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EAE Induction by Passive Transfer of MOG-specific CD4+ T Cells
通过MOG特异性CD4+ T细胞的被动转移诱导EAE   

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Abstract

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS), which is a chronic inflammatory disease of the central nervous system (CNS). It is characterized by focal demyelination and inflammatory responses mediated by myelin-specific autoreactive CD4+ T cells. Using a passive transfer model of EAE in mice, we have demonstrated that regional specific neural signals by sensory-sympathetic communications create gateways for immune cells at specific blood vessels of the CNS, a phenomenon known as the gateway reflex (Arima et al., 2012; Tracey, 2012; Arima et al., 2013; Sabharwal et al., 2014; Arima et al., 2015b). Here we describe protocols for passive transfer model of EAE using freshly isolated (MOG)-specific CD4+ T cells or periodically restimulated MOG-specific CD4+ T cell lines, which are suitable for tracking pathogenic CD4+ T cells in vivo, particularly in the CNS (Ogura et al., 2008; Arima et al., 2012 and 2015b).

Keywords: Experimental autoimmune encephalomyelitis(实验性自身免疫性脑脊髓炎), Pathogenic CD4+ T cells(致病性CD4+ T细胞), Myelin oligodendrocyte glycoprotein(髓鞘少突胶质细胞糖蛋白), Gateway reflex(通路反射), Passive transfer(被动转移)

Background

It is widely accepted that autoreactive CD4+ T cells play a significant role in the pathogenesis of MS and EAE (Reboldi, 2009; International Multiple Sclerosis Genetics et al., 2011; Steinman, 2014), which are chronic inflammatory diseases of the CNS. The CNS is protected by the blood-brain barrier (BBB), which limits immune cell infiltration from the periphery (Liu et al., 2012). Until recently, where and how CD4+ T cells enter the CNS from the peripheral blood was unclear. Although EAE can be induced by immunization of animals with CNS-autoantigens emulsified in complete Freund’s adjuvant (CFA) and pertussis toxin (PTx) (Andreasen et al., 2009; Lu et al., 2016), unwanted systemic inflammation occurs by injection of CFA and PTx, which potentially affects the integrity of the BBB (Schellenberg et al., 2012; Marbourg et al., 2017). Alternatively, we recommend a passive transfer EAE model, in which activated CD4+ T cells specific for MOG are injected into naïve mice without treatments with CFA or PTx. Using this passive transfer EAE model, we have identified the dorsal vessels of the fifth lumbar (L5) cord as an initial gateway for autoreactive CD4+ T cells to reach the CNS (Arima et al., 2012). Mechanistically, gravity-mediated constant activation of sensory neurons in the soleus muscles induces sympathetic nerve activation that connects to the L5 dorsal vessels. The resulting noradrenaline secretion at the vessels enhances NF-κB activity, leading to the production of chemokines that recruit the CNS autoreactive CD4+ T cells (Arima et al., 2012). This sensory-sympathetic communication driven by anti-gravity responses through the soleus muscles is called ‘gravity-gateway reflex’ (Arima et al., 2012; Tracey, 2012; Sabharwal et al., 2014). In addition, this passive transfer EAE model enabled us to discover that other neural activators such as weak electric stimulation or pain sensation create unique gateways for immune cells at different sites (Arima et al., 2015a and 2015b). Here, we describe detailed protocols for the passive transfer EAE model using MOG-specific CD4+ T cells, which are suitable for tracking autoreactive CD4+ T cells in vivo. Although protocols for EAE have been reported (Racke, 2001), we particularly focus on the passive transfer EAE models and describe the methods in detail. The protocols here induce a transient EAE, in which after adoptive transfer, paralyzed tail (score 1) is expected to appear around 7 days, the clinical signs peak around 10-14 days with score 2 (uneven gait) to 2.5 (one paralyzed rear leg), and then the clinical symptoms will disappear around 20-25 days (Arima et al., 2015b). In this remission phase, the mice look healthy. However, activated monocytes remain in the spinal cords, and paralysis returns upon specific neural activation including pain sensation (Arima et al., 2015b).

