Download Tetramer Staining T Cells with Optimized HLA Class II + CD4

Ultrasensitive Detection and Phenotyping of
CD4 + T Cells with Optimized HLA Class II
Tetramer Staining
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of August 12, 2017.
Thomas J. Scriba, Marco Purbhoo, Cheryl L. Day, Nicola
Robinson, Sarah Fidler, Julie Fox, Jonathan N. Weber, Paul
Klenerman, Andrew K. Sewell and Rodney E. Phillips
J Immunol 2005; 175:6334-6343; ;
doi: 10.4049/jimmunol.175.10.6334
http://www.jimmunol.org/content/175/10/6334
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References
The Journal of Immunology
Ultrasensitive Detection and Phenotyping of CD4ⴙ T Cells
with Optimized HLA Class II Tetramer Staining1
Thomas J. Scriba,* Marco Purbhoo,† Cheryl L. Day,* Nicola Robinson,* Sarah Fidler,‡
Julie Fox,‡ Jonathan N. Weber,‡ Paul Klenerman,* Andrew K. Sewell,* and
Rodney E. Phillips2*
T
lymphocytes recognize antigenic peptides presented by
MHC molecules on the surface of APCs with their unique
TCR. The interaction between the TCR complex and peptide-MHC (pMHC)3 is weak and has a fast dissociation rate (1–3).
As a result, soluble recombinant monomeric pMHC molecules do
not stably adhere to the surface of cognate T cells. This limitation
can be overcome by increasing the valency of interactions by multimerization of biotinylated pMHC molecules. Multimerized
pMHC can be conjugated to fluorochromes to allow detection by
FACS (4). Such multimeric pMHC complexes were able to bind
multiple TCRs on Ag-specific cells and thus lowered the dissociation rate sufficiently to be used as an immunological stain. The
development of these peptide-HLA tetrameric complexes marked
the beginning of a new era in the study of Ag-specific T cells, and
HLA class I complexes have revolutionized the study of MHC
class I (MHCI)-restricted CD8⫹ T cell responses. Specifically, the
capacity to physically detect Ag-specific T cells in diverse states of
activation or function makes the use of HLA class I, and potentially class II tetramers, more powerful than techniques that rely on
*The Peter Medawar Building for Pathogen Research, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, United Kingdom; †Avidex Ltd., Abingdon, United Kingdom; and ‡Department of Medicine, Imperial College, St. Mary’s
Hospital, London, United Kingdom
Received for publication July 8, 2005. Accepted for publication September 12, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by SPARTAC, a Wellcome Trust clinical trial, and a
programme grant from the Wellcome Trust (to R.E.P. and J.N.W.). T.J.S. is funded
by the Fogarty Foundation and the South African National Research Foundation.
C.L.D is a Royal Society Research-funded fellow. P.K. is a Wellcome Trust Senior
Clinical Research Fellow, and A.K.S. is a Wellcome Trust Senior Basic Research
Fellow. R.E.P and P.K. are foundation investigators of the James Martin 21st Century
School, University of Oxford.
2
Address correspondence and reprint requests to Prof. Rodney E. Phillips, The Peter
Medawar Building for Pathogen Research, South Parks Road, Oxford, OX1 3SY,
U.K. E-mail address: [email protected]
3
Abbreviations used in this paper: pMHC, peptide-MHC; MHCI, MHC class I;
MHCII, MHC class II; ICS, intracellular cytokine staining; HCV, hepatitis C virus;
7-AAD, 7-aminoactinomycin D; HA, hemagglutinin; TT, tetanus toxoid; MFI, mean
fluorescence intensity; DIC, differential interference contrast.
Copyright © 2005 by The American Association of Immunologists, Inc.
the ability of cells to respond to synthetic Ags; a quality that may
define only a subset of the relevant cell population (5, 6).
The application of this powerful technology to the study of Agspecific CD4⫹ T cells has not been fully realized due to numerous
technical issues. Production of MHC class II (MHCII) complexes
has been more difficult than MHCI. The MHCI H chain can be
expressed in Escherichia coli as a soluble product by truncation of
the membrane-spanning domain and folded readily around the antigenic peptide in the presence of ␤2-microglobulin (4). pMHCII
molecules consist of two polymorphic chains (␣ and ␤), which
have been more difficult to refold around the antigenic peptide. E.
coli expression of the of MHCII alleles has been inefficient (7).
However, correctly folded molecules can be manufactured in insect and mammalian cells (8, 9). These efforts allowed successful
staining of in vitro-stimulated CD4⫹ T cell lines and clones (9 –
16). Despite these advances, the direct detection of Ag-specific
CD4⫹ T cells in PBMC remained difficult, mainly because the
cells are rare in peripheral blood (17–20).
Virtually all studies reporting MHCII tetramer staining used
high concentrations of tetramer (20 ␮g/ml or more), long incubation times (1–5 h) and temperatures of 23 or 37°C (8 –10, 13–17,
21–27). In these studies, HLA class II tetramers stained cognate T
cell clones poorly or not at all at 4°C (12, 21, 25). Cameron et al.
