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In vitro Homeostatic Proliferation of Human CD8 T Cells
人CD8 T细胞的体外稳态增殖   

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Abstract

Long-lived T-cell–mediated immunity requires persistence of memory T cells in an antigen-free environment while also maintaining a heightened capacity to recall effector functions. Such antigen-independent homeostatic proliferation is mediated in part by the common gamma-chain cytokines IL-7 and IL-15. To further explore the mechanisms governing maintenance of effector functions in long-lived memory T cells during antigen-independent proliferation, human naïve and memory CD8 T cells can be sorted from peripheral blood mononuclear cells (PBMCs), labeled with the proliferation-tracking dye carboxyfluorescein succinimidyl ester (CFSE), and then purified based on their levels of cell division. This allows investigators to assess differences in the desired molecular target in cells that have undergone cytokine-driven proliferation. We provide here a protocol for assessing epigenetic programs in divided and undivided human naïve and memory CD8 T cells following 7 days in culture with IL-7 and IL-15 to illustrate how this approach can shed light on the mechanism(s) that governs the preservation of effector functions during homeostasis of long-lived memory CD8 T cells.

Keywords: Homeostatic proliferation(稳态增殖), Epigenetic programs(表观遗传程序), Naïve CD8 T cells(幼稚CD8 T细胞), Memory CD8 T cell subsets(记忆CD8 T细胞亚群), IL-7 and IL-15(IL-7和IL-15)

Background

A cardinal feature of adaptive immunity is the development of immunological memory against previously encountered pathogens (Plotkin et al., 2013). Memory CD8 T cells play a major role in providing life-long protection against pathogens previously encountered by the host, but in order to provide long-lived protection, T cells must have acquired the ability to persist and maintain effector functions in an antigen-free environment. During this time, memory T cells undergo antigen-independent proliferation in response to IL-7/15 cytokines, albeit to different degrees.

The total pool of memory CD8 T cells is a heterogeneous combination of several different cell subsets that respond differently to homeostatic cytokines. For instance, the highly proliferative central memory (Tcm) subset of CD8 T cells expresses the chemokine receptor CCR7, which enables those cells to access lymphoid tissue. Conversely, the low proliferative effector memory (Tem) subset of CD8 T cells lacks CCR7 expression and has limited access to lymphoid tissue (Sallusto et al., 1999; Masopust et al., 2001). Another recently defined subset of memory T cells, stem-cell memory (Tscm), which were named based on their heightened ability to self-renew and give rise to other memory subsets, exhibits the greatest level of cytokine-driven proliferation, compared to the other subsets (Gattinoni et al., 2011). Although phenotypically and functionally distinct, all of these memory T-cell subsets retain their ability to rapidly recall effector function upon antigen re-exposure. Thus, a common defining feature for the development of the various memory CD8 T-cell subsets is the maintenance of effector functions in the absence of antigen. However, the cell-intrinsic mechanisms that maintain memory-associated effector functions remain poorly understood. Below we describe an in vitro homeostatic proliferation assay that enables us to analyze mechanisms that regulate fate commitment during human memory T-cell homeostasis. We specifically describe loci-specific bisulfite-sequencing DNA-methylation analysis to examine the stability of effector DNA-methylation programs in memory CD8 T-cell subsets over several rounds of cell division (Abdelsamed et al., 2017).

Materials and Regents

  1. ART tips 1,000 μl (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 2079G )
  2. ART tips 200 μl (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 2069G )
  3. ART tips 10 μl (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 2139-RT )
  4. 5 ml STRIPETTE (Corning, Costar®, catalog number: 4487 )
  5. 10 ml STRIPETTE (Corning, Costar®, catalog number: 4488 )
  6. 25 ml STRIPETTE (Corning, Costar®, catalog number: 4489 )
  7. 18 G x 1-inch disposable needles (BD, catalog number: 305195 )
  8. 30 ml BD Luer-Lok syringe (BD, catalog number: 302832 )
  9. Falcon conical tubes, 50-ml (Corning, Falcon®, catalog number: 352098 )
  10. Falcon conical tubes, 15-ml (Corning, Falcon®, catalog number: 352057 )
  11. 96-well cell culture plates, round bottom (Corning, Costar®, catalog number: 3799 )
  12. Sterile polystyrene Petri dishes 100 x 15 mm (Fisher Scientific, catalog number: FB0875712 )
  13. Apheresis blood unit
  14. XL10-Gold Ultracompetent cells (Agilent Technologies, catalog number: 200314 )
  15. pGEM-T Vector System I (Promega, catalog number: A3600 )
  16. 1x calcium- and magnesium-free PBS (Lonza, catalog number: 17-516Q )
  17. Ficoll-Paque PLUS (GE Healthcare, catalog number: 17-1440-02 )
  18. Trypan blue solution, 0.4% (Sigma-Aldrich, catalog number: T8154 )
  19. EasySep Human CD8+ T Cell Enrichment Kit (STEMCELL Technologies, catalog number: 19053 )
  20. Antibodies
    CD8 (BioLegend, catalog number: 301033 )
    CD45RO (BioLegend, catalog number: 304210 )
    CD45RA (BioLegend, catalog number: 304134 )
    CCR7 (BD, BD Biosciences, catalog number: 561271 )
    CD95 (BioLegend, catalog number: 305622 )
    Live dead stain (Tonbo Biosciences, catalog number: 13-0870 )
  21. CpGenome Direct Prep Bisulfite Modification Kit (Merck, catalog number: 17-10451 )
  22. Zymoclean Gel DNA Recovery Kit (ZYMO RESEARCH, catalog number: D4002 )
  23. S.O.C. medium (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15544034 )
  24. DirectPrep 96 MiniPrep Kit (QIAGEN, catalog number: 27361 )
  25. Primers for methylation-specific PCR (Purchased from Integrated DNA Technologies) (see Notes for design)
  26. Heat-inactivated fetal bovine serum (FBS) (GE Healthcare, HycloneTM, catalog number: SH30910.03 )
  27. RPMI 1640 with L-glutamine (Mediatech, catalog number: 10-040-CM )
  28. 0.5 M EDTA (Invitrogen, catalog number: 15575-038 )
  29. Sodium azide (Sigma-Aldrich, catalog number: S2002-25G )
  30. Carboxyfluorescein succinimidyl ester (CFSE) (Life Technologies, catalog number: C1165 )
  31. Gentamicin (50 mg/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15750060 )
  32. Penicillin-streptomycin (10,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  33. Human recombinant IL-7 stock, 250 µg/ml (PeproTech, catalog number: AF-200-07 )
  34. Human recombinant IL-15 stock, 250 µg/ml (PeproTech, catalog number: AF-200-15 )
  35. 10x Tris/Boric Acid/EDTA (TBE), nucleic acid electrophoresis buffer (Bio-Rad Laboratories, catalog number: 1610733 )
  36. Agarose (Hoefer, catalog number: GR140-500 )
  37. Ethidium bromide 10 mg/ml (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15585011 )
  38. LB agar miller (Sigma-Aldrich, catalog number: L3027-1KG )
  39. 2x LB broth (Teknova, catalog number: L8080 )
  40. Ampicillin (Sigma-Aldrich, catalog number: A9518-5G )
  41. IPTG (Sigma-Aldrich, catalog number: I6758-10G )
  42. X-gal (Lambda Biotech)
  43. JumpStart Taq ReadyMix 2x (Sigma-Aldrich, catalog number: P2893 )
  44. Heat-inactivated FBS (see Recipes)
  45. RPMI 1640 with glutamine and 4% FBS (see Recipes)
  46. Enrichment buffer (see Recipes)
  47. RPMI 1640 with glutamine, 4% FBS, and 0.02% sodium azide (see Recipes)
  48. FACS buffer (see Recipes)
  49. 4 µM CFSE working solution (see Recipes)
  50. RPMI 1640 with glutamine and 10% FBS (see Recipes)
  51. Culture medium (see Recipes)
  52. IL-7 and IL-15 working solutions (see Recipes)
  53. 1x TBE (see Recipes)
  54. 2% agarose gel (see Recipes)
  55. LB agar plates (see Recipes)