Materials and Reagents

  1. Three-way connector (TERUMO Medical, catalog number: TS-TR1K )
  2. 1 ml syringe (TERUMO Medical, catalog number: SS-01T )
  3. Needle (25 G x 1) (TERUMO Medical, catalog number: NN-2525R )
  4. Needle (27 G x ¾) (TERUMO Medical, catalog number: NN-2719S )
  5. Cell strainer (100 μm) (Corning, Falcon®, catalog number: 352360 )
  6. 50 ml polypropylene conical tube (Corning, Falcon®, catalog number: 352070 )
  7. 2.5 ml syringe (TERUMO Medical, catalog number: SS-02SZ )
  8. 10 cm dish (Corning, catalog number: 430167 )
  9. Needle (18 G x 1 ½) (TERUMO Medical, catalog number: NN-1838R )
  10. 96-well U-bottom plate (Corning, catalog number: 3799 )
  11. Nylon wool
  12. 20 ml syringe
  13. MACS LS columns (Miltenyi Biotec, catalog number: 130-042-401 )
  14. C57BL/6 mouse (Japan SLC)
  15. M. Tuberculosis H37 RA (BD, catalog number: 231141 )
  16. MOG peptide 35-55 (MEVGWYRSPFSRVVHLYRNGK) (Sigma-Aldrich), stock solution = 4 mg/ml
  17. Incomplete Freund’s adjuvant (IFA) (Sigma-Aldrich, catalog number: F5506 )
  18. Isoflurane (Pfizer)
  19. Pertussis toxin from Bordetella pertussis (PTx) (Sigma-Aldrich, catalog number: P7208-50UG )
  20. CD4 (L3T4) Microbeads, mouse (Miltenyi Biotec, catalog number: 130-049-201 )
  21. Saline (Otsuka Pharmaceutical Factory, catalog number: 0815 )
  22. Mouse IL-1β (BioLegend, catalog number: 575102 ), stock solution = 10 μg/ml
  23. Mouse IL-23 (BioLegend, catalog number: 589002 ), stock solution = 10 μg/ml
  24. Human IL-6 (Toray, order-made) stock solution = 100 μg/ml (Commercially available mouse IL-6 will work)
  25. CellBanker (Takara Bio, Clontech, catalog number: CB021 )
  26. Ammonium chloride (NH4Cl) (Sigma-Aldrich, catalog number: A4514 )
  27. DDW
  28. EDTA-2Na
  29. Fetal bovine serum (FBS) (GE Healthcare, HyCloneTM, catalog number: SH30910.03 )
  30. RPMI medium 1640 basic (1x) (Thermo Fisher Scientific, GibcoTM, catalog number: C11875500BT )
  31. Penicillin/streptomycin (Sigma-Aldrich, catalog number: P4333-100ML )
  32. 2-mercaptoethanol (NACALAI TESQUE, catalog number: 21417 )
  33. Iscove’s modified Dulbecco’s medium (Sigma-Aldrich, catalog number: I3390-500ML )
  34. GlutaMAX-1 (100x) (Thermo Fisher Scientific, GibcoTM, catalog number: 35050061 )
  35. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9625-5KG )
  36. Potassium chloride (KCl) (Wako Pure Chemical Industries, catalog number: 163-03545 )
  37. Sodium hydrogen phosphate (Na2HPO4) (Wako Pure Chemical Industries, catalog number: 197-02865 )
  38. Potassium dihydrogen phosphate (KH2PO4) (Wako Pure Chemical Industries, catalog number: 169-04245 )
  39. Red blood cell (RBC) lysis buffer (see Recipes)
  40. Phosphate buffered saline (PBS) (see Recipes)
  41. MACS buffer (see Recipes)
  42. RP10 medium (see Recipes)
  43. Nylon wool column (see Recipes)
  44. IM20 medium (see Recipes)

Equipment

  1. X-ray irradiator (Hitachi, model: MBR-1520R ) or equivalent
  2. Scissors (BONIMED, catalog number: 669-060-72 )
  3. Micro-dissecting scissors (Karl Hammacher, catalog number: HSB 014-11 )
  4. Angled serrated tip forceps (Karl Hammacher, catalog number: HSC 187-11 )
  5. Pipet-aid (Corning, Falcon®, catalog number: 357471 )
  6. Glass syringe (Tsubasa Industry, 5 ml, lock type), autoclaved
  7. Centrifuge (Hitachi, model: CF7D2 )
  8. Cell culture incubator, 37 °C, 5% CO2 (Panasonic Healthcare, model: MCO-175 )
  9. Multichannel pipette
  10. Water bath
  11. Autoclave

Part I. Passive transfer method with MOG-specific CD4+ T cells isolated from MOG immunized mice

Procedure

  1. MOG immunization in mice (Figure 1)


    Figure 1. Making emulsion and immunization in C57BL/6 mice. A. MOG peptide (35-55) and CFA mixed at 1:1 and emulsified; B. Tail base immunization with 200 μg/100 μl emulsion.

    1. Take sufficient volume (2 ml for 30 mice) of 4 mg/ml MOG peptide (35-55) in one glass syringe, take the same volume of CFA in the other glass syringe, and connect two syringes with a two- or three-way connector. Then, emulsify the two solutions by pushing plungers of the two syringes until the MOG/CFA solution becomes white, uniform emulsion (about 50 strokes).
      Note: CFA is prepared by mixing 10 ml IFA and 2 vials of 10 mg M. Tuberculosis H37 RA.
    2. Move all the emulsion to one-side of the glass syringe, unlock the other empty glass syringe, and attach a new disposable 1-ml syringe to the open side of the connector.
    3. Load the emulsion to the 1-ml syringe, and put a 25 G x 1 needle for immunization.
    4. Anesthetize a mouse with isoflurane. It is easier for injections if the body of the mouse is fixed in a restrainer (optional). Intravenously administer (i.v.) 200 ng/200 μl PTx to the tail vein of C57BL/6 mice (6-8 weeks old) with 27 G x ¾ needle, followed by immunization by subcutaneous administration (s.c.) of 200 μg/100 μl MOG peptide emulsified in CFA at tail base on day 0. Perform the PTx injection and MOG immunization on the same day.
    5. Inject i.v. 200 ng/200 μl PTx on days 2 and 7.

  2. Separation of CD4+ T cells from MOG immunized mice by using CD4 Microbeads.
    1. Collect spleens from MOG immunized mice (30 mice) on day 9 or 10.
    2. Homogenize the spleens on a cell strainer attached to a 50-ml tube using a plunger from a 2.5 ml syringe (7-8 spleens/tube, total four strainers and four 50-ml tubes are used for 30 mice). Add plain RPMI medium up to 50 ml during homogenization.
    3. Centrifuge the tubes (600 x g, 5 min, 4 °C) and aspirate the supernatant.
    4. Resuspend the cell pellet in 10 ml/tube (4 tubes if 30 mice are used) of RBC lysis buffer (see Recipe 1) and incubate on ice for about 1 min.
    5. Add plain RPMI medium up to 50 ml/tube.
    6. Centrifuge the tubes (600 x g, 5 min, 4 °C) and aspirate the supernatant.
    7. Resuspend the pellet in 1 ml/tube of MACS buffer (see Recipe 2).
    8. Add 100 μl/tube of CD4 MACS beads and incubate on ice for 30 min.
    9. Add plain RPMI medium up to 50 ml/tube.
    10. Centrifuge the tubes (600 x g, 5 min, 4 °C) and aspirate the supernatant.
    11. Resuspend the pellet in 5 ml/tube MACS buffer (4 tubes if 30 mice are used). Use the same number of MACS LS columns as that of 50-ml tubes used. Apply the cell suspension to MACS LS columns (5 ml/column). Refer to the manufacturer guide for the use of MACS LS columns.
    12. Wash the column with 3 ml/column of MACS buffer twice.
      Note: Do not discard the flow-through cells, which are used as antigen-presenting cells.
    13. Elute the CD4 positive selected cells with 5 ml/column MACS buffer and pool the eluted fractions to a 50 ml tube.
    14. Centrifuge the tubes (600 x g, 5 min, 4 °C) and aspirate the supernatant.
    15. Resuspend the CD4+ T cells in 20 ml RP10 medium (see Recipe 3) and adjust to 8 x 106 cells/ml.

