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Mouse BMDC-dependent T Cell Polarization Assays
小鼠BMDC依赖性T淋巴细胞的极化试验   

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Abstract

In response to exposure to antigen, T cells whose T cell receptor (TCR) are capable of recognizing the self MHC-antigen derived peptide complex, respond to the antigen and differentiate into one of several subsets, namely TH1, TH2, TH17, Treg, etc. characterized by the signature cytokine they secrete, namely IFN-γ, IL-4, IL-17 or IL-10, respectively, referred to as syngeneic polarization as the MHC presenting the foreign antigen/epitope is self-derived.
T cell responses following incubation for defined periods, usually 3 days for mouse splenocytes, are routinely measured by assessing the antigen-stimulated proliferation of T cells by measuring the radiolabeled precursor thymidine incorporated into the genomic DNA of the dividing T cell; the direction of polarization is assessed by measuring the cytokine produced by the proliferating or non-proliferating responding T cells using ELISA of culture supernatants or by intracellular cytokine staining followed by flow cytometry.
In the protocols detailed below, we describe the use of syngeneic mouse bone marrow-derived primary dendritic cells (BMDC) as APC to stimulate spleen derived T cells. The proliferative response of the T cells is measured by incorporation of radiolabeled precursor thymidine into the genomic DNA and their direction of polarization is assessed by measuring the cytokines they secrete, namely IFN-γ, IL-4 and IL-17 over a 72 h period using ELISA. In addition, we used flow cytometry after intracellular cytokine staining to detect IL-17 positive T cells within the CD3+/CD4+/CD25low population. Prior live infection of BMDC with strains of Mycobacterium bovis- Bacille Calmette Guerin (BCG) was used as antigen to pre-condition the BMDC that presented antigens derived therefrom to T cells. We also measured cytokines secreted within 6 to 8 h of BCG infection by BMDC in order to correlate the BMDC cytokine profile with subsequent direction of T cell polarization.

Keywords: Dendritic cells(树突状细胞), GM-CSF(GM-CSF), Primary bone marrow cells(原发性骨髓细胞), Cytokines(细胞因子), T cell polarization(T细胞极化)

Materials and Reagents

  1. 10 cm Petri dishes, non-treated (Thermo Fisher Scientific, FalconTM, catalog number: 08-757-100D )
  2. 6 well 35 mm dia plastic cell culture treated dishes (Thermo Fisher Scientific, FalconTM, catalog number: 353046 )
  3. 0.22 µm filter
  4. BALB/c mice (6 to 8 weeks old female)
  5. Iscove’s modification of Dulbecco’s minimum essential medium (IMDM) (Life Technologies, catalog number: 12200069 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 12200069”.
  6. Sterile PBS
  7. Dulbecco’s Phosphate buffered saline (Sigma-Aldrich, catalog number: D5773 )
  8. Saponin from quillaja bark (wash buffer for flow cytometry staining) (Sigma-Aldrich, catalog number: S7900 )
  9. Murine rGM-CSF (recombinant mouse GM-CSF) (PEPROTECH, catalog number: 315-03 )
    Note: Recombinant GM-CSF from Peprotech Reconstitute powder (recommended by manufacturer).
  10. ELISA antibodies
    Note: We use R&D Systems Duoset capture and detection antibodies or BD paired ELISA antibody sets as detailed below:
    Mouse IFN-γ DuoSet ELISA (R&D Systems, catalog number: DY485-05 )
    Mouse IL-17 DuoSet ELISA (R&D Systems, catalog number: DY421-05 )
    Mouse IL-6 DuoSet ELISA (R&D Systems, catalog number: DY406-05 )
    Mouse TNF-α DuoSet ELISA (R&D Systems, catalog number: DY410-05 )
    Mouse IL-10 DuoSet ELISA (R&D Systems, catalog number: DY417-05 )
    Mouse IL-2 DuoSet ELISA (R&D Systems, catalog number: DY402-05 )
    Mouse IL-12p40 DuoSet ELISA (R&D Systems, catalog number: DY499-05 )
    Mouse IFN-gamma DuoSet ELISA (R&D Systems, catalog number: DY1679-05 )
  11. 1x PBS-1% BSA-0.05% sodium azide
  12. Concanavalin A from Canavalia ensiformis (Jack bean) (Sigma-Aldrich, catalog number: C5275 )
  13. CD11c MicroBeads (Miltenyi Biotech, catalog number: 130-052-001 )
  14. Glutamax-100x (Thermo Fisher Scientific, GibcoTM, catalog number: 35050-061 )
  15. Mitomycin C from Streptomyces caespitosus (Sigma-Aldrich, catalog number: M4287 )
  16. Penicillin-Streptomycin, 100x (Thermo Fischer Scientific, GibcoTM, catalog number: 15070-063 )
  17. Penicillin-Streptomycin (10,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140-122 )
  18. IL-17-APC (eBioscience, clone: eBio17B7 )
  19. 10% FBS
  20. [methyl-3H]
  21. 70% and 100% ethanol
  22. Brefeldin A (BFA) (Sigma-Aldrich, catalog number: B7651 )
  23. Ammonium chloride (NH4Cl) (Sigma-Aldrich, catalog number: A9434 )
  24. Potassium bicarbonate (KHCO3) (Sigma-Aldrich, catalog number: 60339 )
  25. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E6758 )
  26. Tris (pH 7.5)
  27. IMDM complete (see Recipes)
  28. Thymidine (see Recipes)
  29. Brefeldin A (BFA) (Sigma-Aldrich, catalog number: B7651) (see Recipes)
  30. Monensin sodium salt (Sigma-Aldrich, catalog number: M5273 ) (see Recipes)
  31. RBC lysis buffer (100 ml) (see Recipes)
  32. RBC lysis buffer (see Recipes)

Equipment

  1. Sterile scissors and forceps
  2. Cell scrapers (Corning, Costar®, model: 3010 Small Cell Scraper )
  3. Centrifuges (Table top) (Eppendorf)
  4. Water bath set at 37 °C
  5. Flow cytometer [FACS-Canto-II flow cytometer (BD) or any machine with Argon ion Blue laser)
  6. Biosafety cabinet with laminar air flow

Procedure

Note: The protocol outlined below has been adapted from Satchidanandam et al. (2014).

