Download Fluorescent Antigen–Transfected Target Cell Cytotoxic T

MAJOR ARTICLE
Fluorescent Antigen–Transfected Target Cell Cytotoxic
T Lymphocyte Assay for Ex Vivo Detection
of Antigen-Specific Cell-Mediated Cytotoxicity
Carel A. van Baalen, David Kwa, Esther J. Verschuren, Mariska L. Reedijk, Adrianus C. M. Boon,
Gerrie de Mutsert, Guus F. Rimmelzwaan, Albert D. M. E. Osterhaus, and Rob A. Gruters
Department of Virology, Erasmus MC, University Medical Center and Postgraduate School of Molecular Medicine, Rotterdam, The Netherlands
Ex vivo detection of virus-specific cytotoxic T lymphocyte (CTL) responses is limited to the use of methods
assessing cytokine production, degranulation, or perforin contents of antigen-specific CD8+ T cells. Generally,
their cytotoxic activity is detectable only after cultivation. We describe the fluorescent antigen–transfected
target cell–CTL (FATT-CTL) assay, which measures antigen-specific cytotoxicity ex vivo. Target cells were generated by nucleofection with DNA vectors encoding antigen–green fluorescent protein (GFP) fusion proteins.
After coculture at various effector:target (E:T) cell ratios, viable and dead GFP-positive cells were quantified
by flow cytometry, and antigen-specific target-cell elimination was calculated. The assay was validated with
human immunodeficiency virus (HIV)– and influenza virus–specific CTL clones and revealed cytotoxicity at
lower E:T cell ratios than standard 51Cr-release assays. Moreover, antigen-specific cytotoxicity was detected ex
vivo within 1 day in peripheral blood mononuclear cells from HIV-infected individuals. The FATT-CTL assay
provides a versatile tool that will advance our understanding of cell-mediated immunity.
Virus-specific cytotoxic T lymphocytes (CTLs) contribute to the control of virus infection by eliminating infected cells and by producing antiviral cytokines [1].
During past decades, the most widely used method to
study CTL-mediated cytotoxicity has been the 51Cr-release assay [2]. Recently, alternative methods for the
detection of CTL responses have been developed, including enzyme-linked immunospot, intracellular cytokine staining (ICS), CD107 staining, and specific T
Received 2 February 2005; accepted 13 May 2005; electronically published 30
August 2005.
Presented in part: AIDS Vaccine 2004 International Conference, Lausanne,
Switzerland, 30 August–1 September 2005 (abstract P166); HIV Vaccines: Current
Challenges and Future Prospects, Keystone Symposia, Banff, Alberta, Canada, 9–
15 April 2005 (abstract 431); World Health Organization Consultation on Immunologic Assays to Evaluate Efficacy of Influenza Vaccines, Geneva, Switzerland, 25
January 2005.
Potential conflicts of interest: none reported.
Patent application on the technology is pending (Erasmus MC will be the patent
owner).
Financial support: Novaflu; European Union (grant QLRT-01034). R.A.G. is on
sabbatical leave from the Centre National de la Recherche Scientifique.
Reprints or correspondence: Dr. A.D.M.E. Osterhaus, Dept. of Virology, Erasmus
MC, PO Box 1738, 3000 DR Rotterdam, The Netherlands ([email protected]).
The Journal of Infectious Diseases 2005; 192:1183–90
2005 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2005/19207-0009$15.00
cell receptor staining by use of fluorescent major histocompatibility class (MHC) I peptide complexes [3–
6]. These methods are aimed at the detection of virusspecific CTLs in lymphocyte populations and do not
directly measure the outcome of the full cytolytic cascade [7, 8]. Several nonradioactive alternatives for the
51
Cr assay have been developed for the study of the
cytolytic activity of CTLs. These assays use fluorescent
dyes to discriminate target and effector cells and to
assess target-cell elimination [9–11].
