So the main advantage of our novel technique, we termed it E plus E, which stands for enrichment plus expansion, is that we can enrich and expand these cells, rare antigen-specific cells, simultaneously. This allows us to develop sufficient number of cells that we're gonna be able to use to investigate their role in different settings and in different clinical settings or even allows us to develop now large number of cells that we can utilize for immunotherapy in a fashion that we couldn't before. The method that we have developed, this E plus E, can help answer key questions in basic immunology about the presence of neoepitopes.
What are neoepitopes? Those are the personalized epitopes that we know are now very important in terms of cancer immunotherapy and also antigen-specific dynamics and how these factors affect disease states and for effective cellular therapy. The implications of this technique extend towards immunotherapy, as the nanoparticle artificial antigen-presenting cell could be an off-the-shelf product that is easily tailored for individual patient immune responses.
To generate nanoparticle artificial antigen-presenting cells, resuspend the lyophilized particles in one milliliter of resuspension buffer and vigorously vortex the suspension for at least 15 minutes until no aggregates are visible. Particles not sufficiently vortexed and resuspended prior to the reaction with protein will result in larger aggregated particles that are not able to be dissociated. Place the magnetic particles on a magnetic stand to remove the supernatant and resuspend them with 0.5 milliliters of fresh resuspension buffer.
Transfer the particle suspension to a glass scintillation vial for vortexing until no aggregates are visible, and add 0.1 milligrams of total stimulatory signals per one milligram of resuspended particles. After vortexing, allow the suspension to react for 2.5 hours at room temperature with mixing in a rotator, followed by the addition of 0.1 milliliters of quenching buffer for an additional 30-minute incubation at room temperature with mixing. At the end of the second reaction, place the scintillation vial onto the magnetic stand and wash the particles in one milliliter of resuspension buffer four times, removing the buffer when it turns clear after each wash and replacing it with fresh resuspension buffer for the subsequent wash on the magnet.
After the last wash, aspirate the solution and remove the particles from the magnetic stand, then store the particles in one milliliter of fresh resuspension buffer at four degrees Celsius for up to six months. To isolate antigen-specific CD8+T cells, macerate the spleens and lymph nodes from wild type C57 black 6J mice through a sterile, 70-micrometer cell strainer with frequent PBS rinses and use a no-touch CD8+T cell isolation kit according to the manufacturer's instructions to eliminate the non-CD8+T cells. After counting, collect the isolated CD8+T cells by centrifugation and resuspend the pellet in 100 microliters of PBS supplemented with 0.5%Bovine Serum Albumin and two-millimolar EDTA, then incubate the cells with the nanoparticle artificial antigen-presenting cells such that there are one times 10 to the 11th peptide-loaded MHC-ig per one times 10 to the sixth isolated CD8+T cells in a sterile, five-milliliter polystyrene round-bottomed tube for one hour at four degrees Celsius with continual mixing.
At the end of the incubation, wash the nanoparticle cell suspension three times on the magnetic stand as demonstrated, resuspending the artificial antigen-presenting cells and the enriched CD8+T cells in 500 microliters of fresh, supplemented medium with 1%T cell growth factor after the last wash. For maximum cell recovery, it is critical to leave the particles and cells on the magnetic column for at least two minutes and to aspirate the buffer carefully without disrupting the particles and cells subject to the magnetic field. After counting, seed 2.5 times 10 to the fifth enriched CD8+T cells and magnetic particle mixture per 160 microliters of supplemented medium plus 1%TCGF in a 96-well U-bottomed plate.
On Day Three, feed the cells with 80 microliters of supplemented medium with 2%T cell growth factor per well and return the plate to the cell culture incubator for four more days. On Day Seven, harvest the stimulated cells into a five-milliliter round-bottomed tube for counting and collect the cells by centrifugation. Resuspend the pellet in 0.5 milliliters of PBS supplemented with 0.05%sodium azide and 2%fetal bovine serum for counting and aliquot five times 10 to the fourth to five times 10 to the fifth cells into new five-milliliter round-bottomed tubes for antigen-specific staining.
Label the appropriate tubes with biotinylated MHC-ig and anti-mouse CD8a for one hour at four degrees Celsius, followed by a centrifuge wash in fresh PBS to remove any excess biotinylated immunoglobulin, then stain the samples with an appropriate streptavidin-conjugated secondary antibody and an appropriate live-dead fixable dead cell strain for 15 minutes at four degrees Celsius and read the cells on a flow cytometer to determine the specificity and number of the antigen-specific CD8+T cells according to standard protocols. Here a successful protein conjugation was achieved using three different methods of protein attachment to particles. If the aAPC ligand density is too low, there will not be an effective stimulation of the antigen-specific CD8+T cells.
Quality control of the biotinylated dimer can be completed on transgenic CD8+T cells to verify staining. For example, these representative results demonstrate a positive staining with gp100-specific CD8+T cells with non-antigen-specific b6 CD8+T cells as a background control. Further, knowledge of the background staining of the biotinylated dimer is critical, as any percentage lower than this staining in the cognate-stained tubes should be considered a negative result.
After seven days of enrichment and expansion, between two times 10 to the fourth and two times 10 to the fifth antigen-specific and five to 50%CD8+T cells can be expected from an original five times 10 to the sixth endogenous CD8+T cell starting population. The same methodology can be used to isolate and stimulate human antigen-specific CD8+T cells with similar increases in percentages and numbers of antigen-specific CD8+T cells observed after only one week of expansion following enrichment. While attempting this procedure, it's important to remember to perform the appropriate reagent quality checks as described.
Following this procedure, other methods like flow cytometry for intracellular cytokine staining or surface protein expression can be performed to answer additional questions about the functionality or phenotype of the cells. This technique paves the way for researchers in the basic immunology field to explore the breadth, depth, and dynamics of antigen-specific responses in other biomedical fields such as cancer, infectious disease, or autoimmunity research.
