37

Lymphocyte Proliferation Assay

Karl V. Sitz and Deborah L. Birx

1. Introduction
The lymphocyte proliferation assay is used as an in vitro surrogate, similar
to the in vivo delayed-type hypersensitivity assay,to assessthe overall quality
and character of the cellular arm of the immune response. The lymphocyte
proliferation assay is used to assesscongenital immune deficiencies, transient
mrmune compromised states,and more recently, the progressive immune defi-
ciency of HIV infection (see Note 1) (1-19).
This assay is used to assessthe ability of peripheral blood mononuclear
cells (PBMCs) (Note 2) to proliferate in response to various nonspecific (mi-
togen) and specific (recall antigen) stimuli. These stimuli trigger a compli-
cated series of events that ultimately leads to cell division. Although cell
division may be determined by simple microscopic enumeration of cells
before and after stimulation, this technique is both labor intensive and error
prone. Therefore, the incorporation of [3H]-labeled thymidme into newly
synthesized DNA has become a standard surrogate measurement of pro-
liferation (see Note 3). A flow chart (Fig. 1) depicts the steps involved m
this assay.
This assay may be slightly modified for the following studies: T-lympho-
cyte epitope mapping, assessmentof cytokme production, evaluation of growth
enhancing and inhibiting factors, evaluation of crossreactivity between differ-
ent protein sequences,assessmentfor prior exposure to infectious agents, study
of the requirements for signal transductron, and evaluation of factors related to
apoptosis.

From Methods m Molecular Medrone. l/o/ 17 HIV Protocols

Edlted by N L Michael and J H Kim 0 Humana Press Inc , Totowa, NJ

343
344 Sitz and Birx
Proliferation Assav Flow Diagram
Venipuncture to obtain heparinized whole blood

Ficoll-Hypaque isolation of PBMCs

Plating of isolated PBMCs into 96 well culture

plates containing antigenslmitogens

c
Incubation of plates for pre-determined times

c
Pulsing plates with 9[H]-thymidine

c
Harvesting and counting the plates

Acquiring and analysing the data

Fig. 1. Proltferation assay flow diagram.

2. Materials (see Note 4)

2.1. Ficoll-Hypaque Isolation of PBMC
1 Heparmized whole blood or blood collected m acid citrate dextrose (ACD) tubes
(Subheading 3.1. and see Note 5)
2 Phosphate-buffered saline. PBS may be conveniently obtained commercrally in
sterile bottles. If desired, it may be made usmg the followmg recipe and sterile
filtered
a. 1.15 g anhydrous Na*HPO,
b. 0.23 g anhydrous Na*HPO,.
c. 9.00 g NaCl.
d. Distilled HZ0 to 950 mL; adjust pH to between 7.2 and 7.4 with either 1 M
HCl or 1 MNaOH, add distilled HZ0 to 1000 mL
e. Filter sterilize by passage through a 0.45 @4 filter unit
Lymphocyte Proliferation Assay 345

3. Ficoll-Hypaque solution (1.077 g/hter; commercially available).

4 Complete medium (see Note 6).
a. RPM1 1640 medmm.
b 2 mA4 L-glutamme.
c 100 U/mL pemcillin
d 100 mg/mL streptomycin.
e 50 mh4 2-mercaptoethanol.
f 5% heat-inactivated (56°C for 30 mm) normal human AB serum
5 50 mL conical centrifuge tubes.
6 Low-speed centrtfuge.
7 Trypan Blue, 0 4%.
8 Hemocytometer
2.2. Culture Procedure
1. PBMC suspenston (see Note 7).
2. Complete medium
3 Selected mitogens and/or antigens (Figs. 2 and 3 and Note 8)
4. 96-well U-bottom tissue culture plates (Note 9).
5. Incubator with temperature, CO, and humidity controls.
2.3. Pulse and Harvesting Procedure
1 3H-labeled methyl thymidme, 50-75 mCt/mL
2. Semi-automated multiwell harvester with recommended filter paper
3 Scintillation fluid.
4. Liquid scinttllation counter
2.4 Data Analysis
1. Computer with spreadsheet or database software
3. Methods (see Note 4)
3.1. Ficoll+fypaque isolation of PBMC
1 Using standard venipuncture techniques, withdraw 10-15 mL of whole blood
mto a heparmized syringe, green top (sodium heparin) tube (Notes 2 and 5), or
ACD tube To heparimze a syringe, draw 0.25 mL of sodium heparin (1000 U/
mL) into the syringe, coating the inner surface prior to vempuncture
2. Invert the tubes or syringe several times after drawing to mix the heparm through-
out the blood sampie. It is critical to prevent clotting, since clotting activates the
cellular components and also depletes lymphocytes
3. Pipet 10-15 mL of heparmized whole blood into a 50-mL tube
4. Add an equal volume of PBS and gently mix.
5 Underlay with Ficoll-Hypaque, slowly and carefully pipet 10 mL of Ficoll-
Hypaque mto the very bottom of the 50-mL comcal tube containing the blood/
PBS mixture, taking care to maintain a perfect interface between the blood/PBS
mixture and the Ficoll (see Note 10).
346 Sitz and Birx