When the epitopes are known, target cells can be
sensitized for lysis by specific CTLs by pulsing them
with excess peptide representing the epitope. For the
detection of CTLs against unknown epitopes, sets of
overlapping peptides spanning whole proteins can be
used [12, 13]. A disadvantage of this approach is that,
in addition to the costs related to peptide synthesis, the
repertoire of epitopes recognized after the infection of
cells may differ from that identified by exogenously
added peptides [14, 15]. Moreover, differences in avidity and the protective potential of CTLs cannot be
determined [16–18]. To achieve the endogenous processing of proteins, recombinant viral vectors like recombinant vaccinia virus are used for the infection
Ex Vivo Cell-Mediated Cytotoxicity • JID 2005:192 (1 October) • 1183
Figure 1. Construction of plasmid DNA vectors for the expression of antigen–fluorescent protein fusion proteins. A, Construct of HIV genes encoding
Rev, Tat, Gag, and Nef in the codon-optimized subtype B consensus sequence. B, Multiple cloning site of N1 Living Colors vectors. C, Spacer for creating
an in-frame cloning site for the influenza virus genes. D, Influenza virus genes encoding nucleoprotein (NP) strain A/NL/18/94 (NP01), NP strain A/HK/2/
68 (NP02), NP strain A/PR/8/34 (NP03), and matrix protein 1 (M1) strain A/NL/18/94. E, Expression of HIV-1 Rev–green fluorescent protein (GFP), Tat-GFP,
and influenza virus NP01-GFP in nucleofected B lymphoblastoid cells. Left, Live-gated (FSC/TP3) B157 cells, 20 h after nucleofection with 5 mg pRev-GFP
(solid lines, 53.3% in M1) or pTat-GFP (dashed lines, 59.4%) and with untreated B157 cells (dotted lines, 0.71%). Right, Live-gated (FSC/TP3) B3180 cells,
20 h after nucleofection with 4 mg of pNP01-GFP (solid lines, 30.7%) and untreated B3180 cells (dotted lines, 0.9%).
of target cells [19]. In human studies, this use is complicated
by the existence of preexisting immunity against the vector,
which often results in high levels of background CTL immunity
at baseline.
Direct expression of the relevant protein antigens from plasmid
DNA in appropriate MHC-matched target cells would circumvent this problem [3]. However, classic transfection techniques
usually result in poor viability, as well as in low transfection
efficiencies, and long-term cultures of cells in selective media are
required to obtain sufficient numbers of antigen-presenting cells
(APCs).
For the development of a versatile, nonradioactive procedure
to measure T cell–mediated cytotoxicity, we used nucleofection,
a novel transfection technology that enables the rapid and highlevel expression of foreign genes in resting and activated cells
[20, 21]. In this assay, fluorescent antigen–transfected target
cells (FATT) are cocultured with CTL effector cells, and their
killing by CTLs is monitored by flow cytometry.
MATERIALS AND METHODS
Effector cell lines and clones. The generation and culture of
the CD8+ T cell clones used in the present study have been de1184 • JID 2005:192 (1 October) • van Baalen et al.
scribed elsewhere [22–24]. The clones were 709TCC108, which
is specific for the HIV Rev67–75 epitope SAEPVPLQL; TCC-C10,
the influenza A nucleoprotein (NP)418–426 epitope LPFEKSTVM,
which is restricted via HLA-B*3501; TCC3180, the influenza
A NP418–426 epitope LPFEKSTVM, which is restricted via HLAB*3501; TCC1.7, the influenza A NP44–52 epitope CTELKLSDY,
which is restricted via HLA-A*0101; and TCCM1/A2, the influenza A matrix protein 1 (M1)58–66 epitope GILGFVFTL, which
is restricted via HL-A*0201. The cells were cultured for at least
7 days after stimulation with phytohemagglutinin (PHA) and
feeder cells before their use as effector cells in CTL assays.
Vectors. The cloning strategy for the construction of vectors plasmid (p) Rev–green fluorescent protein (GFP), pTatGFP, pGag-GFP, pNef-GFP, pNP01-GFP, pNP02-GFP, pNP03GFP, and pM1-GFP is depicted in figure 1. Genes were cloned
into the multiple cloning site of the Living Colors vector
pEGFP-N1 (BD Biosciences) in frame with the fluorescent protein open-reading frame (ORF) by use of the indicated restriction enzymes. By omitting the stop codon of the cloned genes,
a read through of the fluorescent gene was achieved. HIV genes
were codon-optimized consensus subtype B synthetic genes
(GeneArt). Influenza virus genes were derived from NP strain
A/NL/18/94 (NP01), NP strain A/HK/2/68 (NP02), NP strain
A/PR/8/34 (NP03), and M1 strain A/NL/18/94 (M1) [25, 26].