  1. Bone marrow cell isolation for BMDC culture (all steps to be performed at room temperature) (Figure 1)


    Figure 1. Bone marrow cell isolation for BMDC culture. The figure shows the X-ray picture of a mouse hind limb. The femur and tibia are labelled, The points where the bones have to be cut during the initial dissection are clearly marked by arrows.
    .
    1. We used female BALB/c mice bred in-house in the Institute’s central animal facility. Use of mice (and any other animals) requires prior approval by the Institutional Animal Ethics Committee. The studies were carried out in strict accordance with the Institutional Animal Ethics Committee-approved protocols of the Indian Institute of Science for mouse experiments (CAF/Ethics/220-2011 dated February 10, 2011). For details of Animal Ethics guidelines, please refer to Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines (https://www.nc3rs.org.uk/arrive-guidelines). Mice were administered inhalation anesthesia with chloroform and euthanized by cervical dislocation.
    2. Handle the mice gently and avoid making the animals edgy before they are sacrificed.
    3. Using dissection tweezers and dissection scissors remove the skin on the lower abdomen and both legs (Video 1). This will expose both legs from the hip down to the metatarsals. Proper removal of the skin at this point will prevent contamination of cultures with epidermal cells. Begin by separating the muscle along the entire leg from the skin. Then proceed by using the tweezers to stabilize the leg while removing the muscle with scissors and scalpel blade.

      Video 1. Dissect mouse leg

    4. Remove the femur and tibia as follows:
      1. Make one cut at the hip. Be sure to keep as much of the epiphysis intact as possible on the femur. To do this, position the scissors as close to the hip as possible while cutting (Figure 1).
      2. Make the second cut below the ankle joint, again being careful to leave the epiphysis intact (Figure 1).
      3. Place the harvested femur and tibia (should still be connected at the knee-joint) in a sterile Petri plate in about 5 ml of IMDM containing Penicillin and Streptomycin. Use bacteriological Petri dishes with lid.
    5. Remove all the flesh clinging to the bones using a scalpel blade, scissors and forceps (Video 2).

      Video 2. Clean bones

    6. Transfer bones to a new Petri dish with fresh medium with antibiotics. Repeat removing flesh sticking to bones if necessary.
    7. Collect bone marrow under sterile conditions (Video 3).

      Video 3. Flush bone marrow

      1. Using a scissor first find the site of intersection between the femur and tibia and then very gently separate them from each other. Avoid exposing marrow by cutting off either femur or tibia during this process.
      2. For flushing out the marrow use different size needles for femur and tibia. For tibia use a 26 or 24 gauge needle and for femur-use a 21 gauge needle.
      3. Fill a sterile 5 ml syringe with about 5 ml of IMDM. Place a 26 gauge needle on the end of the syringe and keep ready for the next step. Prepare a fresh sterile non-treated Petri-dish to collect flushed bone marrow in the next step.
      4. Hold the bone with sterile tweezers and using a scissor gently pull each epiphysis off or cut off epiphysis to expose the bone marrow. (Avoid placing the cut bone back into the Petri place after cutting to avoid loss/leaching out of bone marrow.)
      5. Hold the cut bone over a sterile Petri dish and puncture the centre of the bone ends with the needle. It should slide in easily if it is in the center. Slowly flush out the bone marrow with sterile IMDM + antibiotics. If necessary, move the needle up and down in the bone. Repeat this process at the other end of the bone if necessary. Do not break the bone. The bone should appear white or translucent when it is clean.
      6. Repeat this procedure for the other bones. Do not use medium containing flushed marrow cells to repeat flushing of bones. Always suck up fresh medium into the syringe for each flush.
    8. Discard each bone once clean.
    9. Ensure that the bones are always kept submerged under complete IMDM as you handle one bone at a time. To cut open the ends of the bones, use a good sharp pair of scissors and avoid generating splinters. These fine bone pieces are extremely difficult to separate from the cells of the marrow. The differentiating BMDC will engulf them and undergo premature activation.
    10. Once the clumps of cells have been suspended by repeated trituration with a pipette, transfer the cells to a sterile 50 ml conical tube. Let the cell suspension in a volume of about 40 ml, stand in a conical 50 ml tube for 5 min. Use a pipette to aspirate the top 35 ml, leaving behind debris and cell clumps. These clumps can be once again triturated to obtain more cells in suspension. You may alternately filter the cell suspension through a 70 µm filter to remove clumps.

  2. BMDC culture
    1. RBC Lysis. This step is extremely critical and should be carried out with great care. Excessive exposure to the lysis buffer can kill all cells.
      1. Spin cells at 1,000 x g, 10 min at room temperature (RT).
        Collect supernatant after centrifugation in a clean tube. Do not discard. Check this supernatant for cells. This should not have more than a total of 2-4 million cells.
      2. Very gently dislodge cell pellet into a thick slurry by gentle repeated tapping. Add 1 ml RBC lysis buffer for 50 million cells. Mix well to suspend the cells by gentle tapping. Incubate at RT, exactly 1 min 30 sec.
      3. Rapidly bring total volume up to 25 ml (add 24 ml) with plain IMDM and spin at 1,000 x g, 15 min at RT.
      4. Decant supernatant, gently re-suspend cell pellet and add fresh 20 ml IMDM, mix well to wash off any traces of RBC lysis buffer, and pellet cells once again at 1,000 x g, 15 min at RT.
        Decant supernatant, gently re-suspend cell pellet and add 10 ml complete IMDM without GMCSF. Usually, from one mouse, total bone marrow cell yield is 40-70 million cells. Count cells and increase volume if required.
    2. Bone marrow cells were cultured in IMDM-10% FBS containing 2 mM β-mercapto-ethanol essentially according to standard published protocols with 50 U/ml murine rGM-CSF. Some protocols call for 200 to 800 U/ml GM-CSF. We have found these high concentrations give rise to mature BMDC with high surface expression of MHCII and CD86. Standard antibiotics Penicillin-Streptomycin (10,000 U/ml) may be included at recommended concentrations. Seed 0.5 to 1.0 million cells per 35 mm dish in a volume of 3 ml.
    3. 50% of the culture medium was removed and replenished every two days.
    4. Non-adherent cells were harvested on day 7. One can get 1 to 2 million cells for every million cells seeded on day 0.
    5. Harvested cells were analysed by flow cytometry using phycoerythrin (PE)-conjugated antibodies against MHC-II, CD80 and CD86, individually in combination with FITC conjugated anti-mouse CD11c antibody to determine the percentage of immature dendritic cells which were then used for all experiments. A simple flow cytometer with an Argon ion (blue) laser will suffice to detect these two fluors. In mice, all subsets of DC express CD11c. Immature dendritic cells will express intermediate levels of MHC-II, CD80 and CD86. In most preparations, a small proportion, about 2 to 5% of the BMDC by day 7 will also express high levels of these co-stimulatory molecules. We normally obtain 60 to 85% cells being CD11c-positive by day 7. When high purity is required, we purify the CD11c-positive cells using mouse CD11c microbeads from Miltenyi Biotech according to manufacturer’s instructions.
    6. The BMDC thus obtained were infected at 5 multiplicity of infection (MOI) with Mycobacterium bovis BCG for 24 h. BMDC may also be incubated with other protein or peptide antigens; optimum incubation times required will need to be determined for each antigen.