Equipment

  1. Neubauer hemacytometer (Hausser Scientific, catalog number: 3200 )
  2. P1000 (Gilson, catalog number: F123602 )
  3. P200 (Gilson, catalog number: F123601 )
  4. P10 (Gilson, catalog number: F144802 )
  5. Water bath
  6. 37 °C shaker
  7. 37 °C incubator
  8. Sorval Legend XTR centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: SorvalTM LegendTM XTR , catalog number: 75004520)
  9. EasySep STEMCELL Technologies 15-ml and 50-ml magnets
  10. Tissue culture incubator
  11. Biosafety cabinet
  12. Horizontal gel electrophoresis unit (Hoefer, model: SUBHT )
  13. Vacuum manifold
  14. Nikon Eclipse TS100 microscope (Nikon Instruments, model: Eclipse TS100 )
  15. Sony Biotechnology SY3200 sorter (Synergy) (Sony Biotechnology, model: SY3200 )
  16. Autoclave (Getinge, model: 133LS )

Software

  1. FlowJo Software (Version 9.7.6)
  2. QUMA Software (http://quma.cdb.riken.jp/)

Procedure

  1. Isolation of human peripheral blood mononuclear cells (PBMCs)
    1. Collect the blood from an apheresis blood unit by using a 30 ml syringe with an 18-G, 1-inch needle.
      Note: Dispense all blood products, tubes, and solutions in a biohazard bag.
    2. Slowly dispense 10 ml blood on the wall of a 50 ml tube containing 25 ml 1x calcium- and magnesium-free PBS.
    3. With steady flow, overlay the diluted blood on the wall of a 50 ml tube containing 15 ml Ficoll.
    4. Spin at 400 x g for 15 min without brakes.
    5. Collect the interphase, which contains the peripheral blood mononuclear cells, and transfer it to a 50 ml tube.
    6. Wash with RPMI 1640 with glutamine and 4% FBS (see Recipes) at 10x the volume and then spin at 400 x g for 5 min.

  2. Enrichment of human CD8 T cells
    1. Decant the supernatant from step A6, and resuspend the cell pellet in enrichment buffer for counting (see Recipes).
    2. Take 10 µl of the cell suspension, add to 90 µl trypan blue (1:10 dilution), and then use a hemacytometer for counting (expected cell number is 108-109).
    3. After counting, prepare cell suspension at a concentration 5 x 107 cells/ml (if > 4.25 x 108 total cells, then use a 50 ml tube; if < 4.25 x 108 total cells, then use a 15-ml tube).
    4. Add the enrichment cocktail (included in the EasySep kit) at 50 µl/ml cells, mix well by pipetting up and down, and incubate at room temperature for 10 min.
    5. During the 10 min incubation, vortex the EasySep D magnetic particles for 30 sec or until you observe a uniform suspension.
    6. Add the particles at 150 µl/ml cells, mix well, and incubate at room temperature for 5 min.
    7. After a 5 min incubation, use enrichment buffer (see Recipes) to bring the cell suspension to a total volume of 10 ml if cell number < 4.25 x 108 total cells or 20 ml if > 4.25 x 108 total cells.
    8. Place the tube on the EasySep magnet.
    9. After 5 min, use a pipette to collect the negative fraction from the 50 ml tube. If a 15 ml tube was used, pour off the desired fraction, in one continuous motion, into a new 50-ml tube for counting (similar to step B2). For more details about the protocol and this step, please visit https://www.stemcell.com/easysep-human-cd8-t-cell-enrichment-kit.html.

  3. Sorting human naïve and memory CD8 T cell subsets
    1. After counting (expected cell number is 10-15% of the total PBMCs count), in a 50 ml tube wash the cells with RPMI and 4% FBS (till the 50 ml mark) and spin at 400 x g for 5 min.
    2. Decant, resuspend in FACS buffer, and distribute the cells as 50 x 106 cells per FACS tube in a 100 µl final volume.
    3. Stain for CCR7 antigen and then incubate in a water bath at 37 °C for 20 min (see Notes and Materials and Reagents’ above for fluorochromes, clones, manufacturer, catalog numbers and dilutions-Table 1).

