Isolation of CD4+ T-cells from anti coagulated whole blood

CD4 T cells:

CD 4 is a co receptor that associates with MCH class II molecules. CD 4 is expressed in T-

cells. CD 4 consists of a cytosylic domain rich in basic amino acid lysine, a hydrophobic
transmembrane domain and an extra cellular domain that attaches to MCH class II
molecules on the Antigen presenting cells.

Importance of CD4 T-cells:

It is known that CD4 is involved in signal transduction and helps T cell form a strong
association with the MCH class II molecules. One of the main hallmarks of chronic HIV
infections is a decrease in CD4+ T-cells. HIV can attach to CD4, infect and kill T-cells. In this
process billions of T cells are destroyed and billions more produced to replace the lost T-
cells. Counting and analysing CD4+ T-cells are essential to monitor the extent of HIV
infection and even for prognosis of treatments (Busch et al., 2004).

Steps and principles involved in CD4+ T-cell separation

Separation of (Peripheral blood mononuclear cells) PBMC from heparinized blood:

The first step involves isolation of PBMC from the blood. Heparin and EDTA are generally the
anticoagulants added. Ficoll-Paque centrifugation is the primary method employed to
specifically isolate PBMC.

Ficoll-Paque centrifugation:

This separation technique is based on studies conducted by Boyum in 1968. Following his
publications, mixture of Ficoll 400 and an iodinated density gradient medium (sodium
diatrizoate) have been very widely (Amersham Biosciences).

The technique involves layering of anticoagulated blood on the Ficoll-Paque solution. On

centrifugation at 400 G for 30–40 min, different layers are obtained. RBCs and granulocytes
aggregate and sediment at the bottom due to the increase in their density contributed by
the Hypertonic Ficoll- Paque solution. Lymphocytes, monocytes and platelets owing to lower
densities accumulate between the ficoll- paque layer and the plasma. This interface layer
can be collected washed in a balanced salt to remove platelets and other contaminants.

RosetteSep™ Separation:

It is a Stemcell technologies patented separation method. It uses Tetrameric antibody

complexes to tag and crosslink unwanted cells to RBCs. Ideally all unwanted cells pellet to
the bottom along with RBC and lymphocytes containing t-cells can be isolated.
 Figure showing rosette separation (http://www.stemcell.com)

Blood

Plasma
Centrifuge, 800G

PBMC
30-40 min
Ficoll-paque

Ficoll-paque
RBCs
Figure illustrating Ficoll- paque separation.

PBMC isolated from blood contain leucocytes (NK cells, B cells and T cells) and monocytes.
CD4+ T-cells are isolated from this mixture using antibodies. Antibodies targeting CD4 on T-
cell surface can be used. Antibodies coupled with fluorophores can be potentially used to
count and characterise CD4+cells.

Two main methods used for isolating the CD4+T-cells from PBMC are Fluorescence
Activated Cell Sorting (FACS) and Magnetic separation.

Magnetic separation:

It is a fast and reliable technique for isolating a particular group of cell having a specific cell
marker. Magnetic beads conjugated with antibodies specific to a type cell marker are used
(Miltenyi et al., 1989).

The beads used for separation are usually less than 0.5 µM. Magnetic particles over 5µM
could interfere with the viability of the cells and bound cells are difficult to detach from
multiple attachment points (Miltenyi et al., 1989). Dynal separation and Magnetic activated
cell sorting are the two types of magnetic separation available.
Magnetic activated cell sorting:

Magnetic activated cell sorter (MACS) was developed by Miltenyi et al. (1989). Super
paramagnetic particles and a high gradient magnetic field (HGM) were used to separate the
bound cells (modification of Molday and Molday’s work). Fluorescence labelling to the
process and cells could be analysed in a fluorescence microscope or flow cytometer. The
target cells are separated by elution.

Dynal separation:

Dyna beads are used to separate cells. Antibodies specific to the target cells ate coated to
the magnetic beads. The cells are separated in a Magnetic Particle Separator (MPS).
Dynabeads® M-450 are used for CD4 cells separation. Magnetic beads are removed using
Dynal DETACHaBEAD® (polyclonal Ab). They compete with Abs attached to the target cells
and release them (invitrogen).

N S
S

CD4 bound to magnetic beads

Unbound cells
Figure showing positive MACS (adopted from Miltenyi et al., 1989)

Magnet

CD4+ cells Unwanted cells

Figure showing positive magnetic separation using Dynal beads.
Selection:

The selection process may be positive or negative. In Positive selection, target specific
antibodies are coated to magnetic beads, where as in negative selection, non target specific
antibodies are coated to the magnetic beads.

