Detection of low-frequency human antigen-specific CD4+ T cells using MHC class II multimer bead sorting and immunoscope analysis


  • Fabrice Lemaître,

    Corresponding author
    1. Unité de Recherche et d'Expertise Immunité anti-virale, Biothérapie et Vaccins, INSERM U277, Institut Pasteur, Paris, France
    • INSERM U277, Unité de Recherche et d'Expertise Immunité anti-virale, Biothérapie et Vaccins, Institut Pasteur, 25 rue du Docteur Roux, F-75724 Paris Cedex 15, France, Fax: +33-1-4568-8548
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    • The first two authors contributed equally to this work.

  • Manuelle Viguier,

    1. Unité de Recherche et d'Expertise Immunité anti-virale, Biothérapie et Vaccins, INSERM U277, Institut Pasteur, Paris, France
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    • The first two authors contributed equally to this work.

  • Min-Sun Cho,

    1. Unité de Recherche et d'Expertise Immunité anti-virale, Biothérapie et Vaccins, INSERM U277, Institut Pasteur, Paris, France
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  • Jean-Marie Fourneau,

    1. INSERM U580, Institut Necker, Paris, France
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  • Bernard Maillère,

    1. Département d'ingénierie et d'étude des protéines, CEA-Saclay, Gif-sur-Yvette, France
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  • Philippe Kourilsky,

    1. Unité de Recherche et d'Expertise Immunité anti-virale, Biothérapie et Vaccins, INSERM U277, Institut Pasteur, Paris, France
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  • Peter M. van Endert,

    1. INSERM U580, Institut Necker, Paris, France
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  • Laurent Ferradini

    1. Unité de Recherche et d'Expertise Immunité anti-virale, Biothérapie et Vaccins, INSERM U277, Institut Pasteur, Paris, France
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MHC class II tetramers are attractive tools to study antigen-specific CD4+ T cell responses in various clinical situations in humans. HLA-DRA1*0101/DRB1*0401 MHC class II heterodimers were produced as empty molecules using the Drosophila melanogaster expression system. Peptide binding experiments revealed that these molecules could be loaded efficiently with appropriate MHC class II tumor epitopes. Interestingly, MHC class II tetramer staining was influenced by modifications in membrane lipid rafts, and could in itself induce activation changes of stained CD4+ T cells at 37°C. In order to increase the threshold of detection of poorly represented peripheral antigen-specific CD4+ T cells, we combined cell sorting using MHC class II multimer beads together with TCR analysis using the immunoscope technology. This strategy greatly increased the sensitivity of detection of specific CD4+ T cells to frequencies as low as 4×10–6 among peripheral blood mononuclear cells. Such a combined approach may have promising applications in the immunomonitoring of patients under vaccination protocols to tightly follow induced antigen-specific CD4+ T cells expressing previously identified TCR.


Influenza hemagglutinin-A




Lymphoblastoid cell line

1 Introduction

MHC class I tetramers have been found in these last few years to be extremely powerful to study ex vivo both murine and human CD8+ T cells specific for given peptide epitopes during normal and pathological situations 1. The MHC class I heavy chain and the β2-microglobulin can be routinely produced and purified as inclusion bodies from Escherichia coli and then refolded in vitro with cognate peptide epitopes. Given the central role of CD4+ T cells in the control of immune responses 25, similar reagents composed of MHC class II molecules are intensively awaited for the study of Ag-specific CD4+ T cell immune responses. Indeed, ex vivo characterization of such CD4+ T cells is critical to understand their role during immune responses, because of the phenotypic and functional biases potentially introduced by in vitro culture systems.

Only few groups have reported the successful usage of MHC class II tetrameric complexes to follow Ag-specific CD4+ T cells during given pathological situations in mice 68 and humans 911. Several reasons could explain such difficulties, including the lower extent of expansion of specific CD4+ T cell precursors compared to their CD8+ counterpart 12, the heterogeneity of TCR affinities 13, or the particular conditions and molecular interactions needed to efficiently stain CD4+ T cells with such reagents, compared to MHC class I tetramers. Clearly, an intense and stable staining, as well as a low background, will be critical to enable the detection of low-frequency Ag-specific T cells.