Inserts were sequenced completely, to confirm that no errors
had been introduced and that they were expressed in frame
with the fluorescent protein ORF. Sequences have been submitted to GenBank (accession numbers AY936877–AY936886).
Plasmid DNA was prepared by use of the Endo-Free Plasmid
maxi kit from Qiagen, in accordance with the manufacturer’s
instructions. Vector DNA prepared with this kit contains !0.1
endotoxin units (0.046 ng of lipopolysaccharide)/mg of DNA
(see http://www1.qiagen.com for further details).
Target cells. Two Epstein-Barr virus (EBV)–transformed B
lymphoblastoid cell lines (BLCLs), B157 and B3180, were used
as sources of autologous or HLA-matched target cells for the
CTL clones. BLCLs were cultured in RPMI 1640 supplemented
with 2 mmol/L l-glutamine, 100 U/mL penicillin, 100 mg/mL
streptomycin, 1 ⫻ 10⫺5 mol/L 2-mercaptoethanol, and 10% fetal bovine serum (R10F; Greiner, Bio-One). Antigen expression
was achieved by transfecting BLCLs with plasmid DNA vectors by use of the Amaxa Nucleofector technology, in accordance with the manufacturers’ instructions. Briefly, 1–2 ⫻ 10 6
cells in the logarithmic growth phase were resuspended in 100
mL of nucleofection buffer (Amaxa) that contained 2–4 mg of
DNA; they were then subjected to one of the electroporation
programs. Subsequently, cells were cultured overnight in a final
volume of 2–4 mL of R10F in 5% CO2 at 37C. All buffers and
programs of the Cell Line Optimization Nucleofector kit
(Amaxa) were tested, and the combination of buffer V with
program P-16 (Amaxa) resulted in the highest concentration
of viable GFP-expressing cells, combined with high overall viability—that is, 50% after 24 h (data not shown). Target cells
for the ex vivo FATT-CTL assay were generated by nucleofecting freshly isolated peripheral blood mononuclear cells
(PBMCs) by use of the optimized Human T Cell Nucleofector
kit (Amaxa), as described below. This resulted in 30%–70%
GFP-positive events in live-gated populations after 24 h.
FATT-CTL assay (4 h). Target cells were washed and cocultured with effector cells at increasing effector:target cell
(E:T) ratios in 200 mL of R10F in 5% CO2 for 3–4 h at 37C.
Cells were transferred to wells or tubes that contained 5 mL of
EDTA (final concentration, 2.5 mmol/L), to reduce the number
of cell-cell conjugates, and 5 mL of TO-PRO-3 iodide (TP3; final concentration, 25 nmol/L; Molecular Probes), to discriminate between viable and nonviable cells [10]. In some experiments, EDTA/TP3-treated cells were cooled on ice and stained
with anti–CD8–phycoerythrin (PE; BD Biosciences) for 20 min
before acquisition. The 51Cr-release assay was performed as described elsewhere [22]. Samples were acquired on a FACSCalibur device (BD Biosciences) for a fixed period of 60 s/sample.
