T-Cell Isolation and
Propagation in vitro
Cellular immunity is largely based on Tlymphocytes.
Like B cells, T cells also arise from the
bone marrow. However, unlike B cells, they migrate
to the thymus for maturation. A T-cell expresses a
unique antigen-binding molecule called the T-cell
receptor (TCR) on the cell surface. In contrast to
membrane-bound antibodies on B cells, which can
recognize the antigen alone, the majority of TCR recognizes
a complex ligand, comprising an antigenic
peptide bound to a protein called the major histocompatibility
complex (MHC) [known in humans as
human leukocyte antigen (HLA)] molecule (Moss et al
1992). When a T-cell encounters an antigen in the
context of an HLA molecule, it undergoes clonal
expansion and differentiates into memory and various
effector T-cells: CD4+
T-helper cells and CD8+
T-lymphocytes (CTL). Activation of both humoral and
cell-mediated parts of the immune response requires
cytokines produced by T-helper cells. The activation of
T-helper cells is carefully regulated, and naive cells
only become activated when they recognize an antigen
presented by class II HLA molecules in context
with the appropriate costimulatory molecules on the
surface of professional antigen-presenting cells
(macrophages, B cells, and dendritic cells) (Stockwin
et al., 2000).
Although antigen-presenting cells encounter and
incorporate an antigen in many different compartments,
the interaction with T-helper cells is largely
confined to secondary lymphoid organs (Fu et al.,
1999). While the T-helper cells provide help to activate
B-cells, antigen-presenting cells, and CTL, only the
latter has a vital function in monitoring the cells of the body and in eliminating cells that display antigen,
primarily virus-infected cells. However, in addition
to providing protection against infectious agents, CTL
are thought to provide some degree of protection
against spontaneous tumors by virtue of their
ability to detect quantitative and qualitative antigenic
differences in transformed cells (Castelli et al., 2000).
Tumorigenic alterations result in an altered
protein repertoire inside the cell. Class I MHC
molecules sample peptides from protein degradation
inside the cell and present these at the cell surface
to CTL. Hence, this enables CTL to scan for cellular
Until recently, measurements of the levels of cellular
immune responses, i.e., those mediated by CD4+
T-lymphocytes, have depended largely on
culture in vitro
and the subsequent measurement of
specific functions (such as cytolysis). More recently,
new technologies based around tetrameric class I
peptide complexes (tetramers) have allowed immunologists
to isolate and measure CD8+
levels directly ex vivo
. This article describes measures
used to generate and clone specific T-cells in culture as
well as measures to isolate specific T-cells by means
of recombinant HLA/peptide complexes. Finally, it
describes the conventional chrome release assay and
the ELISPOT assay for the measurement of specific Tcell
immunity. It should be noted, however, that T-cells
behave very differently and that a T-cell protocol
should not be considered as the definitive receipt but
rather as a guideline that can be altered depending on
the target or the donor. Finally, the making of dendritic
cells has been described in detail elsewhere in this
volume. However, this is an important first step of
making primary T-cells and is consequently mentioned
II. MATERIALS AND
A. Tissue Culture Medium
X-vivo medium (Cambrex Bio Science, Cat. No.
04-418Q). Store at 4°C.
Human serum (Sigma, Cat. No. H1513). Store at-20°C.
Standard medium: X-vivo, 5% human serum. Store at
RPMI 1640 medium (GIBCO, Cat. No. 61870-010).
Store at 4°C.
Fetal calf serum (FCS) (Sigma, Cat. No. F7524). Store
R10 medium: RPMI 1640 + 10% FCS. Store at 4°C.
Interleukin (IL)-2 [Apodan, Cat. No. 004184]. Store at
-80°C. Aliquot 20 units/µl.
IL-4 [Peprotech (trichem), Cat. No. 200-04]. Store at
-20°C. Aliquot 10 units /µl.
IL-7 [Peprotech (trichem), Cat. No. 200-07]. Store at
-20°C. Aliquot 5 ng/µl.
IL-12 [Peprotech (trichem), Cat. No. 200-12]. Store at
-20°C. Aliquot 20 units/µl.
