Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

All animal procedures were performed in compliance with experimental guidelines approved by the University of California, San Francisco Institutional Animal Care and Use Committee. 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 M. 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 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.¹⁹
   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. Set aside 3-4 x 10⁶ for a proliferation assay (see Section 3 below).
5. Set up polarizing cultures for adoptive transfer (Section 6).
   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 to Section 6 for adoptive transfer. Polarization efficiency may be verified by intracellular cytokine staining (ICS), as described in Section 5.
6. Set up cultures using LN from individual mice for flow cytometric analysis of surface Vβ expression (Section 4).
   1. Prepare 5 x 10⁶ LN cells per mL with 10 µg/mL Ag in TCM.
   2. Add 2 mL (1 x 10⁷ cells) of the culture per well into a 12-well plate. Cultures should be maintained in a humidified incubator at 37 °C with 5% CO₂ for 10 days. Proceed to Section 4.

3. Proliferation Assay

NOTE: This continues from Section 2.4.

1. Prepare 3-4 mL of LN cells in a tube, at 2 x 10⁶ cells per mL in TCM.
2. Gently mix the cells by inverting the tube several times, and then transfer to a sterile trough.
3. Use a multichannel pipettor to plate 100 µL of cells per well into a 96-well round bottom tissue culture plate. Typically, plate 15 wells for triplicate testing of 4 Ag concentrations and a no Ag reference.
4. Dilute the Ag in TCM at various concentrations, typically 80, 20, 5, 1 and 0 µg/mL (for 2x stocks). Add 100 µL of each concentration in triplicate for a final concentration of 40, 10, 2.5. 0.5 and 0 µg/mL. Incubate for approximately 72 h at 37 °C.
   NOTE: Steps 3.5 through 3.7 involve radioactive materials. Proper usage, monitoring and disposal of radioactive materials should be conducted, according to institutional and governmental regulations.
5. Prepare a working stock solution of ³H-thymidine (40 µCi/mL) by adding 1 mCi ³H-thymidine to 25 mL RPMI, and store this at 4 °C. Pipet 0.5 mL of the working solution into a sterile trough.
6. Add 25 µL of ³H-thymidine (1 µCi) per well using a multichannel pipet. Incubate for an additional 18 h at 37 °C.
7. Harvest cells onto a glass filter mat using a 96-well cell harvester and rinse with 70% ethanol. After drying, seal the filter mat into a plastic sample bag with 5 mL scintillation fluid. Measure radioactivity in each well using a scintillation counter.

4. Flow Cytometry Analysis for Cell Surface TCR Vβ Expression

NOTE: This continues from Section 2.6. Antibodies for mouse TCR Vβ 2, 3, 4, 5.1 and 5.2, 6, 7, 8.1 and 8.2, 8.3, 9, 10b, 11, 12, 13, 14, and 17a are available commercially for testing the TCR Vβ expression.

1. For detection of TCR Vβ, dislodge T cells from the culture plate by pipetting several times and transfer cells to a tube.
2. Wash with FACS buffer (FB) containing 2% FBS, 0.1% sodium azide and 2 mM EDTA in PBS.
3. Transfer cells to a V-bottom 96-well plate at a density of 1 x 10⁵ to 3 x 10⁶ cells per well. If the entire Vβ panel is being tested, the cells should be distributed into multiple wells (one well per Vβ being tested).
4. Exclude dead cells from flow cytometric analysis by staining with a LIVE/DEAD viability dye, which reacts with free amines exposed by necrotic cells. Follow the manufacturer's instructions for centrifugation and resuspension in viability dye. Incubate on ice for 10-20 min. Throughout the cell staining procedures, protect cells from light and keep on ice.
5. After incubation, wash the plate once in FB. To detect TCR Vβ, suspend the cells in 100 µL of FB containing a 1:100 dilution of antibodies for CD4 (clone RM4-5) and TCR Vβ. Incubate for 15-60 min on ice.
6. Wash the plate once in FB and fix the cells by adding 200 µL of 4% paraformaldehyde diluted in PBS. Incubate for 20 min on ice. Wash the plate in FB and analyze by flow cytometry.

