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*
Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford, United Kingdom;
Molecular Immunology Group, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, United Kingdom; and
Department of Pathology, Sir William Dunn School, Oxford, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CTLs were derived and maintained as described previously
(5, 24). PBLs that had been cryopreserved were cultured
for 
2 h after thawing before staining with tetramers. Cytolytic
activity was determined using standard 51Cr
release protocols (25).
Peptides
Peptides were synthesized by standard fluorenylmethoxycarbonyl chemistry and subsequently used in chromium release assays and in synthesis of tetramers. The peptides used, referred to subsequently in the text by their first three letters only, included: SLYNTVATL, an HLA-A2.1-restricted epitope from the HIV p17 gag matrix protein, and its naturally occurring variants SLHNTVATL, SLSNTVATL, SLFNTVATL, SLFNAVATL, and SLNYTIAVL (24, 26, 27); ILKEPVHGV, an HLA-A2.1-restricted epitope from the HIV pol protein (2); GLCTLVAML, an HLA-A2.1 epitope from the BMLF1 protein of EBV; and GILGFVFTL, an HLA-2.1-restricted epitope from the influenza matrix protein (IMP) (6).
Tetrameric MHC class I/peptide complexes
Tetrameric peptide-MHC class I complexes were made as described previously (1, 2). Briefly, recombinant ß2-microglobulin and the extracellular portion of the MHC class I heavy chain containing the BirA recognition sequence in frame at its C terminus were expressed in Escherichia coli as insoluble aggregates that formed inclusion bodies. Purified inclusion bodies were solubilized in urea, and monomeric HLA class I complexes were refolded around peptide by dilution of denaturing conditions. After buffer exchange, a specific lysine residue in the heavy chain C-terminal tag was biotinylated with BirA enzyme. Monomeric complexes were purified by gel filtration and anion exchange chromatography. Tetrameric arrays of biotinylated peptide-MHC class I complexes were formed by the addition of PE- or FITC-labeled avidin (extravidin, Sigma, St. Louis, MO). Tetramers are referred to by the first three letters of their peptide (e.g., GIL for the HLA-A2.1 GILGFVFTL tetramer).
Cell staining and FACS analysis
CTL clones and lines were washed in tissue culture medium and
incubated with PE-labeled tetramers (0.1 mg/ml of monomer) at 4°C,
23°C, or 37°C for the times indicated, along with TriColor
anti-human CD8 (Caltag, Burlingame, CA) when cells were
double-stained. For comparisons between PBL staining protocols, cells
were washed in tissue culture medium before incubation with tetramers
for 15 min or 60 min at 4°C or 37°C and subsequently washed at
either 4°C or 37°C before incubation with TriColor anti-CD8 for
15 min on ice. All cells were washed twice after staining with 2 ml
PBS/FCS 1% before fixation in 1% formaldehyde. Stained cells were
analyzed by FACS (Becton Dickinson, Mountain View, CA) using CellQuest
software.
Confocal microscopy
Cloned human CTLs specific for the HLA-A2.1-restricted IMP epitope were incubated with FITC-labeled GIL tetramer for 15 min at 37°C and subsequently adhered to glass slides coated with 50 mg/ml poly-L-lysine (Sigma) and fixed with methanol for 5 min. Some slides were stained for 30 min with monoclonal mouse OKT9 (anti-transferrin receptor Ab), washed three times in PBS/FCS 1%, and incubated with Texas Red-conjugated goat anti-mouse sera (Jackson, West Grove, PA) (diluted 1/100 in PBS/BSA 1% for 30 min). All slides were then washed three times as described previously and a mounted on a coverslip with PBS/glycerol 90% containing 2.5% 1,4-diazabicyclo[2,2,2]octane (Fluka, Buchs, Switzerland). Cell staining was examined with an MRC-1024 confocal microscope (Bio-Rad, Richmond, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The majority of HLA-A*0201 HIV-1-infected patients mount a CTL
response to an immunodominant epitope (SLYNTVATL) encoded in the p17
gag matrix protein (27). We have documented naturally
occurring mutations within this epitope that can lead to escape from,
or altered recognition by, patient CTLs (24, 27, 28, 29). CTL
clones 003 and 5D8 and a CTL line from patient 868 show different
patterns of recognition of naturally occurring SLYNTVATL variants
(Figs. 1
-3),
consistent with their different origins and TCRs (our unpublished data
and Ref. 13). We used these CTL and HLA-A2 tetramers
folded around wild-type and variant peptides to examine the specificity
of direct CTL visualization in vitro.