Plasma, Platelets
and PBS

Mononuclear Cells

Ficoll-Hypaque

RBCs

Fig. 2 Ftcoll-Hypaque denstty gradient.

6 Balance the tube(s) m enclosed centrrfuge containers, place m the centnfbge, and
spin at 900g (2000 rpm for Beckman GH-3.7 rotors) for 20-30 mm at 22°C. Make
certain that the centrifuge brake IS “OFF,” otherwise the interface will be disrupted
7 A representatton of how the specimen will appear post-centnfugation is given m Fig. 2
8. Usmg a IO-mL prpet, carefully aspnate the clear, yellow, plasma/PBS component to a
level just above the mononuclear cell interface. Care should be taken to remove as much
ofthe plasma/PBS, with contanunatmg platelets, asposstble while not losing mononuclear
cells The diluted plasma may be saved for future antrbody studies if desired.
9. Using a 5-mL ptpet, aspirate the cloudy mononuclear cell interface bemg careful
not to aspirate up much of the Ficoll layer. Transfer these cells mto a new 50-mL
conical tube and add approx 40 mL of PBS (see Note 11).
10 Balance the tubes in the centrifuge contamers; place in the centrifuge; and centrt-
fuge at 400g (1300 rpm for Beckman GH-3.7 rotors) for 10 mm at 22°C
11. Discard the supernatant, either by ptpet or single-motion pourmg, taking care to
not disturb the mononuclear cell pellet.
12. Resuspend the pellet by gentle vortexing
13. Add 10 mL of PBS to the 50-mL tube and re-mix.
14. Take a 20-pL aliquot of cells and mix with 20 pL of trypan blue Count the
number of vrable and dead cells using a hemocytometer.
15 Add approx 40 mL of PBS and repeat steps 10-12. These washing steps are used
to remove residual Ftcoll, platelets, and other serum components
16. Add an appropriate volume of complete medmm to achieve a final concentratton
of 1 x lo6 PBMCs/mL
Lymphocyte Proliferation Assay 347

I 1 2,3,4 WV’ 8.9.10 I 11 12

A PBS Only PBS Only PBS Only PBSOnly I PBS Only PBS Only

100,000 PBMC 100,000 PBMC 100,000 PBMC 100,000 PBMC

B1 PBSOnly
PHA-L
5 ug/ml
PWM
2.5 ug/ml
CollA
20 ug/ml Meduun Only PBSOnlv

SamDle#l SamDIe #l Sample #l Samule #I

100,000 PBMC 100,000 PBMC 100,000 PBMC 100,000 PBMC
PHA-L PWM ConA
2 5ug/ml 125ug/ml 10 ug/ml Medrum Ody PBSOnly

Sample #l Samule #l Samwle #I Samole #I

100,000 PBMC 100,000 PBMC 100,000 PBMC 100,000 PBMC
PHA-L PWM ConA
1.25 q/ml 0 625ug/ml 5 u&ml Medium Only PBS Only

Samule #I Samole #l SampleiN

100,000 PBMC

Medmm Only PBSOnlv

Samule #l

PBS Only

PBS Only

PBSOnly PBS Only PBS Only PBS Only PBS Only

Fig. 3 Mitogen plate

17 Gently mix the PBMCkomplete medium and place the tube m the 37”C, 5%
CO*, 95% humid Incubator with the cap loosened until ready to plate
3.2. Culture Procedure
1 Dilute the chosen mltogens and antigens to 2x the final desired concentration
usmg complete medium. The range of final desired concentrations should always
348 Sitz and Brx