(25) further suggested that HLA class II tetramer staining was
dependent on active cellular processes by demonstrating blockade
of tetramer staining when endocytosis and cytoskeletal rearrangements were disrupted. Because the association rates of tetrameric
TCR/pMHCII interactions fall within a similar range to those seen for
TCR/pMHCI (28), the kinetics of HLA class II tetramer staining
should resemble those of HLA class I tetramers. Efficient staining of
CD8⫹ T cells was reported at 4°C, and incubation of cells with tetramer for 10 min is sufficient for complete staining (29, 30).
In this study, we determine the conditions required for optimal
HLA class II tetramer staining of DR1- and DR4-restricted CD4⫹
T cells specific for HIV, CMV, tetanus toxoid (TT), and influenza
virus. We find rapid and efficient staining of CD4⫹ T cells in
conditions similar to those reported for HLA class I staining of
CD8⫹ T cells. Active cellular processes such as internalization are
0022-1767/05/$02.00
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HLA class I tetramers have revolutionized the study of Ag-specific CD8ⴙ T cell responses. Technical problems and the rarity of
Ag-specific CD4ⴙ Th cells have not allowed the potential of HLA class II tetramers to be fully realized. Here, we optimize HLA
class II tetramer staining methods through the use of a comprehensive panel of HIV-, influenza-, CMV-, and tetanus toxoid-specific
tetramers. We find rapid and efficient staining of DR1- and DR4-restricted CD4ⴙ cell lines and clones and show that TCR
internalization is not a requirement for immunological staining. We combine tetramer staining with magnetic bead enrichment to
detect rare Ag-specific CD4ⴙ T cells with frequencies as low as 1 in 250,000 (0.0004% of CD4ⴙ cells) in human PBLs analyzed
directly ex vivo. This ultrasensitive detection allowed phenotypic analysis of rare CD4ⴙ T lymphocytes that had experienced
diverse exposure to Ag during the course of viral infections. These cells would not be detectable with normal flow-cytometric
techniques. The Journal of Immunology, 2005, 175: 6334 – 6343.
The Journal of Immunology
6335
not a requirement for staining. We test and apply an optimized
method in combination with a magnetic bead enrichment technique to detect HIV- and CMV-specific CD4⫹ T cells at frequencies below 1 in 100,000 in direct ex vivo human PBMC samples.
This technology allowed us to compare the phenotypic profile of
rare virus-specific CD4⫹ T cells from different infections.
Gag peptide p24.4 (aa 164 –183; AFSPEVIPMFSALSEGATPQ), and a
DRB1*0401 tetramer complexed to the hepatitis C virus (HCV) NS3 peptide (aa 1248 –1261; GYKVLVLNPSVAATL) was used as control. These
tetramers are referred to as p24.4-DR4 and HCV1-DR4, respectively. All
tetramers were PE-conjugated and supplied at a concentration of 100
␮g/ml.
Materials and Methods
Unless stated otherwise, cell lines and clones were incubated with 2 ␮g/ml
HLA class II tetramer for 30 min at 37°C in PBS/1% FCS or PBS/1% BSA
buffer. The cell surface marker Abs CD4-allophycocyanin or CD4-FITC
and 7-aminoactinomycin D (7-AAD) (all BD Pharmingen) were added for
the last 20 min or for 20 min after the cells were washed with cold PBS/1%
FCS. Stained cells were washed with cold PBS/1% FCS and fixed in 1%
PBS/formaldehyde. At least 30,000 cells were acquired on a FACSCalibur
flow cytometer (BD Biosciences), and data were analyzed using CellQuest
software (BD Biosciences).
Patients and HLA typing
Heparinized blood was obtained from HIV-infected patients who were recruited at St Mary’s Hospital, London, as described elsewhere (20), the
SPARTAC Trial or from healthy, HIV-negative donors. Informed consent
was obtained from all participants, and the study had full ethical approval.
PBMC were isolated by density gradient centrifugation of blood layered
over Lymphoprep (Axis Shield). High-resolution HLA class II genotypes
were determined for each study participant by PCR using sequence-specific
primers (31).
Cell lines and clones
MHCII tetramers
DRB1*0101 and DRB1*0401 iTAg MHCII tetramers were purchased
from Beckman Coulter. DRB1*0101 tetramers were complexed to the HIV
p24 Gag peptide p24.17 (aa 294 –313; FRDYVDRFYKTLRAEQASQD),
the p24 Gag peptide p24.14 (aa 264 –283; KRWIILGLNKIVRMYSPTSI),
the TT830 – 843 peptide (QYIKANSKFIGITE), the influenza hemagglutinin
(HA)307–319 peptide (PKYVKQNTLKLAT) and the CMV pp65108 –127
peptide (LPLKMLNIPSINVH). These tetramers are referred to as p24.17DR1, p24.14-DR1, TT830-DR1, HA307-DR1, and pp65-DR1, respectively. The DRB1*0401 tetramer p24.4-DR4 was complexed to the p24
Cell surface tetramer decay assay
CD4⫹ T cells were preincubated on ice for 5 min with chilled azide buffer
(0.1% NaN3, 0.5% BSA in PBS) and incubated with 1 ␮g/ml tetramer in
azide buffer for 2 h on ice. Stained cells were washed twice in chilled azide
buffer, split into two aliquots, and placed at room temperature. To one
aliquot, 100 ␮g/ml anti-HLA-DR Ab (clone L243; BD Biosciences) was
added, and cells were taken at time points 0, 1, 2, 5, 10, and 30 min,
resuspended in PBS, and acquired on a flow cytometer.
