A Peptide-MHC-I Tetramer Staining Technique to Analyze SIV-Specific CD8+ Memory T Cells

Published: January 31, 2024

Abstract

Source: Gonzalez-Nieto, L., et al. Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining. J. Vis. Exp. (2016)

This video demonstrates a method for characterizing CD8+ memory T cells specific to simian immunodeficiency virus (SIV) using peptide-major histocompatibility complex class I (pMHC-I) tetramers. A pMHC-I tetramer is a complex of four MHC-I molecules bound to SIV-specific peptides and conjugated to a fluorophore. These tetramers selectively bind to T cell receptors (TCRs) on SIV-specific CD8+ T cells, facilitating the characterization of these cells.

Protocol

1. PKI Treatment

NOTE: This is optional, but recommended for Mamu-B*017:01 tetramers.

  1. Resuspend PBMC in R10 medium at a concentration of 1.6 x 107 cells/ml.
  2. Add 50 µl of this cell suspension to the corresponding flow cytometry tubes. Add 50 µl of a 100 nM solution of PKI to each tube.
  3. Vortex each tube. Incubate at 37 °C for 30 min. By the end of this step, each tube should have 100 µl of a cell suspension containing 8.0 x 105 PBMC and 50 nM of PKI.
  4. Proceed to the pMHC-I tetramer staining step.

2. Staining with Fluorochrome-labeled pMHC-I Tetramers

  1. Before preparing the pMHC-I tetramer master mix, centrifuge the pMHC-I tetramer tubes at 20,000 x g for at least 15 min at 4 °C. The goal of this step is to pellet protein aggregates in the pMHC-I tetramer solution that may increase background staining. Once the centrifugation step is done, avoid pipetting from the bottom of the tube where the protein aggregates will have accumulated.
  2. Prepare enough pMHC-I tetramer master mix to stain all experimental tubes. Prepare an excess of 15% of the total volume of this master mix to account for pipetting errors.
  3. Dilute pMHC-I tetramers in stain buffer (e.g., Brilliant Stain Buffer) so that 25 µl of the pMHC-I tetramer master mix is added to each test.
    1. Label PBMC with two pMHC-I tetramers per test; one conjugated to allophycocyanin (APC) and the other to Brilliant Violet (BV) 421.
  4. Add 8.0 x 105 PBMC to the corresponding flow cytometry tubes in a final volume of 100 µl. If the cells were subjected to the PKI treatment described above, they should already be resuspended in 100 µl at this step.
  5. Add 25 µl of pMHC-I tetramer master mix to the corresponding flow cytometry tubes. By the end of this step, the final volume in each tube should be 125 µl.
  6. Vortex each tube in order to homogenize the cell suspension.
  7. Incubate in the dark at room temp for 45 min. Prepare the surface-staining monoclonal antibody (mAb) cocktail during this 45-minute incubation.

3. Surface Staining

  1. Prepare enough mAb master mix to stain all experimental tubes. Prepare an excess of 15% of the total volume of this master mix to account for pipetting errors.
  2. Adjust the volume of the master mix with stain buffer so that 50 µl is added per test.
  3. For proper exclusion of non-CD8+ T-cells and delineation of memory subsets, use titrated amounts of mAbs directed against the following molecules in the surface staining master mix.
    NOTE: The fluorochromes conjugated to each mAbs are provided as a reference: CD14 BV 510, CD16 BV 510, CD20 BV 510, CD8α BV 785, CD28 PE Cy7, and CCR7 FITC. Note that the mAbs against CD14, CD16, and CD20 are conjugated to the same fluorochrome (i.e., BV 510) since they will be included in the "dump" gate.
  4. Include a fixable dye for discriminating dead cells in the surface staining master mix [i.e., amine-reactive dye (ARD)]. Make sure that the ARD reagent is conjugated to a fluorochrome with a similar emission spectrum as the ones used in the "dump" gate. In this case, use ARD Aqua.
  5. Add 50 µl of the staining master mix described in 3.1-3.4 to the corresponding flow cytometry tubes. By the end of this step, the final volume in each tube should be 175 µl.
  6. Vortex each tube. Incubate in the dark at room temp for 25 min.
  7. Wash cells with wash buffer (phosphate-buffered saline (PBS) solution containing 0.1% of bovine serum albumin and 0.45 g/L NaN3).
    CAUTION: Sodium azide (NaN3) is a toxic substance. Exposure to even minute amounts can cause symptoms. Handle this substance according to the guidelines specified by the Environmental Health and Safety (EHS) Office at the institution where these experiments are being performed.
  8. Centrifuge tubes at 510 x g for 5 min. Carefully decant supernatant into a separate waste container. Be sure not to disturb the pellet.
    CAUTION: Do not decant wash buffer supernatant into reservoirs containing bleach as it can react with the sodium azide present in the wash buffer and result in the formation of a toxic gas. Contact the EHS Office at the institution where these experiments are being performed for guidelines on how to dispose of sodium azide.
  9. After decanting, vortex the cells in the leftover liquid retained in each tube.

