origene公司目录号:MR227544,STING TMEM173 NM_198282人力标记ORF克隆,Origene公司目录号:RC208418)O ˚F STING ΔTM蛋白(138至378个氨基酸的小鼠斯汀,139合成了379个氨基酸(用于人类STING的氨基酸)作为gBlock DNA片段(Integrated DNA Technologies),并克隆到了pSH200表达载体(通过Nco I和Not I限制酶线性化)的N端带有一个六组氨酸标签(图1)。PL asmids为STING ΔTM突变体如S365A然后通过位点特异性诱变产生。DE3大肠杆菌(E. coli)用于表达蛋白质(BL21 DE3中的小鼠STINGΔTM,Rosetta DE3中的人STINGΔTM)。含有抗生素氨苄西林(100 mg / L)的溶菌汤(LB)用于细菌培养,摇床条件是37°C 220 rpm进行生长和18°C 220 rpm进行诱导。





图1. (A)pSH200_His6_TEV_human STING (139-379aa)和(B)pSH200_His6_TEV_mouse STING (138-378aa)的质粒图


第一天:细菌培养

从甘油储备液中提取DE3大肠杆菌,并在50 ml LB和氨苄西林中于37°C过夜培养。

第2天:入职

将50 ml过夜培养物转移到装有1 L LB和氨苄青霉素的烧瓶中,并在37°C下培养2 h,直到培养物的OD 600达到0.4。该OD值对于在Rose tta DE3中表达人STINGΔTM至关重要,因为我们发现较高的浓度会损害蛋白质的纯度。对于小鼠STINGΔTM在BL21 DE3中的表达,OD600可以在0.4-0.8的范围内,而不会影响产量和纯度。
在冰上冷却1 L培养物,加入0.5 ml 1 M IPTG,然后在18°C摇动大约20 h。

第三天:蛋白质纯化

诱导后,离心细菌以6,000 × g培养20分钟。
收集沉淀并用30 ml PBS洗涤一次,然后在含有20 mg溶菌酶,200μlTriton X-100、1 mM PMSF (每30分钟补充一次,直至与钴结合)的20 ml蛋白结合缓冲液(配方1 )中裂解,并溶解一片ø ˚F完全蛋白酶抑制剂混合物片剂在室温下以20rpm 20分钟,轻轻旋转。裂解细胞后,应在整个纯化过程中将蛋白质保持冰冷状态,以最大程度地减少降解(使用冷蛋白质结合缓冲液,蛋白质洗脱缓冲液和蛋白质储存缓冲液)。
在冰水上冷却细胞裂解物,并在18 W下与Misonix超声波仪3000进行超声处理(间隔3秒打开和5秒关闭以防止蛋白质热失活),总共5分钟。
超声处理后,在4°C下以14,000 × g离心细胞裂解液30分钟。
用5 ml蛋白质结合缓冲液洗涤0.5 ml HisPur钴树脂,然后将离心分离的细菌上清液与20μlTriton X-114(以消除内毒素)一起加入钴-HisTag结合液(4°C,持续1 h)。以20 rpm缓慢旋转)。
结合后,小心吸出上清液,并用含5μlTriton X-114的5 ml蛋白结合缓冲液洗涤钴树脂两次(每次4°C,每次20分钟,以20 rpm缓慢旋转)。
将洗涤过的钴树脂转移到含有5 ml蛋白结合缓冲液的Poly-Prep色谱柱中。在重力作用下将所有蛋白质结合缓冲液排干后,添加1.5 ml蛋白质洗脱缓冲液(配方2 )并收集洗脱液。
用Zeba TM旋转脱盐柱(40k MWCO 10 ml)对蛋白质洗脱液进行脱盐。在色谱柱中添加10 ml的蛋白质存储缓冲液(配方3 ),并使其在重力作用下完全排干(直到在树脂表面上方没有液体残留)。滴加1.5 ml蛋白质洗脱液到树脂中间,使其完全流干。最后,用蛋白质存储缓冲液收集5个1 ml的馏分(添加1 ml的存储缓冲液,然后将流通液收集为“ Fraction 1”,然后再添加1 ml的存储缓冲液并收集“ Fraction 2” ...一直到“分数5”)。结合小数部分s 3和d 4(包含大部分脱盐蛋白)进行SDS-PAGE表征和BCA定量蛋白质浓度。馏分1、2可能包含极少量的蛋白质,馏分5可能包含具有咪唑的蛋白质。每个级分的浓度也可以通过BCA分析进行定量。
为了储存,加入1 mM DTT,将蛋白质溶液等分,然后储存在-80°C。在这种储存条件下,蛋白质功能可以维持数年,但会因多次冻融循环而受损。

