The overall goals of this protocol are to induce central nervous system or CNS autoimmune disease by the adoptive transfer of aquaporin 4-specific T cells, and to monitor the resulting effects on the CNS and the dynamic visual system. This method will help answer key questions regarding the role of aquaporin 4, commonly known as AQP4, specific T cells in neuromyelitis optica as well as other inflammatory visual disorders. The main advantage of optical coherence tomography is the capacity to do longitudinal monitoring of retinal injury in a noninvasive manner.
Demonstrating the procedure will be Sharon Sagan, a research associate in my laboratory as well as Andreas Cruz Harans, a fellow in RE Greens Laboratory. Begin by attaching a three-way nylon stopcock with two male Luer lock connections to a glass Luer lock syringe without the plunger. Switch the stopcock lever toward the syringe to close the stopcock and position the syringe with the open end facing up.
After vortexing, add a one-to-one volume of peptide, and Complete Freund's Adjuvant solution to the syringe, and reinsert the glass plunger into the syringe. Holding the plunger in place, invert the syringe so that the stopcock is facing up, and switch the lever to the unused female connection. Carefully depress the plunger to remove the excess air from the syringe.
The solution will fill the second male Luer lock connection of the stopcock. Then attach a second glass syringe with the plunger fully inserted into the second male Luer lock connection. To create an emulsion, alternately depress the plungers to pass the solution between the two syringes for about two minutes, and cool the solution for about 10 minutes until there is a visible change in the viscosity of the solution.
When a very stiff white emulsion forms, transfer all of the solution into one of the syringes, and replace the empty syringe with a new one-millimeter Luer lock syringe. Fill the new syringe with the emulsion. Remove the syringe from the stopcock, and attacH a 25-gauge needle.
To water test the emulsion for consistency, expel a drop of the emulsion into a small dish of water. If the emulsion does not disperse, it is suitable for injection. Then inject each donor AQP4 knockout mouse subcutaneously one time into each side of the lower abdomen, and into each side of the chest wall medial to the armpit.
10 to 12 days after the immunization, make a midline incision in the skin from the groin to the neck, and down each leg of the first mouse. Pull the skin away from the peritoneum, and secure it tightly to the board. Then harvest the inguinal and axillary lymph nodes, and place tissues in a cell strainer within a petri dish containing complete medium.
When all of the lymph nodes have been collected, place the cell strainer onto a 50-milliliter conical tube containing fresh complete medium on ice, and use the flat end of a sterile five-milliliter syringe plunger to press the lymph node cells through the filter mesh. Rinse the mesh with 20 to 30 milliliters of complete medium to facilitate the cell recovery followed by two centrifugation washes. After the second centrifugation, resuspend the pellet in T cell medium for counting.
Add the appropriate concentrations of antigen, recombinant mouse interleukin mouse 23, and recombinant mouse IL-6 to the cells, and add two milliliters of cells per well in this Th17 polarizing mixture into each well of a 12-well plate. Then place the plate in a humidified 37 degree Celsius incubator with 5%CO2 for 72 hours. On the third day of polarization, triturate the cultures in each well a few times to dislodge the polarized cells, and pool them in a 50-milliliter conical tube on ice for counting.
After two washes in PBS, dilute the cells to a one times 10 to the eighth cells per milliliter concentration in PBS, and gently mix the cell suspension by swirling. Within one hour of their collection, transfer the cells into a one-milliliter syringe, and deliver 200 microliters of cells to each recipient animal intravenously through the tail vein. Then inject each mouse intraperitoneally immediately and two days later with 200 nanograms of B pertussis toxin diluted in 200 microliters of PBS.
For in vivo retinal imaging of the recipient animals by optical coherence tomography, five minutes prior to imaging, dilate the pupils with one drop of 1%tropicamide per eye, and turn on the heat mat. Place the mouse on the imaging cylinder, and redirect the anesthesia flow accordingly. Protect the eyes with 0.3%hydroxypropyl methylcellulose to keep them moist, and to ensure refraction continuity, and place a custom contact lens onto the eye to be examined.
Guided by the infrared fundus image, direct the laser to the lens-covered eye taking care that the beam is centered on the optic nerve head. Perform 25 B-scans in high resolution mode, and rasterize the images from 30 averaged A-scans. After imaging both eyes, replace the contact lens with ophthalmic gel, and place the mouse in a warm recovery cage.
To analyze the acquired images using the modular imaging software for the automated segmentation, first manually correct the segments corresponding to the inner limiting membrane and the inner plexiform layer to represent the limits of the inner retinal layers. Then verify that the retinal nerve fiber and ganglion cell layers reside within the limits of the inner retinal layer, and that they do not overlap. Subcutaneous immunization with AQP4 peptides that contain pathogenic T cell epitopes elicit strong proliferative T cell responses in the draining lymph nodes of AQP4 knockout mice compared to wild type T cell responses.
Flow cytometric analyses of individual V beta expression demonstrates that T cells specific for AQP4 peptides that contain pathogenic T cell epitopes utilize unique TCR repertoires, and demonstrate a selective hyperproliferation indicating that pathogenic T cell responses to these determinants is normally regulated by thymic negative selection. Approximately six to nine days after polarized T cell injection, nearly all of the recipient mice developed clinical signs of CNS autoimmune disease including limp tail and hind limb paralysis. With Th17-polarized AQP4-specific T cells inducing a more severe clinical disease than Th1-polarized AQP4-specific T cells.
For example, this mouse developed complete hind limb paralysis after Th17-polarized AQP4 peptide-specific T cell administration. Clinical disease induced by AQP4 peptide-specific Th17 cells is also associated with an infiltration of mononuclear cells into the CNS parenchyma and meninges as well as optic nerve inflammation as further evidenced by a swelling and increased thickness of the inner retinal layer that returns to baseline as the mice recover from the illness. After watching this video, you should have a good understanding of how to induce and monitor AQP4-targeted autoimmunity, which will permit further study to understand how T cells participate in neuromyelitis optica.