  3. T cell polarization assay
    1. Splenocytes were isolated from BCG-immunized mice by dissecting the abdomen and removing the spleen within the biosafety cabinet to maintain sterility. The splenocyte suspension is prepared by forcing IMDM into the spleen tissue using a 20 ml syringe and 21 gauge needle. You may also gently press down on the spleen a few times using the flat rear end of a sterile syringe to release splenocytes into the surrounding medium. After counting the cell suspension, splenocytes are collected by centrifugation of the suspension at 1,300 x g for 10 min. RBC lysis was carried out for splenocytes exactly as described for bone marrow cells.
    2. 20 million splenocytes were then incubated in 10 ml complete IMDM for 2 h at 37 °C in 10 cm diameter cell culture dishes. Non-adherent cells were then collected for the assay.
    3. For syngeneic polarization studies, the above mentioned infected BMDC were killed by 50 µg/ml mitomycin C treatment for 1 h followed by extensive washing. BMDC were co-cultured in 200 µl volume in 96 well plates in triplicate with 5 x 105 non-adherent splenocytes per well from BALB/c mice infected intraperitoneally 3 weeks previously with 5 x 108 Mycobacterium bovis-BCG, at varying BMDC: splenocyte ratios of 1:10, 1:20 and 1:100 for 72 h. You may alternately kill infected BMDC by gamma irradiation (15 Gray) if you have access to this machine. Place the harvested BMDC in complete IMDM in a round bottom sterile snap cap tube and place inside the gamma irradiator. Control uninfected BMDC were also cultured with splenocytes similarly.
    4. After 72 h of co-culture of non-adherent splenocytes and killed BMDC, supernatants were collected for cytokine measurements. Identity of cytokine secreting cells can be determined by intracellular cytokine staining and flow cytometry. We routinely measured by ELISA, the cytokines IFN-γ, IL-4 and IL-17 to detect polarization of T cells to give TH1, TH2 or TH17 cells, respectively.
    5. Con-A stimulation of non-adherent splenocytes at 5 µg/ml was included as positive control.

  4. Intracellular cytokine-staining and flow cytometry to detect TH17 cells
    1. 106 non-adherent splenocytes were cultured with 104 or 5 x 103 irradiated BMDC In 24 well tissue culture plates in triplicate wells.
    2. 10 h later, 10 µg/ml brefeldin A and 0.75 µM monensin were added for the next 26 h of culture. At the end of 36 h, harvested splenocytes were washed once with PBS-0.05% azide, and surface stained for CD4 (CD4-PE; clone GK1.5), CD25-PE-Cy7 (clone PC 61.5) and CD3-FITC (clone 145-2C-11) from BD Biosciences in PBS-1% BSA, washed with PBS-0.05% azide, fixed for 10 min. with 2% paraformaldehyde in 1x PBS and permeabilized in 500 µl of PBS-0.05% sodium azide -1% BSA with 0.1% saponin for 30 min.
    3. Intracellular IL-17 was detected using, IL-17-APC in PBS-0.05% N3-1% BSA with 0.1% saponin for 30 min on ice.
    4. Stained cells were washed once with 1 ml PBS-0.05% sodium azide - with 0.1% saponin.
    5. Stained and washed cell pellets were suspended in 100 µl PBS-0.05% sodium azide containing 1% paraformaldehyde, held on ice for 15 min. and diluted to 400 µl with PBS-0.05% azide.
    6. Samples were acquired on a BD FACS-Canto-II flow cytometer. Any flow cytometer with a blue (argon ion, 488 nm) and red (633 nm) laser will be suitable to detect this combination of fluors.
    7. IL-17 producing cells within the CD3-high, CD4-high and CD25-low population were enumerated.
    8. Identity of IL-17 producing cells was confirmed by using an isotype control antibody and more importantly, by staining with a fluorescence minus one (FMO) control missing the IL-17-APC antibody.


      Figure 2. Gating strategy to detect IL-17 secreting T cells. Splenocytes stained as described were displayed on a linear FSC vs. SSC plot and the gate shown was applied to pick up all lymphocytes. Cells staining positive for CD3 from within this population were further selected and displayed for IL-17 and CD25. IL-17-positive cells within the CD25-low subset represent the true TH-17 subset of T cells.

Representative data

Table 1. The table shows the levels of IFN-γ and IL-17 detected by ELISA in the culture supernatants of splenocytes co-cultured with the indicated BMDC samples. The cytokines are shown as pg/ml culture volume secreted by one million non-adherent splenocytes.

