Selective Isolation of CD4+ Vδ1+ γδ T Cells from Human Peripheral Blood Mononuclear Cells

Abstract

Source: Welker, C., et al. Isolation and Ex Vivo Culture of Vδ1⁺CD4⁺γδ T Cells, an Extrathymic αβT-cell Progenitor. J. Vis. Exp. (2015).

This video demonstrates a protocol for the magnetic labeling-based isolation of a low-concentration population of CD4⁺Vδ1⁺γδ T cells from human peripheral blood mononuclear cells (PBMCs). Using a combination of FITC-conjugated anti-Vδ1 antibodies, magnetic anti-FITC microbeads, and anti-CD4 antibody-coated superparamagnetic beads, the CD4⁺Vδ1⁺ T-cells can be successfully purified from the PBMC population.

Protocol

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. Isolation of Vδ1 T Cells

1. Take <1 x 10⁶ isolated lymphocytes for an initial FACS determining the frequency of Vδ1 CD4 T cells.
   NOTE: The population size of this T-cell entity may differ greatly between individuals and according to their immunologic state. Typically, about 1% of T cells are Vδ1⁺ and about 1-5% of Vδ1⁺ cells are CD4⁺.
   1. Dilute the cells with 1 ml FACS buffer, which consists of PBS containing 2% FCS and 5 mM EDTA. Centrifuge at 660 x g for 2 min and decant the supernatant. The reflux volume of about 100 µl FACS buffer is sufficient for the subsequent staining. Briefly vortex and incubate the cells with 5 µl Fc-Blocking Reagent for 5 min at RT for increased specificity of the FACS staining.
   2. Add human monoclonal antibodies directly to the cells without removing the Fc-Blocking Reagent. Make sure to stain the cells with antibodies against Vδ1 (1:50), CD3 (1:50), CD4 (1:200), CD8 (1:200), and TCRαβ (1:25). Prepare the isotype control with the corresponding immunoglobulin isotypes. Incubate cells with antibodies for 20 min at 4 °C in the dark.
   3. Wash the cells in 1 ml FACS buffer (centrifuge for 2 min; at 660 x g), decant the supernatant, and proceed to FACS acquisition in the remaining buffer volume.
2. Pelletize the cells (that are not used in the FACS analysis) by centrifugation for 12 min; at 300 g; 8 °C. Remove the supernatant and resuspend the cell pellet in 60 µl MACS-buffer per 1 x 10⁷ cells. Add 20 µl Fc-Blocking Reagent per 1 x 10⁷ cells and incubate for 5 min at 4°C.
3. Add 10 µl FITC-conjugated anti-human Vδ1 antibody per 1 x 10⁷ cells directly to the cells. Incubate for 12 min at 4 °C in the dark.
4. Wash the cells using 14 ml MACS buffer (centrifuge for 12 min; at 300 g; 8 °C). Discard the supernatant completely and resuspend the cells in 80 µl MACS buffer per 1×10⁷ cells.
5. Take 1-2 x 10⁵ cells and dilute them in 100 µl FACS buffer for verification of the labeling by FACS analysis. No further additives are needed for this step. Make sure that there is a sufficient difference in the brightness for Vδ1^– and Vδ1⁺ cells. If this is not the case, repeat steps 1.3 and 1.4.
6. Add 20 µl anti-FITC microbeads per 1 x 10⁷ cells to the remaining cells, mix well, and incubate for 15 min in the dark at 4 °C. Thereafter, wash the cells using at least 10 ml MACS buffer (centrifuge for 12 min, at 300 x g; 8 °C).
7. During the centrifugation, equilibrate the pre-cooled magnetic column placed in a magnet with 500 µl MACS buffer.
8. Resuspend the cells in 500 µl MACS buffer (for cell numbers higher than 1 x 10⁸, scale up this volume accordingly) and apply the cells carefully onto the column. Collect the flow-through containing the Vδ1^– cells.
9. Wash the column three times with 500 µl MACS buffer and collect the flow-through again in the same tube. Note that the reservoir must be empty before applying buffer onto the column.
10. Remove the column from the magnetic device and place it into a 15 ml conical tube. Quickly flush the column with 1 ml MACS buffer by firmly pushing the plunger into the column. Collect the eluate in a tube.
    NOTE: In order to obtain a purity of >98%, it is usually necessary to repeat steps 1.7-1.9 with a second column.
11. Verify the purity of the cells by FACS analysis with the same antibody panel as before (see 1.1.2) and count the cells.