      Table 1. List of human antibody conjugated fluorochromes used for cell sorting

      Note: All antibody dilutions are 1:100 except for anti-CD95 PECy7 antibody that is 1:20. Final volume is 100 µl. Live dead staining is performed prior to surface stains in serum-free pbs.

    4. Wash the cells with RPMI 4% FBS, and 0.02% sodium azide (see Recipes and Notes) and then spin at 400 x g for 5 min.
    5. Decant and stain for CD8, CD45RO, CD45RA, CD95, and living/dead stain and incubate at room temperature in the dark for 30 min.
    6. Wash the cells with FACS buffer and then spin at 400 x g for 5 min.
    7. Decant and pool all of the stained cells into a single tube for sorting.
    8. Sort live naïve and memory CD8 T cell subsets based on the following surface markers:
      1. Naïve CD8 T cells: CCR7+, CD45RO-, CD45RA+, CD95-
      2. Tem CD8 T cells: CCR7-, CD45RO+
      3. Tcm CD8 T cells: CCR7+, CD45RO+
      4. Tscm CD8 T cells: CCR7+, CD45RO-, CD95+

  4. Labeling sorted T cells with the proliferation-tracking dye CFSE
    1. Wash the sorted cells with prewarmed FACS buffer (see Recipes) and then spin at 400 x g for 5 min.
    2. Decant and resuspend 500,000 cells in 1 ml prewarmed FACS buffer.
    3. Prepare 4 µM CFSE working solution (see Recipes), then add 1 ml working solution to 1 ml cell suspension, and incubate at 37 °C for 5 min.
    4. Wash with 50 ml cold RPMI and 20% FBS (see Recipes) and then place on ice for 5 min.
    5. Spin at 400 x g for 5 min at 4 °C.
    6. Repeat steps D4 and D5.
    7. Decant completely, resuspend in 150 µl culture medium (see Recipes), and transfer the cell suspension into a 96-well, round-bottom plate.
    8. Prepare IL-7 and IL-15 working solutions at 50 ng/ml each (see Recipes), and then add 150 µl working solution to the 150 µl cell suspension in the 96-well plate.
    9. Incubate at 37 °C in 5% CO2.
    10. After 7 days of culture, sort undivided and divided cell populations (third division or more) for DNA-methylation analysis as shown in Figure 1A (Abdelsmed et al., 2017).


      Figure 1. Representative data analysis. A. FACS plots: CFSE dilution from one representative donor showing proliferation of sorted CD8 T cell subsets after IL-7/IL-15 in vitro culture for 7 days. Percentage in each gate represents cells dividing more than 3 divisions. B. Bisulfite sequencing analysis: Each horizontal line represents a clone while vertical lines are different CpG sites in the differentially methylated region. Open circles depict unmethylated CpG while filled circles depict methylated CpG. These data and analyses are representative of published data (Abdelsamed et al., 2017; Ghoneim et al., 2017).

  5. Loci-specific bisulfite-sequencing analysis (DNA-methylation analysis)
    1. Sorted cells are lysed, and DNA undergoes bisulfite treatment using the CpGenome Direct Prep Bisulfite Modification Kit.
    2. Bisulfite-treated DNA is used as a template to amplify a specific target sequence using JumpStart ReadyMix 2x. The Methylation-specific PCR program is as follows:
      1. 95 °C for 5 min
      2. 95 °C for 30 sec
      3. 55 °C 1:30 min
      4. Go to step b 49 times
      5. 4 °C indefinitely
    3. Prepare 2% agarose gel containing ethidium bromide (see Recipes).
    4. Pour the gel into the gel tray and then wait until it solidifies.
    5. Load the PCR product into the gel and then run the gel using 1x TBE (see Recipes).
    6. Cut the appropriate band size, digest the gel, and extract the DNA using the Zymoclean Gel DNA Recovery Kit.
    7. Use the entire PCR product for the ligation reaction using pGEM-T Vector System I kit.
      Note: Overnight incubation at 4 °C is recommended.
    8. Mix the ligation product with 50 µl XL-10–competent bacteria and place on ice for 20 min.
      Note: It is recommended to use one third of the ligation product for transformation and save the rest in -20 °C.
    9. Incubate the tubes containing bacteria/DNA mix at 42 °C for 45 sec.
    10. Place the tubes immediately on ice for 2 min.
    11. Add 400 µl S.O.C. medium to each tube and then incubate the tubes in a shaker (150 rpm) at 37 °C for 90 min.
    12. Spread 150 µl of the bacterial suspension on LB agar plates (see Recipes).
    13. Incubate at 37 °C for 18 h.
    14. In a deep 96-well plate, inoculate one white bacterial colony in each well containing 900 µl LB medium with 0.1 mg/ml ampicillin (see Notes).
    15. Incubate the cell cultures at 37 °C for 18-22 h with shaking (220 rpm).
    16. Spin the 96-deep well plates at 1,600 x g for 15 min.
    17. Decant the supernatant and then extract the plasmid from the bacterial pellet by following the instructions in DirectPrep 96 Miniprep Kit.
    18. Elute the DNA with 60 µl elution buffer, dry the 96-well plate overnight, and then use 300-600 ng DNA for Sanger sequencing using pUC/M13 reverse primers.
    19. Upload the Ab1 sequencing files as zip files to the QUMA website (QUantification tool for Methylation Analysis, http://quma.cdb.riken.jp/).
    20. After analyzing the data by using the QUMA website, its readout is depicted as shown in Figure 1B.

Data analysis

FlowJo Software (Version 9.7.6) was used to analyze flow cytometry data (Figure 1A), while DNA methylation data was analyzed using QUMA Software (http://quma.cdb.riken.jp/). Each sequencing AB1 file represents a clone. Briefly, all AB1 files for each clone were compressed and uploaded to the software then the target sequence of the gene of interest was aligned against the sequenced clones. The software readout is shown in Figure 1B.

