Nanoparticle-Based Enrichment and Expansion of Antigen-Specific CD8+ T Cells

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Enrich Antigen-specific CD8+ T Cells with Prepared Nanoparticle Artificial Antigen-Presenting Cells.

1. Isolate CD8+ T cells.
   1. Euthanize the animals by exposure to isoflurane followed by cervical dislocation.
   2. Remove the spleen and lymph nodes from wild-type C57BL/6j mice and place them in a solution of PBS. Macerate the organs and elute the cells through a sterile 70 µm cell strainer with frequent washes of PBS.
   3. To eliminate non-CD8+ T cells, use a no-touch CD8+ T cell isolation kit and follow the manufacturer's instructions.
      NOTE: Each antigen condition requires at least 3 x 10⁶ CD8+ T cells.
2. Add the nanoparticle aAPCs to bind to the CD8+ T cells.
   1. Following the isolation, concentrate to a volume of 100 µL in PBS with 0.5% bovine serum albumin (BSA) and 2 mM EDTA.
   2. Determine the number of aAPCs to add by calculating based on the ratio of 10¹¹ aAPC-bound, peptide-loaded MHC-Ig for every 10⁶ CD8+ T cells.
   3. Incubate aAPC particles and CD8+ T cells for 1 h at 4 °C with continual mixing in a sterile 5 mL polystyrene round bottom tube.
3. Prepare supplemented media and T cell growth factor (TCGF) to elute and culture CD8+ T cells.
   1. For supplemented media, supplement complete RPMI 1640 media (with glutamine) with 1x non-essential amino acids, 1 mM sodium pyruvate, 0.4x vitamin solution, 92 µM 2-mercaptoethanol, 10 µM ciprofloxacin and 10% fetal bovine serum (FBS).
   2. To make TCGF, follow established protocols.
      NOTE: TCGF is an in-house cocktail of human immune cytokines which is essential to provide T cells with additional stimulation signals needed to grow. TCGF could be exchanged for known T cell stimulatory cytokines such as IL-2, IL-7, or IL-15; however, each may polarize the T cell response accordingly.
4. Wash and enrich aAPC and CD8+ T cell mixture.
   1. Wash the magnetic particle aAPCs, first using the PBS buffer with 0.5% BSA and 2 mM EDTA, second using supplemented media, and third using supplemented media with 1% TCGF.
   2. Elute aAPCs and enriched CD8+ T cells in 500 µL of supplemented media with 1% TCGF.
   3. Count the cells using a hemocytometer and plate in a 96 U-bottomed plate in 160 µL per well of supplemented media with 1% TCGF at a concentration of 2.5 x 10⁵ CD8+ T cells/mL.
   4. If isolating using aAPCs with only peptide-loaded MHC-Ig on the surface (no co-stimulatory signals), then complete Step 4.5. If isolating using aAPCs with both peptide-loaded MHC-Ig on the surface and co-stimulatory signals, then proceed to Step 5.
5. Add the magnetic particles coated with co-stimulatory signals on the surface to the enriched fraction and add a magnetic field to co-cluster the stimulatory signals on the surface of the T cells.
   1. To the enriched fractions, add an equimolar (or greater depending on the application-see section about aAPC properties to control) stimulatory antibody to the number of peptide-loaded MHC-Ig on the particle.
   2. Allow co-stimulatory magnetic particles to bind to enriched CD8+ T cells for 1 h at 4 °C.
   3. Add a magnetic field by placing the culture plate between two neodymium N52 disk magnets of 1.9 cm (0.75 inches) in length.
      NOTE: N52 disk magnets have an extremely strong field. Care should be taken both to store them with spacers between magnets, as it is hard to remove them from one another when putting them on the culture plates. To minimize the magnets from sticking to the metal components of the incubator, place them in 50 mL conical tube Styrofoam containers on both the bottom and the top.

2. Expand and Detect Antigen-specific CD8+ T Cells with Prepared Nanoparticle Artificial Antigen-Presenting Cells

1. Add the 96 U-bottomed well plate with aAPCs and CD8+ T cells in a humidified 5% CO₂, 37 °C incubator for 3 days. On day 3, feed the cells with 80 μL per well of supplemented media with 2% TCGF and place them back into the incubator until day 7.
2. On day 7, harvest the stimulated cells into a 5 mL round bottom tube for counting.
3. Once all the solution is harvested, spin down the harvested cells to resuspend in 0.5 mL of PBS with 0.05% sodium azide and 2% FBS. Count viable cells by staining with trypan blue and counting on a hemocytometer.
4. Remove 50,000-500,000 counted cells into two new 5 mL round bottom tubes for antigen-specific staining. One tube will be used for the cognate peptide-MHC stain, and the other tube will be used for the non-cognate stain to determine background staining.
5. Add 1 μg of biotinylated MHC-Ig to the respective cognate and non-cognate tubes in 100 μL of PBS with 0.05% sodium azide and 2% FBS with allophycocyanin (APC)-conjugated rat anti-mouse CD8a, clone 53-6.7 (dilution ratio of 1:100) for 1 h at 4 °C.
6. Add secondary streptavidin and live dead stain. Wash out excess biotinylated MHC-Ig with PBS through centrifugation. Then stain all samples with a 1:350 ratio of phycoerythrin (PE)-labeled streptavidin and a 1:1000 ratio of a live/dead fixable green dead cell stain for 15 min at 4 °C.
7. Read all samples on a flow cytometer to determine the specificity and the number of antigen-specific cells.
   1. Wash out excess secondary and live/dead stains by centrifugation and resuspend with 150 μL of PBS buffer with 0.05% sodium azide and 2% FBS to read on a flow cytometer.
8. Determine the number and percent of antigen-specific cells with data analysis software.
   1. To determine the percent of antigen-specific cells, use the following gates in the respective order live+, lymphocyte+ (forward scatter by side scatter), CD8+, and Dimer+. Determine the Dimer+ gate by comparing the non-cognate to the cognate stain.
   2. Determine the percentage of antigen-specific cells in a sample by subtracting the percentage of Dimer+ of the cognate MHC-Ig stain from the non-cognate MHC-Ig stain.
   3. Using this percentage of antigen-specific cells, multiply it by the number of cells counted, yielding the number of antigen-specific cells resulting from the enrichment and expansion.
      NOTE: Compensation will have to be set up on the flow cytometer since there is spectral overlap with the fluorophores used in this panel.