The overall goal of this experiment is to determine the ability of adjuvants of interest to generate cytotoxic T lymphocytes by monitoring adoptively transferred CFSE stained OT1 CD8 positive T cell proliferation in the draining lymph nodes and spleens of immunized recipient animals. This method can help to answer key questions in the vaccinology field like the potency of an adjuvant to generate a cytotoxicity cell response. The main advantage of this technique is that you can determine not only the CTL capacity of an adjuvant, but also if this is local or systemic and you can do it only in four days.
Demonstrating the procedure will be a technician in our lab, Elena Reinhard. To isolate thigh 1.1 positive CD8 positive T cells from OT1 mice, first harvest the spleen and inguinal, axillary, and cervical lymph nodes according to standard protocols placing all of the organs into a 100 micrometer pore mesh strainer in a 60 millimeter Petri dish containing three to five milliliters of complete medium on ice per mouse as they are collected. Do not harvest in large OT1 spleens or lymph nodes because the T cells from these lymph tissues may be leukemic and will proliferate even without stimulation confounding the assay results.
When all of the organs have been retrieved from each donor animal, use a syringe plunger to mash the samples through the mesh filter and transfer the resulting single suspensions into one 15 milliliter conical tube per mouse. Pellet the cells by centrifugation, followed by a centrifugation wash in 10 milliliters of cold PBS. Lyse the red blood cell pellet in one milliliter of ammonium chloride buffer per tube on ice.
After 90 seconds, wash the cells in another 10 milliliters of PBS and resuspend the cells in one milliliter of PBS plus 5%FBS per tube. Then use a negative selective kit to isolate the CD8 positive T cells according to standard magnetic bead isolation protocols and count the number of magnetic bead sorted CD8 positive T cells. Next, dilute the T cells to a one to five by 10 to the seventh cells per milliliter of PBS concentration in individual 15 milliliter conical tubes and stain each cell sample with five micromolar CFSE for seven minutes at 37 degrees Celsius and protected from light.
At the end of incubation, quench the staining reaction with an equal volume of FBS and return the cells to a 37 degree Celsius incubator for an additional seven minutes. At the end of the second incubation, wash the cells two times in 10 milliliters of PBS per wash and resuspend the cells in three to five by 10 to the seventh cells per milliliter volumes of PBS. For adoptive transfer of the CFSE labeled OT1 T cells, load the cells into one one milliliter syringe equipped with a 25 gauge needle per experimental cell sample and immobilize the first recipient mouse in a restrainer.
Then when the tail veins have vasodilated, deliver 100 microliters of cells via the lateral or dorsal tail vein. For immunization of the adoptively transferred animals, shave the fur over the gluteus superficialis of the OT1 T cell transplanted mouse and use a 25 gauge needle to subcutaneously inject 50 microliters of endotoxin-free OVA into the shaved area. It's important to use endotoxin-free OVA because traces of endotoxin in OVA can lead you to false results.
Forty-eight hours after the immunization, harvest the inguinal lymph nodes and spleens from each recipient animal according to standard protocols and generate individual single cell suspensions for each lymph node and spleen sample as just demonstrated. Collect the cells by centrifugation and resuspend the pellets in 100 microliters of PBS per sample. To stain the samples for flow cytometric analysis, incubate the cells with 100 microliters of fluorophore conjugated primary antibody master mix cocktail for 30 minutes at four degrees Celsius.
At the end of the incubation, wash the cells two times in 10 milliliters of PBS and resuspend the samples in 0.5 to one milliliter of PBS per tube. Filter the cells through 70 to 100 micrometer strainers into five millimeter flow cytometry analysis tubes and acquire the single staining compensation controls as well as the samples in the flow cytometer. To gate the samples, open a forward scatter height versus forward scatter area plot and a side scatter wide versus side scatter area plot and gate to exclude the doublets.
Generate a FITC versus auto fluorescence plot in order to discriminate the population of true CSFE stained cells from the high auto fluorescent cells. Gate these OT1 CSFE stained cells as the populations that extend mostly on the FITC axis. To distinguish cells derived from donor versus recipient animals, gate the thigh 1.1 plus CD90 0.1 plus CD8 plus T cells and re-gate the CD8 plus population to exclude the CD4 plus T cells.
Then display this proliferated CD8 T cell population in a histogram according to their CSFE expression and gate the proliferated populations from a 10 to the second intensity up to the undivided control population intensity. Acquire at least 5, 000 events for the compensation controls and 10, 000 events for each sample at a low or medium flow analysis rate. In this experiment, two groups of mice were vaccinated with one of two different adjuvants.
Mice that were injected with OVA plus the second adjuvant exhibited a higher cytotoxic T cell generation capacity locally in the draining lymph nodes compared to mice injected with the first adjuvant plus OVA, OVA alone, or the PBS control vaccine. In contrast, in mice injected with the second adjuvant plus OVA did not generate a cytotoxic T cell response in a systemic compartment whereas CD8 positive T cells isolated from the spleens of adjuvant one plus OVA mice demonstrated higher levels of proliferation than splenic CD8 positive T cells isolated from OVA alone, PBS control, and adjuvant two plus OVA vaccinated animals. Once mastered, this technique will allow you to determine the CTL capacity of an adjuvant molecule by using OVA antigen in just four days of work.
After performing this technique, you will be able to further apply all other techniques to the T cells isolated and then to phenotype the cytokine profile or the metabolic profile of the CTL response of your adjuvant.