  3. Irradiation of splenocytes
    1. Collect the flow-through cells obtained in the STEP 12 of the previous section.
    2. Centrifuge the flow-through cells depleted of CD4+ T cells (600 x g, 5 min, 4 °C) and aspirate the supernatant.
    3. Resuspend the cells in 10 ml of RP10 medium.
    4. Irradiate the cells at 35 Gy, centrifuge (600 x g, 5 min, 4 °C) and aspirate the supernatant.
    5. Resuspend the cells in 20 ml of RP10 medium and adjust to 2 x 107 cells/ml.

  4. In vitro restimulation and adoptive transfer
    1. Co-culture 4 x 107 cells CD4+ T cells and 1 x 108 irradiated splenocytes in 10 ml RP10 medium in a 10-cm dish containing 2 ng/ml IL-23 and 25 μg/ml MOG peptide for 2 days.
    2. Add 10 ml RP10 medium in each dish on day 1.
    3. Collect the cells by pipetting up and down on day 2, centrifuge (600 x g, 5 min, 4 °C) and aspirate the supernatant.
    4. Resuspend the cells in 10 ml RP10 medium pre-warmed at 37 °C and add the suspension to a nylon wool column (see Recipe 4) (Video 1).
      Note: Put an 18 G x 1 ½ inches needle to a nylon wool column, and stand the column in a 50 ml tube without a cap. To equilibrate the column, add 20 ml RP10 medium pre-warmed at 37 °C until the medium spreads uniformly in nylon wool. Incubate the nylon wool column with the co-cultured cells in a CO2 incubator (37 °C, 20 min). After 20 min, elute the cells with 30 ml RP10 medium.

      Video 1. Making a nylon wool column. 20 ml of RP10 medium are added to nylon wool and stirred until the medium uniformly spreads in the wool.

    5. Centrifuge the eluted cells (600 x g, 5 min, 4 °C) and aspirate the supernatant.
    6. Resuspend the pellets in 1 ml MACS buffer.
    7. Add 100 μl/tube of CD4 MACS beads and incubate on ice for 30 min.
    8. Add plain RPMI medium up to 50 ml/tube.
    9. Centrifuge the tubes (600 x g, 5 min, 4 °C) and aspirate the supernatant.
    10. Resuspend the pellet in 10 ml/tube MACS buffer and apply the suspensions to two MACS columns (5 ml/column).
    11. Wash with 3 ml/column MACS buffer twice.
    12. Elute the CD4 positive selected cells with 5 ml/column MACS buffer and pool the eluted fractions to a 50 ml tube.
    13. Centrifuge the tube (600 x g, 5 min, 4 °C) and aspirate the supernatant.
    14. Resuspend the eluted CD4+ T cells in sterile saline.
      Note: Resuspended volume should not exceed the maximal volume of i.v. injection allowed in the ethics guideline of your institute.
    15. Inject 1.5 x 107 cells/mouse, i.v.
      Note: Usually, 6-8 mice can be injected using 30 spleens.
    16. Measure clinical scores as described previously (Ogura et al., 2008; Huseby et al., 2001; Arima et al., 2012 and 2015b).

Part II. Passive transfer model with MOG-specific T cells lines

Passive transfer EAE can also be induced using a MOG-specific T cell line generated by periodical antigen stimulations in vitro. Once the T cell line is established, this method is useful to reduce the number of mice used to induce the transfer EAE.

Procedure

  1. MOG immunization
    1. Perform MOG immunization in the same way as Part I. A. MOG immunization in mice, except for the number of mice to be immunized. Usually, 3-4 mice are sufficient to obtain MOG-specific T cells lines.

  2. Culture and passage of T cell lines
    1. Collect inguinal lymph nodes (iLNs) from MOG immunized mice (3-4 mice) on day 9 or 10 (Figure 2).


      Figure 2. The location of the inguinal lymph nodes (iLN). iLN of the immunized C57BL/6 mouse.

    2. Homogenize the iLNs on a cell strainer in a 50 ml tube using the plunger from a 2.5 ml syringe.
    3. Centrifuge the tubes (600 x g, 5 min, 4 °C) and aspirate the supernatant.
    4. Resuspend the cell pellet in 10 ml/tube RBC lysis buffer (see Recipe 1) and incubate on ice for 1 min.
    5. Add plain RPMI medium up to 50 ml/tube.
    6. Centrifuge the tubes (600 x g, 5 min, 4 °C) and aspirate the supernatant.
    7. Resuspend the cells in 2 ml IM20 medium (see Recipe 5).
    8. Filter the cells using a cell strainer.
    9. Count the cells.
    10. Seed 2.5 x 105 cells/well/200 μl iLN cells in the presence of 4 μg/ml MOG peptide, 0.5 ng/ml IL-1β, 5 ng/ml IL-6 and 0.5 ng/ml IL-23 in 96-well U-bottom plates for 10 to 14 days.
    11. Proceed to step C1.

  3. T cell preparation
    1. Collect CD4+ T-cell-rich iLN cells from 96U plates in a 10-cm dish by gently pipetting 2 to 3 times using a multichannel pipette.
    2. Collect the cells into 50 ml tubes and centrifuge (600 x g, 5 min, 4 °C).
    3. Aspirate the supernatant and resuspend the T cells in 5 ml IM20 medium.
    4. Count the cells and adjust the concentration to 2.5 x 105 cells/ml.