Notes

  1. The methylated cytosines are protected from a deamination reaction, while the nonmethylated cytosines are converted to uracils. Using Sanger sequencing, this single-nucleotide–resolution method detects methylated cytosines as cytosines and nonmethylated cytosines as thymines. To learn more about bisulfite conversion, visit the Zymo Research website (www.zymoresearch.com/epigentics).
  2. Primers used in the methylation-specific PCR program should have the following conditions:
    1. Do not contain any CpG sites
    2. Annealing temperature: 56-60 °C
    3. Length: 26-30 bp
    4. All cytosines should be converted to thymines. In case of the forward primer, all cytosines are converted to thymines while in case of reverse primer; all cytosines are converted to thymines followed by reverse complement.
  3. Sodium azide is recommended to be added to the staining buffer since anti-CD95 antibody can induce cell apoptosis.
  4. CCR7 is a chemokine receptor that recycles through the cell membrane. Incubation at 37 °C improves its detection.
  5. To obtain representative data, you need to inoculate 16-24 bacterial colonies per agar plate, which represents one given condition.

Recipes

  1. Heat-inactivated FBS
    Incubate a bottle of FBS in a water bath at 56 °C for 30 min and then prepare 50 ml aliquots and store at -20 °C
  2. RPMI 1640 with glutamine and 4% FBS
    Add 40 ml heat-inactivated FBS to 1 L RPMI with glutamine
  3. Enrichment buffer
    Add 10 ml heat-inactivated FBS to 500 ml 1x PBS without calcium and magnesium + 1 ml EDTA (0.5 M EDTA)
  4. RPMI 1640 with glutamine, 4% FBS, and 0.02% sodium azide
    Add 40 ml heat-inactivated FBS to 1 L RPMI with glutamine + 0.2 g sodium azide
  5. FACS buffer
    Add 20 ml heat-inactivated FBS to 500 ml 1x PBS without calcium and magnesium
  6. 4 µM CFSE working solution
    Add 1.5 µl CFSE from stock (44.8 mM or 25 mg/ml) to 10 ml warm PBS
  7. RPMI 1640 with glutamine and 20% FBS
    Add 100 ml heat-inactivated FBS to 1 L RPMI with glutamine
  8. Culture medium
    In 1 L RPMI with glutamine, add the following:
    100 ml heat-inactivated FBS
    1 ml gentamycin (1:1,000 dilution)
    10 ml penicillin-streptomycin (1:100 dilution)
  9. IL-7 and IL-15 working solutions
    1. Add 1 µl of 250 µg/ml IL-7 and 1 µl of 250 µg/ml IL-15 into 2.5 ml of culture medium
    2. Dilute into culture medium at 1:1 ratio, then use directly 150 µl and add to 150 µl of CFSE labeled cells seeded in a 96-well plate
  10. 1x TBE
    Dilute 10x TBE to 1x TBE using deionized water
  11. 2% agarose gel
    1. Add 2 g agarose LE to 100 ml of 1x TBE and then heat in a microwave until the gel dissolves completely
    2. Add 5 µl ethidium bromide (10 mg/ml) to achieve the final concentration of 5 ng/ml
    3. Mix well and then pour into the gel tray
  12. LB agar plates
    1. Dissolve 40 g LB agar into 1 L of double-distilled water and then autoclave
    2. Bring the temperature down to 45 °C and then add 100 mg ampicillin, 230 mg IPTG, and 100 µl X-gal
    3. Mix well and pour 15 ml into a sterile Petri dish and leave open under a biosafety cabinet
    4. Wait until the agar plates are solidified then store at 4 °C for up to one month

Acknowledgments

We thank Dr. Angela McArthur for scientific editing. This work was supported by the National Institutes of Health grant 1R01AI114442 and ALSAC (to B.Y.). The authors declare no competing financial interests. This protocol was adapted and modified from Lugli et al., 2013-Identification, isolation and in vitro expansion of human and nonhuman primate T stem cell memory cells. Nat Protoc 8(1): 33-42.

References

  1. Abdelsamed, H. A., Moustaki, A., Fan, Y., Dogra, P., Ghoneim, H. E., Zebley, C. C., Triplett, B. M., Sekaly, R. P. and Youngblood, B. (2017). Human memory CD8 T cell effector potential is epigenetically preserved during in vivo homeostasis. J Exp Med 214(6): 1593-1606.
  2. Gattinoni, L., Lugli, E., Ji, Y., Pos, Z., Paulos, C. M., Quigley, M. F., Almeida, J. R., Gostick, E., Yu, Z., Carpenito, C., Wang, E., Douek, D. C., Price, D. A., June, C. H., Marincola, F. M., Roederer, M. and Restifo, N. P. (2011). A human memory T cell subset with stem cell-like properties. Nat Med 17(10): 1290-1297.
  3. Ghoneim, H. E., Fan, Y., Moustaki, A., Abdelsamed, H. A., Dash, P., Dogra, P., Carter, R., Awad, W., Neale, G., Thomas, P. G. and Youngblood, B. (2017). De novo epigenetic programs inhibit PD-1 blockade-mediated T cell rejuvenation. Cell 170(1): 142-157 e119.
  4. Lugli, E., Gattinoni, L., Roberto, A., Mavilio, D., Price, D. A., Restifo, N. P. and Roederer, M. (2013). Identification, isolation and in vitro expansion of human and nonhuman primate T stem cell memory cells. Nat Protoc 8(1): 33-42.
  5. Masopust, D., Vezys, V., Marzo, A. L. and Lefrancois, L. (2001). Preferential localization of effector memory cells in nonlymphoid tissue. Science 291(5512): 2413-2417.
  6. Plotkin, S. A., Orenstein, W. A. and Offit, P. A. (2013). Vaccines. (6th edition). 1550.
  7. Sallusto, F., Lenig, D., Forster, R., Lipp, M. and Lanzavecchia, A. (1999). Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 401(6754): 708-712.