GM-CSF [Peprotech (trichem), Cat. No. 300-03]. Store
at -20°C. Aliquot 800 units/µl.
TNF-α [Peprotech (trichem), Cat. No. 300-01A]. Store
at-20°C. Aliquot 10ng/µl.
PHA (Sigma, Cat. No.). Store at-20°C. Aliquot
Anti-CD28 (eBioscience, Clone 28.8, Cat. No. 16-0288-
81). Store at 4°C.
Anti-CD3 (eBioscience, Clone OKT3, Cat. No. 14-0037-
82). Store at 4°C.
Tricolor-anti-CD8 (Caltag, Burlingame, CA, Cat. No.).
Store at 4°C.
D. Sterile Plastic Plates
96-well plates [Boule (Corning Costar), Cat. No. 3799]
48-well plates [Boule (Corning Costar), Cat. No. 3548]
24-well plates [Boule (Corning Costar), Cat. No. 3526]
6-well plates [Boule (Corning Costar), Cat. No. 3516]
E. Additional Materials
Lymphoprep/Ficoll (Medinor, Cat. No. 30066.03).
Store in the dark at 4°C.
Crom (Amersham, Cat. No. CJS1) 5mCi/1ml. Store
at-20°C. Dilute 1:5 in phosphate-buffered saline
(PBS) before use.
Biotinylated monomers or PE-labeled tetramer (Proimmune,
Oxford, UK). Store at 4°C.
Streptavin-coated magnetic beads (Dynabeads M-280,
Dynal A/S, Cat. No. M-280). Store at 4°C.
F. Materials for ELISPOT
Nitrocellulose plates (Millipore MAIPN 4550)
Coating antibody: Mab anti-hlFN γ clone 1-D1K,
1 mg/ml, MABTECH 3420-3. Store at 4°C. Dilute
to 7.5µg/ml in PBS before use.
Secondary antibody: Biotinylated Mab anti-hlFN-γ,
7-b6-1, 1 mg/ml, MABTECH 3420-6. Store at 4°C. Dilute to 0.75µg/ml in diluting buffer before use.
Streptavidin AP: Calbiochem CAL 189732, 2ml. Add
0 and 2ml glycerol (85%). Store at 4°C. Dilute 1:1000 in diluting buffer before use.
NBT/BCIP substrate system. DAKO, Cat. No. K 0598.
Store at 4°C. Dilute 1:5 in substrate buffer before
In addition to standard cell culture instruments:
Gamma counter (Cobra 5005, Packard Instruments,
ELISPOT counter [ImmunoSpot Series 2.0 analyzer
(CTL Analyzers, LLC, Cleveland, OH]
Magnetic isolator (Dynal A/S, Oslo, Norway)
FACSVantage (Becton-Dickinson, Mountain View, CA)
A. Induction of Specific T Cells as
The following is a description on how to grow
antigen-specific T-cells using peptide-loaded dendrictic
cells as stimulator cells (Pawelec, 2000). This protocol
can also be used to grow tumor-specific T cells if
tumor lysate-loaded dendritic cells are used as stimulator
cells as described.
1. Stimulator Cell (Dendritic Cell) Culture
Dilute 50ml blood 1:1 with RPMI medium and
separate on lymphoprep by centrifugation for 30min
at 1200rpm. Harvest mononuclear cells, mix with an
equal volume of RPMI, and centrifuge at 1500rpm for
10min, followed by two washes in R10 (1200rpm,
5 min). Resuspend cells to 20 × 106
cells/ml and plate out at 3 ml/well in 6-well plates. Incubate the cells for
2h at 37°C. Remove nonadherent cells (lymphocytes)
by gentle suction. If necessary, the lymphocytes may
be frozen. Add 2.5 ml standard medium containing 800
units/ml GM-CSF and 500 units/ml IL-4 to each well.
Add 2.5ml standard medium containing 1600
units / ml GM-CSF and 1000 units / ml IL-4.
