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*
Division of Infectious Diseases and Immunology, Queensland Institute of Medical Research and University of Queensland Joint Oncology Program, The Bancroft Centre, Brisbane, Australia;
Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia; and
Emory Vaccine Center and Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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production following peptide addition (2) and
direct TCR binding assays using MHC-peptide multimers
(3, 4, 5, 6, 7, 8, 9), have demonstrated far larger primary
CD8+ T cell responses to a variety of pathogens
than were generally realized previously. For instance, acute infection
with EBV has been shown to have a dramatic impact on the composition of
the T cell pool of the host, with epitope-specific T cell frequencies
of up to 44% of the CD8+ subset within
peripheral blood being reported (5). The MHC class
I-peptide tetrameric complex is arguably the most important of these
technological advances because of its potential additional application
in sorting live Ag-specific T cells for adoptive transfer in the
clinic. The feasibility of such therapeutic strategies is supported by
the recent demonstration that high avidity CTLs that lyse tumor cells
can be isolated from heterogeneous populations using MHC tetramers
that include peptides from human tumor Ags (10, 11).
Although MHC class I-restricted T cell recognition has sufficient
specificity to discriminate between an enormous variety of antigenic
peptides, it is becoming increasingly evident that a degree of
flexibility in peptide recognition is an inherent property of the
TCR (12). Cytotoxicity assays using either synthetic
peptide analogs of T cell epitopes or complex peptide libraries have
demonstrated that most TCRs can undoubtedly recognize multiple peptide
ligands (13, 14, 15, 16, 17). It is also very clear from these reports
that the number of different stimulatory peptides for a single TCR is
greatly influenced by the concentration at which each peptide is used
in in vitro experiments. Thus many different synthetic peptides will
fully activate a T cell clone, but only those peptide-MHC complexes
with the highest TCR affinity will do so at low peptide concentrations.
The limited levels of antigenic peptide presented naturally on the cell
surface after endogenous processing clearly restrict T cell
cross-reactivity and play a major role in maintaining the fine
specificity of CTL recognition (18).
Because the number of different peptides recognized by a single TCR increases with the concentration of exogenously added peptides, it follows that the number of different TCRs that recognize a given peptide will also increase with peptide concentration. When attempting to identify T cells that will recognize naturally presented levels of an antigenic peptide, it is therefore critical to present the peptide either endogenously on the natural target cell or exogenously at a level roughly equivalent to that presented physiologically. T cells raised against epitope X from one pathogen will thereby be distinguishable from other irrelevant T cell populations that may have been expanded in response to another pathogen but that cross-recognize epitope X only at high, nonphysiological concentrations. With these concerns in mind, together with the knowledge that MHC-peptide tetramers were designed to bind their TCR ligands with higher avidity and slower dissociation rates than individual MHC-peptide complexes (3), the present investigation aimed to assess whether T cell specificity for peptide presented in MHC class I tetramers is equivalent to that for peptide presented on the cell surface at levels roughly analogous to those presented naturally after endogenous processing. Using a panel of HLA B8 tetramers incorporating a highly immunogenic peptide from EBV (FLRGRAYGL) (19), or single amino acid variants of this peptide, together with a series of CTL clones with a range of fine specificities for these peptides, we now demonstrate that tetramer staining faithfully reflects these fine peptide specificities and is therefore a very good surrogate marker for T cell recognition of physiological levels of presented peptide.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Lymphoblastoid cell lines
(LCLs)3 were
established by exogenous transformation of peripheral B cells with EBV
derived either from the QIMR-Wil or the Ag876 cell lines. The QIMR-Wil
EBV strain encodes the CTL epitope sequence FLRGRAYGL, which binds to
HLA B8, whereas the Ag876 EBV strain encodes a variant of this sequence
(LLRGRAYGQ), which does not bind to HLA B8 and is therefore not a
target for CTL recognition (19, 20). CTL clones were
generated by agar cloning as previously described (14)
following initial stimulation with -irradiated (8000 rad) autologous
LCLs transformed with the QIMR-Wil EBV strain, using a
responder-stimulator ratio of 50:1. Clones were amplified in culture
with bi-weekly stimulation with -irradiated autologous LCLs
transformed with QIMR-Wil EBV. CTL clones LC13, CF34, CF4, RL42, CF8,
and WY6 have been described previously (14, 17).
