Inducing Paralysis in a Mouse Model via Transfer of Aquaporin-4-Specific Th17 Cells

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.      

C57BL/6 (H-2^b) female mice, 8 weeks of age, were purchased and C57BL/6 AQP4^-/- mice were provided by A. Verkman.

1. Immunization of Mice with AQP4 Peptides

1. Prepare complete Freund's adjuvant (CFA) working stock.​
   1. Finely grind a desiccated preparation of Mycobacterium tuberculosis (H37Ra) using a mortar and pestle.
   2. Add ground H37Ra to incomplete Freund's adjuvant to a final concentration of 4 mg/mL. CFA can be stored at 4 °C for up to 6 months.
2. Dissolve lyophilized antigen (Ag) AQP4 peptide in phosphate buffered saline (PBS) to a final concentration of 1 mg/mL. Maintain at 4 °C or on ice.
3. Prepare the assembly of 2 luer-lock syringes, joined with a stopcock, containing the CFA/peptide emulsion.
   1. Attach a glass luer-lock syringe without plunger to a 3-way nylon stopcock with 2 male luer-lock connections. Close the stopcock by switching the lever towards the syringe. Position the open end of the syringe up, allowing the syringe to act as a test tube. Place this in a tube rack for added stability.
   2. Vortex the CFA working stock and the Ag solution. Add a 1:1 volume of peptide / CFA to the open syringe. 200 µL of CFA/Ag (4 x 50 µL) is required per mouse.
      NOTE: Typically, about 1 mL of preparation is lost in the stopcock, which will reduce the amount available for immunization. Therefore, it is important to incorporate this into the calculation of total volume required.
      Example: For immunization of 5 mice: (200 µL/animal x 5 animals) + 1 mL extra volume = 2 mL total CFA/Ag required.
   3. Insert the glass plunger into the syringe and while holding it in place, invert the syringe so that the stopcock is oriented upward. Switch the stopcock lever to the unused female connection. Carefully apply pressure to the glass plunger in order to remove excess air from the syringe. The liquid fills the second male luer-lock connection of the stopcock.
   4. Attach a second glass syringe (plunger fully inserted) to the second male luer-lock connection of the stopcock. This should be a closed system of 2 glass syringes connected with a stopcock containing as little excess air as possible.
   5. To create an emulsion, mix by pushing the plungers to pass the liquid alternately between the 2 syringes for about 2 min. Chill the syringes on ice for about 10 min. Repeat the chilling and mixing until there is a clear change in the viscosity of the preparation, resulting in a very stiff, white emulsion. Refrigerate the syringes containing the emulsion at 4 °C or maintain on ice.
4. Transfer all of the emulsion to one glass luer-lock syringe, remove the empty glass syringe and replace it with a 1 mL luer-lock syringe for injection. Fill the new syringe with emulsion, remove it from the stopcock and attach a needle (25G x 5/8).
5. "Water-test" the emulsion for consistency. Expel a drop of the emulsion into a small dish of water. The emulsion should not disperse, indicating that it is suitable for injection.
   1. If the emulsion disperses, it is not ready for injection; transfer the liquid back into the glass syringes and continue to mix and then chill again until the emulsion is stiff.
   2. Test the emulsion again until the proper consistency has been achieved.
6. Inject donor AQP4^-/- mice subcutaneously (s.c.) with emulsion containing the AQP4 peptide. Each mouse receives 4 x 50 µL s.c. injections (200 µL total (100 µg peptide)). The 4 sites include both sides of the lower abdomen, which drain into the inguinal lymph nodes (LN), and both sides of the chest wall medial to each armpit, which drain into axillary LN. The adoptive transfer will require 1-2 immunized donor mice per recipient mouse.