Remove 2.5ml of medium from each well and
replace with 2.5ml of fresh medium containing
1600 units / ml GM-CSF and 1000 units / ml IL-4.
Remove 1 ml of medium from each well and replace
with 1 ml of fresh medium containing 10ng/ml of
Harvest the cultured DC and wash twice in RPMI
medium. Resuspend in 1 ml RPMI medium containing
50µg/ml peptide and 3µg/ml β2
m. Incubate the cells
for 4h at 37°C; gently resuspend every hour. Irradiate
at 25 Gy, wash twice with RPMI medum, and resupend
cells (stimulator cells) at 3 × 105
/ml in standard
2. Initiation of Primary T-Cell Culture
Mix 3 × 106
freshly isolated lymphocytes and 3 × 105
peptide-pulsed stimulator cells in a 24-well plate at
Add IL-7 to a final concentration of 5ng/ml and
100 pg/ml IL-12.
Remove 1 ml of medium and replace with 1 ml of
standard medium containing 20ng/ml of IL-7.
Harvest responder cells, separate over Ficoll, wash
once, and count viable cells. Resuspend at 1.5 × 106
in standard medium and keep tube at 37°C. Thaw
2 × 106
autologous peripheral blood mononuclear
cells (PBMC) per 1.5 × 106
responder cells. Wash the
PBMC once in RPMI medium and irradiate at 60Gy.
Wash again in RPMI and incubate 2h at 37°C with
20µ g/ml peptide and 2µ/ml β2
m. Remove medium
and gently wash once in RPMI. Mix 1.5 × 106
2 × 106
peptide-pulsed autologous PBMC in 2ml of
standard medium. Alternatively, instead of peptidepulsed PBMC, use peptide-pulsed DC prepared as on
Remove 1 ml of medium and replace with 1 ml of
fresh medium containing 40 units/ml IL-2 to each
Restimulate as on day 12. Add IL-2 to the culture as
on day 14. Restimulation is needed four or five times
before a primary response is measurable.
B. Cloning of T Cells by Limiting Dilution
Wash autologous PBMC once in RPMI medium and
irradiate at 30Gy. Wash again in RPMI and incubate
2h at 37°C with 20µg/ml peptide and 2µg/ml β2
Plate T-cells at limiting dilution (featuring 10; 3; 1;
0.3 cells/well) in 96-well round-bottom microtiter
plates containing 105
irradiated, autologous peptidepulsed
PBL, 10 units/ml IL-4, and 40 units/ml IL-2 in
100 µl standard medium.
Add 50µl standard medium containing IL-2 and
IL-4 to a final concentration of 10 and 40 units/ml,
Add 50µl standard medium containing IL-2 and
IL-4 to a final concentration of 10 and 40 units/ml,
Inspect cells for growing cells microscopically.
Transfer growing cells to 48-well plates containing
peptide-pulsed autologous feeder cells and antigen. At
the same time, test clones for antigen specificity by the
chrome release assay or ELISPOT. Incubate plates 7
days at 37°C in a 5% CO2
incubator adding IL-2 and
IL-4 every third day and transfer antigen-specific
T-cells to 24-well plates.
An Alternative Cloning Protocol Using Anti-CD3
and Anti-CD28 Antibodies
This procedure is modified from Oelke et al. (2000).
Coat a 96-well plate with 100ng/ml of anti-CD3
and anti-CD28 antibodies in PBS for 24h at room
Plate T cells at limiting dilution in precoated 96-well
plates containing 105
irradiated, autologous PBL,
10 units/ml IL-4, and 40 units/ml IL-2 in 100 µl standard
medium. Continue as described in previously.
C. Expansion of T-Cell Clones
This is a protocol for the expansion of already established
clones modified from Dunbar et al
. (1998). The
cloning mix described in the following can, however,
also be used as stimulators to clone T-cells instead of
autologous PBL as described previously.