Technical constraints encountered in the production of properly folded MHC class II complexes and the variable ability displayed by these complexes to be appropriately loaded with peptides, may also explain why MHC class II tetramers are not as widely used as their MHC class I counterparts. Indeed, no standardized protocol of production has been set up to date and several strategies have been considered that mainly vary because of the diversity of expression systems and the use of linked or loaded peptide epitopes. One of these approaches, based on the Drosophila melanogaster expression system and the use of fused leucine zipper sequences, has been reported by Kwok's group 9, 1416 to detect CD4+ T cells specific for the influenza hemagglutinin-A (HA) or Herpes simplex virus type 2 epitopes.

We used a similar strategy to produce HLA-DRA1*0101/DRB1*0401 (DR4) MHC class II heterodimers as empty molecules in Drosophila S2 cells. These molecules could be efficiently loaded with appropriate MHC class II tumor epitopes. We report here that cell sorting with DR4 multimer beads followed by TCR immunoscope analysis highly increased the sensitivity of detection of Ag-specific CD4+ T cells present at very low frequencies among PBMC. This approach may be very useful in following Ag-specific CD4+ T cells during immunomonitoring of patients under anti-tumor vaccination protocols, since such CD4+ precursors are likely to be poorly represented in vivo.

2 Results and discussion

2.1 DR4 heterodimers produced in Drosophila S2 cells bind peptides as efficiently as native corresponding molecules purified from lymphoblastoid cell lines

Empty DR4 heterodimers were purified by immunoaffinity from supernatants of D. melanogaster S2 cells as previously described 9, 17. One liter of such supernatant enabled us to obtain 8 mg of empty DR4 heterodimers. The molecular mass of the purified molecule (65 kDa) and the presence of α and β chains were confirmed by gel-filtration chromatography (Fig. 1A) and Western blot analysis (Fig. 1B).

Figure 1.

Analysis of purified DR4 heterodimers produced using the D. melanogaster expression system. (A) DR4 heterodimers were injected through a Superdex 75 gel-filtration column, giving a major peak corresponding to the purified heterodimer (65 kDa) at a 24-min retention time, followed by a shoulder of free unpaired DR α and DR β chains. Molecular weight correlations were made using gel filtration standards (not shown). (B) Affinity-purified DR4 molecules (6.5 µg) were loaded onto a native polyacrylamide gel, before migration and transfer on a nitrocellulose membrane. DR α and DR β chains were immunodetected using specific antisera. Molecular masses are given in kDa. (C) Fc receptors of LCL 676 were first saturated either with conformational L243 Ab (thick line in histogram) or with a mouse IgG2a isotype control (dotted line in histogram). After incubation with DR4-HA-PE tetramers (tet) and anti-CD19-allophycocyanin Ab, an increase in PE fluorescence intensity was observed according to the proper conformation of the DR4 heterodimers. (D) Peptide binding capacity of recombinant and LCL-purified HLA-DR4 molecules. HA p306–318 (square) and NY-ESO-1 p119–143 (triangle) peptides were incubated at various concentrations with recombinant (open symbols) or LCL-purified (filled symbols) HLA-DR4 molecules, in the presence of 30 nM of the biotinylated HA p306–318 peptide, used as a tracer. The percentages of complexes loaded with the biotinylated peptide are represented at various concentrations of the competitor peptides.

In order to test the conformation of our recombinant DR4 heterodimers, we performed a variant of the recently described flow cytometry-based test 18. The DR-specific L243 mAb was bound to Fc receptors of a lymphoblastoid cell line (LCL), to further enable the capture of PE-labeled DR4 tetramers in case of integrity and appropriate conformation of the DR4 heterodimers. As expected, a shift in the fluorescence intensity was observed when using the L243 mAb, but not while using its corresponding isotype as a control (Fig. 1C). This demonstrated the conformational integrity of the produced DR4 heterodimers.