4. Cell Fixation

  1. Add 250 µl of a 2% paraformaldehyde (PFA) solution to all tubes to fix the cells.
    CAUTION: PFA is a toxic substance. Exposure to even minute amounts can cause symptoms. Handle this substance according to the guidelines specified by the EHS Office at the institution where these experiments are being performed.
    1. Since the 2% PFA solution must be isotonic to the cells, use PBS to prepare this solution. One hundred milliliters of a 2% PFA solution is enough for multiple experiments. Thoroughly vortex all tubes immediately after the addition of 2% PFA in order to prevent the formation of cell aggregates.
  2. Incubate in the dark at 4 °C for 20 min.
  3. Wash cells by repeating steps 3.7-3.9. Once this step is completed, the cells can be stored in the dark at 4 °C for 24-48 hr. Before proceeding to the Permeabilization phase, vortex the tubes thoroughly.

5. Permeabilization of Cells

  1. Add 500 µl of permeabilization buffer. Vortex all tubes.
  2. Incubate in the dark at room temp for 10 min.
  3. Wash cells by repeating steps 3.7-3.9.

6. Intracellular Staining

  1. Prepare enough mAb master mix to stain all experimental tubes.
  2. Adjust the volume with PBS or stain buffer so that 50 µl of the intracellular mAb master mix is added per test.
  3. Prepare the mAb master mix described in 6.1 and 6.2 using titrated amounts of mAbs directed against CD3 and granzyme B (Gzm B).
    NOTE: TCR engagement by pMHC-I tetramers can result in CD3 internalization, which can interfere with the detection of tetramer + CD3+ CD8+ T-cells if the anti-CD3 mAb is added to the surface staining master mix. To avoid this, add the anti-CD3 mAb at this stage, that is after the cells are permeabilized. The fluorochromes conjugated to each mAb are listed as a reference: CD3 PerCP Cy5.5 and Gzm B PE.
  4. Add 50 µl of mAb cocktail to the corresponding tubes. Incubate in the dark at room temp for 30 min.
  5. Wash cells by repeating steps 3.7-3.9. The tubes are ready to be acquired in a flow cytometer.

Declarações

The authors have nothing to disclose.

Materials

Dasatinib Axon medchem Axon 1392 Must be resuspended in DMSO and immediately stored at -20°C
RPMI w/ Glutamax gibco/ Life Technologies 61870-036 Must be stored at 4 °C 
Heat Inactivated FBS gibco/ Life Technologies 10082-147 Must be stored at 4 °C 
Penicillin-Streptomycinp-Amphotericin B Lonza 17-745E Must be stored at 4 °C 
DMSO, Anhydrous Life Technologies D12345 Store at room temperature.
5-mL Round-Bottom Polypropylene Tubes VWR 60819-728
Fluorochrome-conjugated pMHC-I tetramers NIH Tetramer Core or MBL, Inc. Must be stored and maintained at  4 °C. Centrifuge at 20,000 x g for 15 min before use. Do not freeze.
Fluorochrome-conjugated mAbs Various companies Must be stored and maintained at  4 °C. Do not freeze.
LIVE/DEAD Fixable Aqua Dead Cell Stain Kit Life Technologies l34957 Must be stored at -20 °C. Resuspend each aliquot in 50 μL of DMSO prior to use.
Brilliant Stain Buffer BD Biosciences 563794 Must be stored at  4 °C 
Phosphate Buffered Saline VWR 97064-158 Store at room temperature
Albumine Bovine VWR 700011-230 Must be stored at  4 °C 
Sodium Azide VWR 97064-646 Store at room temperature. Toxic substance. Do not mix with bleach.
Bleach VWR 89501-620 Corrosive chemical, cannot be mixed with sodium azide. Handle with care
Paraformaldehyde Electron Microscopy Sciences 15714-S Flammable, corrosive, and toxic reagent. Handle with care
Polysorbate 20 (Tween-20) Alfa Aesar L15029 Store at room temperature
Permeabilization Solution 2  BD Biosciences 340973 Toxic and corrosive reagent. Handle with care
Sarsdet Tubes 1.5mL screw top VWR 72.692.005
2.0ml DNA/RNA Low bind Tubes Eppendorf 22431048 The use of Sterile microtubes is preffered 
Vortex mixer
Biosafety Cabinet 
Milli Q Intergral Water Purification system EMD Millipore ZRXQ010WW Molecular Biology grade water from any provider may be used
Microcentrifuge
Centrifuge
4 °C  refrigerator
BD LSR II BD Biosciences Flow cytometer must contain lasers and filters that are compatible with the staining panel used.
Deionized water
Aluminum Foil VWR SCIENTIFIC INC. 89068-738
Incubator Must be able to maintain 37 °C  internal temperature
FACS Diva software BD Biosciences
Flowjo software version 9.6 Flowjo Used to analyze FCS files generated by FACS Diva software
Micropippette tips

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A Peptide-MHC-I Tetramer Staining Technique to Analyze SIV-Specific CD8+ Memory T Cells. J. Vis. Exp. (Pending Publication), e21921, doi: (2024).

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