Microscopy
For microscopy, CD4⫹ T cells were stained at 37°C or on ice in azide
buffer as described for each experiment and washed with PBS/0.5% BSA.
For imaging, stained cells were suspended in PBS/0.5% BSA in eight-well
chambers (Labtek; Nunc). A Zeiss 200M/Universal Imaging system with a
⫻63 objective was used for wide-field fluorescence microscopy. PE fluorescence was detected using a 535/50 excitation, 610/75 emission, and
565LP dichroic filter set (Chroma). To cover the entire three-dimensional
surface of the cell, z-stack fluorescent images were taken (21 individual
planes; 1 mm apart). Data were analyzed using Metamorph software. Data
were evaluated for at least 20 cells in each experimental condition.
Detection of rare cells by magnetic bead enrichment and
phenotypic staining
Magnetic bead enrichment of tetramer-positive CD4⫹ T cells was done as
described previously (20). Briefly, the cells were incubated with 5 ␮g/ml
HLA class II tetramer (to compensate for large cell numbers). Anti-CD4allophycocyanin, anti-CD19-PerCP, and anti-CD14-PerCP Abs and
7-AAD (all from BD Biosciences) were added during the last 20 min for
the 120-min staining at 23°C and for 20 min after a wash for the 20-min
staining at 37°C. For phenotypic staining of tetramer-positive CD4⫹ cells,
anti-CCR7-FITC (R&D Systems) and anti-CD27-FITC and antiCD28-FITC (both from BD Biosciences) were added with the other Abs.
The cells were washed and labeled with anti-PE microbeads according to
the manufacturer’s protocol (Miltenyi Biotec), and 10% of the cells were
reserved for FACS analysis while 90% were subjected to magnetic bead
enrichment of PE-conjugated tetramer-positive cells using a single MACS
column (Miltenyi Biotec). All cells were acquired on a FACSCalibur flow
cytometer (BD Biosciences), gated on CD14⫺, CD19⫺, and 7-AAD-negative lymphocytes, and analyzed using CellQuest software (BD Biosciences). The frequency of tetramer-positive cells was calculated by dividing the number of postenrichment CD4⫹tetramer⫹ cells by the number
Table I. Ag-specific CD4⫹ T cell lines and clones
Cell Line/Clone
Ox24-p24.17
Ox97-clone10
Ox24-p24.14
K37-p24.4
JF-TTox
HA1.7
JF-HA
LA-pp65
a
Line/Clone
Line
Clone
Line
Line
Line
Clone
Line
Line
HLA-DRB1*
Typea
*0101,
*0101,
*0101,
*0401
*0101,
*0101
*0101,
*0101,
*0102
*0401
*0102
*1401
*1401
*1301
HLA restriction of epitope specificity is in boldface type.
Epitope Specificity
Specific Tetramer
Epitope Sequence
HIV Gag p24294 –313
HIV Gag p24294 –313
HIV Gag p24264 –283
HIV Gag p24164 –183
TT830 – 843
Influenza HA307–319
Influenza HA307–319
CMV pp65108 –127
p24.17-DR1
p24.17-DR1
p24.14-DR1
p24.4-DR4
TT830-DR1
HA307-DR1
HA307-DR1
pp65-DR1
FRDYVDRFYKTLRAEQASQD
FRDYVDRFYKTLRAEQASQD
KRWIILGLNKIVRMYSPTSI
AFSPEVIPMFSALSEGATPQ
QYIKANSKFIGITE
PKYVKQNTLKLAT
PKYVKQNTLKLAT
LPLKMLNIPSINVH
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Fresh PBMC were depleted of CD8⫹ cells using anti-CD8-conjugated
magnetic beads (Dynal) and stimulated with 1–2 ␮M peptide in RPMI
1640 medium containing 10% heat-inactivated human AB⫹ serum. When
lines were cultured from HIV-infected patients, 0.4 ␮M indinavir (Merck)
and 0.5 ␮M zidovudine (GlaxoSmithKline) were added to the medium.
