The isolation of antigen-specific T cells has long been of interest to immunologists in vaccine, infectious disease, immuno-oncology and immunotherapy research.

Antigen-specific T cells can be isolated using two methods:

The following protocols describe how to isolate antigen-specific T cells using the AIM and tetramer strategies with EasySep™ Release Human PE Positive Selection Kit (Catalog #17654) or EasySep™ Human PE Positive Selection Kit II (Catalog #17664). EasySep™ Release Human PE Positive Selection Kit is recommended if the positive selection marker is a PE-conjugated HLA class I-peptide tetramer, a PE-conjugated anti-human CD137 antibody, or when particle-free positively selected cells are required for downstream applications. EasySep™ Human PE Positive Selection Kit II can be used for the enrichment of T cells labeled with PE-conjugated HLA class I-peptide tetramers, or PE-conjugated antibodies that bind to CD134, CD137, or CD154. Both protocols have undergone in-house testing and provide a reliable guide to support your antigen-specific T cell isolation experiment design. These protocols may be adjusted to fit your research needs.

For further assistance and information on other possible product choices to isolate antigen-specific T cells, please contact techsupport@stemcell.com.

Protocols

Pre-Isolation Peptide Activation of Antigen-Specific T Cells with AIM Method

If using the AIM method, first perform the in vitro stimulation step with human PBMCs, prior to the antigen-specific T cell isolation. For this step, incubate the PBMCs with the viral peptide pools of choice for the recommended duration to induce expression of activation markers such as CD137 (4-1BB). The protocol below provides suggested steps to stimulate antigen-specific T cells with the CMV (pp65) Peptide Pool.

1. Prepare a PBMC suspension at 1 x 10⁷ cells/mL in ImmunoCult™-XF T Cell Expansion Medium for T cell expansion and seed the cells to either 6-well or 12-well tissue culture plates at a cell density of 5 x 10⁶ cells/cm².
2. Add the viral peptide pool(s) at 1 μg/mL per peptide to the cell suspension to stimulate the T cells. Mix the cells and peptide(s) carefully and incubate at 37°C and 5% CO₂ for the recommended time.
   Note: Antibody stimulation conditions
   Optimal time points differ for each antibody-induced marker, and must be optimized. As a starting point, activate cells for 16 - 24 hours for CD137, 24 - 48 hours for CD134, and 6 - 18 hours for CD154.
3. Harvest the cells after stimulation by gently pipetting up and down to ensure all cells are in suspension, then transfer to an appropriate tube from the tissue culture plates.
   Optional: To improve PBMC recovery, wash each well with 1 - 2 mL of PBS and add the recovered cells to the harvested cells.
4. Centrifuge the cells at 300 x g for 10 minutes (low brake) at room temperature. Discard the supernatant and resuspend the cells in EasySep™ Buffer at the recommended minimal volume for EasySep™ Release Human PE Positive Selection Kit or EasySep™ Human PE Positive Selection Kit II (indicated in the EasySep™ Release Human PE Positive Selection Kit Protocol step 1 below).
5. Count the cells using 3% Acetic Acid with Methylene Blue (see How to Count Cells with a Hemocytometer). Trypan blue may also be used to assess cell viability.
6. Dilute the PBMCs to 1 x 10⁸ cells/mL with EasySep™ Buffer, or use at the recommended minimum volume.

EasySep™ Release Human PE Positive Selection Kit Protocol

This protocol section describes how to isolate antigen-specific T cells from PBMCs using EasySep™ Release Human PE Positive Selection Kit for tetramer or CD137 (AIM method) staining.