Fig. 4. Antigen plate

be experlmentally determined by tltratlon of actual reagents with control PBMC

samples
2 A sample 96-well U-bottom plate diagram IS shown in Figs. 3 and 4
3 Add 0.1 mL of the appropriate antigens and mitogens by Eppendorf repeater pi-
pet to each well followmg a specific protocol, similar to the examples above
4. Remove the PBMC from the incubator. Gently mix the tube to thoroughly resus-
pend the PBMC, then add 0 1 mL (100,000) of cells by repeater plpet to each
designated well of the 96-well plates.
Lymphocyte Proliferation Assay 349
5 Remaining cells may be cryopreserved for future study.
6. Place the plates mto a 37”C, 5% CO,, 95% humid incubator for the predeter-
mined incubation periods This is often 2-3 d for the mitogen plate and 6-7 d for
the anttgen plate (see Note 12)

3.3. Pulse and Harvesting Procedure

1. At the end of the mcubation period, and at a standard time prtor to harvesting the
plate (6-l 8 h), add 1J-l.5 mCi (20 mL of 5&75 mCt/mL m complete medium)
of [3H]-labeled methyl thymtdme to each well of the assay (see Note 13)
2 Place the plates back mto the incubator for the remainmg 6-l 8 h
3 At the end of the [3H]-thymidme pulse, harvest the plates onto filter paper usmg
a semi-automated multiwell harvester. Follow the dtrectlons specified by the
manufacturer (see Note 14).
4. The filter paper containing bound DNA may be drted using either ethanol or by
moderate mtcrowave heatmg.
5 Once the filter paper is dry, transfer the filter paper into a scmtillatton container
appropriate to the automated scintillation counter that IS being used Add scmtil-
lation fluid to the scmtillatton container(s) as spectfied by the counter system
6. Count the samples m the scmnllation counter accordmg to the program’s specifi-
canon wtth a desired variabtlity of ~2%

3.4. Data Analysis (see Note 15)

1 If possible, import the raw data mto a spreadsheet or database to allow for more
automated analysis
2 Determme the mean and standard deviation of the wells representing background
prohferation (wells containing only cells and complete medmm)
3 Determine the mean and standard devtation of all series of replicate experimental
stimulations
4 Data are routinely presented m the following ways:
a. Simple enumeration of both baseline and experimental counts per minute
(cpm) wtth associated standard deviation or standard error of the mean
b. Net stimulation. subtractton of the background mean from the experimental
mean.
c. Sttmulation index. division of the experimental mean by the background mean.

4. Notes
1. A complete discussion of the dtseases associated with defects m lymphocyte prolif-
eration is beyond the scope of this text These are more fully discussed m ref. 20
2. Although PBMCs are described m these methods, other sources of lymphocytes,
such a lymph node homogenates and bone marrow aspirates, may also be used
when available
3 This methodology deals exclusively with [3H]-thymidine incorporatton. Other,
nonradioacttve, techmques involving fluorescence labeling and flow cytometric
enumeration of cells may also be used but are beyond the scope of this text
 Sitz and Birx
4 The lymphocyte proliferation assay 1s dependent on culture stertltty Contamt-
natmg bacteria or fungi will adversely affect the interpretation of the results It is
therefore imperative that sterile materials and sterile technique be used up to the
harvestmg step. Although antibtotics are often added to culture medmm, con-
tamination by mycoplasma and fungi will not be avoided unless scrupulous asep-
ttc technique is used. If contammatton problems occur, it 1s wise to check the
incubators, water baths, hoods, and other equtpment for potential colomzation
5. When working with mfectious material appropriate measures must be undertaken
to minimize the risk of infection of laboratory workers Details of these measures
may be obtained from CDC and OSHA recommendattons and are beyond the
scope of this text
6 Complete medium should be freshly made m quanttttes that will be used in < 1
mo; otherwtse, pH changes and potenttal degradation of serum components and
anttbtottcs may ensue Anttbtottcs may be omttted tf the mononuclear cell source
is sterile and scrupulous sterile technique is observed 10% normal human AB
serum or lO-20% fetal calf serum (FCS) may be required for more fasttdtous
long term cultures. Since serum components may contam factors that cause sttmu-
latxon or inhibttton of culture growth, tt is tmperattve to screen several lots of
various sera at different concentrattons m order to determine the lot/concentra-
tion that provides maximal growth with mimmal background stimulation. Once a
desirable lot 1sidentified tt should be purchased m a quanttty sufficient to be used
throughout the experimental pertod; once the serum 1s heat-macttvated, aliquots
should be stored at -20°C or lower temperature until ready to be used.
7. It 1scrtttcal to use PBMC of high vtabtltty PBMC that have been freshly isolated
from blood samples that are less than 24 h old are usually highly viable (>95%).
Cryopreserved PBMC may contam slightly fewer viable cells but tf the cryo-
preservatton and thawing techmques are performed correctly, cryopreservatton
should not adversely affect proltferative capacity However, care must be taken
m mterpretatton of expertments m which PBMC of low viability are used since
damaged cells and cellular debris may inhibit proliferative responses.
8 Depending on the goal of the experiment a variety of mttogens and/or antigens
may be tested m the proltferatton assay Figs. 3 and 4 simply depict panels that
have been recently utilized in our laboratory. Once the mttogens/antigens are
selected it 1s imperative that preltmmary tttratton experiments be performed to
determme the antigen concentratton that causes peak specific proltferatton m an
ahquot of control cells (tf known and tf available) under the identical expertmen-
tal condttions (i.e., fresh or cryopreserved cells). Because indtvtdual vartatton
may occur, tt 1soften wise to bracket the wells contammg the opttmal concentra-
tion wtth wells that contain 2x and 1/2x the optimal concentratton.
9 U-bottom (round bottom) wells allow cells to better physically associate with
other cells m the culture Thts 1susually an advantage when dealing wtth moder-
ate numbers of cells (1 x 105) with a relatively low precursor frequency for the
stimulus, as occurs m most PBMC assays Assays that use rapidly dividing cell
lines often prefer flat-bottom wells that allow expansion without crowding
Lymphocyte Proliferabon Assay 351