For preparing the cloning mix
isolate fresh lymphocytes
from at least three individuals, resuspend them
in standard medium, and irradiate (20Gy). Count the
lymphocytes and mix them together to give a final
total concentration of 1 × 106
/ml. Add PHA to a final
concentration of lµg/ml and leave in the incubator
while the clone is thawed and counted. Thaw the
clone, count, and resuspend in prewarmed cloning
mix at between 105
and 5 × 105
cellsml. Plate out into
a U-bottomed, 96-well plate at 100µl/well.
Add 100ml medium and 40 units/ml IL-2 to each
Prepare fresh cloning mix, plate 1 ml per well into
24-well plates and prewarm in an incubator.
Transfer I well of the 96-well plate into I well of the
prewarmed 24-well plate.
Add 1 ml of medium and 40 units/ml IL-2 to each
Pool the wells for each individual clone, count, and
remove the required amount of cells for a chrome
release assay to check if the clones have maintained
antigen specificity. Freeze the remaining cells in
aliquots of 2 to 7 million per vial.
D. Isolation of Peptide-Specific T Cells
Momomeric or tetrameric MHC/peptide complexes
can be bought commercially, e.g., proimmune
(Oxford, UK). They can, however, also be made as
described by Pedersen et al. (2001). However, these rather complex procedures are not the scope of this
1. Isolation of Specific T-Cells Using MHC Class
I/Peptide Complexes Coupled to Magnetic Beads
This procedure is performed according to Andersen et al
. (2001; Schrama et al
., 2001). Incubate 2.5µg
biotinylated monomer of a given HLA/peptide
complex with 5 × 106
streptavin-coated magnetic beads
in 40µl PBS for 20min at room temperature. Wash the
magnetic complexes three times in PBS using a magnetic
PBL or lymphocytes in 100µl PBS with 5%
bovine serum albumin (BSA) and rotate very gently for
I h. Wash the antigen-specific T-cells associating with
the magnetic complexes gently three times in PBL.
Incubate for 2 h at 37°C and resuspend several times in
standard medium to release the cells from the magnetic
Assay the antigen specificity of isolated antigenspecific
T-cells in an ELISPOT or chrome release
assay after at least 5 days in culture.
2. Isolation of Specific T-Cells by FACS
This protocol is modified from Dunbar et al
Culture PBL overnight in standard medium before
sorting. Alternatively, pulse PBL with 10 µM
standard medium, plus 10U/ml IL-7 and culture for
10 days before sorting. Stain cells with PE-labeled
tetrameric HLA/peptide complex for 15min at 37°C before the addition of tricolor-anti-CD8 for 15min on
ice. Wash the cells six times in PBS before analysis on
Gate specific T-cells according to tetramer/CD8
double staining by forward and side scatter profile.
Sort single cells directly into U-bottom, 96-well plates
and stimulate these as described in Section III,B.
E. Examination of Antigen Specificity
1. Crome Release Assay
Cr release assays for CTL-mediated
cytotoxicity can be used to test the specificity of CTL
lines against relevant target cells, e.g., autologous EBVtransformed
B-cell lines or cancer cell lines. This procedure
is performed according to Brunner et al
target cells in 50 µlR10 mediumd with [51
(100µCi) in a round-bottomed well of a 96-well plate
at 37°C for 60min. If necessary, add 4µg of peptide.
Wash the target cells four times and plate out in 96-
well plates in 100µl R10 medium. Add T-cells at
various effector:target ratios in another 100µl R10 and
incubate at 37°C for 4 h. Aspirate 100 µl of medium and count [51
Cr] release in a gamma counter. Determine
the maximum [51
Cr] release in separate wells by the
addition of 100µl 10% Triton X-100 and the spontaneous
release by the addition of 100µl R10 only to
Calculate the specific lysis using the following
[(experimental release - spontaneous release)/
(maximium release- spoontaneous release)] × 100.
For the measurement of specific T-cell immunity,
ELISPOT analysis, involving the incubation of primary
PBMC with one or more peptide epitopes, is probably
the most sensitive and reliable assay (Pittet et al
ELISPOT is based on the detection of the peptideinduced
release of cytokines such as interferon (IFN)-
γ by single T-cells upon stimulation with a peptide
antigen (Scheibenbogen et al
Single T-cells can be detected and quantified as
cytokine spots on special nitrocellulose filter plates.