Some difficulties in loading empty molecules have been reported arguing that peptide exchange from linked peptides may be a better option to produce specific MHC class II tetramers 18. Using a previously described peptide binding assay 19, we could show that MHC class II molecules produced in D. melanogaster S2 cells bind distinct peptides as efficiently as MHC class II molecules directly purified from EBV LCL. This is illustrated in Fig. 1D for the HA p306–318 and NY-ESO-1 p119–143 peptides, and similar data were obtained using other tumor Ag-derived peptides as MAGE3 p171–185, NY-ESO-1 p119–130 and MelanA/MART1 p51–73 (data not shown). As already reported by Kwok's group 9, 1416, these data confirm that the use of empty MHC class II molecules produced in insect cells, which can be easily loaded with distinct peptides, represents an attractive and more flexible alternative to the peptide-linked tetramer approach widely used in mice 7, 8, 20, 21 and humans 10, 18, or to the recently reported CLIP peptide-exchange strategy 11.

2.2 Peptide-loaded DR4 tetramers can specifically detect poorly represented Ag-specific CD4+ T cells among polyclonal T cell populations, but induce ex vivo some degree of activation

In order to determine the optimal staining conditions, purified DR4 heterodimers were loaded with an excess of HA p306–318 peptide according to Novak et al. 9, and tetramerized with streptavidin-PE. The corresponding DR4-HA tetramers gave positive staining of the two HA1.7 and LR51 CD4+ T cell clones which are specific for the HA p306–318 peptide presented by DR4 molecules, whereas DR4 tetramers loaded with an irrelevant peptide did not (Fig. 2A). Optimal staining intensities were obtained using a 20-μg/ml concentration of tetramer and a 1-h incubation at 37°C. Increasing tetramer concentrations only slightly improved the level of staining. On the other hand, decreasing the temperature of incubation lowered the fluorescence intensity, with almost no staining at 4°C (Fig. 2B). It has been reported that T cells bearing high-affinity TCR could be stained with MHC class II tetramers independently of the incubation temperature, while T cells with low-affinity TCR require an incubation at 37°C 13.

Figure 2.

DR4-HA tetramer staining of HA p306–318-specific CD4+ T cell clones. (A) HA1.7 and LR51 HA p306–318-specific CD4+ T cells were incubated 1 h at 37°C with the appropriate DR4-HA-PE tetramers at a concentration of 20 μg/ml (thick lines) or with an irrelevant tetramer (thin lines). (B) Staining of the HA1.7 T cells was performed at 37°C (thick line), 25°C (thin line), and 4°C (dotted line) incubation temperatures. Unstained cells are represented by pointed line. (C) Pretreatment of HA1.7 T cells was performed with various concentrations of βMCD: 20 mM (pointed lines), 10 mM (dotted lines), 5 mM (thin lines), and 0 mM (thick lines). DR4-HA tetramer and anti-CD4 Ab stainings are reported. (D) Intracellular IFN-γ expression was measured by FACS analysis after incubation of HA1.7 T cells with DR4-HA-PE tetramer or an irrelevant tetramer. As a positive control, intracellular IFN-γ expression after PMA/ionomycin stimulation is shown. Percentages of IFN-γ-positive cells among tetramer-positive HA1.7 T cells are indicated in the upper right quadrant.

Taken together, these observations suggest that membrane mobility may be required to achieve appropriate MHC class II tetramer staining of T cells with various ranges of TCR affinities. To address the influence of membrane components on MHC class II tetramer staining, we treated HA1.7 T cells with methyl-β-cyclodextrine (βMCD), which is known to extract part of the cholesterol from the cell membrane, destabilizing lipid raft structures 22. We observed that pretreating HA1.7 T cells with βMCD decreased the intensity of tetramer staining in a dose-dependent manner, while CD4 expression levels remained unchanged (Fig. 2C). As it has been reported for MHC class I tetramer binding 23, this suggests that the integrity of lipid raft structures may also be required for optimal staining with MHC class II tetramers.