The forward scatter (FSC) acquisition threshold was set to include nonviable events. Debris was excluded by gating in FSC-
TP3 dot plots during data analyses. The flow rate was plotted
in a time-event histogram and generally proved to be constant
in each of the samples per experiment. If it was not, we defined
a region to select a shared period of constant flow rate. A region
to exclude GFP-negative events was defined in GFP-TP3 or
GFP-FL3 dot plots of the data acquired from cultures that
contained BLCLs that had not been nucleofected. GFP-positive
events derived from cultures that contained nucleofected BLCLs
were displayed in FSC-TP3 or GFP-TP3 dot plots for the definition of viable GFP-positive (VG) events—that is, TP3-negative events—and nonviable or dead GFP-positive (DG) events—
that is, TP3-positive events (figure 2A). Percentages of dead
GFP-positive events (%DG) were calculated with the formula
100 ⫻ (number of DG)/(number of VG + number of DG). CTLmediated target-cell elimination was calculated with the for-
Figure 2. Antigen-specific killing of fluorescent antigen–transfected B
lymphoblastoid cells and peripheral blood mononuclear cells (PBMCs) by
cloned cytotoxic T lymphocyte (CTL) populations. A, Green fluorescent protein
(GFP)– and TP3-fluorescence intensities of pRev-GFP– (top) or pTat-GFP–
nucleofected (bottom) B157 cells that had been cocultured with or without
cells of a Rev-specific CTL clone at the indicated effector:target (E:T) ratios
for 4 h. The mean fluorescence intensity of control GFP-negative events
was ∼4. Nos. of viable and dead GFP-positive events detected during a
fixed acquisition period of constant flow rate are indicated. The percentage
of dead GFP-positive events is shown in parentheses. B, left, Percentage
CTL-mediated lysis with values as shown in panel A. Initial E:T ratios were
calculated from nos. of CD8+ and GFP-positive events detected in cultures
that contained effector or target cells only, at 0 h. B, right, Antigen-specific
lysis of pNP01-GFP–nucleofected PBMCs by cells of the influenza virus
nucleoprotein (NP)–specific CTL clone TCC-C10.
Ex Vivo Cell-Mediated Cytotoxicity • JID 2005:192 (1 October) • 1185
Figure 3. Comparison between 51Cr-release and fluorescent antigen–
transfected target cell–cytotoxic T lymphocyte (FATT-CTL) assays. B157 cells
were nucleofected with pRev–green fluorescent protein (GFP) or pTat-GFP;
after overnight incubation, one-half of the cells were labeled with 51Cr and
used as target cells in a standard 4-h 51Cr-release assay. The other half
was tested in a 4-h FATT-CTL assay. Target cells were cocultured with Revspecific CTLs at indicated effector:target (E:T) ratios, and percentages of
specific lysis were determined as described in Materials and Methods. T*,
calculation of the initial E:T ratio in the FATT-CTL assay including GFPpositive and GFP-negative target cells, to allow a direct comparison between
the 2 assays.
mula 100 ⫻ (%DG+E ⫺ %DG⫺E )/(100 ⫺ %DG⫺E ), where +E and
⫺E denote the presence and absence, respectively, of effector
cells in the cultures.
Ex vivo FATT-CTL assay (18–24 h). PBMCs were isolated
by density centrifugation (Lymphoprep; Nycomed) of heparinized blood (28–30 mL) obtained from 4 HIV-1–seropositive
individuals attending the Erasmus MC who had received no
antiretroviral therapy, had CD4 counts of 1300 cells/mm3, and
had a viral load between 50 and 1 ⫻ 10 5 RNA copies/mL. Informed consent was obtained from all patients. We isolated
PBMCs from buffy coats obtained from healthy blood donors
as controls. Freshly isolated PBMCs (2 ⫻ 10 6 cells/cuvette) were
nucleofected with plasmid DNA vectors (2 mg) by use of the
Human T Cell Nucleofector kit (Amaxa), in accordance with
the manufacturer’s instructions, and incubated in a humidified
incubator in 1.5– 2.0 mL of R10F in 5% CO2 at 37C. Four
hours later, a 50-mL sample was diluted 4-fold in R10F and
acquired for 60 s for the determination of the number of GFPpositive events per time unit. The volume per time unit (flow
rate) was determined in parallel for each experiment by use of
tubes that contained a calibrated number of beads (TruCOUNT
tubes; BD Biosciences) and 2 mL of FACSFlow sheath fluid
(BD Biosciences), without antibodies. The concentration of viable GFP-positive events was generally between 1 ⫻ 10 5 and
3 ⫻ 105 cells/1.6 mL. Between 3 ⫻ 10 3 and 5 ⫻ 10 3 GFP-positive
events were seeded per well and were cocultured with or without untreated PBMCs at a PBMC:GFP-positive cell ratio of
∼150 (triplicates) in conical 96-microwell Thermo-Fast 96 de1186 • JID 2005:192 (1 October) • van Baalen et al.