Cytokine-specific antibodies are coated to the filters to
capture the secreted cytokine; peptide-pulsed target
cells are added together with cells containing the precursor precursor
T-cells. If a T-cell recognizes the peptide epitope
examined, the cell releases cytokine. This can be
detected as a spot by a colorimetric reaction with secondary
antibodies, which represents the cytokine after
secretion by a single activated cell. The principle of
ELISPOT is illustrated in Fig. 1. If case responses are
weak, a single round of in vitro
stimulation can be
used. For cytotoxic T-cells, IFN-γ, or granzyme B,
ELISPOT can be performed; for T-helper cells, IFN-γ,
or IL-4 (for T-helper 1-type and T-helper 2-type immunity,
respectively) can be performed.
Washing buffer: PBS and 0.05% Tween 20; store at
Diluting buffer: PBS, 1% BSA, and 0.02% NaN3
at room temperature
Substrate buffer: 0.1M NaCl, 50mM
, and 0.1M
Tris-HCl, pH 9.50; store at room temperature
Coating of Plates
Add 75µl 7.5 ug/ml coating antibody. The antibody
concentration may change depending on the cytokine
target. Leave overnight at room temperature (if coating 3-5 days in advance, leave at 4°C). Wash the plate with
6 × 200 µl PBS. Block the plate with 200 µl media. Leave
in incubator for 2h.
Setting up the ELISPOT
|FIGURE 1 (A) Schematic illustration of the ELISPOT assay. Cytokine-specific antibodies are coated onto
nitrocellulose filter plates to capture the secreted cytokine; a peptide-pulsed target cell is added together with
cells containing the precursor T-cells. If a T-cell recognizes the peptide epitope examined, the cell releases
cytokine. This can be detected as a spot by a colorimetric reaction with secondary antibodies. Thus, the spot
represents the cytokine after secretion by a single activated cell. (B) ELISPOT wells after incubation with
T-cells that were either nonreactive (left) or reactive (right) against the antigen examined.
Prepare the serial dilutions of the cells (sterile) at relevant
concentrations in order to add the cells to each
well in 200 µl media. Poor off the blocking media, and
add 200 µl cells and 0.5 µl peptide (2mM
) to each well.
Leave overnight in incubator. (The plate must not
shake during incubation.) Wash the plate with 6× 200 µl washing buffer. Add 75 µl of the secondary antibody
(1 µg/ml) to each well. The antibody concentration
may change depending on the cytokine target.
Incubate for 2h at room temperature. Wash the plate
with 6 × 200 µl washing buffer.
Add 75 µl streptavidin (diluted 1:1000) to each well.
Incubate for 1 h at room temperature.
Wash the plate with 6 × 200 µl washing buffer and 1 × 200µl substrate buffer. Mix the substrate: 10ml substrate
buffer + 44 µl NBT + 33 µl BCIP.
Add 75 µl fresh substrate to each well.
Leave at room temperature for 2-20min.
Stop the reaction with tap water when spot development
Count the spots using the ImmunoSpot Series 2.0
analyzer and calculate the peptide-specific T-cell frequency
from the number of spot-forming cells.
IV. COMMENTS AND PITFALLS
It is always optimal to prepare T-cell cultures from
fresh material. T-cells behave very differently, and it is
always important to carefully inspect T-cell cultures
daily in a microscope. Thus, it is always possible to
alter the T-cell protocol depending on the target or the
donor, e.g., the concentration of cytokines, the addition
of new cytokines, the amount of antigen-presenting
cells, the day of restimulation, and the addition of antibodies
such as anti-CD28 anti-CD3, or other costimulatory
factors. Additionally, T-cells should never be
kept at too low cell densities, as cell-to-cell contact is
very important. This is also why round-bottom wells
are used instead of flat-bottom wells during the
cloning of T-cells. Furthermore, when T-cells are transferred
from small to larger wells, it is important to
carefully inspect the cells microscopically to ensure
that the cells are in good condition. In this regard,
feeder cells (e.g., irradiated, autologous PBL) may be
added if needed.