It has also been reported that MHC class I tetramers may specifically induce T cell activation under defined staining conditions 24, 25, modifying ex vivo the functional characteristics of the T cells studied. We thus tested whether staining with MHC class II tetramers could similarly activate CD4+ T cells. Intracellular production of IFN-γ by HA1.7 T cells was measured after a 1-h incubation at 37°C with DR4-HA MHC class II tetramers. Staining with DR4-HA tetramers slightly induced IFN-γ intracellular staining while an irrelevant DR4 tetramer did not (Fig. 2D). As a positive control, PMA/ionomycin stimulation of HA1.7 T cells induced very high levels of intracellular IFN-γ expression. This observation confirms that staining with MHC class II tetramers may also induce different levels of T cell activation and cytokine production 20, 2628.

We then tested the ability of our DR4 tetramer to detect tumor Ag-specific CD4+ T cells. For this purpose, a T cell line specific for the melanoma Ag NY-ESO-1 was generated in vitro from PBMC of a DRB1*0401 healthy donor. Its specificity was assessed by proliferation (Fig. 3A) and IFN-γ secretion (not shown) assays in the presence of NY-ESO-1 p119–130 peptide-loaded APC. When used on this T cell line, DR4 tetramers loaded with the corresponding NY-ESO-1 p119–130 peptide effectively stained around 30% of the CD4+ T cells, whereas no staining was observed when using an irrelevant DR4-HA tetramer (Fig. 3B). These data revealed that our MHC class II tetramers are effective in detecting tumor Ag-specific CD4+ T cells among in vitro enriched T cells.

Figure 3.

DR4-NY-ESO-1 tetramer staining of the 840–1 NY-ESO-1-specific T cell line. (A) Proliferation assay assessing the specificity of the NY-ESO-1 p119–130 DR4-restricted CD4+ T cell line (840–1). Incorporation of [3H]thymidine was measured after 72 h of culture of 840–1 T cells, either in monoculture (open bar), or in co-culture with the T2 LCL unloaded (grey bar) or loaded with NY-ESO-1 p119–130 (black bar). (B) 840–1 T cells were stained under previously described conditions with anti-CD4-FITC Ab and DR4-NY-ESO-1-PE or DR4-HA-PE tetramers. Percentages of positive cells among CD4+ T cells are indicated in the upper right quadrant.

To assess the ability of DR4 tetramers to detect poorly represented Ag-specific CD4+ T cells among PBMC, we used the HA1.7 T cell clone as a tool to define the lowest percentage of T cells detected by the corresponding DR4 tetramer. For this purpose and as previously described 15, 18, 29, we tested DR4-HA tetramer staining on serial dilutions of the HA1.7 T cells in allogeneic PBMC from a DR4-expressing donor. As shown in Fig. 4, the percentage of detected tetramer-positive T cells closely correlated with the number of HA1.7 T cells present in the corresponding dilutions (correlation coefficient R2=0.9928). The background of staining observed in these experiments on the PBMC alone was around 0.15%. This indicated that the lowest frequency of HA1.7 diluted T cells which could unambiguously be detected was around 1/700. This frequency is close to the one reported by Cunliffe et al. 18 with DR1-HA tetramers and the HA1.7 clone, but clearly differs from the observations reported by Danke et al. 15, who were able to directly detect ex vivo HA-specific T cells at frequencies as low as 1/30,000 among PBMC in patients under influenza vaccination. Differences in the levels of activation and in TCR affinities for the same MHC class II/peptide complex might explain these discrepancies between in vitro studies using defined T cell clones and ex vivo observations on various Ag-specific populations in vaccinated patients.

Figure 4.

Detection of HA1.7 T cells diluted among PBMC using DR4-HA tetramers. The percentages of DR4-HA-PE tetramer-positive T cells detected by flow cytometry are expressed according to the percentage of HA1.7 T cells added to the PBMC. CD4+ T cells were gated and dead cells were excluded. The correlation coefficient (R2) is given.