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, SLS in
Fig. 2, and SLF in Fig. 3). Surprisingly,
APL-MHC tetramers of ligands that do not stimulate CTL-mediated lysis,
such as the SLH variant with 868 CTL, can also
stain CTLs efficiently at 4°C (Fig. 3). We were unable to demonstrate
antagonism of these CTLs with the SLH variant.
None of these A2 gag CTLs were observed to stain with A2 tetramers of
the unrelated epitopes ILKEPVHGV (
Figs. 1–3), GILGFVFTL, or GLCTLVAML
(data not shown).
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The binding of TCRs to their peptide-MHC ligands at 25°C is
characterized by low affinity, slow kinetics, and a high degree of
cross-reactivity (20). The dramatic increase in binding
kinetics with temperature (30) prompted us to investigate
the effect of temperature on the ability of peptide-MHC tetramers to
form stable complexes with cell surface TCR. At 4°C, both CTL clones
stained with tetramers containing SLYNTVATL variant peptides that they
recognized only weakly in chromium release (see variant
SLH in Fig. 1 and SLS in
Fig. 2). For both clones, increasing the incubation temperature to
37°C reduced the staining by tetramers incorporating weakly
recognized peptide variants without a reduction in the strong staining
by tetramers incorporating strongly recognized peptide variants (Fig. 1, B–C, and Fig. 2B). The hierarchy of staining
with peptide variant tetramers at 37°C reflected the hierarchy of
recognition of each of the variant peptides in the chromium release
assay (e.g., for the CTL clone 5D8 both tetramer staining and chromium
release show the hierarchy SLH >
SLY > SLS (Fig. 2)). Staining with variant tetramers at 4°C did not correlate with
recognition of variant peptides in the chromium release assay for
either clone.
The potential effect of this phenomenon on the staining of biological
samples is seen with a polyclonal CTL line from patient 868 that
also recognizes SLYNTVATL peptide variants to different degrees (Fig. 3). This polyclonal line contains 
33% of SLYNTVATL-specific CTLs,
as seen by staining with the SLY tetramer (Fig. 3B). When incubated at 4°C, tetramers made from the
SLH variant peptide, which does not stimulate
CTL-mediated lysis, stain a population of similar size (Fig. 3B). Increasing the temperature of the tetramer
incubation reduces the proportion of this line that stains with the
SLH tetramer, so that at 37°C, there is no
staining with this unrecognized peptide variant (Fig. 3, B
and C). The results for three different TCRs and several
APLs in
Figs. 1–3 show that improvements in staining specificity for
strongly recognized ligands with increasing temperature are neither
TCR- nor APL-dependent.
In addition to increased specificity for well-recognized peptide
variants, staining at 37°C also increased staining intensity with
strongly recognized ligands (see the SLY
tetramer in Fig. 1B and the SLH
tetramer in Fig. 2B).
It is important to note that all SLYNTVATL variant tetramers used are
sufficiently stable at 37°C to stain appropriate CTLs brightly (
Fig. 1–3). Hence where increasing temperature reduces tetramer staining, in
the case of variant peptides that are poorly recognized by the CTLs,
this is not due to tetramer instability at higher temperature (e.g.,
the SLF tetramer stains 003 CTLs brightly at
37°C but does not stain 868 CTLs at this temperature). Preincubation
of tetramers at 37°C for 60 min before staining CTLs also had no
demonstrable effects on subsequent staining (data not shown),
confirming the stability of the reagents under the experimental
conditions employed.
Tetramers can be internalized by CTL at 37°C
Incubation of the 003 CTL clone (Fig. 4) and the 868 CTL line (data not shown)
for between 0 min and 20 min at 37°C showed that staining was
complete after 5 min and did not increase significantly with longer
incubations. Staining occurred in minutes in the presence of 0.1%
azide during staining (Fig. 4) and was indistinguishable from staining
without the inclusion of azide (data not shown).
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). These vesicles
overlapped in distribution with vesicles containing the transferrin
receptor, although not all of these early endosomes were labeled with
tetramer. Internalized tetramer did not colocalize to secretory
lysosomes containing perforin or granzyme B (data not shown).