1. Prepare a PBMC suspension (containing antigen-specific T cell population) in EasySep™ Buffer at 1 x 10⁸ cells/mL (maximum volume of 2 mL) in a 5 mL (12 x 75 mm) polystyrene round-bottom tube. If starting with fewer than 5 x 10⁷ cells, resuspend cells in 0.5 mL.
2. Add EasySep™ Anti-Human CD32 (Fc gamma RII) Blocker (Component #18520) to the cell suspension at 200 μL/mL of sample. Mix and incubate at room temperature for 5 minutes.
3. Label the cells of interest with desired antibodies:
   1. For the AIM method, label the T cells with PE-conjugated antibodies against the target activation marker(s) of interest; e.g. if using CD137 as the selection marker, add PE-conjugated anti-human CD137 antibody (clone 4B4-1). The primary antibody concentration needs to be optimized for each PE-conjugated reagent; however, 1 μg/mL of PE-conjugated antibody is recommended as a starting point. Incubate for 10 minutes at room temperature, protected from light.
   2. For the tetramer staining method, add PE-conjugated tetramer such as the CMV pp65-specific PE-conjugated tetramer (iTAg Tetramer/PE – HLA-A*02:01 CMV pp65 (NLVPMVATV)). The tetramer concentration needs to be optimized for each PE-conjugated tetramer; however, 10 μL/mL of PE-conjugated tetramer is recommended as a starting point. Incubate for 30 minutes at room temperature, protected from light.
4. Add 2 volumes of EasySep™ Buffer and centrifuge the cells at 300 x g for 10 minutes (low brake).
5. Carefully aspirate and discard supernatant, and resuspend the cells with EasySep™ Buffer at the same volume as in step 1. Once resuspended, return the sample to the tube used for step 1.
   Note: If needed, the wash step can be performed in a larger tube.
6. Add EasySep™ Release PE Positive Selection Cocktail (Component #17654C) to the sample at 100 μL/mL. Mix and incubate for 10 minutes at room temperature.
7. Vortex EasySep™ Releasable Rapidspheres™ (Component #50201) for 30 seconds.
   Note: Particles should appear evenly dispersed.
8. Add EasySep™ Releasable Rapidspheres™ to the sample at 75 - 100 μL/mL. Mix and incubate for 5 minutes at room temperature.
   Note: Adjust EasySep™ Releasable Rapidspheres™ volume depending on whether high purity or high recovery is the priority; recommended volume is 75 μL/mL for purity and 100 μL/mL for recovery.
9. Add EasySep™ Buffer to top up the sample to 2.5 mL. Mix by gently pipetting up and down 2 - 3 times.
10. For the magnetic separations, place the tube without a lid into the purple EasySep™ Magnet and incubate for 10 minutes (first magnet incubation step).
    Note: Cells are subjected to four magnet incubation steps, the first for 10 minutes.
11. Pick up the magnet and, in one continuous motion, invert the magnet and tube, pouring off the supernatant. Remove the tube from the magnet; this tube contains the desired positively selected cells.
12. Resuspend cells in 2.5 mL of EasySep™ Buffer.
13. Place the tube (without lid) into the purple EasySep™ Magnet and incubate for 5 minutes (second magnet incubation step).
14. Pick up the magnet, and, in one continuous motion, invert the magnet and tube, pouring off the supernatant. Remove the tube from the magnet; this tube contains the desired positively selected cells.
15. Resuspend cells in 2.5 mL of EasySep™ Buffer.
16. Repeat Steps 13 and 14 twice for a third and fourth separation step.
    Optional: Reduce magnetic steps for higher recovery (but reduced purity) or when the start frequency of the desired cells is > 5%.
17. Dilute EasySep™ Release Buffer (Component #20165) 1 in 40 with EasySep™ Buffer.
    Note: EasySep™ Release Buffer (1X) must be prepared on the day of use.
18. Add EasySep™ Release Buffer (1X) to top up the positively selected cell sample to 2.5 mL. Mix by gently pipetting up and down 2 - 3 times, and incubate for 3 minutes.
19. Place the tube (without lid) into the magnet and incubate for 5 minutes. This step will remove the magnetic particles.
20. Pick up the magnet, and in one continuous motion, invert the magnet and tube, pouring the enriched cell suspension into a new tube.
21. Isolated cells (in the new tube) are now ready for use in downstream applications.
    Optional: Centrifuge the cells at 300 x g for 10 minutes (low brake) to concentrate the isolated cells. Decant the supernatant and resuspend the isolated cells in the desired medium.

EasySep™ Human PE Positive Selection Kit II Protocol

This protocol section describes how to isolate antigen-specific T cells from PBMCs using EasySep™ Human PE Positive Selection Kit II for tetramer, CD134, CD137, or CD154 stainings.