The plates were incubated at 37°C in 5% CO2, and after 3 days, the medium was supplemented with 100 U/ml IL-2 (Proleukin; Chiron) and 5%
human T cell culture supplement (T-STIM) (BD Biosciences). Medium
was exchanged as necessary. Cell lines were restimulated with peptide,
IL-2, human T cell culture supplement (T-STIM), and irradiated autologous PBMC every 12–14 days. For generation of CD4⫹ T cell clones, cell
lines were stimulated with 4 ␮M peptide for 4 h, and IFN-␥- and IL-2producing cells were selected using a cytokine detection and enrichment
method (Miltenyi Biotec). Ag-specific (IFN-␥⫹ and IL-2⫹ cells) CD4⫹
cells were plated at limiting dilution in medium containing 10% human
AB⫹ serum, 2 ⫻ 105 irradiated mixed lymphocytes/ml, 100 U/ml IL-2, 5%
T-STIM, and 1 ␮g/ml PHA (Sigma-Aldrich). Cell lines and clones were
tested for Ag specificity by IFN-␥ ELISPOT, intracellular cytokine staining
(ICS), or HLA class II tetramer staining. ELISPOT and ICS assays were
performed with either peptides or recombinant proteins as Ag as described
(20). The cell lines and clones used in this study are listed in Table I. All
cell lines were used for experiments after two or three rounds of antigenic
stimulation, and all experiments were done at least 7 days after restimulation of cell lines and clones. The influenza HA307-319-specific CD4⫹ T
cell clone HA1.7 (32) was kindly donated by J. Lamb (Medical Research
Council Center for Inflammation Research, University of Edinburgh, Edinburgh, U.K.).
Tetramer staining and flow cytometry
ULTRASENSITIVE DETECTION AND PHENOTYPING OF CD4⫹ T CELLS
6336
of CD4⫹ cells in the pre-enrichment sample multiplied by 9 (to account for
the fact that 90% of the cells were used for the enrichment).
Results
Staining of Ag-specific CD4⫹ T cells with HLA class II
tetramers is highly specific
⫹
Optimizing the use of HLA class II tetramers for staining T cells
A wide range of HLA class II tetramer concentrations are reported
to be required for adequate staining of specific CD4⫹ T cells.
Some investigators have used tetramer concentrations of 20 ␮g/ml
or more (8 –10, 12, 13, 15–18, 21, 23–26). Others have used lower
concentrations (11, 14, 20, 22, 33, 34). Meyer et al. (17) found that
certain Borrelia-specific CD4⫹ T cell clones could be stained with
as little as 0.2 ␮g/ml. To assess the optimal HLA class II tetramer
staining concentrations for a range of cell lines and clones, we
incubated CD4⫹ T cells with tetramer ranging from 0.01 to 10
␮g/ml in a total volume of 100 ␮l (Fig. 2) for 30 min at 37°C.
Although some variation was seen, 2 ␮g/ml tetramer was sufficient
HLA class II tetramer staining is rapid
CD8⫹ T cells can readily be labeled with HLA class I tetramers by
incubation at 37°C for 10 min (29, 35) and staining can be detected
as early as after 30 s (29). To test the rate at which HLA class II
tetramers stained CD4⫹ T cells, we incubated all cell lines and
clones with 2 ␮g/ml HLA class II tetramer at 37 and 23°C for
1–120 min. We found efficient staining of CD4⫹ T cells of all
specificities within 10 min at 37°C. This was observed with the
TT, the influenza HA and CMV pp65 DR1 tetramers as well as the
HIV Gag p24 DR1 and DR4 tetramers when used to stain all
CD4⫹ cell lines and clones (Fig. 3, A and B). Although all cells
were stained after 10 min, the mean fluorescence intensity (MFI)
continued to increase up to 120 min after starting incubation with
the tetramer (data not shown). Notably, the proportion of
tetramer⫹CD4⫺ cells also increased with time, suggesting that internalization of pMHCII-TCR complexes was taking place (data
not shown). At 23°C, tetramer staining was slower for most specificities, although the HIV-specific Ox24-p24.17 and K37-p24.4
cell lines and the influenza-specific HA1.7 clone were fully stained
after 10 min. At 23°C, complete staining of all CD4⫹ T cells was
seen only after 30 – 60 min for the other cell lines and clones (Fig.
3C), and the MFI was generally lower than when cells were stained
at 37°C (data not shown).
HLA class II tetramer staining does not require internalization
Labeling of Ag-specific CD8⫹ T cells with HLA class I tetramers
can be efficient at 4°C (30, 35). However, some earlier investigators failed to label Ag-specific CD4⫹ T cells with HLA class II
FIGURE 1. HLA class II tetramer staining of Ag-specific CD4⫹ T cell lines and clones. A, Testing the frequency of Ag-specific CD4⫹ T cells by
intracellular IFN-␥ staining. FACS dot plots showing Th cell lines Ox24-p24.17 and K37-p24.4 either unstimulated (No peptide) or stimulated with 2 ␮M
peptide. The percentage of CD4⫹ cells producing IFN-␥ is indicated in each plot. B, FACS dot plots showing the cell lines stained with their matched (upper
plots) and unmatched (lower plots) HLA class II tetramer. The percentage of tetramer⫹CD4⫹ cells is indicated in each plot. The histograms represent the
HIV-p24-specific Ox97-clone10 and the influenza HA-specific clone HA1.7 stained with matched (in red) and three unmatched HLA class II tetramers.