Incubation with tetramer for 15 min at 4°C did not induce detectable
internalization of tetramer (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peptide-MHC complexes need not bind with high affinity to trigger the TCR. A recent report shows that there is little correlation between the equilibrium binding affinity (Kd) of a ligand and its potency for T cell activation, and that the potency of a ligand correlates much better with its off-rate (31). Small changes in the sequence of an antigenic peptide (APL) can lead to slightly faster off-rates; relatively small changes in the off-rate can greatly affect the ability of a ligand to trigger T cells (32). To activate T cells, a single peptide-MHC needs to serially trigger multiple TCRs (33). Thus, strongly recognized ligands fall within a "kinetic window" representing a compromise between enough time to allow all the molecular steps required to trigger a TCR and a rapid enough dissociation from the triggered TCR to make it available for further cycles of binding and triggering (32). Assuming a fast enough on-rate, the ability of peptide-MHC class I tetramers to stain CTLs for FACScan analysis will be exclusively dependent upon a sufficiently slow off-rate. Our data demonstrate that the dramatic temperature dependence of the TCR/peptide-MHC interaction (30, 34) can be exploited to increase the specificity of tetramer staining for strongly recognized ligands. At higher temperatures, it appears that the off-rate can be increased beyond the threshold at which the avidity of tetrameric forms of weakly recognized MHC ligands can compensate to allow the formation of stable complexes with cell surface TCRs.
Moderate changes in peptide sequence may allow sufficient interaction
at low temperature for formation of stable complexes with cell surface
TCR when tetramerized but fall outside the kinetic window for
activation. This appears to be the case for the
SLH variant with the 868 CTL (Fig. 3).
Consequently, the use of tetramers to stain CTLs within PBL samples at
a low temperature may result in background staining due to the binding
of tetramers to TCRs that only recognize the peptide-MHC very weakly.
This may result in an overestimation of the frequency of functional
Ag-specific CTLs, especially when tetramer-low cells are counted as
tetramer-positive. Importantly, however, we have no evidence that CTLs
will bind tetramers incorporating completely unrelated peptides, even
at 4°C. All clones and polyclonal lines reported here showed no
background staining with tetramers based around any other epitope,
confirming that tetramers demonstrate a high specificity for CTLs
recognizing their peptide-MHC class I complex.
Although the cross-reactivity of tetramers at 4°C probably relates to only fine differences in CTL specificity for highly similar epitopes, the effects of this phenomenon on tetramer-generated data may be potentially important. The possibility of cross-staining with tetramers is of most concern with pathogens of variable antigenicity such as HIV-1 or hepatitis C virus (26). The phenomenon of original antigenic sin or epitope imprinting, whereby the exposure to an Ag influences subsequent responses to similar Ags, was recently extended to CTL epitopes (35). This study showed that mice primed with lymphocyte choriomeningitis virus respond to a subsequent infection by lymphocyte choriomeningitis virus containing CTL epitope variants with a CTL response directed against the initial epitope rather than against the new variant. Maintenance of an initial CTL response in the face of a poorly recognized variant CTL epitope could severely compromise the effectiveness of the overall CTL response. It was noted that pathogens of variable antigenicity may exploit this phenomenon (35). The incubation of tetramers at both 4°C and 37°C may allow distinction between CTLs that recognize variant epitopes to different degrees and may prove to be a useful tool for tracking CTL responses as epitopes mutate.
Cross-reactive staining may be problematic in other systems. When Yee et al. (19) used tetramer incubations at 4°C to study CTLs specific for the melanoma Ag Melan-A, tetramer-low CTLs lysed peptide-pulsed cells but had insufficient avidity to lyse tumor cells. Substitution of a brief 37°C incubation for a longer 4°C incubation reduces the detection of this potentially nontumoricidal tetramer-low population relative to the highly lytic tetramer-high population (data not shown). Such effects may be particularly important for this HLA-A2.1-restricted Melan-A26–35 epitope, where CTLs specific for epitopes from a number of common viruses may cross-react with the Melan-A peptide (36). Given the huge array of nonself peptides to which the human immune system must be exposed, such epitope mimicry may not be an uncommon phenomenon. Although staining at 4°C may not be problematic in many systems, our results suggest that cross-reactive tetramer staining of CTLs specific for other epitopes that may closely mimic the epitope of interest will be lower at 37°C. If performing low temperature staining, inclusion of a high temperature control and confirmation of Ag specificity after cell sorting (5, 6, 17) are recommended. Exploitation of our findings to isolate both high- and low-avidity CTLs may prove useful to some researchers.
In addition to improvements in specificity, we have demonstrated that
staining at 37°C consistently produces higher intensity staining of
both clones (
Figs. 1–2) and Ag-specific CTLs within polyclonal
populations, and allows clearer separation of these cells from
tetramer-negative cells (data not shown). Interestingly, our findings
of tetramer internalization at 37°C suggest an important new
therapeutic possibility. Because tetramers show such fine specificity
for high-avidity CTLs at 37°C, multimerized peptide-MHC class I
complexes may make highly specific probes for targeting CTLs in vivo.