tection plates (ABgene), in a total volume of 200 mL/well with
or without recombinant interleukin (rIL)–2 (50 IU/mL). After overnight incubation, the cultures were transferred to 1.4mL U-tubes (Micronic) that contained 5 mL of EDTA (final
concentration, 2.5 mmol/L) and 5 mL of TP3 (final concentration, 25 nmol/L), incubated for 20 min at 37C, transferred
to melting ice, and acquired on a FACSCalibur within 2 h. To
prevent event count rates being 12000 total events/s, we set an
FL1 threshold during acquisition to exclude the majority of
GFP-negative events, in addition to an FSC threshold to exclude
debris. Because many killed GFP-positive cells can no longer
be detected as TP3 positive, for GFP-positive events after an
overnight incubation period (data not shown), we used the
difference between the number of VG events in cultures with
(VG+E) and without (VG⫺E) effector PBMCs to calculate the
percentage of PBMC-mediated antigen-specific target-cell elimination: 100 ⫻ (VG⫺E ⫺ VG+E )/VG⫺E.
ICS. ICS was performed with the BD FastImmune CD8 cytokine 4-color kit (BD Biosciences), in accordance with the manufacturer’s instructions. Overlapping peptide sets spanning Gag
or Nef (15-mers with 11 overlap, HXB2; National Institutes of
Health AIDS Research and Reference Reagent Program) were
added to 1 mL of whole blood each (final concentration, 4 mg/
mL for each peptide) and incubated for 6 h in the presence of
a CD28/CD49d monoclonal antibody mixture and brefeldin A
(both of which were included in the kit). Unstimulated (1 mL)
and staphylococcal enterotoxin B–stimulated (1 mg/mL; Sigma)
blood samples were included as negative and positive controls,
respectively. After the lyse/fix and permeabilization steps, cells
were stained with CD8 peridinin-chlorophyll-protein complex–
Cy5.5/CD3 allophycocyanin and anti–human interferon (IFN)–
g fluorescein isothiocyanate/CD69 PE or isotype controls, ac-
Figure 4. Cytotoxic T lymphocyte (CTL)–mediated killing of target cells
expressing recombinant influenza virus nucleoprotein (NP)— or matrix protein 1 (M1)–green fluorescent protein (GFP). B3180 cells were nucleofected
with pNP01-GFP, pNP02-GFP, pNP03-GFP, or pM1-GFP. The next day, these
cells were cocultured for 3 h with or without TCC1.7, TCC-C10, TCC3180,
and TCCM1/A2 cells at CD8+ :GFP-positive cell ratios of 10, 10, 5, and 2,
respectively. CTL-mediated target-cell elimination was determined as described in Materials and Methods.
Table 1. Specific lysis of nucleoprotein (NP)–green fluorescent
protein–positive cells by different CD8+ T cell clones.
CD8+ T cell clone
Gene
Epitope
NP01/02/03
NP01
NP02
NP03
M1
NOTE.
CTELKLSDY
LPFEKSTVM
---D-P-I---DRT-IGILGFVFTL
TCC1.7 TCC-C10 TCC3180 TCCM1/A2
a
50
…
…
…
…
…
…
0.8
0.5
15 ⫻ 103
26
1104
1100
…
…
…
…
…
…
50
M1, matrix protein 1.
a
Functional avidity: EC50 (nanomolar) of the cytotoxic T lymphocyte
clones for the epitope variants, as determined in a 51Cr-release assay [24].
quired on a FACSCalibur, and analyzed by use of CellQuest
software (version 4.0.2; BD Biosciences).
RESULTS
CTL-mediated lysis of fluorescent antigen–transfected BLCLs
and PBMCs. Genes encoding viral proteins of HIV (rev and
tat) and influenza A virus (nucleo- and matrix-protein) were
inserted in frame with GFP in the pEGFP-N1 plasmid, as depicted in figure 1. Nucleofection of cells of the EBV-transformed lymphoblastoid cell line (BLCL) B157 with pRev-GFP
and pTat-GFP resulted in 50%–60% GFP-positive cells (figure
1E). Antigen processing and the presentation of antigen-fluorescent fusion proteins (Ag-FP) was first assessed by coculturing
pRev-GFP–transfected B157 cells with cells of the Rev-specific
CTL clone (709TCC108) at increasing E:T ratios. We used pTatGFP–transfected B157 cells as negative controls. After 4 h of
incubation, percentages of dead target cells—that is, TP3-positive, GFP-positive cells—increased from 20% to 84% in an E:
T ratio–dependent fashion. The proportion of nonviable control target cells did not increase (figure 2A). After we corrected
the values for spontaneous background dead cells, the antigenspecific cytolytic activity of the Rev-specific CTLs at E:T ratios
of ∼0.3, ∼1, and ∼3 were 18%, 58%, and 80%, respectively
(figure 2B, left). Next, we explored the use of fresh PBMCs as
target cells. The nucleofection efficiency of unstimulated PBMCs
or CD8+ cell–depleted PBMCs was typically between 30% and
70% (data not shown), which proved to be sufficient for their
use as target cells. MHC class I–matched PBMCs, nucleofected
with pNP01-GFP, were lysed by the CTL clone TCC-C10 and
thus could be used as target cells in the FATT-CTL assay (figure
2B, right).