  4. Irradiation of splenocytes
    1. Collect the spleens of naive C57BL/6 mice and homogenize using the plunger from a 2.5 ml syringe.
    2. Add plain RPMI medium up to 50 ml/tube and centrifuge (600 x g, 5 min, 4 °C).
    3. Aspirate the supernatant and add 1 ml/spleen RBC lysis buffer.
    4. Add plain RPMI medium up to 50 ml and centrifuge (600 x g, 5 min, 4 °C).
    5. Irradiate the cells at 35 Gy, centrifuge (600 x g, 5 min, 4 °C) and aspirate the supernatant.
    6. Suspend the irradiated splenocytes in 10 ml IM20 medium, count and adjust the concentration to 2.5 x 106 cells/ml.

  5. In vitro stimulation
    1. Mix 2.5 x 105 cells/ml CD4+ T-cell-rich iLN cells and 2.5 x 106 cells/ml irradiated splenocytes at 1:1.
    2. Seed the cells at 200 μl/well containing 4 μg/ml MOG peptide, 0.5 ng/ml IL-1β, 5 ng/ml IL-6 and 0.5 ng/ml IL-23 in 96-well U-bottom plates.
    3. Incubate the co-cultured cells at 37 °C, 5% CO2 for 10-14 days.
    4. Repeat steps C1 to E3 to enrich the percentage of MOG-specific CD4+ T cells from total iLN cells (Figure 3). Use fresh irradiated splenocytes in each repeating cycle.
      Notes:
      1. T cells can be frozen after step E3 in CellBanker or equivalent solution at -80 °C. When thawing, use a 37 °C water bath, and wash the cells in plain RPMI medium. Then, the T cell line can be used for culture (start from step E1).
      2. Typically, more than 95% of live cells will be CD4+ T cells after four rounds of in vitro stimulation (Figure 3).


        Figure 3. Representative FACS plots of a T cell line. Most living cells (> 95%) are CD4+ T cells (CD19- T cell receptor (TCR)β+CD90.2+CD4+ T cells) after 5 days of the fourth round of in vitro stimulation (Procedure E). Irradiated splenocytes can be seen as forward scatter (FSC)low, side scatter (SSC)low dead cells.

  6. Adoptive transfer of T cell lines
    1. Set up culture the same way as ‘E. In vitro stimulation’.
      Note: Do not forget to add 4 μg/ml MOG peptide, 0.5 ng/ml IL-1β, 5 ng/ml IL-6 and 0.5 ng/ml IL-23.
    2. Seed these cells at 200 μl/well in 96-well U-bottom plates (about 5 plates for 1 mouse).
    3. Incubate the co-cultured cells in 37 °C, 5% CO2 for 2 days.
    4. Collect the cells from the 96-well U-bottom plates in a 10-cm dish by gently pipetting 2 to 3 times with a multichannel pipette.
    5. Collect the cells in 50 ml tubes and centrifuge (600 x g, 5 min, 4 °C).
    6. Aspirate the medium and resuspend the cells in 1 ml plain RPMI medium.
    7. Count the cells and adjust the concentration to 2.5-3.75 x 107/ml.
      Note: Count only living cells.
    8. Inject 1-1.5 x 107 cells i.v. into C57BL/6 mice.
    9. Measure clinical scores as described previously (Huseby et al., 2001; Ogura et al., 2008; Arima et al., 2012 and 2015b).

Data analysis

The clinical symptoms of EAE are evaluated as follows: grade 1, paralyzed tail; grade 2, uneven gait; grade 2.5, one paralyzed rear leg; grade 3, rear limb paralysis; grade 4, paralyzed front and rear legs; and grade 5, moribund (Huseby et al., 2001; Ogura et al., 2008; Arima et al., 2012 and 2015b).

Recipes

  1. Red blood cell (RBC) lysis buffer (500 ml)
    4.41 g NH4Cl
    500 ml DDW, then autoclave
  2. Phosphate buffered saline (PBS)
    1.46 g NaCl
    1.86 g KCl
    3.5 g Na2HPO4
    3.4 g KH2PO4
  3. MACS buffer (1,000 ml)
    950 ml PBS
    1.86 g EDTA-2Na
    Add 50 ml heat-inactivated FBS after autoclaving
  4. RP10 medium (500 ml)
    500 ml RPMI medium 1640 basic
    50 ml heat-inactivated FBS
    5 ml 100x penicillin/streptomycin
    1.86 μl 2-mercaptoethanol
  5. Nylon wool column
    Nylon wool (1.2 g) is unraveled with a brush, put into a 20 ml syringe, and sterilized by autoclave
  6. IM20 medium (600 ml)
    500 ml Iscove’s modified Dulbecco’s medium
    100 ml FBS
    5 ml GlutaMAX-1 (100x)
    1.8 μl 2-mercaptoethanol
    5 ml 100x penicillin/streptomycin

Acknowledgments

We appreciate the excellent technical assistance provided by Ms. Ezawa, and Ms. Nakayama, and thank Ms. Fukumoto for her excellent secretarial assistance. We thank Dr. P. Karagiannis (CiRA, Kyoto University, Kyoto, Japan) for carefully reading the manuscript and important discussion. The protocol Part I was used in our previous reports (Arima, 2012 and 2015b; Mori, 2014). This work was supported by KAKENHI (D. K., Y. A., T. A., and M. M.), Takeda Science Foundation (M. M.), Institute for Fermentation Osaka (M. M.), Mitsubishi Foundation (M. M.), Mochida Memorial Foundation for Medical and Pharmaceutical Research (D. K.), Suzuken Memorial Foundation (Y. A.), Japan Prize Foundation (Y. A.), Ono Medical Research Foundation (Y. A.), Kanzawa Medical Research Foundation (Y. A.), Kishimoto Foundation (Y. A.), Nagao Takeshi Research Foundation (Y. A.), Japan Multiple Sclerosis Society (Y. A.), Kanae Foundation (Y. A.), Tokyo Medical Research Foundation (M. M. and Y. A.), Uehara Memorial Foundation (Y. A.), Japan Brain Foundation (Y. A.), Kao Foundation(Y. A.), Nagao Memorial Fund(Y. T.), Suzuken Memorial Foundation (D. K.), Suhara Memorial Foundation (D. K.), Yasuda Memorial Foundation (D. K.), and Novartis Pharma Research Grants (D. K.).