Internalization of these probes might allow toxins to be carried to the
intracellular compartments of CTLs with very high specificity. In
particular, toxins such as saporin (37), which are
biologically active only when internalized through receptor-mediated
internalization, might be conjugated onto multimerized peptide-MHC
class I complexes or similar structures to allow specific immunolysis
of CTLs involved in pathology such as autoimmune diseases.
Our results may be broadly relevant to all studies using peptide-MHC class I tetrameric complexes to quantify CTLs ex vivo. If the interaction between peptide-MHC class II and the TCR is also influenced by temperature, then similar considerations will likely apply to the use of tetramer technology in MHC class II-restricted systems (38, 39).
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Acknowledgments |
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Footnotes |
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2 J.A.W. and P.R.D. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Andrew Sewell, Nuffield Department of Clinical Medicine, Room 7508, John Radcliffe Hospital, Headington, Oxford OX3 9DU, U.K. E-mail address:
4 Abbreviations used in this paper: APL, altered peptide ligand; IMP, influenza matrix protein.
Received for publication June 1, 1999. Accepted for publication August 5, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-encoded tyrosine for T cell recognition of an HLA B27/peptide complex. Eur. J. Immunol. 28:2704.[Medline]
ß T cell receptors. Annu. Rev. Immunol. 16:523.[Medline]
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T. M. Lawson, S. Man, E. C. Y. Wang, S. Williams, N. Amos, G. M. Gillespie, P. A. Moss, and L. K. Borysiewicz Functional differences between influenza A-specific cytotoxic T lymphocyte clones expressing dominant and subdominant TCR Int. Immunol., November 1, 2001; 13(11): 1383 - 1390. [Abstract] [Full Text] [PDF]
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G. Denkberg, C. J. Cohen, and Y. Reiter Critical Role for CD8 in Binding of MHC Tetramers to TCR: CD8 Antibodies Block Specific Binding of Human Tumor- Specific MHC-Peptide Tetramers to TCR J. Immunol., July 1, 2001; 167(1): 270 - 276. [Abstract] [Full Text] [PDF]
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N. H. Gruener, F. Lechner, M.-C. Jung, H. Diepolder, T. Gerlach, G. Lauer, B. Walker, J. Sullivan, R. Phillips, G. R. Pape, et al. Sustained Dysfunction of Antiviral CD8+ T Lymphocytes after Infection with Hepatitis C Virus J. Virol., June 15, 2001; 75(12): 5550 - 5558. [Abstract] [Full Text]
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M. A. Derby, J. Wang, D. H. Margulies, and J. A. Berzofsky Two intermediate-avidity cytotoxic T lymphocyte clones with a disparity between functional avidity and MHC tetramer staining Int. Immunol., June 1, 2001; 13(6): 817 - 824. [Abstract] [Full Text] [PDF]
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F. Gays, K. P. Fraser, J. A. Toomey, A. G. Diamond, M. M. Millrain, P. J. Dyson, and C. G. Brooks Functional Analysis of the Molecular Factors Controlling Qa1-Mediated Protection of Target Cells from NK Lysis J. Immunol., February 1, 2001; 166(3): 1601 - 1610. [Abstract] [Full Text] [PDF]
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T. O. Cameron, J. R. Cochran, B. Yassine-Diab, R.-P. Sekaly, and L. J. Stern Cutting Edge: Detection of Antigen-Specific CD4+ T Cells by HLA-DR1 Oligomers Is Dependent on the T Cell Activation State J. Immunol., January 15, 2001; 166(2): 741 - 745. [Abstract] [Full Text] [PDF]
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T. Tolfvenstam, A. Oxenius, D. A. Price, B. L. Shacklett, H. M. L. Spiegel, K. Hedman, O. Norbeck, M. Levi, K. Olsen, M. Kantzanou, et al. Direct Ex Vivo Measurement of CD8+ T-Lymphocyte Responses to Human Parvovirus B19 J. Virol., January 1, 2001; 75(1): 540 - 543. [Abstract] [Full Text]
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H. W. H. G. Kessels, M. D. van den Boom, H. Spits, E. Hooijberg, and T. N. M. Schumacher Changing T cell specificity by retroviral T cell receptor display PNAS, December 19, 2000; 97(26): 14578 - 14583. [Abstract] [Full Text] [PDF]