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We studied the labeling of Ag-specific CD4 T cells with HLA
class II tetramers at different experimental conditions. Cell lines
and clones specific for antigenic peptides encoded by HIV type 1
(HIV-1) p24 Gag, influenza HA, TT, or human CMV pp65 were
generated and tested for specificity by IFN-␥ ELISPOT assay or
ICS (Fig. 1A). Clones and CD4⫹ T cell lines were stained with
their corresponding peptide-HLA class II tetramer (Fig. 1B). Although not identical, the frequencies of HLA class II tetramerpositive CD4⫹ cells were comparable with the frequencies of IFN␥-producing cells as measured by ICS (Fig. 1 and data not shown).
Staining of all cell lines and clones with peptide-mismatched,
DRB1-allele-matched tetramers yielded CD4⫹tetramer⫹ populations that never exceeded 0.2% (Fig. 1B). The cell lines and clones
and peptide-matched HLA class II tetramers are shown in Table I.
to label Ag-specific cells within their respective cell lines and to
label true clones as well. At 5–10 ␮g/ml and higher concentrations,
background staining increased for all tetramers as nonspecific cells
were also stained (Fig. 2A and data not shown).
The Journal of Immunology
6337
FIGURE 2. Concentration titration of HLA class II
tetramers. A, FACS dot plots of the CD4⫹ T cell line
Ox24-p24.17 stained with the indicated concentrations
of p24.17-DR1 tetramer at 37°C for 30 min. The percentage of cells falling into the upper right quadrant is
indicated in each plot. B, Graphical representation of
the MFI obtained from concentration titrations of five
HLA class II tetramers. C, Graphical representation of
the percentage CD4⫹tetramer⫹ cells recorded for each
tetramer.
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FIGURE 3. HLA class II tetramer staining is rapid. FACS density plots (A) and histograms (B) showing efficient HLA class II tetramer staining of
Ag-specific CD4⫹ cell lines and clones after a 10-min incubation at 37°C. C, Graphical representation of the percent maximum CD4⫹tetramer⫹ cells
measured when cells were stained at 37°C (upper chart) and 23°C (lower chart) for the indicated time periods.
6338
ULTRASENSITIVE DETECTION AND PHENOTYPING OF CD4⫹ T CELLS
of HLA-DR1-restricted, HIV p24-specific Ox97-clone10 stained
with tetramer at 37°C or in azide buffer on ice. When cells were
labeled at 37°C for 60 min, clear PE-clusters were visible that were
not associated with the cell membrane when the mid-cell z-section
of PE-fluorescence and differential interference contrast (DIC) images were overlaid (Fig. 6). These clusters typically localized to
compartments within the cytoplasm, suggesting internalized TCR/
tetramers. In contrast, when azide-treated cells were stained on ice,
PE-clusters of lower fluorescence intensity than those seen at 37°C
were only visible along the outer perimeter of the cells. These
clusters aligned with the plasma membrane when the DIC and
mid-cell z-section of PE-fluorescence images were overlaid, suggesting that visible tetramers were membrane-bound and not
internalized.
Sensitive detection of rare CD4⫹ T cells
The frequencies of most CD4⫹ Th cell populations are markedly
lower than those seen for CD8⫹ T cells (36). Because the lower
detection limit of flow cytometric techniques is ⬃0.02% (17, 37,
38), confident detection of many Ag-specific CD4⫹ T cell responses with conventional HLA class II tetramer staining and flow
cytometry is impossible. Recently, the use of HLA class I and II
tetramers and a magnetic bead enrichment technique has improved
FIGURE 4. Staining of cell lines and clones with HLA class II tetramer at 4°C. A, FACS density plots showing staining of cell lines for 120 min at 4°C.
B, Histograms of the DR1-restricted CD4⫹ clones Ox97-clone10 and HA1.7 showing staining for 120 min at 4°C. C, Graphical representation of the percent
CD4⫹tetramer⫹ cells (upper chart) and the MFI (lower chart) when cells were stained at 4°C for the indicated time periods expressed as a percentage of
the maximum recorded at 37°C.
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tetramers at 4°C (12, 21, 25). By contrast, we stained CD4⫹ T cell
lines and clones with tetramer at 4°C (Fig. 4). The HIV p24-specific cell lines Ox24-p24.17 and K37-p24.4 and Ox97-clone10
could be stained at 4 and at 0°C. Successful HLA class II tetramer
staining was also seen when the TT-specific cell line JF-TT830
and the influenza-specific cell line JF-HA were incubated with
their cognate tetramers at 4°C. Staining at 4°C was markedly
slower than at 23 or 37°C and resulted in considerably lower fluorescence intensities (Fig. 4C).
Successful staining at 4°C suggests that CD4⫹ T cells can be
labeled with surface-bound tetramer and that this process does not
require receptor internalization. To assess this further, we stained
prechilled, azide-treated cells with HLA class II tetramer in azide
buffer on ice. Following removal of unbound tetramer and blocking of tetramer rebinding, the decay of cell surface-bound tetramer
was followed by monitoring the decrease of the PE-fluorescence.