Cytotoxicity assay
CTL clones were tested in duplicate for cytotoxicity in the
standard 5-h chromium release assay (E:T ratio of 2:1). To test
synthetic peptides for activity as agonists, facilitating CTL lysis,
each peptide was added directly to 51Cr-labeled
HLA B8+ LCLs transformed with the Ag876 strain of
EBV, incubated for 1 h before CTL addition, and remained present
throughout the assay. To test peptides as antagonists of CTL lysis, the
synthetic peptides were added to HLA B8+ LCLs
transformed with the QIMR-Wil strain of EBV, incubated for 1 h
before CTL addition, and remained present throughout the assay.
Toxicity testing of all peptides was performed by adding peptide to
51Cr-labeled LCLs in the absence of CTL
effectors. A Topcount Microplate scintillation counter (Packard,
Meriden, CT) was used to measure 51Cr levels in
assay supernatant samples. The mean spontaneous lysis for target cells
in culture medium was <20%, and the variation about the mean specific
lysis was <10%. Peptides were made by Mimotopes (Clayton, Victoria,
Australia).
Generation of HLA-B8-peptide tetramers
Tetrameric HLA-B8-peptide complexes were prepared essentially as
previously described (3). Briefly, recombinant HLA B8 and
human 2 microglobulin, produced in
Escherichia coli, were solubilized in urea and injected
together with each synthetic peptide into a refolding buffer consisting
of 100 mM Tris (pH 8.0), 400 mM arginine, 2 mM EDTA, 5 mM reduced
glutathione, and 0.5 mM oxidized glutathione. Refolded complexes were
purified by anion exchange chromatography using DE52 resin (Whatman,
Tewksbury, MA) followed by gel filtration through a Superdex 75 column
(Amersham Pharmacia Biotech, Piscataway, NJ). The refolded
HLA-B8-peptide complexes were biotinylated by incubation for 16 h
at 30°C with the BirA enzyme (Avidity, Denver, CO). Tetrameric
HLA-peptide complexes were produced by the stepwise addition of
extravidin-conjugated PE (Sigma, St. Louis, MO) to achieve a 1:4 molar
ratio (extravidin-PE:biotinylated class I). Tetramer stock
concentrations were 
200 ng/µl.
Cell staining and FACS analysis
CTL clones or PBMCs (2–5 x 105) were incubated for 50 min at 4°C or 37°C with each tetramer (1:100 diluted) in 100 µl of 10% FCS/RPMI 1640 and then washed twice in PBS containing 1% FCS. The PBMCs were then incubated at 4°C with TriColor anti-human CD8 Ab (1:100 diluted; Caltag, Burlingame, CA). Stained cells were analyzed on a FACScalibur (Becton Dickinson, Mountain View, CA) using CellQuest software. Logical gating on forward and side scatter was selected for activated T cells with minimal autofluorescence and cell death.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Eight EBV-specific CTL clones that recognize the HLA-B8-binding
peptide FLRGRAYGL, from the nuclear Ag EBNA3A, were raised from five
unrelated individuals and used in this study. The and TCR
chains of six of these CTL clones (LC13, CF34, CF4, RL42, CF8, and WY6)
were sequenced at the junctional regions in a previous investigation
and are known to be very different for each clone (14, 17). The two other CTL clones (BK10 and CF19) that have not been
described previously were raised in the same way as the other clones
from healthy HLA-B8+,
EBV-sero+ individuals against autologous cells
presenting the EBV epitope. Each CTL clone was tested in a
chromium-release assay for lysis of target cells that had been
preincubated with various concentrations of peptide FLRGRAYGL. These
target cells were HLA-B8+ LCL that had been
transformed with the Ag876 strain of EBV, which are not recognized by
these CTL clones in the absence of exogenous peptide because this EBV
strain encodes a variant of the CTL epitope that does not bind to HLA
B8 (19). As shown in Fig. 1
A, the different CTL clones
recognized the EBV peptide with similar efficiencies, with half-maximum
lysis values of between 30 nM (for LC13 and CF4) and 150 nM (for WY6)
of exogenously added peptide.