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简介

实验性自身免疫性脑脊髓炎(EAE)是多发性硬化症(MS)的动物模型,其是中枢神经系统(CNS)的慢性炎性疾病。其特征在于由髓磷脂特异性自身反应性CD4 + T细胞介导的局灶性脱髓鞘和炎症反应。在小鼠中使用EAE的被动转移模型,我们已经证明,通过感觉交感通信的区域特异性神经信号在CNS的特定血管上产生免疫细胞的网关,这被称为网关反射(Arima等, ,2012; Tracey,2012; Arima等人,2013; Sabharwal等人,2014; Arima等人。 >,2015b)。在这里,我们描述了使用新鲜分离的(MOG)特异性CD4 + T细胞或周期性再刺激的MOG特异性CD4 + T细胞系的EAE的被动转移模型的方案,其是适用于体内追踪致病性CD4 T细胞,特别是CNS(Ogura等人,2008; Arima等人) 。,2012和2015b)。
【背景】广泛接受的是,自身反应性CD4 + T细胞在MS和EAE的发病机理中起重要作用(Reboldi,2009; International Multiple Sclerosis Genetics,et al。,2011; Steinman,2014),它们是CNS的慢性炎性疾病。 CNS受到血脑屏障(BBB)的保护,其限制了外周免疫细胞浸润(Liu et al。,2012)。直到最近,CD4 + T细胞从外周血进入CNS的方式和方式尚不清楚。尽管EAE可以通过在完全弗氏佐剂(CFA)和百日咳毒素(PTx)(Andreasen等人,2009; Lu等人)中乳化的CNS-自身抗原免疫动物诱导, ,2016),通过注射可能影响BBB完整性的CFA和PTx发生不期望的全身炎症(Schellenberg等人,2012; Marbourg等人, ,2017)。或者,我们建议使用被动转移EAE模型,其中将特异于MOG的活化的CD4 + T细胞注射到未经初步的小鼠中,而不用CFA或PTx进行处理。使用这种被动转移EAE模型,我们已经确定了第五条腰椎(L5)线的背侧血管,作为自体反应性CD4 + T细胞到达CNS的初始途径(Arima等, ,2012)。机械地,重力介导的比目鱼肌中的感觉神经元的恒定活化诱导与L5背侧血管连接的交感神经活化。血管形成的去甲肾上腺素分泌增强NF-κB活性,导致募集CNS自身反应性CD4 + T细胞的趋化因子(Arima等人,2012) 。这种通过比目鱼肌的反重力反应驱动的感觉交感交流称为“重力网关反射”(Arima et al。,2012; Tracey,2012; Sabharwal等人, ,2014)。此外,这种被动转移EAE模型使我们能够发现其他神经激活因子,如电刺激或疼痛感觉,为不同部位的免疫细胞创造独特的网关(Arima等,2015a和2015b) 。在这里,我们描述了使用MOG特异性CD4 + T细胞的被动转移EAE模型的详细方案,其适合于在体内跟踪自体CD4 + sup + T细胞。虽然已经报告了EAE协议(Racke,2001),但我们特别关注被动传输EAE模型,并详细描述了这些方法。这里的协议诱导暂时性EAE,其中在过继转移后,瘫痪尾巴(1分)预计出现约7天,临床症状在10-14天达到峰值,得分2(步态不均)至2.5(一个瘫痪后腿),然后临床症状将在20-25天左右消失(Arima等人,2015b)。在这个缓解阶段,老鼠看起来很健康。然而,活化的单核细胞保留在脊髓中,并且麻痹在包括疼痛感觉的特定神经激活(Arima等人,2015b)时返回。