5. Flow Cytometric Analysis for Intracellular Cytokine Production

1. For intracellular cytokine staining (ICS), treat T cell cultures with a 1:1,000 dilution of protein transport inhibitor reagent (e.g., GolgiPlug) in TCM, 4 h prior to ICS to prevent cytokine secretion. At that time, activate T cells with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) and 500 ng/mL ionomycin in order to induce protein production.
2. After 4 h of culture at 37 °C, dislodge cells from the culture plate by pipetting up and down, and transfer to a centrifuge tube (15 or 50 mL).
3. Wash with FB. Resuspend cells in FB and transfer to a V-bottom 96-well plate at a density of 5 x 10⁵ to 3 x 10⁶ cells per well.
4. Stain cells with viability dye as described above (section 4.2). After the incubation, wash the plate once in FB.
5. Add antibodies for detection of surface markers as follows:
   1. For detection of surface markers, suspend the cells in 100 µL of FB containing a 1:100 dilution of antibody for CD4 (clone RM4-5).
   2. Optionally, include antibodies for B220 (clone 30-F11) and CD11b (clone M1/70) at a 1:100 dilution to improve gating of B cells and monocytes/macrophages, respectively. Generally, PE-Cy7 anti-CD4, FITC anti-B220 and PerCP-Cy5.5 anti-CD11b work well. Choice of antibody/fluorochrome conjugates should be guided by configuration of flow cytometer to be used for analysis, and optimal concentrations of antibodies should be determined empirically.
   3. Incubate for 15-60 min on ice.
6. Wash the plate with FB and resuspend the cells in 200 µL of Fixation/Permeabilization solution for 20 min on ice. This will fix the cells and permeabilize the membranes.
7. In order to maintain the permeabilization of the cell membranes during the intracellular staining, use Perm/Wash buffer (PW) (diluted 1:10 from the 10X stock) instead of FB. Wash the wells in 200 µL of PW.
8. For ICS, prepare a 1:100 dilution of the antibodies for interferon (IFN)-γ and IL-17A using 1x PW. One option is APC anti-IFN-γ (clone XMG1.2) and PE anti-IL-17A (clone eBio17B7) which works well. Resuspend cells in 100 µL of the intracellular antibody cocktail and then incubate for at least 15 min. It is also possible to continue the staining overnight at 4 °C.
9. Wash the wells in 200 µL of PW and resuspension in FB for flow cytometry.
10. In parallel, prepare an unstained sample (cells in FB without antibodies) to optimize detector voltages, and single-stained samples for each individual fluorochrome (i.e., cells or compensation beads stained with only one antibody/fluorochrome) to determine compensation spillover values.
11. Using a flow cytometer, acquire ≥10,000 cells (events), gating on viable (LIVE/DEAD-negative) CD4⁺ cells. To ensure accurate gating of T cells, exclude the B220⁺ and CD11b⁺ cells (B cells and monocytes/macrophages, respectively). Within the CD4⁺ T cell subpopulation, determine the percentage of cells expressing IFN-γ (Th1 lineage) or IL-17A (Th17 lineage).

6. 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 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 and transfer 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.

7. Clinical Assessment of ATCA

1. Examine recipient mice daily for clinical signs of CNS autoimmunity. In general, mice will show signs of CNS autoimmunity 5-8 days after adoptive transfer of AQP4-specific T cells. The mice will show clinical symptoms of neurological dysfunction. It is helpful to perform the evaluations on a naive mouse to define the normal ability of a mouse.
2. Evaluate mice for loss of tail tone. Hold the mice gently at the base of the tail and lift upright. A tail that droops continuously is described as loss of tail tone (score of 1). Loss of tail tone is often the first sign of clinical disease.
3. Test for righting reflex by placing mouse on its back on a flat surface. Slow righting is a sign of truncal incoordination (score of 2). A naïve mouse will completely resist any attempt to place it on its back, so any slowness in the ability to right is scored as a dysfunction. It is typical to test the mouse 3-4 times for this reflex.
4. Evaluate posture and ambulation. Mice begin to show a change in posture resulting in a lowering or dragging of the hips during ambulation (score of 2.5). Further disease results in monoplegia, complete paralysis of one hind limb (score of 3.0) and paraplegia, full paralysis of both hind limbs (score of 3.5). Moderate quadraparesis, weakness of all four extremities, results in very slow forward movement (score of 4). Severe quadraparesis allows almost no forward movement (score of 4.5). Finally, with severe disease they may become moribund or die (score of 5).
5. Administer to mice that lose the ability to acquire fluid or solid food 1 mL of 5% glucose in saline i.p. Alternatively, use supplementation with fluid gel cups, high caloric gel and bacon softies to counteract dehydration and food deprivation. Euthanize moribund mice.