Azide-treated CD4⫹ T cells from the HIV-Gag p24-specific Ox97clone10 and JF-TT830 were labeled with HLA class II tetramer on
ice (Fig. 5 and not shown). When rebinding of unbound tetramer
was blocked with anti-HLA-DR Ab, the fluorescence of tetramerlabeled cells fell, suggesting tetramer dissociation from the cell
surface. This effect was seen to a lesser degree when no competitor
Ab was added (Fig. 5).
To confirm that HLA class II tetramer can bind CD4⫹ T cells
without being internalized, we performed fluorescence microscopy
The Journal of Immunology
the sensitivity of detection considerably (18 –20, 37). This technique enriches the cells of interest and reduces background staining by ensuring that only cell surface-stained cells are enriched
(thereby eliminating nonspecific tetramer internalization). We previously demonstrated that combination of these techniques yielded
a linear enrichment recovery from human PBMC (20). The application of the optimized tetramer labeling techniques developed in
this report to the study of CD4⫹ T cell populations directly ex vivo
may allow HLA class II tetramers to become more useful. To
optimize the combination of HLA class II tetramer staining and
magnetic bead enrichment, a balance must be met between obtaining tetramer staining intense enough to allow discrimination between tetramer-positive and -negative populations. Internalization
of tetramers must also be prevented so that magnetic beads can
bind to cell surface-associated tetramers. To address this, we compared the detection of Ag-specific CD4⫹ T cells by HLA class II
tetramer staining and magnetic bead enrichment with two methods: staining at 23°C for 120 min and 37°C for 20 min. The mean
and SEs obtained for triplicate stainings at two defined spike frequencies (0.8 and 1.6%) before and after magnetic bead enrichment are shown in Fig. 7. The mean pre- (Fig. 7A) and postenrichment (B) CD4⫹tetramer⫹ frequencies obtained with the
different staining protocols were virtually identical. When the enrichment recovery was determined (expected postenrichment frequency of CD4⫹tetramer⫹ cells as a percentage of observed
postenrichment frequency), the two methods were also indistinguishable (Fig. 7C). Notably, the recovery of tetramer⫹CD4⫹ cells
was ⬍50%, indicating that a considerable number of tetramerstained cells are lost during the enrichment process.
FIGURE 6. HLA class II tetramers are internalized at 37°C but remain
surface bound at 0°C. A, FACS histogram representing the fluorescence
intensity seen when Ox97-clone10 cells were stained with p24.17-DR1
tetramer for 60 min at 37°C or on ice in the presence of azide. B, Fluorescence microscopy of Ox97-clone10 cells stained with the PE-labeled
p24.17-DR1 tetramer at 37°C or on ice in the presence of azide. The DIC
and mid-cell z section of PE-fluorescence recordings as well as an overlay
of the two are shown. Images were recorded at a ⫻63 magnification.
Direct ex vivo HLA class II tetramer staining of Ag-specific
CD4⫹ cells from PBMC at frequencies that fall below the typical
threshold of detection (0.02%) are shown in Fig. 8. Although a few
CD4⫹tetramer⫹ cells can be identified by normal flow cytometric
detection, the very low frequency does not allow confident quantification of such responses. However, after magnetic bead enrichment, the number and proportion of CD4⫹tetramer⫹ cells is increased, greatly enhancing the detection sensitivity and reducing
background staining (Fig. 8A). We performed cross-sectional
staining of HIV-infected individuals with the HIV-specific tetramers p24.4-DR4, p24.14-DR1, and p24.17-DR1, as well as the
CMV-specific pp65-DR1 tetramer. Patients with CD4⫹tetramer⫹
T cell responses ⬍0.02% were studied (Fig. 8). Tetramer staining
combined with magnetic bead enrichment detected Ag-specific
CD4⫹ responses reliably at frequencies as low as 0.0004% (1 in
250,000 CD4⫹ cells; Fig. 8B). No HLA class II tetramer staining
of HLA-mismatched or HLA-matched, HIV-negative and CMVnegative control PBMCs was seen with the p24.17-DR1 and
p24.4-DR4 or pp65-DR1 tetramers, respectively (data not shown
and Ref. 20).
Phenotypic analysis of virus-specific CD4⫹ T cells
Memory virus-specific CD4⫹ T cells have different functions and
different phenotypes that are thought to be associated with virus
persistence and re-exposure to Ag. In infections such as influenza
where virus is cleared from the host, virus-specific CD4⫹ T cells
have been shown to predominantly produce IL-2 and display a
CCR7⫹ phenotype. In contrast, the persistent Ag exposure seen in
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FIGURE 5. HLA class II tetramer can dissociate from the cell surface.
Azide-treated JF-TTox cells were stained with the TT830-DR1 tetramer in
ice. The fluorescence of surface-bound tetramer was then measured 1, 2, 5,
10, 20, and 30 min later in the presence or absence of a competitor Ab
(anti-HLA-DR). A, FACS density plots showing TT830-DR1 staining of
the JF-TTox cell line 30 min after the competitor was added. The percentage of CD4⫹tetramer⫹ cells is indicated in the top right quadrant of each
plot. B, Graphical representation of the percent CD4⫹tetramer⫹ cells detected at each time point.