Bernard et al., (2002) adopted various selection systems to isolate CD 4 cells. Positive
selection included coating magnetic beads with alpha mAb using the dynal beads and also
using micro beads (Miltenyi).

Classical negative selection included incubation with a cocktail of mAbs (against CD-8, 14, 16,
19 and 56) and then incubation with goat antimouse coated magnetic beads. Also negative
selection by incubating blood with a cocktail of mAbs ( against CD- 8,16,18,36 and 56) and
holding them in a tetrameric array with a murine Ab recognizing human glycophorin A on
RBC ( Rosette sep., stem sep).

They found that positive selection gave higher purity as compared to the negative selection.
However negative rosette separation gave the highest cell yield though the purity was not
very high.

Flow Cytometry and Fluorescence activated cell sorting (FACS):

Flow cytometry is the study of the physical and/ or chemical characteristics of cells passing
through a measuring apparatus (Mandy, Bergeron and Minkus, 1995). Fluorescence
activated cell sorting is an extension of the flow cytometer with the capacity to separate a
subset of cells using flourochrome conjugated to antibodies specific to a cell surface marker.
FACS is a trademark of Becton Dickinson Immunocytometry Systems (BDIS).

FACS is usually carried out to isolate CD4 T-cells after obtaining PBMC (McCune et al., 2000;
Busch et al., 2004). Anti CD4mAbs are conjugated to fluorochromes. Phycoerythrin is usually
conjugated to the antibodies. Labelling can be direct or indirect.

As a droplet is being formed at the nozzle of the cytometer, laser directed to the droplet
cause scattering of light. The scatter can be forward or side scatter. Simultaneously the
Phycoerythrin (tagged to the antibodies bound to CD4+T-cells) gets excited and emits
fluorescence light. These emissions are detected by optic detectors connected to analysers.
The analysers pick the fluorescence emitting CD4cells and the charger gives the CD4 cells a
charge. These charged cells are deflected and can be separated. All unlabelled cells do not
receive any charge and are not deflected by the charge plates (Herzenbergetal., 2002;
Rieseberg et al., 2001).
Selection and labelling:

Both positive and negative selection can be employed to separate CD4 cells. Positive
selection generally involves direct labelling of CD4 and indirect labelling for negative
selection.

Analysis of data:

FlowJo and FCS Express are some softwares used for analysis.

FSC Vs. SSC (Busch et al., 2004).

The figure shows the PBMC data analysed using FACS. The first fugure illustrates Forward
scatter vs. Side scatter gate. CD4+ T-cells tagged with PE can be differentiated from the
other cells. Here the monocytes are tagged with FITC (Fluorescein isothiocyanate). The
lumo of cells left to lymphocytes are cell debri. In HIV patients it is known that the there is a
larger cell debri.

Cell Affinity Chromatography

Affinity chromatography could be used to separate CD4 T-cells from blood. Cell affinity
chromatography involves a column coated with a ligand having affinity to cell surface
receptors. Usually the antibodies are coated to beads attached to the columns. Shibusawa
(2001) used cell surface affinity chromosome on polyethylene glycol (PEG) and
polypropylene glycol (PPG) chemically bonded columns (sepharose 6B). The system is very
selective and tends to retain lymphocytes strongly.

Open-Tubular Capillary Cell Affinity Chromatography (OT-CAC)

OT-CAC technique was used to separate CD4 T-cells from blood. OT-CAC is efficient in cell
capture, enrichment, and recovery. Open tubular capillary coated with anti CD4 antibodies.
The cells could be eluted using force or bubble. The viability of the cells after separation was
85.83 ± 7.34% (Wang et al., 2008).
 Cell loading, non target cells washed away

bubble Using bubble for recovering target

cells
Detachment of concentrated cells

Figure showing the steps in OT-CAC (Wang et al., 2008).

Analysis of purity and yield in the methods used:

Once the CD4 T-cells are separated, the purity and the yield of all the methods used could
be analysed. The total cell yields could be determined by dye exclusion test using a
haemocytometer. The purity can be assessed using a FACS. CD4 purity can be expressed in
terms of CD4/CD3 after labelling the cells with anti-CD4-PE and anti-CD3-FITC (Busch et al.,
2004).

By Kamaal Rayees
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