IntroductionThe frequency of HBV-specific CD8+ T cells can be very low, depending on the specificity. Therefore, they cannot always be detected by conventional ex vivo tetramer staining using 106 PBMCs. Tetramer-based magnetic enrichment enables the detection of these rare virus-specific CD8+ T-cell population by concentration of tetramer-labeled cells. Additionally, if an ex vivo response by conventional tetramer staining can be detected, this enrichment approach can increase the frequency of tetramer-positive cells for the following analysis.
Materials and Reagents
- Tetramers of HLA-A*02-restricted HBV-derived epitopes (core18:FLPSDFFPSV, pol455: GLSRYVARL, env183: FLLTRILTI) (The tetramers are not commercially available but gained from a cooperation with David Price (Cardiff University, UK.)
- Anti-fluorochrome MACS MicroBeads
- Anti-PE, order no: 130-048-801
- Anti-APC, order no: 130-090-855
- MACS LS columns (Miltenyi Biotec, order no.: 130-042-401)
- MACS manual separator (MidiMACS™ or QuadroMACS™ Separators)
- MACS buffer (500 ml PBS, 2.5 g BsA (0,5%), 2 ml EDTA 0,5 M (2 mM))
- RPMI media (RPMI supplemented with 1640 with 10% fetal bovine serum, 1% penicillin- streptomycin and 1.5% 1 M HEPES)
- Benzonase nuclease (Milipore, order no.: 70746-3)
- Prepare the cells you want to use for enrichment.
- Use freshly isolated peripheral blood mononuclear cells (PBMCs) or thaw frozen PBMCs in pre-warmed RPMI.
- Thawing can be performed with Bezonase nuclease (e.g. if cells are frozen for >10 years or cell clumping is expected for whatever reason).
- Use at least 107 PBMCs
- Depending on the frequency of the analyzed virus-specific CD8+ T cells, enrichment can be performed also with less cells.
- Centrifuge cells (500xg, 10 minutes) and discard the supernatant.
- Optional: Incubation of cells with Benzonase-containing RPMI for 10-30min (50Uml -1 Benzonase) at 37 °C, 5% CO2 (after incubation wash cells once with RPMI (500xg, 10 minutes)).
- Resuspend cell pellet in 100μl MACS buffer.
- Centrifuge tetramers at full speed for 4 min at 4°C before use. Add optimized volume of tetramers (labelled with APC or PE), resuspend, incubate 30 min at room temperature in the dark.
- Avoid light exposure when working with fluorochrome-conjugated tetramers!
- Consider tetramer titration for the appropriate amount you have to add!
- After incubation add 5ml MACS buffer.
- Centrifuge the cells (500xg, 10 minutes), discard the supernatant.
- Add 50μl anti-APC and/or anti-PE beads (Miltenyi Biotec) and fill up to a final volume of 250μl with MACS buffer (the optimal concentration of beads should be titrated).
- If you use more cells (e.g. 8x107 PBMCs) or the frequency of your virus-specific CD8+ T cells is very high you probably have to adjust the volume of beads to catch all your tetramer+ cells (the original enrichment protocol used 100μl of beads and a final volume of 500μl).
- Incubate 20 minutes at 4°C in the dark.
- Add 5ml MACS buffer and centrifuge the cells (500xg, 10 minutes), discard the supernatant.
- Resuspend cell pellet in 1ml MACS buffer.
- Remove 5μl for counting the “pre”-fraction if you want to calculate your frequency.
- Caution! Your total volume is about 1.2ml (because the cells also have a volume), consider this in your calculation of the total “pre” cell number.
- Remove 5μl for staining of the “pre”-fraction and plate them in your staining plate.
- before removing the cells you can add 5µl into the “pre” wells of the staining plate to prevent evaporation of the low volume of 5μl.
- in general, staining of the pre-fraction is required for the calculation of the frequency of enriched virus-specific CD8+ T cells (number of tetramer+ cells/ number of CD8+ cells).
- Perform a magnetic separation with according to the manufacturer’s instructions (use a LS column even if your initial cell count was very low – the enrichment does not really work well with MS columns).
- Place the column into the MACS magnet.
- Place a 15ml falcon tube under the column.
- Equilibrate LS column with 3ml of MACS buffer, let the whole buffer run through the column.
- Discard the falcon tube and place a new tube under the column.
- Add your cell suspension onto the column, let run through.
- Add 3ml of MACS buffer onto the column, let run through -> the cells collected in the falcon represent the “depleted” fraction.
- Place the column onto a fresh 15ml falcon tube (outside of the MACS magnet) and add 5ml MACS buffer.
- Elute the labeled cells with the plunger -> this is your “enriched” fraction.
All further steps are for staining the enriched population (for flow cytometric analysis).
Handling & storage
- Centrifuge eluted cells (500xg, 10 minutes).
- During centrifugation remove cells from the depleted fraction for the single stains you may need for compensation (about 100μl each).
- After centrifugation discard supernatant (but leave about 150μl).
- resuspend cells in the leftover MACS buffer and transfer them to your staining plate (you can also rinse the falcon once again with another 100-150μl MACS buffer).
All reagents used for this protocol should be stored at 4°C. In the case of tetramers and antibodies: avoid light exposure. The benzonase is stored at -20 °C.
ReferencesFor more information visit homepage of manufacturer: https://www.miltenyibiotec.com