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chronic, viremic HIV infection is most often associated with
CCR7⫺ HIV-specific CD4⫹ T cells that produce IFN-␥ (39, 40).
Most studies that have compared phenotypes of virus-specific
CD4⫹ T cells have relied on techniques that identify Ag-specific
cells by the detection of cytokines in response to Ag. These methods may fail to detect all of the cells with the defined specificity (5,
6). These methods may also alter the phenotype of the specific
cells during Ag-induced activation (41, 42). By using the sensitive
combination of HLA class II tetramer staining and magnetic bead
enrichment, we compared the phenotype of rare CD4⫹ T cells
specific for HIV p24 Gag, CMV pp65, influenza HA, and HCV by
costaining with HLA class II tetramers and surface-marker Abs.
The phenotypes of CD4⫹ T cells were previously analyzed with
the DR1-restricted, HIV p24 Gag-specific p24.17-DR1 tetramer in
viremic HIV infection (20); the DR1-restricted, influenza HA-specific HA307-DR1 tetramer in resolved influenza infection (19);
and the DR4-restricted, HCV nonstructural protein 3- and 4-specific DR4-HCV 1248, 1579, 1770 tetramers in resolved HCV infection (18). In this study, we measured the expression of CCR7,
CD27, and CD28 on CD4⫹ T cells with the DR4-restricted, HIV
p24 Gag-specific tetramer p24.4-DR4 in viremic HIV infection,
FIGURE 8. Detection of rare Ag-specific CD4⫹ T cell populations with
HLA class II tetramer staining and magnetic bead enrichment. A, Representative FACS dot plots showing normal tetramer staining (left plots) and
tetramer staining in combination with magnetic bead enrichment (right
plots). The percentage of cells falling into the top right quadrant is indicated for each plot. B, Cross-sectional quantification of Ag-specific CD4⫹
T cell frequencies by HLA class II tetramer staining and magnetic bead
enrichment from patients for whom normal tetramer staining yielded responses below the accepted limit for detection (0.02%). The data points in
B that correspond to the FACS plots shown in A are demarcated by matching shapes.
and the DR1-restricted, CMV pp65-specific pp65-DR1 tetramer in
HIV-negative individuals (Fig. 9). Memory CD4⫹ T cells of all
specificities expressed CD28 (HCV NS3- and NS4-specific tetramer data were not available). CD4⫹ Th cells specific for both
HIV p24 epitopes (p24.17-DR1 and p24.4-DR4), the influenza HA
epitope 307–319, and the HCV NS3 and NS4 epitopes were predominantly CD27-positive. However, a lower proportion of CMV
pp65-specific CD4⫹ Th cells expressed CD27. Interpatient variation of CD27 expression on CMV-specific CD4⫹ T cells (as indicated by error bars representing SEM) was also markedly higher
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FIGURE 7. Comparison of direct tetramer staining and magnetic bead
enrichment methods. PBMC were spiked at 0.8 and 1.6% with Ox97clone10 cells and stained with p24.17-DR1 tetramer for 120 min at 23°C
(f) or 20 min at 37°C (䡺). The mean frequency of tetramer⫹CD4⫹ cells
obtained from three repeat analyses before (A) and after (B) magnetic bead
enrichment is shown. Error bars represent SEM. C, The bars represent the
mean and SEM of the observed CD4⫹tetramer⫹ cell frequency recovered
following magnetic bead enrichment expressed as a percentage of the expected frequency calculated from the pre-enrichment sample.
The Journal of Immunology
than that measured for other specificities and surface markers (Fig.
9). A comparison of CCR7 expression on tetramer-positive cells
revealed significant differences. Virtually all HA-specific CD4⫹ T
cells from individuals with cleared influenza infection and HCVspecific Th cells from patients with resolved HCV infection were
CCR7⫹. In contrast, HIV-specific CD4⫹ Th cells in persistently
infected patients expressed significantly lower levels of CCR7.
Approximately 30% of CMV-specific CD4⫹ T cells expressed
CCR7 (Fig. 9).
Discussion
In this study, we optimized the conditions required to label Agspecific CD4⫹ T cells with HLA class II tetramers and developed
techniques for detection of HLA class II staining in direct ex vivo
samples. Many investigators have reported HLA class II tetramerstaining methods that differ substantially from those used to stain
HLA class I-restricted CD8⫹ T cells. CD8⫹ T cells can readily be
labeled with HLA class I tetramers by incubation with them at
37°C for 5–10 min as shown by FACS analysis and confocal microscopy (29, 30, 35). Because the association rates of tetrameric
TCR/pMHCI and TCR/pMHCII interactions are similar (28), HLA
class II tetramer staining of Ag-specific CD4⫹ T cells should be
rapid. Yet, most studies which report HLA class II tetramer staining used long incubation times (1–5 h) (8 –10, 13–17, 21–27, 33).