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A). Each CTL clone
displayed a distinct pattern of fine specificity for the panel of
closely related peptides. For example, comparing data for CTL clones
LC13 and CF34, the relative impact of the different amino acid
substitutions on the activation of each clone was inverse, i.e., the
most tolerated substitutions for LC13 were the least well tolerated
by CF34.
The eight CTL clones were also tested in cytotoxicity assays against a
target panel of four HLA B8+ (Fig. 1B)
and four HLA B8- (Fig. 1C) LCLs that
were transformed with the QIMR-Wil strain of EBV which is known to
encode the FLRGRAYGL epitope. As has been shown in previous studies
using CTL clones (22, 23), lysis levels of LCL target
cells presenting endogenously processed EBV epitopes are rarely as high
as are seen with saturating amounts of exogenously added peptide (Fig. 1B). EBV-latent Ag peptides are presumably presented at
relatively low density on LCLs after endogenous processing, and CTLs
are unable to achieve the very high avidity of interaction that is
possible with target cells preincubated with synthetic peptide. This
experiment was also performed at higher E:T ratios (up to 20:1), but
LCL lysis levels never reached those observed with exogenous synthetic
peptide (data not shown). Although lysis levels of HLA
B8+ LCLs were relatively low, they were certainly
significant compared with the negligible lysis of HLA
B8- LCLs (Fig. 1C).
The dashed lines in Fig. 1A correspond to the mean level of
lysis by each clone of the four HLA B8+ LCLs
transformed with the QIMR-Wil EBV strain, calculated from Fig. 1B, and the concentration of exogenously added peptide
FLRGRAYGL required to induce this level of lysis in HLA
B8+ LCLs transformed with the Ag876 strain of
EBV. For most of the clones, this peptide concentration is close to the
point of inflection on the titration curve where a small
drop in peptide concentration results in a large fall in activity.
Therefore, it seems unlikely that analogs of FLRGRAYGL that
require over 10 times this peptide concentration to promote T cell
lysis will be activating ligands if presented naturally on LCLs
(assuming the single amino acid changes would not improve processing
efficiency significantly). For example, CTL clone LC13 lysed the four
HLA B8+ LCLs transformed with the QIMR-Wil EBV
strain at an average of 33%, which is roughly equivalent to the level
of lysis observed after adding 30 nM of synthetic FLRGRAYGL peptide to
target cells transformed with the Ag876 EBV strain (Fig. 1A,
dashed line on LC13 graph). Extrapolating from this data, analogs of
this EBV peptide that do not facilitate lysis by clone LC13 when added
at 30 nM are unlikely to be agonists for this CTL clone if presented at
physiological levels. Thus, peptide FLRSRAYGL, which is a
weak activating ligand for LC13 (Fig. 1A), is unlikely to
facilitate CTL lysis if processed endogenously and presented naturally
on an HLA B8+ LCL infected with a hypothetical
EBV variant encoding a Gly to Ser mutation at this position. Therefore,
these data have defined the fine specificity of these eight CTL clones
for five closely related peptides over a range of concentrations,
including an estimated physiologically relevant concentration, for
comparison with subsequent tetramer staining experiments.
Tetramer staining accurately reflects the fine specificity of CTL clones for peptides presented at physiological levels on target cells
HLA B8 tetramers were produced that incorporated either the
FLRGRAYGL epitope or one of the four analogs described above. CTL
clone LC13 was stained with the tetramers, and the most intense
staining was observed with the FLRGRAYGL tetramer (Fig. 2). The only other tetramer that bound
significantly was that made with the TLRGRAYGL peptide,
which stained with slightly less intensity than the FLRGRAYGL tetramer.
This result was consistent with our earlier cytotoxicity data showing
the TLRGRAYGL peptide to be a very strong
agonist for CTL clone LC13 (Fig. 1A). Also consistent with
the chromium release assay data was the lack of staining with the
FLRGRAYVL tetramer because this peptide was a null ligand
for this clone. Surprisingly, tetramers made with peptides
FLRGRAFGL and FLRSRAYGL, which were recognized by
clone LC13 in cytotoxicity assays 8- and 100-fold, respectively, less
efficiently than the parent viral peptide, did not stain the clone
above background levels. As an additional specificity control for this
experiment, a CTL clone with known specificity for another HLA
B8-binding EBV epitope (QAKWRLQTL) did not stain with the tetramers
(data not shown).