The FATT-CTL assay was compared with a standard 51Crrelease assay by use of the same target- and effector-cell populations in both assays. Again, 55%–60% viable GFP-positive
events were detected among pRev-GFP– and pTat-GFP–transfected B157 cells. Under the assumption that CTL epitopes were
generated in the GFP-positive cells only, this would be the maximum level of specific lysis that could be achieved in the 51Cr-
release assay. Indeed, 58% specific lysis was observed at the highest E:T ratio, 10 (figure 3). When the FATT-CTL assay was used,
190% of the GFP-positive cells were lysed by Rev-specific CTLs
after 4 h at the highest E:T ratio. Specific lysis of pTat-GFP–
positive cells was !3% for all E:T ratios tested in both assays
(data not shown). Overall, the FATT-CTL assay detected cytotoxicity at significantly lower E:T ratios than the 51Cr-release assay
(figure 3).
Part of the increased sensitivity is most likely due to the ability
to discriminate between cells expressing the gene of interest from
cells that do not. This was supported by the results of separate
experiments with influenza A virus NP-GFP constructs and the
NP-specific CTL clone TCC-C10. Maximal CTL-mediated 51Cr
release never exceeded the fractions of NP-GFP–expressing target
cells, which were ∼20% in these experiments (data not shown).
Parallel flow-cytometric analyses confirmed that the number of
effector cells added to these cultures was sufficient to eliminate
190% of the GFP-positive cells (data not shown).
CTL assays with influenza A virus–specific CTLs and epitope variants. To study the effects of epitope variation on the
outcome of the FATT-CTL assay, we generated expression vectors encoding various influenza A virus NP- and M1-GFP fusion proteins. Three vectors were generated from NP-genes
derived from distinct influenza virus strains: pNP01-, pNP02-,
and pNP03-GFP. These genes contained the same HLA-A*0101
epitope NP44–52 sequence but differed in the HLA-B*3501 epitope NP418–426 sequence (figure 4 and table 1). The pM1-GFP
vector encoded the HLA-A*0201–restricted epitope M158–66.
B3180 cells, which express HLA-A*0101, -A*0201, and -B*3501,
were nucleofected with the different vectors (figure 1E) and were
cocultured the following day for 3 h with or without cells of 3
different NP-specific CTL clones—TCC1.7, TCC-C10, and
TCC3180—or the M1-specific CTL clone M1/A2.