10. As an alternate technique, the blood/PBS mixture may be overlaid into a 50-mL
conical tube contammg 10 mL of Ficoll-Hypaque. Slowly and carefully, ptpet
the blood/PBS mixture onto the top of the Ficoll, letting the liquid run down the
side of the tube at a 45” angle, again maintaining a perfect interface between the
two, Experience with thts technique is critical to maintammg an interface; nov-
ices often find the underlaying techmque (Subheading 3.5.) easier to perform.
11. PBS should be m at least 3x excess durmg the imttal wash, otherwise the cells
may not be pelleted due to contammating Ficoll.
12. The optimal period of mcubation depends on a combmation of the strength of the
stimulus, the precursor frequency of the responding cells, and potential toxictty
associated with the stimulus. Each stimulus may have different optimal mcuba-
tion times for maximal proliferation, which must be determined experimentally
for novel sttmuh. In general, as a compromtse, all mitogens may be placed on the
same plate wtth a 2-3-d incubation period and all antigens may be placed on
another plate wtth a 67-d mcubatton period. Because of potential problems with
evaporatton from the outside wells of the plate, parttcularly those incubated for
greater than five days, it may be preferable to till the outside wells wtth PBS and
use only the interior wells for culture of cells (as demonstrated in Figs. 3 and 4).
13 The optimal length of pulsing with [3H]-thymidine is usually between 6-18 h.
Although 6 h may give excellent results for a large subpopulation of proliferating
cells, 18 h of pulsing may give more conststent incorporatton for more weakly
proltferating cells. Although the length of pulsing is often determmed by conve-
mence of laboratory schedule, tt must be consistent over the course of an expert-
mental protocol.
14. In general, the harvester asptrates each individual well of the plate onto a speci-
fied region of filter paper and lyses the cells using distilled water. Once the cells
lyse, the DNA, with incorporated [3H]-thymtdme, IS bound to the filter paper
while unincorporated [3H]-thymidine is washed away.
15. A detatled discussion of the stattsttcal considerations and quahty control issues
involved m the lymphoprohferative assay is beyond the scope of thts text. An
excellent revtew of these toptcs IS found in refs. 20 and 21.

5. References
1 Ballet, J. J., Couderc, L. J., Rabtan, H. C., Duval, R. C., Jamer, M., Danon, F.,
Clauvel, J. P , and Seligmann, M (1988) Impaired T-lymphocyte-dependent
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Characterization of T lymphocyte responses during primary infection with human
tmmunodeficiency virus. J In&?& Du 157,889-896.
3. Hoy, J. F., Lewis, D. E., and Miller, G. G. (1988) Functional versus phenotyptc
analysis of T cells in subjects seropositive for the human immunodeficiency virus:
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Dis. 158, 1071-1078
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Lymphocyte Proliferation Assay 353

16 Kelker, H. C., Setdlm, M , Vogler, M., and Valentine, F. T. (1992) Lymphocytes

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