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A and 2). Weak agonist peptides that are
unlikely to be activating ligands for the CTL clones if presented at
physiological levels on target cells did not stain at high intensity
when incorporated into B8 tetramers.
The other five CTL clones were then stained with the five tetramers,
and this data is presented in Fig. 3, B and C, as median fluorescence intensity,
together with data from clones LC13, CF34, and WY6. A recent study
indicated that the specificity of CTL interaction with peptide-MHC
class I tetrameric complexes is temperature dependent and that much
more specific staining is obtained by incubating tetramers with T cells
at 37°C rather than 4°C (24). Therefore, we stained
all the clones using either a 4°C (Fig. 3B) or a 37°C
(Fig. 3C) incubation. Also presented in Fig. 3A
for comparison with the tetramer staining data are values of the
exogenous peptide concentration required by each clone for half-maximal
lysis of HLA B8+ LCLs transformed with the Ag876
EBV strain, as determined from the chromium release assay data shown in
Fig. 1A.
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). Considering data from all the
clones, in the 19 cases where a peptide had >10-fold less activity
than the wild-type EBV peptide in cytotoxicity assays, 17 (using 4°C)
or 18 (using 37°C) of the corresponding tetramers stained with
>10-fold less intensity compared with the EBV epitope tetramer. These
data suggest that MHC class I peptide tetramers bind T cells with an
avidity sufficiently low to discriminate between strong stimulatory
agonists and weak agonists that may not induce full T cell activation
at physiological levels of presentation. In additional specificity
controls for these experiments, none of these CTL clones stained with
an HLA B8 tetramer incorporating another EBV epitope, RAKFKQLL (data
not shown).
Peptides with close homology to activating TCR ligands can sometimes
act as antagonists of T cell effector function (25).
Therefore, we considered the possibility that tetramer staining may, in
some cases, be reflecting such peptide interactions. The four peptide
analogs of the FLRGRAYGL EBV epitope were each screened at a range of
concentrations (from 1 nM to 100 µM) for their ability to reduce
lysis of an HLA B8+ LCL, transformed with the
QIMR-Wil EBV strain, in chromium release assays using the eight CTL
clones (data not shown). There were just two peptide-CTL combinations
where antagonism was evident. Lysis by CTL clone RL42 was reduced from
20 to 1% by peptide FLRSRAYGL at 10 µM, and lysis by CTL
clone WY6 was reduced from 15 to 1% by peptide FLRGRAFGL at
1µM. Interestingly, these peptide-CTL combinations were the only ones
where a peptide that had no detectable activity as an agonist was
staining as a tetramer with a median fluorescence intensity above 10
(Fig. 3). However, the relatively low tetramer staining intensity
observed here suggests that HLA B8 molecules presenting these
antagonist peptides bind their TCR ligands with much lower avidity
compared with peptides that promote CTL lysis.
Interestingly, there were two examples (CTL clones CF4 and CF19) where a peptide (FLRGRAYVL) had little activity as either an agonist or an antagonist in the functional assays, and yet quite strong staining with a tetramer incorporating this peptide was observed. This demonstrates that a peptide-MHC complex that binds the Ag receptor of a T cell with quite high avidity can be a null ligand for that T cell. Perhaps such ligands simultaneously trigger both the activation and the antagonism pathway, thereby negating any detectable T cell function. Supporting this notion, it is known that many T cell antagonist peptides can act as agonists of T cell activation at high concentrations (25).