2. Isolation of Vδ1CD4⁺ T Cells

1. Resuspend 25 µl of CD4 beads with a vortex for >30 sec. Wash the beads (the minimum amount as depicted by the manufacturer) in 1 ml MACS buffer in a 1.5 ml reaction tube by placing the tube into a magnet for 1 min. Discard the supernatant carefully with a small-scale pipette (200 µl) and resuspend the beads in the original volume of MACS buffer.
2. Pelletize Vδ1⁺ cells (centrifuge for 12 min; at 300 x g) and aspirate the supernatant and resuspend all the Vδ1⁺ cells in 500 µl MACS buffer and add them to the tube containing the beads. Mix vigorously and incubate for 20 min at 4 °C with constant tilting (e.g., in a rotator).
3. Place the tube containing the cells in a magnet for 2 min. Make sure that there are no remnants in the lid.
   NOTE: CD4⁺ cells will have bound the magnetic beads and attach to the side of the tube facing the magnet.
4. Carefully open the lid while keeping the tube inside the magnetic device and collect the supernatant which contains the CD4^–Vδ1⁺ cells using a small-scale pipette. Place CD4^–Vδ1⁺ cells into a separate tube and place this tube into the magnet again to avoid possibly remaining CD4 cells or beads from the population.
   1. Wash the CD4^– cells in 5 ml MACS buffer (centrifuge for 12 min; at 300 x g) and resuspend them in a concentration of 1 x 10⁵ cells per 100 µl media. CD4^– cells can be readily cultivated under suitable conditions.
5. Place the tube with the CD4⁺ cell targets outside of the magnetic field, resuspend the cells in 500 µl MACS buffer, and put them back into the magnetic device. Repeat steps 2.3-2.5 twice to obtain a higher purity. Remove supernatant by pipetting.
6. Resuspend the CD4⁺ cells in 100 µl culture media (RPMI 1640, 10 % FCS, 1% L-Glutamine, 1% Penicillin/Streptomycin) and add 10 µl of a bead-detaching solution. Incubate at RT for 45 min with constant tilting (e.g., in a rotator).
7. Place the cells in a magnet for 1 min. Carefully collect the supernatant containing bead-free Vδ1⁺CD4⁺ cells using a small-scale pipette.
8. Outside of the magnet, resuspend the beads with 100 µl culture media and repeat steps 2.6 and 2.7 twice to obtain higher cell numbers of CD4⁺ cells.
9. Pelletize the cells (centrifuge at 300 x g, for 12 min) and discard the supernatant completely by pipetting. Resuspend cells in fresh, pre-warmed media and count them. Examine the purity of the Vδ1⁺CD4⁺ cells by FACS analysis depicted in steps 1.1.1-1.1.3.

Materials

Phosphate-buffered Saline	Sigma Aldrich	D8537
MACS buffer	Miltenyi Biotec	130-091-222	supplement with BSA and pre-cool before use
BSA	Miltenyi Biotec	130-091-376	not mandatorily from this supplier
anti-human Vd1 FITC (clone: TS8.2)	Thermo Scientific	TCR2730	not mandatorily from this supplier
anti-human CD3 PerCP (clone: SK7)	BD BioSciences	345766	not mandatorily from this supplier or this fluorochrome
anti-human TCRab PE (clone: T10B9.1A-31)	BD BioSciences	555548	not mandatorily from this supplier or this fluorochrome
anti-human CD4 VioBlue (clone: MT466)	Miltenyi Biotec	130-097-333	not mandatorily from this supplier or this fluorochrome
anti-human CD8 APC-H7 (clone: SK1)	BD BioSciences	641400	not mandatorily from this supplier or this fluorochrome
Anti-FITC MultiSort Kit	Miltenyi Biotec	130-058-701	yields better results than anti-FITC MicroBeads
MS columns	Miltenyi Biotec	130-042-201	pre-cool before use
MiniMACS Separator	Miltenyi Biotec	130-042-102
CD4 Positive Isolation Kit	life technologies	11331D