关键字:实验性自身免疫性脑脊髓炎, 致病性CD4+ T细胞, 髓鞘少突胶质细胞糖蛋白, 通路反射, 被动转移

材料和试剂

  1. 三通连接器(TERUMO Medical,目录号:TS-TR1K)
  2. 1 ml注射器(TERUMO Medical,目录号:SS-01T)
  3. 针(25 G x 1)(TERUMO医疗,目录号:NN-2525R)
  4. 针(27 G x¾)(TERUMO医疗,目录号:NN-2719S)
  5. 细胞过滤器(100μm)(Corning,Falcon ®,目录号:352360)
  6. 50ml聚丙烯锥形管(Corning,Falcon ®,目录号:352070)
  7. 2.5 ml注射器(TERUMO Medical,目录号:SS-02SZ)
  8. 10厘米盘(康宁,目录号:430167)
  9. 针(18 G x 1½)(TERUMO医疗,目录号:NN-1838R)
  10. 96孔U型底板(Corning,目录号:3799)
  11. 尼龙羊毛
  12. 20毫升注射器
  13. MACS LS列(Miltenyi Biotec,目录号:130-042-401)
  14. C57BL / 6小鼠(日本SLC)
  15. 结核分枝杆菌H37 RA(BD,目录号:231141)
  16. MOG肽35-55(MEVGWYRSPFSRVVLYRNGK)(Sigma-Aldrich),储液= 4mg / ml
  17. 不完全弗氏佐剂(IFA)(Sigma-Aldrich,目录号:F5506)
  18. 异氟烷(辉瑞)
  19. 百日咳博德特氏菌的百日咳毒素(PTx)(Sigma-Aldrich,目录号:P7208-50UG)
  20. CD4(L3T4)微珠,小鼠(Miltenyi Biotec,目录号:130-049-201)
  21. 盐水(大冢制药厂,目录号:0815)
  22. 小鼠IL-1β(BioLegend,目录号:575102),储液=10μg/ ml
  23. 小鼠IL-23(BioLegend,目录号:589002),储液=10μg/ ml
  24. 人IL-6(东丽,订购)储液=100μg/ ml(市售的小鼠IL-6将起作用)
  25. CellBanker(Takara Bio,Clontech,目录号:CB021)
  26. 氯化铵(NH 4 Cl)(Sigma-Aldrich,目录号:A4514)
  27. DDW
  28. EDTA-2Na
  29. 胎牛血清(FBS)(GE Healthcare,HyClone TM,目录号:SH30910.03)
  30. RPMI培养基1640碱性(1x)(Thermo Fisher Scientific,Gibco TM,目录号:C11875500BT)
  31. 青霉素/链霉素(Sigma-Aldrich,目录号:P4333-100ML)
  32. 2-巯基乙醇(NACALAI TESQUE,目录号:21417)
  33. Iscove改良的Dulbecco's培养基(Sigma-Aldrich,目录号:I3390-500ML)
  34. GlutaMAX-1(100x)(Thermo Fisher Scientific,Gibco TM,目录号:35050061)
  35. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S9625-5KG)
  36. 氯化钾(KCl)(Wako Pure Chemical Industries,目录号:163-03545)
  37. 磷酸氢钠(Na 2 HPO 4)(和光纯药,目录号:197-02865)
  38. 磷酸二氢钾(KH 2 PO 4)(Wako Pure Chemical Industries,目录号:169-04245)
  39. 红细胞(RBC)裂解缓冲液(参见食谱)
  40. 磷酸盐缓冲盐水(PBS)(见食谱)
  41. MACS缓冲区(请参阅配方)
  42. RP10培养基(参见食谱)
  43. 尼龙羊毛柱(见食谱)
  44. IM20培养基(见食谱)

设备

  1. X射线照相机(日立,型号:MBR-1520R)或等效物
  2. 剪刀(BONIMED,目录号:669-060-72)
  3. 微解剖剪刀(Karl Hammacher,目录号:HSB 014-11)
  4. 斜角锯齿镊子(Karl Hammacher,目录号:HSC 187-11)
  5. 吸管(康宁,Falcon ®,目录号:357471)
  6. 玻璃注射器(Tsubasa工业,5ml,锁式),高压灭菌
  7. 离心机(日立,型号:CF7D2)
  8. 细胞培养箱37℃,5%CO 2(Panasonic Healthcare,型号:MCO-175)
  9. 多通道移液器
  10. 水浴
  11. 高压灭菌器

第一部分从MOG免疫小鼠分离的具有MOG特异性CD4 + T细胞的被动转移方法

实验流程

  1. 小鼠中的MOG免疫(图1)


    图1.在C57BL / 6小鼠中进行乳液和免疫。:一种。 MOG肽(35-55)和CFA以1:1混合并乳化; B.用200μg/100μl乳剂进行尾巴免疫。

    1. 在一个玻璃注射器中,取4mg / ml MOG肽(35-55)的足够体积(30ml小鼠2ml),在另一玻璃注射器中取相同体积的CFA,并将两个或三个方式连接器。然后,通过推动两个注射器的柱塞乳化两种溶液,直到MOG / CFA溶液变成白色,均匀的乳液(约50次)。
      注意:CFA是通过混合10ml IFA和2瓶10 mg结核分枝杆菌H37 RA。
    2. 将所有乳液移动到玻璃注射器的一侧,解开另一个空的玻璃注射器,并将新的一次性1 ml注射器连接到连接器的开口侧。
    3. 将乳液装入1 ml注射器,放入25 G x 1针进行免疫
    4. 用异氟烷麻醉一只老鼠。如果鼠标的主体固定在限制器(可选)中,则更容易进行注射。静脉给予(iv)200ng /200μlPTx到具有27G×3针的C57BL / 6小鼠(6-8周龄)的尾静脉,随后通过皮下给药(sc)200μg/100μlMOG进行免疫肽在第0天在尾巴的CFA中乳化。在同一天进行PTx注射和MOG免疫。
    5. 注射i.v在第2天和第7天200ng /200μlPTx。

  2. 通过使用CD4 Microbeads从MOG免疫的小鼠中分离CD4 + T细胞.
    1. 在第9天或第10天从MOG免疫的小鼠(30只小鼠)收集脾脏
    2. 使用来自2.5ml注射器的柱塞(7-8脾/管,总共四个过滤器和四个50ml管用于30只小鼠)将附着在50ml管的细胞过滤器上的脾匀化。在均质化过程中加入高达50ml的普通RPMI培养基。
    3. 离心管(600 x g,5分钟,4°C)并吸出上清液。
    4. 将细胞沉淀重悬于10ml /管(使用30只小鼠的4管)RBC裂解缓冲液(参见方法1),并在冰上孵育约1分钟。
    5. 加入普通RPMI培养基至50ml /管。
    6. 离心管(600 x g,5分钟,4°C)并吸出上清液。
    7. 将沉淀重悬于1ml /管的MACS缓冲液中(参见方法2)
    8. 加入100μl/管的CD4 MACS珠,并在冰上孵育30分钟
    9. 加入普通RPMI培养基至50ml /管。
    10. 离心管(600 x g,5分钟,4°C)并吸出上清液。
    11. 将沉淀物重悬于5ml /管MACS缓冲液(4只,如果使用30只小鼠)。使用与使用的50ml管相同数量的MACS LS色谱柱。将细胞悬浮液应用于MACS LS柱(5ml /柱)。有关使用MACS LS色谱柱的信息,请参阅制造商指南。
    12. 用3ml /柱的MACS缓冲液洗涤柱子两次。
      注意:不要丢弃用作抗原呈递细胞的流通细胞。
    13. 用5ml /柱MACS缓冲液洗脱CD4阳性选择的细胞,并将洗脱的级分汇集到50ml管中
    14. 离心管(600 x g,5分钟,4°C)并吸出上清液。
    15. 将CD4细胞重悬于20 ml RP10培养基(见方案3),并调整至8×10 6个细胞/ ml。