简介

长寿T细胞介导的免疫需要在无抗原环境中持续存在记忆T细胞,同时还保持提高的回忆效应器功能的能力。这种不依赖于抗原的体内平衡增殖部分由常见的γ链细胞因子IL-7和IL-15介导。为了进一步探索在抗原非依赖性增殖过程中维持长效记忆T细胞中效应子功能的机制,可以从外周血单核细胞(PBMCs)中分选人幼稚和记忆CD8T细胞,用增殖跟踪染料羧基荧光素琥珀酰亚胺酯(CFSE),然后根据它们的细胞分裂水平进行纯化。这使得研究人员能够评估经历细胞因子驱动的增殖的细胞中期望的分子靶点的差异。我们在这里提供了一个协议,用于评估在分裂和未分裂的人类幼稚和记忆CD8 T细胞中的表观遗传程序,在用IL-7和IL-15培养7天之后,以说明这种方法如何阐明控制在长寿记忆CD8T细胞的稳态期间保持效应子功能。
【背景】获得性免疫的一个主要特征是对先前遇到的病原体的免疫记忆的发展(Plotkin等人,2013)。记忆CD8 T细胞在为宿主以前遇到的病原体提供终生保护方面发挥重要作用,但为了提供长寿命的保护,T细胞必须已经获得了在无抗原中持续并保持效应功能的能力环境。在此期间,记忆T细胞响应于IL-7/15细胞因子进行抗原非依赖性增殖,尽管程度不同。

记忆CD8 T细胞的总池是几种不同的细胞亚群的异质组合,其对稳态细胞因子的反应不同。例如,CD8 T细胞的高度增殖性中枢记忆(Tcm)子集表达趋化因子受体CCR7,其使这些细胞进入淋巴组织。相反地,CD8T细胞的低增殖效应记忆(Tem)子集缺乏CCR7表达,并且对淋巴组织的获取有限(Sallusto等人,1999; Masopust等人 >,2001)。记忆T细胞的另一个最近定义的子集,即干细胞记忆(Tscm),其基于其提高的自我更新能力并产生其他记忆亚群而命名,与细胞因子驱动的增殖相比,显示最高水平的其他子集(Gattinoni et al。,2011)。虽然表型和功能不同,但是所有这些记忆T细胞亚群都保留了在抗原再次暴露时快速回忆效应器功能的能力。因此,开发各种记忆CD8 T细胞亚群的共同定义特征是在不存在抗原的情况下维持效应子功能。然而,维持记忆相关效应功能的细胞内在机制仍然知之甚少。下面我们描述一个体外稳态增殖试验,使我们能够分析在人类记忆T细胞稳态期间调节命运承诺的机制。我们具体描述基因座特异性亚硫酸氢盐测序DNA甲基化分析,以检查记忆CD8T细胞亚群中效应子DNA甲基化程序在多轮细胞分裂中的稳定性(Abdelsamed等人,2017年)。

关键字:稳态增殖, 表观遗传程序, 幼稚CD8 T细胞, 记忆CD8 T细胞亚群, IL-7和IL-15

材料和摄取物

  1. ART提示1,000μl(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:2079G)
  2. ART提示200μl(Thermo Fisher Scientific,Thermo Scientific TM,目录号:2069G)
  3. ART提示10μl(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:2139-RT)
  4. 5ml STRIPETTE(Corning,Costar,目录号:4487)
  5. 10ml STRIPETTE(Corning,Costar ,目录号:4488)
  6. 25ml STRIPETTE(Corning,Costar ,目录号:4489)

  7. 18 G x 1英寸一次性针(BD,目录号:305195)
  8. 30毫升BD Luer-Lok注射器(BD,目录号:302832)
  9. 猎鹰锥形管,50毫升(Corning,Falcon ,目录号:352098)
  10. 猎鹰锥形管,15毫升(Corning,Falcon ,目录号:352057)
  11. 96孔细胞培养板,圆底(Corning,Costar ,目录号:3799)