We find efficient and rapid staining of CD4⫹ T cells with HLA
class II tetramers. Specifically, incubation at 37°C for 10 min was
sufficient to allow complete labeling of all Ag-specific cells. At
23°C, staining was slower, and although some lines and clones
were stained after 10 min, complete staining for all cell specificities required 30 – 60 min.
Ag-specific CD8⫹ T cells can also be labeled with HLA class I
tetramers at 4°C (30, 35). Moreover, MHCI tetramers bind Agspecific T cells independent of a cellular response and do not require an active cellular process (4, 43, 44). However, little or no
HLA class II tetramer staining of CD4⫹ T cell clones was observed at 4°C in previous studies (12, 21, 25). These findings led
to the hypothesis that HLA class II tetramer staining is dependent
on active cellular processes. This was supported by experiments
that showed blockade of tetramer staining of the HA1.7 clone
when endocytosis and cytoskeletal rearrangements were disrupted
(25). In contrast to these studies, we demonstrate successful staining of the TT-, influenza HA-, and HIV Gag-specific cell lines and
the HIV Gag-specific Ox97-clone10 at 4°C. The JF-TTox cell line
and Ox97-clone10 were also labeled with tetramer in the presence
of azide on ice as detected by flow cytometry and wide-field microscopy. Furthermore, when the JF-TTox cell line and Ox97clone10 were prestained with HLA class II tetramer, PE-fluorescence decreased upon addition of the competitor Ab anti-HLADR, suggesting dissociation of cell surface-bound tetramer. These
results show that HLA class II tetramer can be surface-bound and
that active cellular processes such as TCR/pMHCII internalization
are not an absolute requirement for HLA class II tetramer staining
of Ag-specific CD4⫹ T cells.
Fluorescence microscopy showed a clear accumulation of PEconjugated HLA class II tetramer clusters that are internalized at
37°C. This finding is in agreement with Cameron et al. (25), who
reported tetramer clusters that colocalized with endocytic compartments. At 4°C or on ice, bound tetramer remained on the cell
surface. These surface-bound tetramers clustered. This suggested
that the TCRs were in close apposition before the tetramers were
added. This is in agreement with previous work, demonstrating
that TCRs are asymmetrically localized to distinct regions of the
plasma membrane that contain the highest concentration of lipid
rafts (45).
We were unable to completely stain some CD4⫹ T cells at 4°C.
Specifically, the HA-specific clone HA1.7 could only be partially
stained and the CMV-specific line LA-pp65 could not be stained.
These findings are in accordance with previous studies (15, 25,
27). The differential staining at low temperatures may be due to
differences in the binding affinities of the TCR/pMHC complexes
as suggested by Reichstetter et al. (27). Data supporting the hypothesis that only “high”-affinity TCRs are bound by tetramers was
published by Falta et al. (46). They reported a strong correlation
between functional avidity and intensity of HLA class II tetramer
staining in human cartilage-specific T cell hybridomas.
Direct flow-cytometric detection of cell populations is too insensitive to reliably identify cell frequencies of 0.02% or lower
(17, 37, 38). HLA class II-restricted Ag-specific CD4⫹ T cells are
rare, so confident detection of these cells with conventional HLA
class II tetramer staining and flow cytometry is impossible. When
HLA class I or II tetramer staining is combined with a magnetic
bead enrichment technique, the sensitivity of detection is considerably improved (18 –20, 37). This advance has made possible the
study of very rare Ag-specific cells without reliance on functional
readouts or cellular responses that may detect only a subset of
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FIGURE 9. Phenotypic analysis of rare virus-specific CD4⫹ T cells detected with HLA class II tetramers. PBMC were stained ex vivo with HLA
class II tetramer and surface marker Abs and subjected to magnetic bead
enrichment. A, Representative postenrichment FACS dot plots showing
costaining of PBMC with anti-CD27 Ab and the HLA class II tetramers
p24.4-DR4 (left plot) and pp65-DR1 (right plot). The percentage of
CD4⫹tetramer⫹ cells expressing CD27 is indicated in parentheses in the
upper right quadrant. B, Phenotypic profile of tetramer-stained CD4⫹ T
cells specific for different viruses. The number of subjects analyzed (n) and
infection state are indicated for each HLA class II tetramer. Data for the
p24.17-DR1, HA307-DR1, and HCV NS3,4-DR4 tetramers have been
published previously (18 –20). Error bars represent SEM. Statistical differences were calculated with the Mann-Whitney U test, and only significant differences are shown. nd, Not done.
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ULTRASENSITIVE DETECTION AND PHENOTYPING OF CD4⫹ T CELLS
fined, so the utility of HLA class II tetramers is expanding. This
technique will advance our knowledge of the Th immune response.
This form of immunity can be critical as a means of curbing infections but remains poorly defined. It is certainly part of antiviral
immune control and should be evaluated when protective immunity is the aim of vaccination.
Acknowledgments
We thank Jonathan Lamb for the HA1.7 clone.
Disclosures
The authors have no financial conflict of interest.
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