用于体外STING激活的细胞系
人类胚胎肾293T(HEK293T)细胞缺乏cGAMP合酶(cGAS)和STING蛋白,但在下游STING信号传导中表达其他必需蛋白,包括TANK结合激酶1(TBK1 )和干扰素调节因子3(IRF3)。因此,它为研究cGAMP-STINGΔTM的功能而与内源性cGAMP或STING蛋白没有相互作用提供了一个很好的模型(图2)。广告ditionally,它可被转染以过表达全长突变STING蛋白(例如HAQ STING)。为了检测STING的激活,我们通过整合一个IFN刺激的响应元件(ISRE)来产生干扰素(IFN)报告细胞系,该元件通过稳定转染潮霉素中选择的pGL4.45 [luc2p / ISRE / Hygro]质粒来驱动荧光素酶表达。 200微克/毫升)。HEK293T细胞不能直接摄取cGAMP-STINGΔTM复合物,因此需要商业转染试剂(我们主要使用TransIT-X2,尽管其他类似Lipofectamine也起作用)才能将cGAMP-STINGΔTM复合物递送到细胞中。HEK293T细胞在含10%胎牛血清(FBS)和1%青霉素/链霉素的Dulbecco改良Eagle培养基(DMEM)中培养。

小鼠巨噬细胞RAW264.7和树突状细胞DC2.4均表达cGAS和STING以及TBK1和IRF3(图2)。它们还能够在不借助转染试剂的情况下内化cGAMP-STINGΔTM复合物。为了检测STING激活,我们可以通过酶联免疫吸附测定(ELISA)测量培养基中细胞分泌的CXCL10浓度,或者通过qPCR定量测定干扰素-βmRNA的水平。上述介质不包含任何单元格-包含单元格的介质简称为“单元格”。在此外,从RAW264.7衍生的报告细胞系:RAW-蓝TM ISG细胞可用于研究STING激活的动力学在体外,并且由于分泌的胚胎碱性磷酸酶(SEAP)- IFN测定只需要20微升的媒体,可以在治疗后的多个时间点执行。RAW264.7细胞在含10%胎牛血清(FBS)和1%青霉素/链霉素的Dulbecco改良Eagle培养基(DMEM)中培养。对于SEAP-IFN分析,将RAW-Blue TM ISG细胞在DMEM中用10%热灭活(56°C,30分钟)FBS和1%青霉素/链霉素进行培养。DC2.4细胞在含有10%FBS和1%青霉素/链霉素的罗斯威尔公园纪念学院(RPMI)培养基中培养。

所有细胞均在37°C,5%CO 2的培养箱中培养,以低传代次数使用,并测试支原体污染呈阴性。





图2.来自HEK293T,RAW264.7和DC2.4细胞的cGAS,STING,TBK1和IRF3蛋白内源性表达的免疫印迹


用HEK293T-luc2p / ISRE / Hygro进行的IFN-萤光素酶测定
第一天:准备细胞

将HEK293T-luc2p / ISRE / Hygro细胞以每孔100μl培养基中3 × 10 5细胞/ ml的密度接种到96孔板(用于荧光素酶测定的透明底部平白板)中。

第二天:细胞治疗

对于每个孔,将1μgSTINGΔTM蛋白与0.025μgcGAMP混合,然后在OptiMEM培养基中加入1μlTransIT-X2至20μl的总体积,并在室温下孵育15分钟。
将混合物添加到细胞培养基中,而无需上下吹打。每种治疗至少应重复3次。
孵育处理的细胞24小时。