Between 60% and 70% specific lysis was detected among
NP01-, NP02-, and NP03-GFP–positive cells in cultures that
contained HLA-A*0101–restricted TCC1.7 CTLs specific for
the conserved NP44–52 epitope (figure 4). HLA-B*3501–restricted TCC-C10 cells also specifically lysed NP01-GFP–positive cells (70%) but did not lyse NP02- and NP03-GFP–
positive cells, in concordance with previously determined
EC50 values of the corresponding peptide variants, ∼0.8,
15000, and 110,000 nmol/L, respectively [24]. NP01-GFP–
positive cells were lysed with similar efficiency by TCC3180
cells that recognized the NP01 peptide variant, with an EC50
value of 0.5 nmol/L. The lower avidity of these cells for the
NP02-variant peptide (EC50, 26 nmol/L) was reflected by an
∼4-fold lower level of specific lysis of NP02-GFP–positive
cells, compared with NP01-GFP–positive cells (table 1). Cells
expressing the NP03 variant (EC50, 1100 nmol/L) were not
lysed by TCC3180. The M1-specific TCC-M1/A2 CTLs did
Ex Vivo Cell-Mediated Cytotoxicity • JID 2005:192 (1 October) • 1187
Figure 5. Ex vivo antigen-specific peripheral blood mononuclear cell
(PBMC)–mediated elimination of HIV-1 Gag–green fluorescent protein (GFP)–
or Nef-GFP–expressing lymphocytes. PBMCs obtained from 4 HIV-1–seropositive individuals were nucleofected with pEGFP-N1, pGag-GFP, or pNefGFP after a 4-h coculture with autologous untreated PBMCs in the absence
(individual RH1-021) or presence (individuals RH1-022, RH1-028, and RH1029) of 50 IU/mL recombinant interleukin–2 at PBMC:GFP-positive ratios
of ∼150. After overnight incubation, viable GFP-positive cells were quantified
by flow cytometry and used to calculate percentages of cell-mediated targetcell elimination. Values represent the average +SE of triplicates. Vertically
arranged below the fluorescent antigen–transfected target cell–CTL (FATTCTL) assay results from individuals RH1-022, RH1-028, and RH1-029 are
dot plots showing CD69 and interferon (IFN)–g expression of CD3+CD8+gated lymphocytes, 6 h after stimulation with no peptides or with HIV-1
Gag or Nef peptide pools. Nos. representing percentages of CD69+IFN-g–
positive cells are indicated in each dot plot.
not specifically lyse the NP-GFP–expressing cells but lysed
50% of M1-GFP–positive cells (table 1).
Detecting antigen-specific cytotoxicity ex vivo. We tested
whether the FATT-CTL assay could be applied to detect antigen-specific cell-mediated cytotoxicity directly ex vivo. To this
end, PBMCs were obtained from 4 highly active antiretroviral therapy–naive HIV-seropositive individuals and 4 HIV-seronegative individuals. Some of the cells were used to generate target cells by nucleofection with pGag-GFP, pNef-GFP,
or pEGFP-N1 as a control. Gag and Nef were chosen as antigens because they are among those most frequently recognized. Four hours later, nucleofected and autologous untreated
1188 • JID 2005:192 (1 October) • van Baalen et al.
PBMCs were cocultured at PBMC:GFP-positive cell ratios of
∼150 with or without the addition of rIL-2. After overnight
incubation, concentrations of viable GFP-positive events were
used to calculate antigen-specific target-cell elimination.
The specific elimination of Gag-GFP– and/or Nef-GFP–expressing cells, compared with that of GFP-expressing cells, was
observed in the absence (individual RH1-021) or presence (individuals RH1-022, RH1-028, and RH1-029) of exogenous IL2 (figure 5). For individuals RH1-022, RH1-028, and RH1-029,
no significant cytotoxicity was observed in the absence of IL2 (data not shown). Because of a limited number of cells, we
could not determine cytotoxicity in the presence of IL-2 for individual RH1-021. No Gag- or Nef-specific cytotoxicity, compared with that of GFP alone, was observed for any of the 4
seronegative control subjects, irrespective of the presence of exogenous IL-2 (data not shown). These data illustrate the practical utility of the FATT-CTL assay to directly measure virus-specific CTL activity ex vivo.
Next, we tested whether the PBMC-mediated elimination of
target cells correlated with the presence of antigen-specific CD8+
T cells. To this end, we stimulated cells obtained from individuals RH1-022, RH1-028, and RH1-029 with pools of overlapping
peptides spanning Gag or Nef and determined the percentages
of CD3+CD8+ cells that responded by up-regulating CD69-expression and IFN-g production, using a standard ICS assay. Figure 5 shows that, when lysis was detected in the FATT-CTL assay,
antigen-specific CD3+CD8+ cells were readily observed.
DISCUSSION
In the present article, a novel method for the measurement of
CTL-mediated cytotoxicity, the FATT-CTL assay, has been described. This method is based on the use of target cells that
have been transfected with plasmid DNA encoding the protein
of interest as a fluorescent fusion protein. The procedure was
evaluated with freshly isolated PBMCs and with HIV-1– and
influenza virus–specific CTL clones, by use of autologous PBMCs
or MHC class I–matched BLCLs as target cells.
Compared with other methods for the measurement of CTL
responses, the FATT-CTL assay offers a number of advantages.