Notes

  1. Use of infection with live BCG strains to condition antigen presenting BMDC. BCG ensures robust stimulation of BMDC to produce a host of inflammatory and regulatory cytokines, including IL-2, IL-6, IL-10, IL-12p40 and p70, TNF-α and TGF-β. We ensured that in setting up the polarization assays, spleen-derived T cells were exposed to the antigen presenting BMDC along with the cytokines secreted by the BMDC. BMDC infected for 24 h prior to addition of splenocytes were therefore not washed to remove culture medium before 3-day incubation with splenocytes so that the T cells were provided with the first, second and third signals from the APC. In fact, we made sure to carry out gamma irradiation of BMDC in their conditioned medium prior to splenocyte addition.
    Other antigens such as purified proteins or synthetic peptides can also be added to BMDC in lieu of live BCG infection and used for T cell polarization assays.
  2. Our protocol therefore limited the cytokines that we could measure by ELISA to assess T cell response, since several cytokines such as IL-2, IL-10 and TNF-α are secreted both by BMDC and T cells. However, cytokine secretion by T cells can be detected by flow cytometry following intracellular cytokine staining.
  3. Other populations of APC such as DC from spleens or lymph nodes and macrophages obtained from bone marrow cells or tissues such as spleen or peritoneal exudates can also be used as APC in T cell polarization assays. Similarly, T cell lines or clones may be used in lieu of splenocytes. In such cases, the appropriate numbers of cells to be used per well and the ratio of APC to T cells for optimal results should be empirically determined.
  4. Thymidine, [methyl-3H], 20 Ci/mmol in 70% ethanol, Perkin Elmer, stored at -20 °C. Dilute in complete medium to give 0.5 µCi in 20 µl. Dispense 20 µl per well of the 96 well plate. Avoid adding the radiolabel in very small volumes per well. This will minimize well to well variation.
  5. BMDC culture. Handle bone marrow cells very gently. Some protocols recommend using an 18 to 20 gauge needle for suspending the clumps of marrow. We have found this reduces viability. The surviving differentiating BMDC are highly phagocytic and engulf these dead cells, leading to their premature and excessive activation.
  6. GM-CSF. Commercially available native GM-CSF is extremely expensive. Several published papers use recombinant GM-CSF purified from E. coli expressing the protein, which works reasonably well, based on percentage of CD11c-high BMDC by day 7 of culture. However, the BMDC produced with this recombinant GM-CSF can sometimes show undesirably high level of maturity based on surface expression of MHC-II and CD86. We have used culture supernatants of HEK293_T cells stably transfected with a plasmid expressing the mouse GM-CSF and obtained satisfactory preparations of immature BMDC.
  7. Culture media. Ensure that the IMDM used for culturing BMDC are stringently clean and totally devoid of even traces of endotoxin. We collect water fresh into glass bottles baked at 200 °C, dissolve powdered medium in it and immediately filter through double layer of 0.2 µm membranes inside a biosafety cabinet.
  8. Fetal bovine serum: Test the batch of serum to ensure that BMDC cultures are immature on day 7. Batches of serum containing high levels of urea, creatinine etc. can cause BMDC activation. Make sure the supplier of the FBS provides data for levels of some 30 odd serum components. Once you find a good batch, order sufficient amounts to last for the duration of these experiments.
  9. FACS staining for ICC. Check to see if the surface marker-specific antibody conjugate in your panel will recognize its cognate antigen on formaldehyde-fixed cells. If not, you will have to carry out the surface staining first, then fix and permeabilize the cells for intracellular staining. Always titrate all FACS antibodies and use the appropriate quantity to minimize background expansion and spill into other channels. Choice of flours on the FACS antibody should be decided based on the flow cytometer configuration and the bandwidth of filters available on the machine.
  10. Treatment times with secretion inhibitors brefeldin-A and monensin need to be determined for each antigen used. Potent protein or peptide antigens may require as little as 6 to 12 h stimulation of T cells while our experiments required longer stimulations. While brefeldin-A is not toxic to cells, monensin exposure for prolonged periods can cause cell death. We therefore reduced the monensin concentration from the standard 3 mM to 0.75 mM.

Recipes

  1. IMDM complete
    10% FBS, 2 mM β-mercapto-ethanol, glutamax
  2. Thymidine
    [methyl-3H], 20 Ci/mmol in 70% ethanol, stored at -20 °C
  3. Brefeldin A (BFA)
    Dissolve 1 mg of BFA in 1 ml of 100% ethanol, and store at -80 °C in aliquots of 50 µl
  4. Monensin
    Mol wt: 670 free and 693 for Na salt
    Prepare stock of 30 mM (20 mg/ml) in absolute ethanol and store in aliquots of 100 µl at -80 °C
  5. RBC lysis buffer (100 ml)
    Ammonium chloride (NH4Cl) 8.99 g
    Potassium bicarbonate (KHCO3) 1.0 g
    Ethylenediaminetetraacetic acid (EDTA) 37 mg
    Dissolve in 80 ml of autoclaved DD water
    Adjust the pH to 7.3 using conc. HCl
    Make up the volume to 100 ml
    Filter using 0.22 µm filter
    Do not autoclave
    Stored at 2-8 °C
  6. RBC lysis buffer
    0.9% NH4Cl in 10 mM Tris (pH 7.5)
    Keep sterile at 4 °C

Acknowledgments

This work was funded under the grant BT/PR7240/INF/22/52/2006 from Department of Biotechnology, Ministry of Science and Technology, Government of India. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank Shoba Elangovan and Rajni Nyodu for help with producing the videos. This protocol was adapted from Satchidanandam et al. (2014).

References

  1. Lutz, M. B. and Rossner, S. (2007). Factors influencing the generation of murine dendritic cells from bone marrow: the special role of fetal calf serum. Immunobiology 212(9-10): 855-862.
  2. Satchidanandam, V., Kumar, N., Jumani, R. S., Challu, V., Elangovan, S. and Khan, N. A. (2014). The glycosylated Rv1860 protein of Mycobacterium tuberculosis inhibits dendritic cell mediated TH1 and TH17 polarization of T cells and abrogates protective immunity conferred by BCG. PLoS Pathog 10(6): e1004176.