8. Tissue Preparation and Histology

1. Anesthetize mice with 100 mg/kg ketamine and 10 mg/kg xylazine, and then pin each footpad to a dissection board.
2. Open up the chest, exposing the heart. Incise the liver to permit blood outflow. Attach a butterfly needle to the tubing of a miniflow variable speed pump using a separate reservoir for PBS and 10% formalin. Insert the needle into the left ventricle of the heart and perfuse with 5 mL of PBS, followed by 25 mL of 10% formalin.
3. Remove the head and then the eyes. Process eyes and retinas as described previously²⁰. Cut across the eye sockets, insert scissors at the nose and cut the skull anterior to posterior on each side.
   1. Remove the skull, expose the optic nerves, cut the optic chiasm and place in a cassette between 2 foam pads.
   2. Immerse in 10% formalin (24 h), and then 50% ethanol (24 h) and then transfer to 70% ethanol.
4. Remove brain and spinal column and place in 10% formalin. Serially section brains in the coronal plane; section spinal cords in sagittal (approximately half) and serial coronal planes (approximately 20 cross-sections per mouse).
5. Process CNS samples routinely for paraffin embedding. Stain 8 µm thick sections with hematoxylin and eosin (optic nerves) or Luxol fast blue-hematoxylin and eosin (brain and spinal cord).
6. Evaluate optic nerves for the presence of inflammation within the nerve and surrounding meninges (optic neuritis) or predominantly in the surrounding meninges (optic perineuritis).
7. Count meningeal and parenchymal inflammatory foci (>10 clustered mononuclear cells) in the brain and spinal cord samples for each mouse.

9. In Vivo Retinal Imaging by Optical Coherence Tomography (OCT)

NOTE: Spectral domain OCT retinal imaging of mice is performed using commercial equipment (e.g., Spectralis with TruTrack eye tracker) to achieve consistent ocular orientation and reduce motion artifacts.

1. Five min prior to imaging, dilate pupils with 1% tropicamide (one drop per eye) and place mouse to be imaged in the anesthesia induction chamber, providing a steady flow of 2 liters per min (1.5% isoflurane). Turn on heat mat and prepare the post-anesthesia recovery cage.
2. Place the mouse on the imaging cylinder and redirect the anesthesia flow accordingly. Protect eyes with 0.3% hydroxypropyl methylcellulose to keep the eye moist and to ensure refraction continuity. Place a custom contact lens on the eye to be examined.
3. Guided by the infra-red fundus image, direct the laser to the eye, ensuring the beam is centered on the optic nerve head. Vertical and horizontal OCT scans should confirm that the retina lays perpendicular to the laser.
4. Perform 25 B-scans in high-resolution mode and rasterize from 30 averaged A-Scans.
5. After imaging both eyes, remove contact lens and apply ophthalmic gel. Leave mouse in the warm recovery cage.

10. Processing and Analysis of OCT

1. Use the modular imaging software for automated segmentation.
   1. Manually correct segments corresponding to the inner limiting membrane (ILM) and inner plexiform layer (IPL), representing the limits of the inner retinal layers (IRL)²¹.
   2. Verify that the retinal nerve fiber layer (RNFL) and the ganglion cell layer (GCL) reside within the limits of the IRL and do not overlap.
2. Calculate IRL thicknesses using the Early Treatment Diabetic Retinopathy Study (ETDRS)² grid with diameters of 1, 2, and 3 mm centered on the optic disc, and export into a spreadsheet file. The software calculates thickness of each retinal layer by averaging each grid sector. Therefore, the central segment, corresponding to the optic nerve head, is excluded.
3. Analyze statistical differences between groups at each time point. Analyze both eyes for each mouse, using generalized estimating equations with an exchangeable correlation matrix and adjustments for intra-subject inter-eye correlations²¹.