This method can help answer key questions in the field of immunology, such as how are white blood cells activated, and how do allergies to individual allergens develop? The main advantage of this robust and reproducible allergy mouse model is that fewer mice can be used to yield a lower variance with an experimental population. New immunotherapies to control immune responses require robust mouse models as an initial means of testing in vivo efficacy.
Thus, the implications of this technique extend toward allergy immunotherapy. Though this method can provide insight into allergies, it can also be applied to other systems, such as autoimmunity in which undesired antibody responses play a key role in disease. Generally, individuals new to this method will struggle because of lack of experience in working with liposomal nanoparticles or an inexperience with the techniques required in the mouse work.
Begin by adding 2.5 molar equivalent of the heterobifunctional crosslinker to the protein and placing the reaction on an oscillating shaker at room temperature for approximately one hour. After a one-hour incubation with SPDP, desalt the protein on a column equilibrated in 100-millimolar sodium acetate, and collect the fractions. Determine the 280-nanometer absorbance, and pool the top fractions.
Wash the column in PBS, and add 25-millimolar dithiothreitol, or DTT, to the pooled protein fractions for a five to 10-minute incubation. Then, measure the 280 and 343-nanometer absorbances of the protein to allow calculation of the linking ratio based on the molarity of protein and the linker. Next, run the pooled fractions of the PBS-washed column, and collect the fractions for measurement of the 280-nanometer and top fraction pooling as demonstrated.
Add the appropriate volume of 100x DSPE-PEG2000-Maleimide to the protein to achieve a 1x, 10-fold molar excess final concentration with gentle swirling. Then, run the reaction overnight under nitrogen in a sealed round-bottom flask. The following day, run the protein over a crosslinked dextran gel bead column, and store the final pooled fractions of lipid-linked protein at four degrees Celsius until their use.
Once the correct amount of each lipid has been calculated, combine all of the lipids into a 12-milliliter borosilicate glass test tube, and use a three-milliliter syringe and tubing to carefully blow the chloroform off with nitrogen. Then, lyophilize the solution with 100 microliters of DMSO overnight. The next morning, add one milliliter of lipid-linked protein to the 12-milliliter lipid tube, and sonicate the solution three to four times for 30 to 60 seconds per sonication in a sonication water bath with at least five-minute rests between sonications.
After the last sonication, load the sample into one of the extruding syringes placed into the extruder, and place the extruder syringe into the other end of the extruder. The empty syringe will fill as the lipid is extruded through the polycarbonate 0.8-micrometer membrane. Place the fully assembled extruder into a heating block, and gently depress the plunger to empty the syringe.
After the last extrusion, transfer the liposomes into a clean vial, and extrude the lipids through polycarbonate 0.2 and 0.1-micrometer membranes as just demonstrated. Then, store the liposomes at four degrees Celsius. To monitor B-cell response to antigenic liposome activation, resuspend 1.5 times 10 to the seventh splenocytes in calcium flux loading buffer, and add 1.5-micromolar Indo-1 to the cells, inverting the solution in the tube several times to mix.
After a 30-minute water bath incubation at 37 degrees Celsius, protected from light, add five times the volume of calcium flux loading buffer, and centrifuge the Indo-1-labeled cells. For B-cell gating, stain cells with anti-CD5 and anti-B220 antibody in 0.5 milliliters of loading buffer at four degrees Celsius for 20 minutes, protected from light. At the end of the incubation, wash the cells in fresh calcium flux loading buffer, and resuspend the pellet at one to two times 10 to the seventh cells per milliliter of fresh calcium flux running buffer for storage on ice, protected from light until analysis.
Next, add 0.5 milliliters of cells to a capped, five-milliliter, round-bottom, polystyrene tube, and warm the cells to 37 degrees Celsius in a water bath. After three to five minutes, transfer the tube to a 37-degree Celsius water-jacketed chamber connected to a recirculating water bath, and run the tube in the water-jacket on the flow cytometer. After collecting 5, 000 to 10, 000 events per second, allow the cells to stabilize for 15 to 30 seconds, and reinitiate the data acquisition, collecting the data for at least 10 seconds to establish a background reading.
At the 10-second mark, quickly remove the tube from the flow cytometer, and add the appropriate experimental concentration of antigenic liposomes. Then, pulse vortex the cells, and read the tube on the cytometer for an additional three to five minutes. To sensitize the animals, deliver 200 microliters of peanut extract containing cholera toxin via oral gavage to each four to five-week-old BALB/c female mouse recipient once a week for three weeks and 300 microliters of diluted peanut extract in the fourth week.
On day 28, prepare peanut extract to a final concentration of one milligram per milliliter in PBS, and use a rectal thermometer to measure the baseline body temperature of each sensitized animal. When all of the temperatures have been measured, administer 200 microliters of peanut extract to each recipient via intraperitoneal injection, and measure the body temperature with the rectal thermometer every 15 minutes for one hour after injection. A dip in body temperature indicates an allergy to peanut.
After isolating splenocytes from the peanut-allergic mice, use a one-milliliter insulin syringe equipped with a 27-gauge, 5/8-inch needle to intravenously inject 1.5 times 10 to the seventh of the extracted allergic splenocytes into the tail veins of naive, unsensitized animals. One day after adoptive transfer, intravenously inject 200 microliters of Ara h 2 antigenic liposomes into the tail vein of each BALB/c mouse that received the allergic splenocytes. Two weeks after the liposome injection, boost the mice with an i.p.
injection of soluble Ara h 2, followed by a 200-microliter Ara h 2 challenge on day 61. Then, measure the body temperatures with the rectal probe every 15 minutes as demonstrated. Protein conjugation can be demonstrated by running a reducing gel showing an increase in molecular weight compared to the unconjugated protein.
To assess the antigenic liposome-stimulated B-cell calcium flux, gate the live single cells to allow selection of the B220-positive CD5-negative B cells. The ratio of Indo-1 violet versus Indo-1 blue fluorescence over time can then be analyzed to assess the amount of liposome-induced B-cell activation. The quantification of Ara h 2-specific IgE and IgG1 in the serum of peanut extract-sensitized mice by ELISA indicates that mice with conferred sensitivity that are boosted with Ara h 2 exhibit measurable Ara h 2-specific IgE and IgG1 in their serum.
Body temperatures recorded during the Ara h 2 challenge reveal that allergic mice demonstrate decreased body temperatures following the challenge, while body temperatures in naive mice remain consistent. While attempting this procedure, it's important to remember to carefully keep track of the amount of lipid-linked protein being incorporated into the liposomes, as too much protein can make extrusion difficult. Following this procedure, other methods like the tracking and sorting of allergen-specific B and T cells can be performed to answer additional questions about how these allergen-specific cells respond to therapies.
This technique will pave the way for researchers in the allergy field to explore new immunotherapies that combat allergic disease using this mouse model. Don't forget that working with cholera toxin can be hazardous and that the guidelines for its use should be followed according to your local institutional biological safety standards.