简介

响应于暴露于抗原,其T细胞受体(TCR)能够识别自身MHC抗原衍生的肽复合物的T细胞应答抗原,并分化为几个亚类之一,即TH1,TH2,TH17,Treg, 等。其特征在于它们分泌的特征性细胞因子,即IFN-γ,IL-4,IL-17或IL-10,分别称为同基因极化,因为呈递外源抗原/表位的MHC是自身衍生的。通常通过测量掺入分裂T细胞的基因组DNA中的放射性标记的前体胸腺嘧啶,通过评估抗原刺激的T细胞增殖来常规测量对于确定的时间段(通常为小鼠脾细胞3天)孵育后的T细胞应答;通过使用培养上清液的ELISA或通过细胞内细胞因子染色随后通过流式细胞术测量由增殖或非增殖应答性T细胞产生的细胞因子来评估极化方向。在下面详述的方案中,我们描述使用的同源小鼠骨髓源性树突状细胞(BMDC)作为APC刺激脾源性T细胞。通过将放射性标记的前体胸苷掺入基因组DNA中来测量T细胞的增殖反应,并通过在72小时期间测量它们分泌的细胞因子即IFN-γ,IL-4和IL-17来评估其极化方向使用ELISA。此外,我们在细胞内细胞因子染色后使用流式细胞术检测CD3 + /CD4 + /CD25低人群中的IL-17阳性T细胞。使用牛分枝杆菌(Mycobacterium bovis) - Bacille Calmette Guerin(BCG)菌株的先前活体感染作为抗原,以预处理呈递由其衍生的抗原的BMDC至T细胞。我们还测量了在BMDC感染的6至8小时内分泌的细胞因子,以便将BMDC细胞因子谱与随后的T细胞极化方向相关联。

关键字:树突状细胞, GM-CSF, 原发性骨髓细胞, 细胞因子, T细胞极化

材料和试剂

  1. 10cm皮氏培养皿,未处理(Thermo Fisher Scientific,Falcon ,目录号:08-757-100D)
  2. 6孔35mm dia塑料细胞培养处理的皿(Thermo Fisher Scientific,Falcon ,目录号:353046)
  3. 0.22μm过滤器
  4. BALB/c小鼠(6至8周龄雌性)
  5. Iscove对Dulbecco最低必需培养基(IMDM)的修饰(Life Technologies,目录号:12200069)
    注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:12200069"。
  6. 无菌PBS
  7. Dulbecco磷酸盐缓冲盐水(Sigma-Aldrich,目录号:D5773)
  8. 来自皂树树皮的皂苷(用于流式细胞术染色的洗涤缓冲液)(Sigma-Aldrich,目录号:S7900)
  9. 鼠rGM-CSF(重组小鼠GM-CSF)(PEPROTECH,目录号:315-03)
    注意:来自Peprotech的重组GM-CSF重组粉末(由制造商推荐)。
  10. ELISA抗体
    注意:我们使用R& D Systems Duoset捕获和检测抗体或BD配对ELISA抗体组,详情如下:
    小鼠IFN-γDuoSet ELISA(R& D Systems,目录号:DY485-05) 小鼠IL-17 DuoSet ELISA(R& D Systems,目录号:DY421-05) 小鼠IL-6 DuoSet ELISA(R& D Systems,目录号:DY406-05) 小鼠TNF-αDuoSet ELISA(R& D Systems,目录号:DY410-05) 小鼠IL-10 DuoSet ELISA(R& D Systems,目录号:DY417-05)
    小鼠IL-2 DuoSet ELISA(R& D Systems,目录号:DY402-05) 小鼠IL-12p40 DuoSet ELISA(R& D Systems,目录号:DY499-05)
    小鼠IFN-γDuoSet ELISA(R& D Systems,目录号:DY1679-05)
  11. 1x PBS-1%BSA-0.05%叠氮化钠
  12. 来自Canavalia ensiformis的菊粉脂蛋白A(Jack bean)(Sigma-Aldrich,目录号:C5275)
  13. CD11c MicroBeads(Miltenyi Biotech,目录号:130-052-001)
  14. Glutamax-100x(Thermo Fisher Scientific,Gibco TM ,目录号:35050-061)
  15. 丝裂霉链霉菌的丝裂霉素C(Sigma-Aldrich,目录号:M4287)
  16. 青霉素 - 链霉素,100x(Thermo Fischer Scientific,Gibco< sup>,目录号:15070-063)
  17. 青霉素 - 链霉素(10,000U/ml)(Thermo Fisher Scientific,Gibco TM,目录号:15140-122)
  18. IL-17-APC(eBioscience,克隆:eBio17B7)
  19. 10%FBS
  20. [甲基 - 叔丁基]
  21. 70%和100%乙醇
  22. 布雷菲德菌素A(BFA)(Sigma-Aldrich,目录号:B7651)
  23. 氯化铵(NH 4 Cl)(Sigma-Aldrich,目录号:A9434)
  24. 碳酸氢钾(KHCO 3)(Sigma-Aldrich,目录号:60339)
  25. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:E6758)
  26. Tris(pH 7.5)
  27. IMDM完成(请参阅配方)
  28. 胸苷(见配方)
  29. 布雷菲德菌素A(BFA)(Sigma-Aldrich,目录号:B7651)(参见Recipes)
  30. 莫能菌素钠盐(Sigma-Aldrich,目录号:M5273)(参见Recipes)
  31. RBC裂解缓冲液(100ml)(参见配方)
  32. RBC裂解缓冲液(参见配方)

设备

  1. 无菌剪刀和镊子
  2. 细胞刮刀(Corning,Costar ,型号:3010小细胞刮刀)
  3. 离心机(桌面)(Eppendorf)
  4. 水浴设置在37℃
  5. 流式细胞仪[FACS-Canto-II流式细胞仪(BD)或任何具有氩离子蓝激光的机器)
  6. 带有层流气流的生物安全柜

