Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

The overall goal of the following experiment is to synthesize and characterize lipid-based vaccine nanoparticles and to assess their ability to elicit antigen-specific cytotoxic T lymphocyte expansion in vivo. This is achieved by engineering pathogen mimicking inter bilayer, cross-linked multilam vesicles or I CMVs co loaded with antigen and adjuvant for vaccination of naive mice. Adoptively transferred with luciferase expressing antigen specific CD eight positive T cells.

In the second step, the expansion of the antigen-specific CD eight positive T cells is evaluated by whole animal in vivo imaging. Peripheral blood mononuclear cells harvested from the ICMV immunized animals are then stained with fluorescently tagged tetramers and analyzed by flow cytometry to quantify the frequency of the endogenous antigen-specific CD eight positive T cells within the systemic compartment. Ultimately, bioluminescent imaging can be used to track the expansion of the antigen-specific cytotoxic T lymphocytes in response to the nanoparticle vaccination.

These methods can help answer key preliminary questions in vaccine design, such as what is the extent of cytotoxic tial lymphocyte expansion and how do these cells traffic to various tissues in response to nanoparticle vaccination? The main advantage of this technique over existing methods, like in-situ T cell analysis after tissue necropsy, is that in vivo imaging of TT cell response is non-invasive. Allowing longitudinal monitoring of the same animal Begin by mixing a one-to-one molar ratio of DOPC and MPB in chloroform, maintaining a total lipid quantity of 1.26 micromoles per batch in a 20 milliliter glass vial.

Next, add lipophilic cargo to the lipid solution at the desired concentration and purge the solution with extra dry nitrogen gas to thoroughly remove the organic solvent. Place the sample under vacuum overnight the next morning at 200 microliters of 10 millimolar BTP containing the water soluble cargo of interest to hydrate the lipid film vortex the resulting solution for 10 seconds every 10 minutes for one hour at room temperature. Then transfer the contents to a 1.5 milliliter micro centrifuge tube.

Next place the tube in an ice water bath with continuous sonication using the 40%intensity setting on a 125 watt 20 kilohertz probe tip soat. After five minutes, add four microliters of 150 millimolar DTT to the tube vortex to mix and spin down the sample in a micro centrifuge. Then mix 40 microliters of 200 millimolar calcium chloride into the sample, followed by cross-linking of the MPB containing lipid layers with DTT at 37 degrees Celsius for one hour.

At the end of the incubation, spin down the solution three times at 200 microliters of double distilled water during the second and third centrifugation to remove the remaining calcium chloride unre reacted DTT and Unencapsulated cargo materials from the supernatant. After the last spin, we suspend the I CMVs in 100 microliters of freshly prepared pegol for 30 minutes at 37 degrees Celsius. Then wash the vesicles two more times in double distilled water.

Resus suspending the final pellet in PBS and store the ICM VS at four degrees Celsius. To characterize the particles, mix the cold ICMV suspension before diluting a small aliquot of the sample in a total volume of one milliliter of double distilled water. Then place the I CMVs in a zeta sizer cell and use a dynamic light scattering and zeta potential measuring system to measure the particle size poly dispersity index and zeta potential of the samples according to the manufacturer's instructions.

To isolate ova specific luciferase expressing CD eight positive T cells. First harvest the spleens from OT one luciferase transgenic mice. Place the tissues in five milliliters of ice cold PBS and 2%FBS and then in a tissue culture hood.

Use the plunger from a three milliliter syringe to grind up to three spleens at once through a 70 micron strainer into a 15 milliliter conical tube. Wash the plunger and the strainer with PBS and 2%FBS. Then bring the total volume of the cyte suspension to 10 milliliters per spleen.

Determine the total cell number and then spin down the cells. Next, use a commercially available magnetic negative selection kit to isolate the CD eight positive spleen cells according to the manufacturer's instructions. Then wash the T cells in PBS.

Count them and incubate two to three times 10 to the fifth of the cells in 20 microliters of mouse CD 1632 blocking antibody. After 10 minutes, add 20 microliters of anti CD eight A PC antibody to the cells for 30 minutes and assess the purity of the magnetic bead isolated CD eight positive T-cell population by flow cytometry. Next adoptively transfer one to 10 times 10 to the fifth OT one luciferase CD eight positive T cells into naive shaved albino C 57 black six mice in 200 microliters of PBS via intravenous tail vein injection 24 hours later, administer the vaccine at the appropriate number of days post vaccination.

Inject the animals with 150 milligrams per kilogram of Lucifer after 10 minutes, acquire the bioluminescence signal for five to 10 minutes with a whole animal imaging system to visualize the OT one Lucifer CD eight positive T cells to determine the number of antigen specific CD eight positive T cells at the appropriate time. Post vaccination, collect approximately 100 microliters of blood from the vaccinated mice via the submandibular bleeding technique into a tube coated with potassium EDTA, invert the tube several times to prevent clotting. Then transfer 100 microliters of blood to a micro centrifuge tube and add one milliliter of a CK lysis buffer.

After two to three minutes, centrifuge the cells and remove the supernatant. Wash the remaining pbmc in one milliliter of fax buffer and block the nonspecific binding with CD 1632 as just demonstrated. After 10 minutes, divide the cell suspension into four milliliter round bottom fax tubes and add 20 microliters of H two KB over tetramer syn fecal PE solution to each sample according to the manufacturer's specifications.

After 30 minutes on ice, add 20 microliters of the appropriate antibody cocktail to each experimental sample and the single fluoro four control antibodies for each fluoro four tag tetramer or antibody to each control sample. Finally, after another 20 minute incubation, wash all of the samples in fax buffer resuspend the pellets in fax buffer supplemented with DPI, and analyze the samples by flow cytometry I CMVs co loaded with fluoro four tact protein antigens and fluorescent lipophilic dyes allow the visualization of antigen and nanoparticle delivery in vivo. For example, confocal microscopy imaging indicates that soluble antigen delivered subcutaneously at the base of the tail, reaches the draining lymph nodes within four hours of administration and is cleared by 24 hours.

In contrast, overloaded I CMVs are first detected at the periphery of the draining lymph nodes by 24 hours after administration with a continued accumulation that results in a large deposit of ova I CMVs within the draining lymph tissue. By day four, indeed bioluminescence imaging of C 57 BL six mice adoptively transferred with OT one Luciferase CD eight positive T cells on the day of vaccination demonstrates a minimal background level of OT one luciferase tcell by day four post vaccination. However, mice immunized with the ova MPI CMVs exhibit a robust bioluminescence signal within the draining inguinal lymph nodes.

The soluble ova vaccinated mice, on the other hand demonstrate a much reduced expansion of the OT one Lucifer CD eight positive T-cell within the draining lymph nodes. Further mice immunized with soluble ova and MPLA without any adoptive transfer of antigen Specific T cells do not exhibit an appreciable expansion of ova specific CD eight positive T cells as indicated by weekly monitoring of the pbmc ICMV vaccination, however, elicits a significantly stronger and endogenous CD eight positive T cell response achieving a peak 28%of the syn fecal tetramer positive T cells within the CD eight positive T cell compartment among pbmc by day 41. After watching this video, you should have a good understanding of how to synthesize lipid-based nanoparticles, perform immunizations, and visualize and quantify the CTL expansion in vivo.

These methods can be further modified to reveal additional information about the CTL response. For example, the teer staining essay can be supplemented with additional markers such as CD 1 27, BCL two, and K lrg one to assess the memory phenotype of the cells.