第三天:发光测量

ř从培养箱中,吸介质EMOVE板,并添加25μl的裂解缓冲液(5倍与FR DI水稀释奥姆所提供的5 ×萤火虫荧光素酶裂解缓冲液)到每个孔中,然后在室温下孵育15分钟,伴随轨道摇动。
向每个孔中添加50μl萤光素酶测定缓冲液(新鲜添加D-萤光素,终浓度为0.2 mg / ml),在Tecan微孔板读数器上读数板进行生物发光。

含RAW264.7和DC2.4的mCXCL10 ELISA
第一天:准备细胞

将RAW264.7或DC2.4细胞以2 × 10 5细胞/ ml的密度接种到96孔板中,每孔100μl培养基中。

第二天:细胞治疗

对于每个孔,在20μlOptiMEM培养基中混合1μgSTINGΔTM蛋白与0.025μgcGAMP和1μlT ransIT-X2 ,然后在室温下孵育15分钟。或者,对于免费车辆处理,混合5微克的STINGΔTM蛋白0.125在20μlOptiMEM培养基微克cGAMP在室温下孵育15分钟。
将混合物添加到细胞培养基中,而无需上下吹打。每种治疗至少应重复3次。
孵育处理的细胞48小时。

第三天:制备ELISA板

ELISA根据制造商的协议进行:小鼠CXCL10 ELISA试剂盒(R&D,DY466)

将PBS中的CXCL-10捕获抗体稀释至2μg/ ml 。
将100μl捕获抗体溶液添加到ELISA分析板的每个孔中。在4°C下孵育过夜。

第四天:ELISA

吸出ELISA分析板的每个孔,用洗涤缓冲液(含0.05%Tween 20的PBS)洗涤3次。
向每个孔中加入300μl试剂稀释剂(含1%BSA的PBS)封闭板。在室温下以220 rpm的频率振荡孵育1 h。
吸板并用洗涤缓冲液洗涤3次。
处理后48小时,不稀释地添加100μlRAW264.7或DC2.4细胞培养基。在220 rpm的轨道摇动下,在室温下孵育2小时。
吸板并用洗涤缓冲液洗涤3次。
向每个孔中加入100μl100 ng / ml的检测抗体(在试剂稀释剂中稀释)。在220 rpm的轨道摇动下,在室温下孵育2小时。
吸板并用洗涤缓冲液洗涤3次。
向每个孔中加入100μl用试剂稀释液稀释40倍的链霉亲和素-HRP储备液。在室温下孵育20分钟,避开光照,并以220 rpm的速度进行轨道摇动。
吸板并用洗涤缓冲液洗涤3次。
添加100μlTMB底物溶液,该溶液是通过将等体积的TMB底物A与TMB底物B混合而新鲜制备的。在室温下孵育20分钟,在220 rpm的轨道振动下避光。
向每个孔中添加50μl终止溶液(2 N H 2 SO 4 )。轻轻拍打板进行混合。用分光光度计/酶标仪测量450 nm处的吸光度。

使用RAW264.7和DC2.4的qPCR定量检测mIFN-β的表达
第一天:准备细胞

将RAW264.7或DC2.4细胞以3 × 10 5细胞/ ml的密度接种到24孔板中,每孔400μl培养基。

第二天:细胞治疗

对于每个孔,在50μlOptiMEM培养基中混合5μgSTINGΔTM蛋白与0.125μgcGAMP和5μlT ransIT-X2 。在室温下孵育15分钟。或者,对于免费车辆处理,混合25微克的STINGΔTM蛋白0.625在50μlOptiMEM培养基微克cGAMP在室温下孵育15分钟。
将混合物添加到细胞培养基中,而无需上下吹打。
孵育处理的细胞24小时。

第3天:RNA提取,逆转录和qPCR

按照RNeasy Micro Kit提供的协议进行RNA提取:

通过将10μlβ-巯基乙醇添加到1 ml RLT缓冲液中来制备新鲜的裂解缓冲液。
吸出培养基,用PBS洗涤细胞一次,然后每孔添加350μl裂解缓冲液。在室温下孵育5分钟。
将细胞裂解液转移到Eppendorf管中,涡旋振荡20 s,然后加入350μl70%乙醇,上下吸管,转移至试剂盒中提供的旋转柱中,并在8,000 × g下离心15 s 。
丢弃流通液,添加350μlRW1缓冲液,并以8,000 × g离心15 s 。
在玻璃瓶中从冻干形式提供的DNase制备DNase缓冲液。使用注射器将500μl水注入玻璃瓶中以溶解粉末。然后打开小瓶,并向柱中添加10μlDNase溶液+ 70μlRDD缓冲液。在室温下孵育15分钟。然后将350μlRW1缓冲液添加到离心柱中,以8,000 × g离心15 s,并丢弃收集管。
将离心柱置于随附的新的2 ml收集管中。加入500μlRPE缓冲液,以8,000 × g离心15 s ,并丢弃流经的溶液。
向旋转柱中加入500μl80%乙醇,以8,000 × g离心2分钟,并丢弃收集管。
将离心柱放入提供的另一个新的2 ml收集桶e中。以8,000 × g离心5分钟,弃去收集管。
将离心管放入随附的新的1.5 ml收集管中,向离心柱中心添加14μl无RNase的水,并以8,000 × g离心1分钟以洗脱RNA。
用Nanodrop分光光度计测量每个细胞样品的RNA浓度。

反转录

用水将1μg提取的RNA稀释至总体积为10μl。
对于每个RNA样品,在PCR管中准备以下混合物:
稀释的RNA 10μl                         
10 × RT缓冲液2μl           
dNTP 0.8微升           
随机引物2μl           
RNase抑制剂1μl                         
酶1微升           
水3.2微升           
总体积20μl           

使用以下程序用热循环仪处理样品:
25°C 10分钟           
37°C 2小时           
85°C 5分钟           
4°C无限保持           

定量PCR

将qPCR引物稀释至10μM工作浓度。
将80μl水加至上一步骤的20μl逆转录产物中(此5倍稀释液在下一步骤中称为“模板”)
对于每个反应,在qPCR 96孔板上添加以下物质(先添加引物,然后添加预混液):
模板   1微升           
2 x SYBR混合物  10μl           
正向引物   0.5μl           
反向引物   0.5μl           
水8微升                           
总体积20μl           

RAW-Blue ISG细胞的IFN-SEAP测定
第一天:准备细胞

将RAW264.7或DC2.4细胞以2 × 10 5细胞/ ml的密度接种到96孔板中,每孔100μl培养基。

第二天:细胞治疗

处理前2小时,移出培养基,并用100μlDMEM补充培养基,其中含有10%热灭活的FBS和1%青霉素/链霉素预热至37°C,以降低随后的QUANTI-Blue测定的噪音水平。
对于每个孔,混合在20 1微克STINGΔTM蛋白质与0.025微克cGAMP然后将1μl的TransIT-X2的微升OptiMEM中我dium ,在室温下温育15分钟。或者,对于免费车辆处理,混合5微克的STINGΔTM蛋白0.125在20μlOptiMEM培养基微克cGAMP在室温下孵育15分钟。
将混合物添加到细胞培养基中,而无需上下吹打。每种治疗至少应重复3次。
将处理过的细胞在37°C的培养箱中培养。

d AY小号3 -4:IFN-SEAP测定法

从处理过的细胞孔中取出20μl培养基,并在单独的96孔板中与180μlQUANTI-Blue分析缓冲液混合。(可以在12到48小时之间使用多个时间点来研究动力学。)
将板在37°C下孵育6至10 h,直到观察到可见的色差(图3)。
通过使用分光光度计/酶标仪在635 nm处测量吸光度来确定IFN-SEAP活性。