程序

(2014) 注意:下面列出的协议已从Satchidanandam

  1. BMDC培养的骨髓细胞分离(所有步骤在室温下进行)(图1)


    图1. BMDC培养的骨髓细胞分离。 该图显示了鼠标后肢的X光照片。股骨和胫骨被标记。在初始切开期间骨头必须切割的点用箭头清楚地标记。

    1. 我们使用在研究所中心内部培育的雌性BALB/c小鼠 动物设施。使用小鼠(和任何其他动物)需要事先 批准由机构动物伦理委员会。研究是 严格按照机构动物伦理进行 委员会批准的印度科学院协议 小鼠实验(2011年2月10日的CAF/Ethics/220-2011)。对于 详细的动物伦理指南,请参考动物研究: 体内实验报告(ARRIVE)指南 (https://www.nc3rs.org.uk/arrive-guidelines)。施用小鼠 吸入麻醉与氯仿和宫颈安乐死 错位。
    2. 轻轻处理小鼠,避免在处死前使动物前行。
    3. 使用解剖镊子和解剖剪刀去除皮肤 ?下腹部和两腿(视频1)。这将暴露两腿 从髋关节到跖骨。正确去除皮肤在这 ?点将防止文化与表皮细胞的污染。 首先从整个腿分离肌肉从皮肤。然后 继续使用镊子稳定腿,同时删除 肌肉与剪刀和手术刀刀片 <! - flashid1721开始 - >

      视频1.剖析鼠标腿
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    4. 按如下方式取出股骨和胫骨:
      1. 在臀部做一个切口。一定要保持尽可能多的骨。 在股骨上尽可能完整。为此,将剪刀定位为 在切割时尽可能接近臀部(图1)
      2. 在踝关节下方进行第二次切割,再次小心翼翼地留下骨ysis(图1)。
      3. 放置收获的股骨和胫骨(应该仍然连接 膝关节)在约5ml IMDM中的无菌培养皿中 含有青霉素和链霉素。使用细菌培养皿 ?带盖。
    5. 使用手术刀刀片,剪刀和镊子(视频2)清除骨骼上附着的所有肉。
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      视频2.清除骨骼
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    6. 转移骨头到一个新的培养皿与新鲜的培养基 抗生素。如果需要,重复去除粘在骨头上的肉。
    7. 在无菌条件下收集骨髓(视频3)。
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      视频3.冲洗骨髓
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      1. 使用剪刀首先找到的交叉点 股骨和胫骨,然后非常轻柔地将它们彼此分开。 在此期间通过切断股骨或胫骨避免骨髓 过程
      2. 用于冲洗骨髓使用不同大小的针 为股骨和胫骨。对于胫骨使用26或24号针和 股骨 - 使用21号针。
      3. 填充无菌5毫升注射器 约5ml IMDM。将26号针放在注射器的末端 并准备好下一步。准备新鲜无菌未处理 培养皿在下一步收集冲洗骨髓。
      4. 保持 用无菌镊子的骨头和使用剪刀轻轻地拉每个 骨ysis或切断骨to以暴露骨髓。 (避免 在切割后将切割的骨放回到Petri地方以避免 损失/浸出骨髓。)
      5. 抓住切开的骨头 无菌培养皿和穿刺骨头的中心 针。如果它在中心,它应该容易滑动。慢慢冲洗 用无菌IMDM +抗生素切除骨髓。如有必要,移动 针在骨头上下。在另一端重复此过程 ?的骨骼。不要打破骨头。骨应该出现 ?白色或半透明,当它是干净的
      6. 重复此过程 为其他骨头。不要使用含有冲洗的骨髓细胞的培养基 重复冲洗骨头。总是吸入新鲜的介质 注射器每次冲洗。
    8. 清洁后丢弃每个骨头。
    9. 确保骨骼始终保持浸没在完全IMDM下 你一次处理一个骨头。要切开骨头的两端,使用a 良好的锋利的剪刀和避免产生碎片。这些罚款 骨片极难从细胞分离 骨髓。区分BMDC将吞噬它们并且过早 激活
    10. 一旦细胞团被悬浮 用移液管重复研磨,将细胞转移至无菌的50 ml锥形管。使细胞悬浮液的体积为约40ml, 在锥形50ml管中放置5分钟。使用移液器吸出 顶部35 ml,留下碎片和细胞团块。这些团块可以 再次研磨以获得更多的悬浮细胞。你可以 交替地通过70μm过滤器过滤细胞悬浮液以除去 团块。

  2. BMDC文化
    1. RBC裂解。这一步骤是非常关键的,应该进行 非常小心。过度暴露于裂解缓冲液可以杀死所有 细胞
      1. 在室温(RT)下以1,000×g /分钟旋转细胞10分钟 在干净的管中离心后收集上清液。不要 丢弃。检查这种上清液的细胞。这应该没有更多 比总共2-4百万个细胞。
      2. 非常轻轻地移动细胞 通过温和地重复敲击而成为稠浆料。加入1ml RBC 裂解缓冲液为5000万细胞。充分混匀以悬浮细胞 轻轻敲击。在室温下孵育,正好1分30秒。
      3. 使用纯IMDM快速使总体积达到25ml(加入24ml),并在室温下以1,000×g旋转15分钟。
      4. 倾析上清液,轻轻地重悬细胞沉淀并加入新鲜的20 ml IMDM,充分混合以洗去任何痕量的RBC裂解缓冲液,并沉淀 ?细胞再次以1000×g ,在室温下15分钟。
        倾析上清液, 轻轻地重悬细胞沉淀和添加10毫升完整IMDM没有GMCSF。 ?通常,从一只小鼠,总的骨髓细胞产量是40-70百万 细胞。计数单元格,如果需要增加音量。
    2. 骨髓 细胞在含有2mMβ-巯基乙醇的IMDM-10%FBS中培养 基本上根据标准公布的方案用50U/ml 鼠rGM-CSF。一些方案需要200至800U/ml GM-CSF。我们有 发现这些高浓度产生具有高的成熟BMDC 表面表达的MHCII和CD86。标准抗生素 建议加入青霉素 - 链霉素(10,000 U/ml) 浓度。在体积中每35mm培养皿种0.5至1.0百万个细胞 ?为3ml。
    3. 取出50%的培养基,每两天补充一次。
    4. 在第7天收获非贴壁细胞。在第0天,每播种一百万个细胞可获得1至200万个细胞。
    5. 使用藻红蛋白通过流式细胞术分析收获的细胞 (PE) - 缀合的抗MHC-II,CD80和CD86的抗体 与FITC缀合的抗小鼠CD11c抗体结合 确定然后是未成熟树突细胞的百分比 用于所有实验。一种带有氩离子的简单流式细胞仪 (蓝色)激光器将足以检测这两种荧光剂。在小鼠,所有 DC的子集表达CD11c。不成熟的树突细胞会表达 中间水平的MHC-II,CD80和CD86。在大多数制备中,a 小比例,约7%的BMDC约2?5%也会表达 高水平的这些共刺激分子。我们通常获得60 85%的细胞在第7天是CD11c阳性。当需要高纯度时, 我们使用小鼠CD11c微珠从中纯化CD11c阳性细胞 Miltenyi Biotech根据制造商的说明
    6. 的 由此获得的BMDC在感染复数(MOI) 用牛分枝杆菌BCG 培养24小时。 BMDC也可与其孵育 其它蛋白或肽抗原;最佳孵育时间 将需要为每种抗原确定。