In contrast to the traditional 51Cr-release assay, it is performed
without the use of radioactive isotopes. Target cells are discriminated from effector cells without the need to stain these
cell populations with fluorescent dyes like PKH-26 or carboxyfluorescein diacetate succinimidyl ester [9–11]. The FATT-CTL
assay facilitates the detection of CTL responses directed against
entire proteins without the need for the generation of recombinant viral vectors or the use of sets of overlapping peptides.
Potential high background CTL activity found against EBV and
recombinant vectors, like vaccinia virus, which have been used
in other assays to generate target cells expressing the antigen of
choice, does not interfere with the outcome of the FATT-CTL
assay. In contrast to the ex vivo detection of virus-specific CTLs
with fluorescent oligomers of HLA class I peptide complexes or
ICS of cytokines on the stimulation of CTLs with peptides representing known CTL epitopes, HLA typing of study individuals or the selection of common haplotypes is not required. The
processing and presentation of endogenously expressed antigens mimic natural conditions more closely than does the use
of exogenous synthetic peptides. In the experiments presented,
HIV-1 subtype B genes were used, which may differ antigenetically from autologous viruses. Therefore, CTL responses to
some epitopes may not have contributed to the elimination of
target cells. Given the relative ease of generating new Ag-FP
plasmids, analysis of the CTL repertoire against autologous
variants and against rapidly changing epitopes and of the role
that CTL-mediated immune pressure plays in individual patients is feasible. In vaccine trials, cell-mediated cytotoxicity can
be assessed by use of Ag-FP plasmids that contain the vaccine
antigen–encoding genes.
The FATT-CTL assay with NP-specific TCC-C10 and
TCC3180 cells reflected differences in their functional avidity
for variant epitope sequences. TCC3180 cells killed autologous
cells pulsed with saturating amounts of the LPFDKPTIM peptide (EC50, 26 nmol/L), but most of the cells expressing NP02,
the NP variant containing the corresponding epitope sequence,
were not killed. This could not be explained by limiting levels of NP expression in the target cells or the condition of the
effector cells, as evinced by the killing of NP02-expressing cells
by TCC1.7 cells or of NP01-expressing cells by TCC3180 cells,
respectively. These data show that, according to the results of
the FATT-CTL assay, differences in T cell–mediated cytotoxicity
toward various epitope variants can be detected. Recent studies
by other researchers have indicated that the use of saturating
peptide concentrations may detect CTLs of low avidity that are
unable to recognize tumor cells [27, 28]. Similar studies have
been reported for the association between avidity and effective
virus control [29, 30].
In HIV infection, virus control is associated with the induction and maintenance of antigen-specific CD8+ cells, especially if they express perforin [8, 31, 32], which indicates that
their cytotoxic activity is an important effector mechanism.
With the FATT-CTL assay, we can now directly assess the outcome of the cytolytic potential of CTLs, including those for
which no fluorescent oligomers of HLA class I peptide complexes are available. For 3 of the 4 individuals that we studied,
Gag- and Nef-specific cytotoxicity was found to be IL-2 dependent. The improvement of HIV-specific cytotoxicity by exogenous IL-2 has been reported to be associated with an increase in CD3z expression on CD8+ cells in HIV infection [33].
As for other ex vivo CTL detection methods, studies of the
influence of costimulatory factors on ex vivo cytotoxicity can
elucidate requirements for the optimal detection of cytotoxic
T cell responses and of the mechanisms of T cell dysfunction
in HIV infection [8].
In conclusion, the FATT-CTL assay offers unique opportunities for the specific and sensitive analysis of functional aspects of T cell–mediated cytotoxicity. This versatile method,
with its practical and direct ex vivo format, will allow moreefficient studies of the role of CTL-mediated immunity as a
correlate of protection in human and animal virus infections
and in vaccine trials.
Acknowledgments
We acknowledge M. E. van der Ende (Department of Internal Medicine,
Erasmus MC) and the individuals who donated blood, for their contribution to the study. The HIV-1 subtype B Gag and Nef (15-mer with 11
overlap, HXB2) peptides, complete set reagent was obtained through the
AIDS Research and Reference Reagent Program, Division of AIDS, National
Institute of Allergy and Infectious Diseases.
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