Andersen, M. H., Pedersen, L. O., Capeller, B., Brocker, E. B., Becker,
J. C., and thor, S. R (2001). Spontaneous cytotoxic T-cell responses
against survivin-derived MHC class I-restricted T-cell epitopes
in situ as well as ex vivo in cancer patients. Cancer Res
Brunner, K. T., Mauel, J., Cerottini, J. C., and Chapuis, B. (1968).
Quantitative assay of the lytic action of immune lymphoid cells
on 51-Cr-labelled allogeneic target cells in vitro
; inhibition by
isoantibody and by drugs. Immunology 14
Castelli, C., Rivoltini, L., Andreola, G., Carrabba, M., Renkvist, N.,
and Parmiani, G. (2000). T-cell recognition of melanomaassociated
antigens. J. Cell Physiol. 182
Dunbar, P. R., Chen, J. L., Chao, D., Rust, N., Teisserenc, H., Ogg,
G. S., Romero, P., Weynants, P., and Cerundolo, V. (1999).
Cutting edge: Rapid cloning of tumor-specific CTL suitable
for adoptive immunotherapy of melanoma. J. Immunol
Dunbar, P. R., Ogg, G. S., Chen, J., Rust, N., van der Bruggen, P., and
Cerundolo, V. (1998). Direct isolation, phenotyping and cloning
of low-frequency antigen-specific cytotoxic T-lymphocytes from
peripheral blood. Curr. Biol
Fu, Y. X., and Chaplin, D. D. (1999). Development and maturation
of secondary lymphoid tissues. Annu. Rev. Immunol. 17
Moss, P. A., Rosenberg, W. M., and Bell, J. I. (1992). The human T-cell
receptor in health and disease. Annu. Rev. Immunol
Oelke, M., Moehrle, U., Chen, J. L., Behringer, D., Cerundolo, V.,
Lindemann, A., and Mackensen, A. (2000). Generation and purification
melan-A-specific cytotoxic T-lymphocytes for
adoptive transfer in tumor immunotherapy. Clin. Cancer Res
Pawelec, G. (2000). New methods to approach immunotherapy of
cancer-and strategies of tumours to avoid elimination. Conference
report, on behalf of EUCAPS. European Cancer Research
Consortium. Cancer Immunol. Immunother
Pedersen, L. O., Nissen, M. H., Nielsen, L. L., Lauemoller, S. L.,
Hansen, N. J. V., Blicher, T., Hansen, A., Hviid, C. S., Thomsen,
A. R., and Buus, S. (2001). Efficient assembly of recombinant
major histocompatibility complex class I molecules with preformed
disulfide bonds. Eur. J. Immunol
Pittet, M. J., Valmori, D., Dunbar, P. R., Speiser, D. E., Lienard, D.,
Lejeune, E, Fleischhauer, K., Cerundolo, V., Cerottini, J. C., and
Romero, P. (1999). High frequencies of naive Melan-A/MART-1-
) T-cells in a large proportion of human histocompatibility
leukocyte antigen (HLA)-A2 individuals. J. Exp. Med
Scheibenbogen, C., Lee, K. H., Mayer, S., Stevanovic, S., Moebius, U.,
Herr, W., Rammensee, H. G., and Keilholz, U. (1997). A sensitive
ELISPOT assay for detection of CD8+
T-lymphocytes specific for
HLA class I-binding peptide epitopes derived from influenza
proteins in the blood of healthy donors and melanoma patients. Clin. Cancer Res. 3
Schrama, D., Andersen, M. H., Terheyden, P., Schroder, L., Pedersen,
L. O., thor Straten, P., and Becker, J. C. (2001). Oligoclonal
T-cell receptor usage of melanocyte differentiation antigen-reactive
T-Cells in stage IV melanoma patients. Cancer Res
Stockwin, L. H., McGonagle, D., Martin, I. G., and Blair, G. E. (2000).
Dendritic cells: Immunological sentinels with a central role in
health and disease. Immunol. Cell Biol