  12. 无菌聚苯乙烯培养皿100×15毫米(Fisher Scientific,目录号:FB0875712)
  13. 血液采集单位
  14. XL10-Gold Ultracompetent电池(Agilent Technologies,目录号:200314)
  15. pGEM-T载体系统I(Promega,目录号:A3600)
  16. 1x不含钙和镁的PBS(Lonza,目录号:17-516Q)
  17. Ficoll-Paque PLUS(GE Healthcare,目录号:17-1440-02)
  18. 台盼蓝溶液,0.4%(Sigma-Aldrich,目录号:T8154)
  19. EasySep人CD8 + T细胞富集试剂盒(STEMCELL Technologies,目录号:19053)
  20. 抗体
    CD8(BioLegend,目录号:301033)
    CD45RO(BioLegend,目录号:304210)
    CD45RA(BioLegend,目录号:304134)
    CCR7(BD,BD Biosciences,目录号:561271)
    CD95(BioLegend,目录号:305622)
    活死的污渍(Tonbo Biosciences,产品目录号:13-0870)
  21. CpGenome直接制备亚硫酸氢盐修饰试剂盒(Merck,目录号:17-10451)
  22. Zymoclean凝胶DNA回收试剂盒(ZYMO RESEARCH,目录号:D4002)
  23. S.O.C.培养基(Thermo Fisher Scientific,Invitrogen TM,产品目录号:15544034)
  24. DirectPrep 96 MiniPrep Kit(QIAGEN,目录号:27361)
  25. 甲基化特异性PCR引物(购自Integrated DNA Technologies)(见设计说明)
  26. 热灭活胎牛血清(FBS)(GE Healthcare,Hyclone TM,目录号:SH30910.03)
  27. 具有L-谷氨酰胺的RPMI 1640(Mediatech,目录号:10-040-CM)
  28. 0.5M EDTA(Invitrogen,目录号:15575-038)
  29. 叠氮化钠(Sigma-Aldrich,目录号:S2002-25G)
  30. 羧基荧光素琥珀酰亚胺酯(CFSE)(Life Technologies,目录号:C1165)
  31. 庆大霉素(50mg / ml)(Thermo Fisher Scientific,Gibco TM,产品目录号:15750060)
  32. 青霉素 - 链霉素(10,000U / ml)(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  33. 人重组IL-7储备液,250μg/ ml(PeproTech,目录号:AF-200-07)
  34. 人重组IL-15储备液,250μg/ ml(PeproTech,目录号:AF-200-15)
  35. 10×Tris /硼酸/ EDTA(TBE),核酸电泳缓冲液(Bio-Rad Laboratories,目录号:1610733)
  36. 琼脂糖(Hoefer,目录号:GR140-500)
  37. 溴化乙锭10mg / ml(Thermo Fisher Scientific,Invitrogen TM,目录号:15585011)
  38. LB琼脂磨(Sigma-Aldrich,目录号:L3027-1KG)
  39. 2x LB肉汤(Teknova,目录号:L8080)
  40. 氨苄青霉素(Sigma-Aldrich,目录号:A9518-5G)
  41. IPTG(Sigma-Aldrich,目录号:I6758-10G)
  42. X-gal(Lambda Biotech)
  43. JumpStart Taq ReadyMix 2x(Sigma-Aldrich,目录号:P2893)
  44. 热灭活FBS(见食谱)
  45. 含有谷氨酰胺和4%FBS的RPMI 1640(见食谱)
  46. 富集缓冲液(见食谱)
  47. 含谷氨酰胺,4%FBS和0.02%叠氮钠的RPMI 1640(见配方)
  48. FACS缓冲液(见食谱)
  49. 4μMCFSE工作解决方案(见食谱)
  50. 含谷氨酰胺和10%FBS的RPMI 1640(见食谱)
  51. 培养基(见食谱)
  52. IL-7和IL-15工作解决方案(见食谱)
  53. 1倍TBE(见食谱)
  54. 2%琼脂糖凝胶(见食谱)
  55. LB琼脂平板(见食谱)

设备

  1. Neubauer血球计(Hausser Scientific,目录号:3200)
  2. P1000(Gilson,目录号:F123602)
  3. P200(Gilson,目录号:F123601)
  4. P10(吉尔森,目录号:F144802)
  5. 水浴
  6. 37°C摇床
  7. 37°C培养箱
  8. Sorval Legend XTR离心机(Thermo Fisher Scientific,Thermo Scientific TM,型号:Sorval TM Legend TM XTR,目录号:75004520) >
  9. EasySep STEMCELL Technologies 15毫升和50毫升磁铁
  10. 组织培养孵化器
  11. 生物安全柜
  12. 卧式凝胶电泳仪(Hoefer,型号:SUBHT)
  13. 真空歧管
  14. Nikon Eclipse TS100显微镜(Nikon Instruments,型号:Eclipse TS100)
  15. 索尼生物技术SY3200分选机(Synergy)(索尼生物技术公司,型号:SY3200)
  16. 高压灭菌器(Getinge,型号:133LS)

软件

  1. FlowJo软件(版本9.7.6)
  2. QUMA软件( http://quma.cdb.riken.jp/

程序

  1. 分离人外周血单核细胞(PBMC)
    1. 通过使用30毫升注射器和18-G 1英寸针头从采血单位收集血液。
      注意:将所有血液制品,试管和溶液分装到生物危害包中。
    2. 在含有25ml 1x无钙和无镁PBS的50ml试管壁上慢慢分配10ml血液。
    3. 在稳定的流动下,将稀释的血液覆盖在含有15ml Ficoll的50ml管的壁上。

    4. 在400 x g的条件下旋转15分钟,不用刹车
    5. 收集含有外周血单核细胞的间期,并将其转移到50ml管中。
    6. 用含谷氨酰胺和4%FBS的RPMI 1640(参见食谱)以10倍体积洗涤,然后在400×g下旋转5分钟。

  2. 富集人类CD8 T细胞
    1. 滗析来自步骤A6的上清液,并将细胞沉淀物重悬于富集缓冲液中进行计数(参见食谱)。
    2. 取10μl细胞悬液,加入90μl台盼蓝(1:10稀释),然后用血细胞计数器计数(预期的细胞数为10 -8 -10 -9, )。
    3. 计数后,制备浓度为5×10 7细胞/ ml(如果> 4.25×10 8个细胞总数的细胞悬液,然后使用50ml管;如果&lt; ; 4.25×10 8个细胞,然后使用15毫升管)。
    4. 添加浓度为50μl/ ml细胞的浓缩混合液(包含在EasySep试剂盒中),上下移液混匀,室温孵育10分钟。
    5. 在孵育10分钟的过程中,将EasySep D磁性颗粒涡旋30秒,或直到观察到均匀的悬浮液。
    6. 加入150μl/ ml细胞的颗粒,充分混合,室温孵育5分钟。
    7. 孵育5分钟后,使用富集缓冲液(参见食谱)使细胞悬浮液的总体积为10ml,总细胞数为4.25×10 8个,或者如果> 4.25×10 8个细胞。
    8. 将管放在EasySep磁铁上。
    9. 5分钟后,使用移液管从50毫升管中收集阴性部分。如果使用15ml试管,则将连续运动中所需的部分倒入新的50ml试管中(类似于步骤B2)。有关协议和此步骤的更多详细信息,请访问 https ://www.stemcell.com/easysep-human-cd8-t-cell-enrichment-kit.html

  3. 排序人类幼稚和记忆CD8 T细胞亚群
    1. 计数后(预计细胞数为总PBMC计数的10-15%),在50ml管中用RPMI和4%FBS(直到50ml标记)洗涤细胞并以400xg < > 5分钟。
    2. 倒出,重悬于FACS缓冲液中,并以100μl最终体积将细胞分配成每个FACS管50×10 6个细胞。
    3. 染色CCR7抗原,然后在37℃的水浴中孵育20分钟(见上文关于荧光染料,克隆,制造商,产品目录号和稀释度的注释和材料和试剂,见表1)。