  3. T细胞极化测定
    1. 通过解剖从BCG免疫的小鼠分离脾细胞 腹部和去除生物安全柜内的脾脏以维持 ?不育。通过强制IMDM制备脾细胞悬浮液 脾组织使用20ml注射器和21号针。你可以 也可以使用平后端轻轻按下脾脏几次 ?的无菌注射器释放脾细胞到周围 中。在计数细胞悬浮液后,收集脾细胞 ?在1,300×g离心悬浮液10分钟。 RBC裂解 ?对于与用于骨髓细胞完全相同的脾细胞进行
    2. 然后将20万个脾细胞在10ml完全IMDM中温育 在37℃下在10cm直径的细胞培养皿中孵育2小时。非粘附 收集细胞用于测定
    3. 同基因 极化研究,上述被感染的BMDC被杀死 50μg/ml丝裂霉素C处理1小时,接着大量洗涤。 BMDC在200μl体积的96孔板中一式三份共培养 与来自感染的BALB/c小鼠的每孔5×10 5个非粘附脾细胞 ?腹腔内,3周前用5×10 8个分枝杆菌 bovis -BCG,在不同的BMDC:脾细胞比例为1:10,1:20和1:100 72小时。您可以选择通过γ辐射杀死感染的BMDC (15灰色),如果您可以访问此机器。放置收获的BMDC 在完全IMDM中在圆底无菌按扣帽管和地方 在γ辐射器内。也培养对照未感染的BMDC 与脾细胞类似
    4. 72小时后共培养 非粘附脾细胞和死亡BMDC,收集上清液 用于细胞因子测量。细胞因子分泌细胞的同一性可以是 通过细胞内细胞因子染色和流式细胞术测定。我们 通过ELISA常规测定细胞因子IFN-γ,IL-4和IL-17 检测T细胞的极化以得到TH1,TH2或TH17细胞, 分别
    5. 包括5μg/ml的非粘附脾细胞的Con-A刺激作为阳性对照。

  4. 细胞内细胞因子染色和流式细胞术检测TH17细胞
    1. 在一式三份的孔中的24孔组织培养板中培养10×10 6个非粘附脾细胞与10×10 4或5×10 8个照射过的BMDC。 br />
    2. 10小时后,加入10μg/ml布雷菲德菌素A和0.75μM莫能菌素 ?接下来26小时的文化。在36 h结束时,收获脾细胞 用PBS-0.05%叠氮化物洗涤一次,并对CD4进行表面染色 (CD4-PE;克隆GK1.5),CD25-PE-Cy7(克隆PC 61.5)和CD3-FITC(克隆 145-2C-11)从BD Biosciences在PBS-1%BSA中洗涤,用PBS-0.05% 叠氮化物,固定10分钟。用1%PBS中的2%多聚甲醛 在500μlPBS-0.05%叠氮化钠-1%BSA和0.1% 皂苷30分钟。
    3. 使用具有0.1%皂苷的PBS-0.05%N 3 -1%BSA中的IL-17-APC在冰上检测细胞内IL-1730分钟。
    4. 染色的细胞用1ml PBS-0.05%叠氮化钠 - 用0.1%皂苷洗涤一次
    5. 将染色和洗涤的细胞沉淀悬浮在100μlPBS-0.05% 叠氮化钠含1%多聚甲醛,在冰上保持15分钟。和 ?用PBS-0.05%叠氮化物稀释至400μl
    6. 在上获取样品 ?BD FACS-Canto-II流式细胞仪。任何带有蓝色的流式细胞仪 (氩离子,488nm)和红(633nm)激光将适合于检测 这种荧光体的组合
    7. 列举了CD3高,CD4高和CD25低群体中的IL-17产生细胞
    8. 通过使用同种型证实IL-17产生细胞的同一性 对照抗体,更重要的是通过用荧光染色 减去一个(FMO)对照缺失IL-17-APC抗体

      图2。 检测IL-17分泌T细胞的门控策略。脾细胞染色 如所描述的在线性FSC对SSC图和门 显示用于拾取所有淋巴细胞。细胞染色阳性 进一步选择和显示来自该群体内的CD3 对于IL-17和CD25。 CD25低亚组内的IL-17阳性细胞 表示T细胞的真实TH-17亚型。

代表数据


表1.该表显示通过ELISA在与指定的BMDC样品共培养的脾细胞的培养物上清液中检测到的IFN-γ和IL-17的水平。细胞因子显示为pg/ml培养物体积由一百万非粘附脾细胞分泌。