  3. 脾细胞照射
    1. 收集上一节STEP 12中获得的流通池
    2. 离心除去CD4 + sup细胞(600 x g,5分钟,4°C)的流通细胞,并吸出上清液。
    3. 将细胞重悬于10ml RP10培养基中
    4. 以35Gy,离心(600×g,5分钟,4℃)照射细胞并吸出上清液。
    5. 将细胞重悬于20ml RP10培养基中并调节至2×10 7个细胞/ ml。

  4. 再次进行体外复制和过继转移
    1. 共培养4×10 7细胞CD4 + sup + T细胞和1×10 8个照射的脾细胞在10ml 10cm的RP10培养基含有2ng / ml IL-23和25μg/ ml MOG肽的培养皿2天
    2. 在第1天在每个盘中加入10ml RP10培养基。
    3. 通过在第2天上下移液收集细胞,离心(600 x g,5分钟,4°C)并吸出上清液。
    4. 将细胞重悬在10ml在37℃预温热的RP10培养基中,并将悬浮液加入尼龙羊毛柱(参见方案4)(视频1)。
      注意:将18 G x 1½英寸的针头插入尼龙羊毛柱,并将柱子放在一个没有盖子的50ml管中。为了平衡柱,加入在37℃下预温热的20ml RP10培养基,直到培养基均匀地铺展在尼龙羊毛中。将培养的母细胞与尼龙羊毛柱孵育在CO 培养箱(37℃,20分钟)中。 20分钟后,用30ml RP10培养基洗脱细胞。

      Video 1. Making a nylon wool column. 20 ml of RP10 medium are added to nylon wool and stirred until the medium uniformly spreads in the wool.

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player


    5. 共培养4×10 7细胞CD4 + T细胞和1×10 8个照射的脾细胞在10ml 10cm的RP10培养基含有2ng / ml IL-23和25μg/ ml MOGept的培养皿2天
    6. 在第1天在每个盘中加入10ml RP10培养基。
    7. 通过在第2天上下移液收集细胞,离心(600 x g,5分钟,4°C)并吸出上清液。
    8. 将细胞重悬在10ml在37℃预温热的RP10培养基中,并将悬浮液加入尼龙羊毛柱(参见方案4)(视频1)。
      注意:将18 G x1½英寸的针头插入尼龙羊毛柱,并将柱子放在一个没有盖子的50ml管中。为了平衡柱,加入在37℃下预温热的20ml RP10培养基,直到培养基均匀 地毯展在尼龙羊毛中。将培养的母细胞与尼龙羊毛柱孵育在CO培养箱(37℃,20分钟)中20分钟后,用30ml RP10培养基洗脱细胞。

Part II. Passive transfer model with MOG-specific T cells lines

Passive transfer EAE can also be induced using a MOG-specific T cell line generated by periodical antigen stimulations in vitro. Once the T cell line is established, this method is useful to reduce the number of mice used to induce the transfer EAE.

Procedure

  1. MOG immunization
    1. Perform MOG immunization in the same way as Part I. A. MOG immunization in mice, except for the number of mice to be immunized. Usually, 3-4 mice are sufficient to obtain MOG-specific T cells lines.

  2. T细胞系的培养和通过
    1. 在第9天或第10天从MOG免疫小鼠(3-4只小鼠)收集腹股沟淋巴结(iLN)(图 2).


      图2.腹股沟淋巴结(iLN)的位置。 免疫的C57BL / 6小鼠的iLN.

    2. 使用来自2.5ml注射器的柱塞在50ml管中将细胞过滤器上的iLN均质化。
    3. 离心管(600 x g,5分钟,4°C)并吸出上清液。
    4. 将细胞沉淀重悬于10ml /管RBC裂解缓冲液(参见方案1),并在冰上孵育1分钟。
    5. 加入普通RPMI培养基至50ml /管。
    6. 离心管(600 x g,5分钟,4°C)并吸出上清液。
    7. 将细胞重悬于2ml IM20培养基中(见方案5)
    8. 使用细胞过滤器过滤细胞。
    9. 计数细胞。
    10. 在4μg/ ml MOG肽,0.5ng / ml IL-1β,5ng / ml IL-6和0.5ng / ml的MOG肽存在下,种子2.5×10 5个细胞/孔/200μliLN细胞, ml IL-23在96孔U底板中10至14天
    11. 继续步骤C1。

  3. T细胞准备
    1. 通过使用多通道移液管轻轻吸取2〜3次,从10 cm的培养皿中取出96U板中的CD4 + T细胞富集的iLN细胞。
    2. 将细胞收集到50ml管中并离心(600×g,5分钟,4℃)。
    3. 吸出上清液并将T细胞重悬于5ml IM20培养基中
    4. 计数细胞并调整浓度至2.5×10 5细胞/ ml。

  4. 脾细胞照射
    1. 收集天然C57BL / 6小鼠的脾脏,并使用2.5ml注射器的柱塞匀浆。
    2. 加入高达50ml / ml的普通RPMI培养基,离心(600 x g,5分钟,4℃)。
    3. 吸出上清液,加入1ml /脾的RBC裂解缓冲液
    4. 加入高达50ml的普通RPMI培养基,离心(600 x g,5分钟,4℃)。
    5. 以35Gy,离心(600×g,5分钟,4℃)照射细胞并吸出上清液。
    6. 将照射的脾细胞悬浮在10ml IM20培养基中,计数并将浓度调节至2.5×10 6细胞/ ml。