2. T Cell Culture and Proinflammatory T cell Polarization

1. 10-12 days after immunization, collect LN from the mice.
   1. In an ice tray, prepare 60 mm Petri dishes fitted with a cell strainer (mesh size 70 µm) and containing chilled Roswell Park Memorial Institute (RPMI) media with 10% heat-inactivated fetal bovine serum (FBS) and 100 U/mL penicillin-100 µg/mL streptomycin (pen-strep) (RFPS).
   2. Euthanize mice by exposure to CO₂, followed by cervical dislocation. Immerse mice in 70% ethanol and then pin each footpad to a dissection board.
   3. Cut a midline skin incision from the groin to the neck and down each leg. Pull the skin away from the peritoneum and pin it tautly to the board. Dissect inguinal and axillary LN and place into the Petri dish containing RFPS, on ice.
   4. For adoptive transfer experiments, combine LN from all animals within the same immunization group. For flow cytometric analyses of Vβ usage, separate LN from individual animals.
2. Place the cell strainer containing the LN onto a 50-mL centrifuge tube containing RFPS. Use the flat end of a sterile 5 mL syringe plunger to press the LN cells through the strainer, washing with 20-30 mL of RFPS to facilitate recovery of the cells. Maintain LN cells on ice throughout the processing until time for culture.
3. Wash twice, centrifuging at 393 x g for 5 min. After the second wash, resuspend the LN cells in T cell media (TCM). TCM contains RPMI with 10% heat-inactivated FBS, 292 µg/mL L-glutamine, 110 µg/mL sodium pyruvate, and 55 µM 2-mercaptoethanol and pen-strep. Typically, a volume of 5 mL TCM per donor mouse is used.
4. Count the LN cells. Expected yield is approximately 3-6 x 10⁷ LN cells per immunized mouse donor.
5. Set up polarizing cultures for adoptive transfer.
   1. Prepare a Th17 polarizing culture of 5 x 10⁶ LN cells per mL with 10 µg/mL Ag, 20 ng/mL recombinant mouse interleukin (IL)-23 and 10 ng/mL recombinant mouse IL-6 in TCM.
   2. Alternatively, for Th1 polarization, prepare a culture of 5 x 10⁶ LN cells per mL with 10 µg/mL Ag and 10 ng/mL recombinant mouse IL-12 in TCM.
   3. Add 2 mL (1 x 10⁷ cells) per well of the polarizing culture mixture into a 12-well plate. The LN cells from 2-4 donor mice will fill one 12-well plate. Maintain cultures in a humidified incubator at 37 °C with 5% CO2 for 72 h.
   4. Visually check the LN cells in culture for activation at 48 h and 72 h. Activated cells will form extensive clusters and the T cells will increase in size, becoming more pleomorphic. The increase in metabolism will cause a lowering of the pH in the media, changing from peach to orange. If the pH is low enough to turn the media yellow, add 1 mL of fresh TCM to prevent toxicity to the cells in the remaining 24 h.
   5. Proceed for adoptive transfer.

3. Adoptive Transfer of Pathogenic AQP4-specific T cells

NOTE: This step follows from Section 2.5: T cell culture and proinflammatory T cell polarization.

1. Recover polarized cells from 12-well plates by pipetting up and down to dislodge, and transferring them to a 50 mL tube. Maintain on ice until injections.
2. Count the cells, wash them twice in cold PBS, and prepare a suspension of 1 x 10⁸ cells/mL in PBS. Generally, inject cells within the hour.
3. Gently mix the cells by swirling them and transfer them to a 1 mL syringe at the time of injection. Administer 200 µL of the cells (2 x 10⁷) intravenously (i.v.) to each mouse, through the tail vein.
4. Inject each recipient mouse i.p. with 200 ng of B. pertussis toxin diluted in 200 µL of PBS that day and 2 days later. Monitor mice daily for signs of clinical disease.

Materials

Name	Company	Catalog Number	Comments
M. tuberculosis H37Ra	BD Difco	231141	Dessicated, killed M. tuberculosis
Incomplete Freund's Adjuvant	BD Difco	263910
AQP4 peptide p135-153	Genemed	Custom Synthesis	Peptide sequence: LVTPPSVVGGLGVTMVHGN
AQP4 peptide p201-220	Genemed	Custom Synthesis	Peptide sequence: HLFAINYTGASMNPARSFGP
MOG peptide p35-55	Genemed / Auspep	Custom Synthesis	Peptide sequence: MEVGWYRSPFSRVVHLYRNGK
3-way Stopcock	Kimble	420163-4503
HyClone Fetal Bovine Serum (Characterized)	GE Healthcare Life Sciences	SH30071
Recombinant Mouse IL-23	R&D Systems (BioTechne)	1887-ML
Recombinant Mouse IL-6	R&D Systems (BioTechne)	406-ML
Recombinant Mouse IL-12	R&D Systems (BioTechne)	419-ML
Pertussis Toxin from B. pertussis	List Biological Laboratories	181
Variable-Flow Peristaltic Pump	Fisher Scientific	13-876-2