图3.经处理的RAW-Blue ISG细胞与QUANTI-Blue分析缓冲液混合培养基的IFN-SEAP分析示例,显示由于STING信号转导引起的颜色差异


数据一nalysis


数据可以用GraphPad棱镜进行分析和统计分析与(ANOVA)单因子变异数分析进行其次是学生的牛逼-检验统计学意义。


[R ecipes


蛋白乙inding乙uffer
50 mM磷酸钠

0.5 M氯化钠

10毫米咪唑

pH值7.4

蛋白质è lution乙uffer
50 mM磷酸钠

0.5 M氯化钠

150 mM咪唑

pH值7.4

蛋白质小号torage乙uffer
20毫米HEPES

150毫米氯化钠

10%甘油

pH值7.4


致谢


这项工作得到了美国国防部国会指导医学研究计划(CDMRP)的卵巢癌研究计划,麻省理工学院David H. Koch综合癌症研究所,士兵纳米技术研究所癌症中心支持补助金(CCSG)试点奖的支持。麻省理工学院(ISN),大理石纳米癌症医学中心,东北大学学院启动资金以及国防部国会指导性医学研究计划(W81XWH18PRMRPDA)的同行评审医学研究计划。


利益争夺


作者宣称他们没有竞争利益。


参考文献


Ahn,J.,Konno,H.和Barber,GN(2015)。STING依赖信号在癌症发展中的不同作用。癌基因34(41):5302-5308。              
Fu,J.,Kanne,DB,Leong,M.,Glickman,LH,McWhirter,SM,Lemmens,E.,Mechette,K.,Leong,JJ,Lauer,P.和Liu,W.(2015)。STING激动剂配制的癌症疫苗可以治愈已建立的对PD-1阻断具有抵抗力的肿瘤。Sci Transl Med 7(283):283ra252-283ra252。              
              他(Y.,Hong,C.,Yan,EZ,Fletcher,SJ,Zhu,G.,Yang,M.,Li,Y.,Sun,X.,Irvine,DJ和Li,J.(2020)。自组装的cGAMP-STINGΔTM信号复合物可作为cGAMP递送的生物启发平台。Sci Adv 6(24):eaba7589。              
Jin,L.,Xu,L.,Yang,IV,Davidson,EJ,Schwartz,DA,Wurfel,MM和Cambier,JC(2011)。鉴定和表征功能丧失的人MPYS变异体。基因我MMUN 12(4):263 -269 。              
T.Ohkuri,A.Kosaka,K.Ishibashi,Kumai,T.Hirata,Y.Ohara,K.Nagato,T.Oikawa,K.,Aoki,N。和Harabuchi ,. 2017)。肿瘤内给予cGAMP会在小鼠肿瘤部位短暂积聚有效的巨噬细胞以产生抗肿瘤免疫力。Cancer Immunol Immunother 66(6):705-716。              
Patel,S.,Blaauboer,SM,Tucker,HR,Mansouri,S.,Ruiz-Moreno,JS,Hamann,L.,Schumann,RR,Opitz,B.和Jin,L.(2017)。常见的R71H-G230A-R293Q人类TMEM173是无效等位基因。免疫学杂志198(2):776-787。              
Shae,D.,Becker,KW,Christov,P.,Yun,DS,Lytton-Jean,AK,Sevimli,S.,Ascano,M.,Kelley,M.,Johnson,DB和Balko,JM(2019)。内溶酶多聚体增加环二核苷酸STING激动剂的活性,以增强癌症的免疫治疗。Nat纳米技术:1。              
Sivick,KE,Surh,NH,Desbien,AL,Grewal,EP,Katibah,GE,McWhirter,SM和Dubensky,TW(2017)。关于“常见的R71H-G230A-R293Q人类TMEM173是无效等位基因”的评论。免疫学杂志198(11):4183-4185。              
              Xia,T.,Konno,H.和Barber,GN(2016)。黑色素瘤中STING信号的反复丢失与病毒溶瘤的敏感性相关。癌症- [R上课76(22):6747-6759。              
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Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
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.
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