      表1.用于细胞分选的人抗体缀合的荧光染料的列表

      注意:除抗CD95 PECy7抗体1:20外,所有抗体稀释度均为1:100。最终体积是100μl。活死亡染色是在无血清pbs表面污渍之前进行的。

    4. 用RPMI 4%FBS和0.02%叠氮化钠洗涤细胞(见配方和注释),然后在400×g下旋转5分钟。
    5. 滗析并染色CD8,CD45RO,CD45RA,CD95和活的/死的污点,在室温下在黑暗中孵育30分钟。
    6. 用FACS缓冲液洗涤细胞,然后在400×g下旋转5分钟。
    7. 滗析并将所有染色的细胞集中成一个管子进行分选。
    8. 根据以下表面标记对活的幼稚和记忆CD8 T细胞亚组进行分类:
      1. 幼稚的CD8 T细胞:CCR7 +,CD45RO +,CD45RA +,CD95-,+ / +
      2. Tem CD8 T细胞:CCR7 - , CD45RO
      3. Tcm CD8 T细胞:CCR7 + / CD45RO + /

      4. Tscm CD8 T细胞:CCR7 +,CD45RO +,CD95 + + / +
  4. 用增殖追踪染料CFSE标记分选的T细胞
    1. 用预热的FACS缓冲液清洗分选的细胞(见食谱),然后在400×g下旋转5分钟。
    2. 在1毫升预热的FACS缓冲液中倾倒并重悬500,000个细胞。
    3. 准备4μMCFSE工作解决方案(见食谱),然后添加1毫升的工作解决方案,1毫升细胞悬液,并在37°C孵育5分钟。
    4. 用50ml冷RPMI和20%FBS洗涤(见食谱),然后放在冰上5分钟。

    5. 在400℃旋转5分钟,4分钟
    6. 重复步骤D4和D5。
    7. 完全沉淀后,重悬于150μl培养基中(见食谱),并将细胞悬液转移到96孔圆底培养皿中。
    8. 准备IL-7和IL-15各50 ng / ml的工作溶液(见配方),然后将150μl工作溶液加到96孔板中的150μl细胞悬液中。

    9. 5%CO 2在37°C孵育。
    10. 培养7天后,如图1A(Abdelsmed等人,2017)所示,对未分裂和分裂的细胞群(第三或更多)进行DNA甲基化分析。


      图1.代表性数据分析A.FACS图:来自一个代表性供体的CFSE稀释物显示IL-7 / IL-15体外分选的CD8T细胞亚群的增殖培养7天。每个门中的百分比表示细胞划分多于3个分区。 B.亚硫酸氢盐测序分析:每条水平线表示一个克隆,而垂直线是差异甲基化区域中不同CpG位点。空心圆表示未甲基化的CpG,而实心圆表示甲基化的CpG。这些数据和分析是已发表数据的代表(2017年,Abdelsamed等人,2017; Ghoneim等人,,2017)。

  5. 基因座特异性亚硫酸氢盐测序分析(DNA-甲基化分析)
    1. 将分选的细胞裂解,并使用CpGenome直接制备亚硫酸氢盐修饰试剂盒对DNA进行亚硫酸氢盐处理。
    2. 使用重亚硫酸盐处理的DNA作为模板以使用JumpStart ReadyMix 2x扩增特定的靶序列。甲基化特异性PCR程序如下:
      1. 95°C 5分钟
      2. 95°C 30秒
      3. 55°C 1:30分钟
      4. 去步骤B 49次
      5. 4°C无限期
    3. 准备含有溴化乙锭的2%琼脂糖凝胶(见食谱)。
    4. 将凝胶倒入凝胶盘,然后等待凝胶。
    5. 将PCR产物加载到凝胶中,然后使用1x TBE运行凝胶(见食谱)。
    6. 剪下合适的条带,消化凝胶,然后用Zymoclean Gel DNA Recovery Kit提取DNA。
    7. 使用pGEM-T Vector System I试剂盒将整个PCR产物用于连接反应。
      注意:建议在4°C下过夜孵育
    8. 将连接产物与50μlXL-10感受态细菌混合并置于冰上20分钟。
      注意:建议使用三分之一的连接产物进行转化,并保存在-20°C以下。

    9. 在42°C孵育含有细菌/ DNA混合物的试管45秒
    10. 立即放置在冰上2分钟
    11. 加入400μlS.O.C。培养至每管,然后在37℃的摇床(150转)孵育90分钟。
    12. 在LB琼脂平板上涂抹150μl细菌悬液(见食谱)。

    13. 在37°C孵育18小时
    14. 在深96孔板中,每孔接种一个白色细菌菌落,每孔含有900μl含有0.1mg / ml氨苄青霉素的LB培养基(见注)。

    15. 在37°C孵育细胞培养18-22小时,摇动(220转)。

    16. 旋转96孔深孔板,1600×g×15分钟
    17. 滗析上清液,然后按照DirectPrep 96 Miniprep Kit中的说明从细菌沉淀中提取质粒。
    18. 用60μl洗脱缓冲液洗脱DNA,将96孔板干燥过夜,然后使用300-600ng DNA进行Sanger测序,使用pUC / M13反向引物。
    19. 上传Ab1测序文件作为zip文件到QUMA网站(甲基化分析定量工具, http://quma.cdb .riken.jp / )。
    20. 通过使用QUMA网站分析数据后,其读数如图1B所示。

数据分析

使用FlowJo软件(版本9.7.6)分析流式细胞术数据(图1A),同时使用QUMA软件分析DNA甲基化数据( http://quma.cdb.riken.jp/ )。每个测序AB1文件代表一个克隆。简而言之,将每个克隆的所有AB1文件压缩并上载到软件中,然后将目标基因的靶序列与测序的克隆进行比对。软件读数显示在图1B中。