笔记

  1. 使用感染活BCG菌株来调节呈递BMDC的抗原。 BCG确保强烈刺激BMDC以产生一系列炎症和调节细胞因子,包括IL-2,IL-6,IL-10,IL-12p40和p70,TNF-α和TGF-β。我们确保在设置极化测定中,将脾来源的T细胞暴露于呈递BMDC的抗原以及由BMDC分泌的细胞因子。因此,在加入脾细胞之前24小时的BMDC感染未被洗涤以除去培养基,然后与脾细胞孵育3天,使得向T细胞提供来自APC的第一,第二和第三信号。事实上,我们确保在添加脾细胞之前在其条件培养基中进行BMDC的γ辐射 也可以将其他抗原如纯化的蛋白质或合成肽加入BMDC中以代替活的BCG感染并用于T细胞极化测定。
  2. 因此,我们的方案限制我们可以通过ELISA测量的细胞因子以评估T细胞应答,因为几种细胞因子如IL-2,IL-10和TNF-α由BMDC和T细胞分泌。然而,细胞内细胞因子染色后,可通过流式细胞术检测T细胞的细胞因子分泌
  3. APC的其他群体如来自脾或淋巴结的DC和从骨髓细胞或组织获得的巨噬细胞如脾或腹膜渗出物也可用作T细胞极化测定中的APC。类似地,可以使用T细胞系或克隆代替脾细胞。在这种情况下,应该根据经验确定每孔使用的细胞数量和APC与T细胞的比例以获得最佳结果。
  4. 胸腺嘧啶,[甲基 - [3 H],20Ci/mmol,在70%乙醇中,Perkin Elmer,储存在-20℃。在完全培养基中稀释,在20μl中得到0.5μCi。分配每孔20微升的96孔板。避免每孔加入极小体积的放射性标记。这将最小化到井的变化。
  5. BMDC培养。非常轻轻地处理骨髓细胞。一些方案建议使用18至20号针头来悬浮骨髓块。我们发现这降低了活力。存活的分化BMDC高度吞噬并吞噬这些死细胞,导致它们过早和过度激活。
  6. GM-CSF。市售的天然GM-CSF是非常昂贵的。几个公开的论文使用从E纯化的重组GM-CSF。大肠杆菌表达蛋白质,其工作相当良好,基于CD11c-高BMDC在培养第7天的百分比。然而,用这种重组GM-CSF产生的BMDC有时可以基于MHC-II和CD86的表面表达显示不期望的高水平成熟。我们已经使用用表达小鼠GM-CSF的质粒稳定转染的HEK293_T细胞的培养物上清液,并获得令人满意的未成熟BMDC的制备物。
  7. 培养基。确保用于培养BMDC的IMDM严格清洁,完全没有内毒素痕迹。我们将新鲜的水收集到在200°C烘烤的玻璃瓶中,将粉状介质溶解在其中,并立即在生物安全柜内通过双层0.2μm膜过滤。
  8. 胎牛血清:测试批次的血清以确保BMDC培养物在第7天不成熟。含有高水平的尿素,肌酐等的血清批次可以引起BMDC激活。确保FBS的供应商提供大约30个奇数血清组分的水平的数据。一旦找到一个好的批次,订购足够的金额持续这些实验的持续时间。
  9. ICC的FACS染色。检查您的组中的表面标志物特异性抗体结合物是否会识别甲醛固定的细胞上的同源抗原。如果没有,您将必须先进行表面染色,然后固定和透化细胞进行细胞内染色。始终滴定所有FACS抗体,并使用适当的量以最小化背景扩增和溢出到其他渠道。 FACS抗体上面粉的选择应根据流式细胞仪配置和机器上可用的过滤器的带宽来决定。
  10. 需要对每种使用的抗原测定分泌抑制剂布雷菲德菌素A和莫能菌素的处理时间。强效的蛋白质或肽抗原可能需要少至6至12小时的T细胞刺激,而我们的实验需要更长的刺激。虽然布雷菲德菌素A对细胞无毒性,但莫西汀长时间暴露可导致细胞死亡。因此,我们将莫能菌素浓度从标准3mM降至0.75mM

食谱

  1. IMDM完成
    10%FBS,2mMβ-巯基乙醇,谷氨酰胺
  2. 胸苷
    [甲基 - ] 3 H],20Ci/mmol在70%乙醇中,储存在-20℃下
  3. 布雷菲德菌素A(BFA)
    将1mg BFA溶解于1ml的100%乙醇中,并在-80℃下以50μl的等分试样储存。
  4. 莫能菌
    分子量:670游离和693的钠盐
    准备30毫米(20毫克/毫升)在无水乙醇的股票,并存储在-80℃的100微升等份
  5. RBC裂解缓冲液(100ml) 氯化铵(NH 4 Cl)8.99g
    碳酸氢钾(KHCO 3)1.0g
    乙二胺四乙酸(EDTA)37mg
    溶解在80ml高压灭菌的DD水中
    使用浓缩液将pH调节至7.3。 HCl
    将体积补偿到100 ml
    使用0.22μm过滤器进行过滤
    不要高压灭菌
    储存在2-8°C
  6. RBC裂解缓冲液
    0.9%NH 4 Cl在10mM Tris(pH 7.5)中的溶液 保持无菌于4°C

致谢

这项工作由印度政府科学技术部生物技术部授权的BT/PR7240/INF/22/52/2006资助。资助者在研究设计,数据收集和分析,决定发布或准备手稿方面没有任何作用。我们感谢Shoba Elangovan和Rajni Nyodu帮助制作视频。该方案改编自Satchidanandam等人(2014)。

参考文献

  1. Lutz,M.B。和Rossner,S。(2007)。 影响从骨髓中产生鼠树突细胞的因素:胎牛血清的特殊作用。/a> Immunobiology 212(9-10):855-862。
  2. Satchidanandam,V.,Kumar,N.,Jumani,R.S.,Challu,V.,Elangovan,S.and Khan,N.A。(2014)。 结核分枝杆菌的糖基化Rv1860蛋白可抑制树突状细胞介导的TH1和TH17 T细胞的极化并消除BCG赋予的保护性免疫。 PLoS Pathog 10(6):e1004176。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2016 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. Satchidanandam, V. (2016). Mouse BMDC-dependent T Cell Polarization Assays. Bio-protocol 6(3): e1721. DOI: 10.21769/BioProtoc.1721.
  2. Satchidanandam, V., Kumar, N., Jumani, R. S., Challu, V., Elangovan, S. and Khan, N. A. (2014). The glycosylated Rv1860 protein of Mycobacterium tuberculosis inhibits dendritic cell mediated TH1 and TH17 polarization of T cells and abrogates protective immunity conferred by BCG. PLoS Pathog 10(6): e1004176.
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