Prior to separation, cells are treated with an Fc receptor (FcR) blocker. Cells are then labeled with PE-conjugated primary antibodies or PE-tetramer at room temperature, followed by the addition of the EasySep™ Positive Selection reagents. Following magnetic separation, isolated cells are resuspended in the desired medium and are ready for use in downstream applications.

1. Prepare a PBMC suspension (containing antigen-specific T cell population) in EasySep™ Buffer at 1 x 10⁸ cells/mL (maximum volume of 2 mL) in a 5 mL (12 x 75 mm) polystyrene round-bottom tube. If starting with fewer than 2 x 10⁷ cells, the minimum PBMC cell suspension volume is 0.2 mL at 1 x 10⁸ cells/mL.
2. Add EasySep™ Anti-Human CD32 (Fc gamma RII) Blocker (Component #18520) to the cell suspension at 200 μL/mL of sample. Mix and incubate at room temperature for 5 minutes.
3. Label cells of interest with desired antibodies:
   1. For the AIM method, label the T cells with PE-conjugated antibodies against the target activation marker(s) of interest; e.g. if using CD137 as the selection marker, add PE-conjugated anti-human CD137 antibody (clone 4B4-1). The primary antibody concentration needs to be optimized for each PE-conjugated reagent; however, 1 μg/mL of PE-conjugated antibody is recommended as a starting point. Incubate for 10 minutes at room temperature, protected from light.
   2. For the tetramer staining method, add PE-conjugated tetramer such as the CMV pp65-specific PE-conjugated tetramer (iTAg Tetramer/PE – HLA-A*02:01 CMV pp65 (NLVPMVATV)). The primary tetramer concentration needs to be optimized for each PE-conjugated tetramer; however, 10 μL/mL of PE-conjugated tetramer is recommended as a starting point. Incubate for 30 minutes at room temperature, protected from light.
4. Add 2 volumes of EasySep™ Buffer and centrifuge the cells at 300 x g for 10 minutes (low brake).
5. Carefully aspirate and discard supernatant, and resuspend the cells with EasySep™ Buffer at the same volume as in step 1. Once resuspended, return the sample to the tube used for step 1.
   Note: If needed, the wash step can be performed in a larger tube.
6. Add EasySep™ PE Positive Selection Cocktail (Component #18151) to sample at 100 μL/mL. Mix and incubate for 10 minutes at room temperature.
7. Vortex RapidSpheres™ for 30 seconds.
   Note: Particles should appear evenly dispersed.
8. Add EasySep™ Dextran Rapidspheres™ (Component # 50100) to sample at 12.5 - 50 μL/mL. Mix and incubate for 5 minutes at room temperature.
   Note: Adjust EasySep™ Dextran Rapidspheres™ volume depending on whether high purity or high recovery is the priority; recommended volume is 12.5 μL/mL for purity and 50 μL/mL for recovery.
9. Add EasySep™ Buffer to top up the sample to 2.5 mL. Mix by gently pipetting up and down 2 - 3 times.
10. For the magnetic separations, place the tube without a lid into the purple EasySep™ Magnet and incubate for 10 minutes (first magnet incubation step).
11. Pick up the magnet and, in one continuous motion, invert the magnet and tube, pouring off the supernatant. Remove the tube from the magnet; this tube contains the desired positively selected cells.
12. Resuspend cells in 2.5 mL of EasySep™ Buffer.
13. Place the tube (without lid) into the purple EasySep™ Magnet and incubate for 5 minutes (second magnet incubation step).
14. Pick up the magnet and, in one continuous motion, invert the magnet and tube, pouring off the supernatant. Remove the tube from the magnet; this tube contains the desired positively selected cells.
15. Resuspend cells in 2.5 mL of EasySep™ Buffer.
16. Repeat Steps 13 and 14 twice for a third and fourth separation step.
    Optional: Reduce magnetic steps for higher recovery (but reduced purity) or use when the start frequency of the desired cells is > 5%.
17. Resuspend cells in desired medium. Be sure to collect cells from the sides of the tube. Isolated cells are now ready for use in downstream applications.

Data Figures

The protocols above describe the isolation of antigen-specific T cells with EasySep™ Release Human PE Positive Isolation Kit and EasySep™ Human PE Positive Isolation Kit II. The flow cytometry data figures below show the antigen-specific T cell enrichment achieved by these two EasySep™ kits, illustrating the effectiveness of each method.