  5. 体外刺激
    1. 将2.5×10 5个细胞/ ml CD4 +细胞富集的iLN细胞和2.5×10 6个细胞/ ml照射的脾细胞在1 :1。
    2. 在96孔U底板中含有4μg/ ml MOG肽,0.5ng / ml IL-1β,5ng / ml IL-6和0.5ng / ml IL-23的200μl/孔的细胞接种细胞。
    3. 将共培养细胞在37℃,5%CO 2孵育10-14天。
    4. 重复步骤C1至E3以丰富来自总iLN细胞的MOG特异性CD4 +细胞的百分比(图3)。 在每个重复循环中使用新鲜辐射的脾细胞。
      笔记:
      1. T细胞可以在CellBanker的步骤E3或-80℃的等效溶液中冷冻。 解冻时,使用37°C水浴,并在普通RPMI培养基中清洗细胞。 然后,T细胞系可用于培养(从步骤E1开始)。
      2. 通常,超过95%的活细胞将是四轮体外刺激后的CD4细胞(图3),CD4细胞是CD4 +


        图3.T细胞系的代表性FACS图。大多数活细胞(> 95%)是CD4细胞T细胞(CD19 T细胞受体 (TCR)β + > CD90.2 + CD4 + T细胞)第四轮体外5天后, (程序E)。 照射的脾细胞可以看作是前向散射(FSC)低,侧向散射(SSC)低死细胞。

  6. 过继转移T细胞系
    1. 以“E.”的方式设定文化。 体外刺激“ 注意:不要忘记加入4μg/ ml MOG肽,0.5ng / ml IL-1β,5ng / ml IL-6和0.5ng / ml IL-23。
    2. 将这些细胞在96孔U底板(约5板1只小鼠)中以200μl/孔种子
    3. 将共培养细胞在37℃,5%CO 2中孵育2天。
    4. 通过使用多通道移液管轻轻吸取2〜3次,从10厘米盘中的96孔U型底板中收集细胞。
    5. 将细胞收集在50ml管中并离心(600×g,5分钟,4℃)。
    6. 吸出培养基并将细胞重悬于1ml纯RPMI培养基中
    7. 计数细胞并将浓度调节至2.5-3.75×10 7 / ml / ml 注意:只计数活细胞。
    8. 注入1-1.5 x 10 7 cell i.v.进入C57BL / 6小鼠
    9. 测量如前所述的临床评分(Huseby等人,2001; Ogura等人,2008; Arima等人,2012和2015b )

数据分析

EAE的临床症状评估如下:1级,瘫痪尾巴; 2级,步态不均匀; 2.5级,瘫痪后腿; 3级,后肢麻痹; 4级,瘫痪前后腿; 和等级5,濒死(Huseby等人,2001; Ogura等人,2008; Arima等人,2012和2015b)。

配方

  1. 红细胞(RBC)裂解缓冲液(500 ml)
    4.41g NH 4 Cl
    500毫升DDW,然后高压灭菌
  2. 磷酸盐缓冲盐水(PBS)
    1.46克NaCl
    1.86克KCl
    3.5g Na 2 HPO 4
    3.4g KH 2 PO 4
  3. MACS缓冲区(1,000毫升)
    950毫升PBS
    1.86g EDTA-2Na
    高压灭菌后加入50 ml热灭活的FBS
  4. RP10培养基(500ml)
    500毫升RPMI培养基1640基本的
    50ml热灭活FBS
    5 ml 100x青霉素/链霉素
    1.86μl2-巯基乙醇
  5. 尼龙羊毛柱
    将尼龙羊毛(1.2克)用刷子拆开,放入20ml注射器中,并用高压灭菌器灭菌
  6. IM20培养基(600毫升)
    500毫升Iscove修改后的Dulbecco的培养基 100毫升FBS
    5 ml GlutaMAX-1(100x)
    1.8微升2-巯基乙醇
    5 ml 100x青霉素/链霉素

致谢

我们感谢泽泽女士和中山女士提供的出色的技术援助,并感谢富康女士提供了出色的秘书协助。感谢P. Karagiannis博士(日本京都京都大学CiRA)仔细阅读手稿和重要讨论。我们以前的报告(Arima,2012和2015b; Mori,2014)中使用了协议第一部分。这项工作得到了KAKENHI(DK,YA,TA和MM),武田科学基金会(MM),大阪发酵研究所(MM),三菱基金会(MM),魔田纪念基金会医药研究(DK))的支持,铃鹿纪念基金会(YA),日本奖基金会(YA),大野医学研究基金会(YA),Kanzawa医学研究基金会(YA),本岛基金会(YA),日本多发性硬化症协会),Kanae基金会(YA),东京医学研究基金会(MM和YA),上原纪念基金会(YA),日本大脑基金会(YA),高雄基金会(Y。 A.),Nagao纪念基金(Y。铃鹿纪念基金会(D.K。),Suhara纪念基金会(D.K。),Yasuda纪念基金会(D.K。)和Novartis Pharma Research Grants(D.K。)。

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Copyright Tanaka et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Tanaka, Y., Arima, Y., Higuchi, K., Ohki, T., Elfeky, M., Ota, M., Kamimura, D. and Murakami, M. (2017). EAE Induction by Passive Transfer of MOG-specific CD4+ T Cells. Bio-protocol 7(13): e2370. DOI: 10.21769/BioProtoc.2370.
  2. Arima, Y., Kamimura, D., Atsumi, T., Harada, M., Kawamoto, T., Nishikawa, N., Stofkova, A., Ohki, T., Higuchi, K., Morimoto, Y., Wieghofer, P., Okada, Y., Mori, Y., Sakoda, S., Saika, S., Yoshioka, Y., Komuro, I., Yamashita, T., Hirano, T., Prinz, M. and Murakami, M. (2015b). A pain-mediated neural signal induces relapse in murine autoimmune encephalomyelitis, a multiple sclerosis model. Elife 4.
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