笔记

  1. 甲基化的胞嘧啶被保护免于脱氨基反应,而非甲基化的胞嘧啶被转化成尿嘧啶。使用Sanger测序,这种单核苷酸分辨方法检测甲基化胞嘧啶作为胞嘧啶和非甲基化胞嘧啶作为胸腺嘧啶。要了解有关亚硫酸氢盐转化的更多信息,请访问Zymo Research网站( www.zymoresearch.com/epigentics )。< br />
  2. 甲基化特异性PCR程序中使用的引物应具有以下条件:
    1. 不包含任何CpG网站
    2. 退火温度:56-60°C
    3. 长度:26-30 bp
    4. 所有的胞嘧啶都应该转化为胸腺嘧啶。在正向引物的情况下,在反向引物的情况下,所有的胞嘧啶转化为胸腺嘧啶;所有的胞嘧啶被转换成胸腺嘧啶,然后被反向补充。
  3. 因为抗CD95抗体可以诱导细胞凋亡,所以建议将叠氮钠添加到染色缓冲液中。
  4. CCR7是通过细胞膜循环的趋化因子受体。
    在37°C培养可以提高检测效果
  5. 要获得有代表性的数据,你需要接种每个琼脂板16-24细菌菌落,代表一个给定的条件。

食谱

  1. 热灭活的FBS
    在56°C的水浴中孵育一瓶FBS 30分钟,然后准备50毫升等分试样,并储存在-20°C。
  2. 含谷氨酰胺和4%FBS的RPMI 1640
    加入40ml热灭活的FBS到含有谷氨酰胺的1L RPMI中
  3. 富集缓冲液
    添加10毫升热灭活的FBS到500毫升1x PBS没有钙和镁+ 1毫升EDTA(0.5 M EDTA)
  4. 含有谷氨酰胺,4%FBS和0.02%叠氮钠的RPMI 1640
    添加40毫升热灭活的FBS到1升RPMI与谷氨酰胺+ 0.2克叠氮化钠
  5. FACS缓冲区
    添加20毫升热灭活FBS到500毫升1x PBS没有钙和镁
  6. 4μMCFSE工作解决方案

    加入1.5μlCFSE(44.8 mM或25 mg / ml)至10 ml温热PBS
  7. 含谷氨酰胺和20%FBS的RPMI 1640
    加入100ml热灭活的FBS到含有谷氨酰胺的1L RPMI中
  8. 培养基
    在含谷氨酰胺的1升RPMI中,加入以下物质:
    100毫升热灭活FBS
    1毫升庆大霉素(1:1,000稀释)
    10 ml青霉素 - 链霉素(1:100稀释)
  9. IL-7和IL-15工作解决方案
    1. 将1微升250微克/毫升IL-7和1微升250微克/毫升IL-15加入到2.5毫升的培养基中
    2. 以1:1的比例稀释到培养基中,然后直接使用150μl,加入150μlCFSE标记的细胞接种于96孔板中。
  10. 1x TBE
    使用去离子水稀释10倍TBE至1倍TBE
  11. 2%琼脂糖凝胶
    1. 加2克琼脂糖LE到100毫升的1×TBE,然后加热微波直到凝胶完全溶解
    2. 加入5μl溴化乙锭(10mg / ml)以达到5ng / ml的最终浓度
    3. 充分混合,然后倒入凝胶盘
  12. LB琼脂平板
    1. 将40克LB琼脂溶解在1升双蒸水中,然后高压灭菌器中
    2. 将温度降至45°C,然后加入100 mg氨苄青霉素,230 mg IPTG和100μlX-gal
    3. 充分混合,倒入无菌培养皿15毫升,并在生物安全柜
      下开放
    4. 等到琼脂平板凝固,然后在4°C储存长达一个月

致谢

我们感谢Angela McArthur博士的科学编辑。这项工作得到了美国国立卫生研究院资助1R01AI114442和ALSAC(致B.Y.)的支持。作者声明没有竞争的财务利益。该方案已经从Lugli等人的2013年 - 识别,分离和体外扩增人和非人灵长类动物T干细胞记忆细胞中进行了修改和修改。 Nat Protoc 8(1):33-42。

参考

  1. Abdelsamed,H.A.,Moustaki,A.,Fan,Y.,Dogra,P.,Ghoneim,H.E.,Zebley,C.C.,Triplett,B.M.,Sekaly,R.P.and Youngblood,B。(2017)。 人类记忆CD8 T细胞效应物潜能在体内体内平衡期间被表观遗传学保存。 J Exp Med 214(6):1593-1606。
  2. Gattinoni,L.,Lugli,E.,Ji,Y.,Pos,Z.,Paulos,CM,Quigley,MF,Almeida,JR,Gostick,E.,Yu,Z.,Carpenito,C.,Wang,E 。,Douek,DC,Price,DA,June,CH,Marincola,FM,Roederer,M。和Restifo,NP(2011)。 具有干细胞特性的人类记忆T细胞亚群 Nat Med 17(10):1290-1297。
  3. Ghoneim,HE,Fan,Y.,Moustaki,A.,Abdelsamed,HA,Dash,P.,Dogra,P.,Carter,R.,Awad,W.,Neale,G.,Thomas,PG和Youngblood,B (2017)。 重新表观遗传程序抑制PD-1阻断介导的T细胞再生。 > Cell 170(1):142-157 e119。
  4. Lugli,E.,Gattinoni,L.,Roberto,A.,Mavilio,D.,Price,D.A.,Restifo,N.P.and Roederer,M.(2013)。 人类和非人类灵长类动物T的鉴定,分离和体外扩增细胞记忆细胞。 Nat Protoc 8(1):33-42。
  5. Masopust,D.,Vezys,V.,Marzo,A.L。和Lefrancois,L.(2001)。 在非淋巴组织中优先定位效应记忆细胞 科学 > 291(5512):2413-2417。
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引用:Abdelsamed, H. A., Zebley, C. C. and Youngblood, B. (2017). In vitro Homeostatic Proliferation of Human CD8 T Cells. Bio-protocol 7(22): e2619. DOI: 10.21769/BioProtoc.2619.
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