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

JoVE Encyclopedia of Experiments

Immunologie

0 weergaven • 5:41 min • July 8th, 2025

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Macerate mouse spleen and lymph nodes on a cell strainer. Flush the strainer with a suitable buffer to collect splenocytes and lymphocytes.

Incubate the cells with a biotinylated antibody cocktail and magnetic anti-biotin microbeads that bind to the non-CD8+ cells.

Pass the cell suspension through a magnetic column. Owing to the magnetic force, the bead-bound non-CD8+ cells bind to the column wall while the CD8+ T cells elute out.

Incubate the eluted cells with artificial antigen-presenting cells, aAPCs. These are magnetic nanoparticles functionalized with antigen-loaded major histocompatibility complex-immunoglobulin fusion protein, MHC-Ig, and anti-CD28 antibody.

The antigen-loaded MHC-Igs bind to distinct receptors on the antigen-specific CD8+ T cells. The anti-CD28 antibodies interact with the T cell CD28 receptors, initiating T cell activation.

Place the tube containing magnetic nanoparticles and cell mixture on a magnetic stand. The aAPC-bound CD8+ T cells get attracted to the magnetic field, while non-specific CD8+ T cells remain suspended. Remove the supernatant.

Remove the tube from the magnetic stand. Resuspend the aAPC-bound cells in T cell growth factor-containing media. Seed them in a multi-well plate.

The growth factors induce antigen-specific CD8+ T cell expansion. 

To isolate antigen-specific CD8-positive T cells, macerate the spleens and lymph nodes from wild-type C57Black/6j mice through a sterile 70-micrometer cell strainer with frequent PBS rinses, and use a no-touch CD8-positive T cell isolation kit, according to the manufacturer's instructions, to eliminate the non-CD8-positive T cells.

After counting, collect the isolated CD8-positive T cells by centrifugation, and resuspend the pellet in 100 microliters of PBS, supplemented with 0.5% bovine serum albumin and 2-millimolar EDTA.

Then, incubate the cells with the nanoparticle artificial antigen-presenting cells such that there are 1 x 1011 peptide-loaded MHC-Ig, per 1 x 106 isolated CD8-positive T cells in a sterile 5-milliliter polystyrene round-bottom tube for 1 hour at 4 degrees Celsius with continual mixing.

At the end of the incubation, wash the nanoparticle cell suspension three times on the magnetic stand as demonstrated, re-suspending the artificial antigen-presenting cells and the enriched CD8-positive 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 in cells on the magnetic column for at least 2 minutes, and to aspirate the buffer carefully without disrupting the particles and cells subject to the magnetic field.

After counting, seed 2.5 x 105 enriched CD8-positive 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 5-milliliter round-bottom 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 5 x 104 to 5 x 105 cells into new 5-milliliter round bottom tubes for antigen-specific staining.

Label the appropriate tubes with biotinylated MHC-Ig and anti-mouse CD8a for 1 hour at 4 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 stain for 15 minutes at 4 degrees Celsius, and read the cells on a flow cytometer to determine the specificity and number of the antigen-specific CD8-positive T cells, according to standard protocols.

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Laatst bijgewerkt: 22 augustus 2026