Fresh peripheral blood T cells stain with MHC class I tetramers that incorporate strong, but not weak, agonist peptides
Previous studies have shown that the FLRGRAYGL EBV epitope often
induces a strong and exceptionally restricted memory response in
healthy EBV-sero+, HLA B8+
individuals (14, 26, 27). For reasons that are not yet
clear, a single public TCR dominates this response in some
people. This dominant TCR is the same as that expressed by the LC13 CTL
clone (26). This unusual feature of the memory response to
the FLRGRAYGL epitope provided an opportunity to examine whether the
fine specificity of tetramer staining, evident using CTL clones, is
also observed with fresh T cells. PBMCs from a healthy
EBV-sero+, HLA B8+ donor
who was known to carry a high frequency of FLRGRAYGL-reactive T cells
that express this conserved TCR (14, 26, 27) were stained
with each of the tetramers, followed by an anti-CD8 Ab. The
tetramer incubations were performed at 37°C or 4°C, but only data
using 37°C is shown (Fig. 4) because
the frequencies of tetramer-positive cells did not vary significantly
between the two protocols. PBMCs from an HLA B8-
donor were also used as a control and were found not to stain with the
tetramers above the background level (data not shown).
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This report has addressed any lingering doubts about the Ag specificity of the huge T cell expansions that have been detected using MHC-peptide tetramers. Although these past studies have included quite convincing specificity controls of lymphocytes from individuals unexposed to the relevant Ag, the numbers of these negative controls for each study have been quite limited. If MHC-peptide tetramers stained with broad specificity, chance cross-reactivity with irrelevant T cells leading to overestimates of epitope-specific T cell numbers may only have been expected in a proportion of individuals, particularly in adult humans, where large clonal CD8+ T cell expansions of unknown origin are often observed in the absence of acute infections (28). Such concerns, which prompted our detailed investigation into tetramer specificity, are certainly alleviated by the data presented herein.
The issue of tetramer fine specificity/degeneracy will be particularly critical if using this technology to analyze T cell responses to genetically unstable pathogens, such as HIV-1 or hepatitis C, in which immune-pressure-driven point mutations often arise within viral antigenic determinants during infection of a single host (24). The results of this study suggest that, regardless of the incubation temperature used, class I MHC-peptide tetramers should have the capacity to distinguish CTLs specific for a variant viral strain from those specific for the original infecting strain.
The recent report from Whelan et al. (24) has raised some doubts about the interpretation of previously published tetramer data by suggesting that staining of T cells with tetramers at 4°C, but not 37°C, leads to a degree of promiscuity in binding. HIV-specific CTL clones, which failed to recognize certain variants of their HIV target epitopes in cytotoxicity assays even at very high synthetic peptide concentrations, were shown to strongly stain with tetramers incorporating these variants when a 4°C incubation was used. In contrast, our EBV-reactive T cells failed to stain strongly with tetramers incorporating nonactivating peptides, regardless of whether tetramer incubations were performed at 4°C or 37°C. The basis for the inconsistency between these two studies is unclear.
This report should encourage the broad future use of class I MHC-peptide tetramers to quantify or isolate peptide-specific CTLs ex vivo in the laboratory and in the clinic. These reagents were specifically designed to bind T cells with far greater avidity than the sum of the individual monomeric affinities. Fortuitously and fortunately, this avidity level appears to be optimal for accurately reflecting the fine specificity of T cells for peptides presented on the cell surface at physiological levels.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Scott R. Burrows, Division of Infectious Diseases and Immunology, Queensland Institute of Medical Research, The Bancroft Center, 300 Herston Road, Brisbane 4029, Australia.
3 Abbreviation used in this paper: LCL, lymphoblastoid cell line.
Received for publication June 30, 2000. Accepted for publication September 5, 2000.
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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P. F. McKay, J. E. Schmitz, D. H. Barouch, M. J. Kuroda, M. A. Lifton, C. E. Nickerson, D. A. Gorgone, and N. L. Letvin Vaccine Protection Against Functional CTL Abnormalities in Simian Human Immunodeficiency Virus-Infected Rhesus Monkeys J. Immunol., January 1, 2002; 168(1): 332 - 337. [Abstract] [Full Text] [PDF]
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V. Rubio-Godoy, V. Dutoit, D. Rimoldi, D. Lienard, F. Lejeune, D. Speiser, P. Guillaume, J.-C. Cerottini, P. Romero, and D. Valmori Discrepancy between ELISPOT IFN-gamma secretion and binding of A2/peptide multimers to TCR reveals interclonal dissociation of CTL effector function from TCR-peptide/MHC complexes half-life PNAS, August 28, 2001; 98(18): 10302 - 10307. [Abstract] [Full Text] [PDF]
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