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S. Reichstetter, R. A. Ettinger, A. W. Liu, J. A. Gebe, G. T. Nepom, and W. W. Kwok Distinct T Cell Interactions with HLA Class II Tetramers Characterize a Spectrum of TCR Affinities in the Human Antigen-Specific T Cell Response J. Immunol., December 15, 2000; 165(12): 6994 - 6998. [Abstract] [Full Text] [PDF]
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S. M. Smith, R. Brookes, M. R. Klein, A. S. Malin, P. T. Lukey, A. S. King, G. S. Ogg, A. V. S. Hill, and H. M. Dockrell Human CD8+ CTL Specific for the Mycobacterial Major Secreted Antigen 85A J. Immunol., December 15, 2000; 165(12): 7088 - 7095. [Abstract] [Full Text] [PDF]
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M. V. Dhodapkar, J. W. Young, P. B. Chapman, W. I. Cox, J. F. Fonteneau, S. Amigorena, A. N. Houghton, R. M. Steinman, and N. Bhardwaj Paucity of Functional T-Cell Memory to Melanoma Antigens in Healthy Donors and Melanoma Patients Clin. Cancer Res., December 1, 2000; 6(12): 4831 - 4838. [Abstract] [Full Text]
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S. R. Burrows, N. Kienzle, A. Winterhalter, M. Bharadwaj, J. D. Altman, and A. Brooks Peptide-MHC Class I Tetrameric Complexes Display Exquisite Ligand Specificity J. Immunol., December 1, 2000; 165(11): 6229 - 6234. [Abstract] [Full Text] [PDF]
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P. R. Dunbar, C. L. Smith, D. Chao, M. Salio, D. Shepherd, F. Mirza, M. Lipp, A. Lanzavecchia, F. Sallusto, A. Evans, et al. A Shift in the Phenotype of Melan-A-Specific CTL Identifies Melanoma Patients with an Active Tumor-Specific Immune Response J. Immunol., December 1, 2000; 165(11): 6644 - 6652. [Abstract] [Full Text] [PDF]
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B. Schuler-Thurner, D. Dieckmann, P. Keikavoussi, A. Bender, C. Maczek, H. Jonuleit, C. Roder, I. Haendle, W. Leisgang, R. Dunbar, et al. Mage-3 and Influenza-Matrix Peptide-Specific Cytotoxic T Cells Are Inducible in Terminal Stage HLA-A2.1+ Melanoma Patients by Mature Monocyte-Derived Dendritic Cells J. Immunol., September 15, 2000; 165(6): 3492 - 3496. [Abstract] [Full Text] [PDF]
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N. Kienzle, M. Buck, S. L. Silins, S. R. Burrows, D. J. Moss, A. Winterhalter, A. Brooks, and R. Khanna Differential Splicing of Antigen-Encoding RNA Reduces Endogenous Epitope Presentation That Regulates the Expansion and Cytotoxicity of T Cells J. Immunol., August 15, 2000; 165(4): 1840 - 1846. [Abstract] [Full Text] [PDF]
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M.-B. Nielsen, V. Monsurro, S. A. Migueles, E. Wang, A. Perez-Diez, K.-H. Lee, U. Kammula, S. A. Rosenberg, and F. M. Marincola Status of Activation of Circulating Vaccine-Elicited CD8+ T Cells J. Immunol., August 15, 2000; 165(4): 2287 - 2296. [Abstract] [Full Text] [PDF]
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V. Appay, D. F. Nixon, S. M. Donahoe, G. M.A. Gillespie, T. Dong, A. King, G. S. Ogg, H. M.L. Spiegel, C. Conlon, C. A. Spina, et al. HIV-Specific Cd8+ T Cells Produce Antiviral Cytokines but Are Impaired in Cytolytic Function J. Exp. Med., July 3, 2000; 192(1): 63 - 76. [Abstract] [Full Text] [PDF]
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T. L. Whiteside Monitoring of Antigen-Specific Cytolytic T Lymphocytes in Cancer Patients Receiving Immunotherapy Clin. Vaccine Immunol., May 1, 2000; 7(3): 327 - 332. [Full Text] [PDF]
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T. M. Allen, T. U. Vogel, D. H. Fuller, B. R. Mothe, S. Steffen, J. E. Boyson, T. Shipley, J. Fuller, T. Hanke, A. Sette, et al. Induction of AIDS Virus-Specific CTL Activity in Fresh, Unstimulated Peripheral Blood Lymphocytes from Rhesus Macaques Vaccinated with a DNA Prime/Modified Vaccinia Virus Ankara Boost Regimen J. Immunol., May 1, 2000; 164(9): 4968 - 4978. [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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S. Reignat, G. J.M. Webster, D. Brown, G. S. Ogg, A. King, S. L. Seneviratne, G. Dusheiko, R. Williams, M. K. Maini, and A. Bertoletti Escaping High Viral Load Exhaustion: CD8 Cells with Altered Tetramer Binding in Chronic Hepatitis B Virus Infection J. Exp. Med., May 6, 2002; 195(9): 1089 - 1101. [Abstract] [Full Text] [PDF]
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