2.3 Increased sensitivity of detection is obtained by combining MHC class II multimer bead sorting with TCR immunoscope analysis

Because the frequency of Ag-specific CD4+ T cells is known to usually be very low, our observations clearly reveal that the detection threshold in classical flow cytometry experiments using DR4 tetramers is probably not sufficient to be widely applied in human physiological or even pathological conditions without previous in vitro expansions of the precursors under study, possibly introducing some bias in their further characterization. They also indicate that ex vivo FACS sorting of rare CD4+ T cells will represent a major challenge. Several strategies have been developed to increase the sensitivity of detection of Ag-specific CD4+ T cells, such as in mice the use of MHC class II-bearing fluorescent liposomes 30 or the pretreatment of T cells with neuraminidase 20, and in humans the use of anti-PE magnetic beads coated with MHC class II tetramers 11, 31 or the use of 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling before in vitro expansion followed by tetramer staining 9, 15.

In order to improve the detection of low-represented Ag-specific T cells among polyclonal populations, we assessed another strategy 32 which combines the use of DR4 peptide monomers coated on magnetic beads to sort HA-specific CD4+ T cells with the immunoscope analysis, which is a reverse transcription (RT)-PCR-based approach to analyze TCR repertoires 29. For this purpose, as described above, we performed further serial dilutions of HA1.7 T cells to obtain frequencies which ranged from 2.5×10–3 (1/400) down to 4×10–6 (1/250,000), and positive cells were sorted or not using DR4-HA multimer beads after a 4-h incubation at room temperature. The TCR expressed by T cells in sorted and unsorted fractions were then analyzed with the immunoscope technology in order to detect the presence of the HA1.7 TCR.

In all the dilutions studied, the ten-amino acid peak corresponding to the CDR3 length of the BV3 TCR β chain expressed by HA1.7 T cells 33 was clearly identified in the sorted fraction (Fig. 5). This characteristic peak was detectable down to the 4×10–6-dilution in the sorted fractions, while it was only clearly detectable down to 0.5×10–3 in the unsorted fractions. When no HA1.7 T cells were added to PBMC, no such a characteristic peak could be identified in the sorted fraction, whereas a normal Gaussian distribution of the BV3 TCR β chain CDR3 length was observed in the unsorted fraction as expected. When cells were sorted using irrelevant multimers, the HA1.7 TCR β chain could not be detected (Fig. 5). The CDR3 profiles corresponding to the other BV families were not modified before sorting with respect to the distinct HA1.7 T cell dilutions, and no additional CDR3 peaks could be detected in other BV than BV3 after bead sorting (data not shown).

Figure 5.

The combination of DR4-HA multimer bead sorting with TCR immunoscope analysis greatly increases the sensitivity of detection of rare Ag-specific CD4+ T cells. T cells from each HA1.7 T cell dilution were sorted or not with DR4-HA or irrelevant multimer magnetic beads and their TCR were analyzed using the immunoscope technology. The CDR3 length distributions obtained with the BV3-BC primers are displayed. For each profile, fluorescence intensity in arbitrary units is represented as a function of the CDR3 length in amino acids. The CDR3 length of ten amino acids corresponding to the BV3 TCR β chain used by HA1.7 T cells is indicated below each immunoscope profile. The absolute numbers and the frequencies of HA1.7 T cells are also indicated for each dilution.

Interestingly, we have shown here that combining the use of MHC class II multimer bead sorting with TCR immunoscope analysis greatly increased the sensitivity of detection of poorly represented HA-specific HA1.7 CD4+ T cells among total PBMC, with detectable frequencies as low as 1/105 to 1/106, and provided a 125-fold increase in sensitivity compared to the use of immunoscope analysis alone. Such a detection threshold is close to the one reported using the ELISPOT technology in similar dilution experiments of activated CD4+ Ag-specific T cells (1/300,000) 34 and is much lower than the limit of detection obtained using intracytoplasmic cytokine staining in the same experiments (1/5,000) 34. These two latter technologies only enable to measure the frequency of cytokine-secreting activated T cells, whereas the MHC class II multimer and TCR immunoscope combined technologies enable the detection of Ag-specific T cells expressing a given TCR whatever their functional status is.

As this approach is applied ex vivo, it avoids cell culture procedures known to potentially bias the T cell repertoire 35, 36, and provides the advantage that all specific peripheral T cells, irrespective of their functional state and potential for proliferation, have an equal chance to be detected. We have already reported the effectiveness of such an approach for the study of specific CD8+ T cells with MHC class I multimers 29. In the case of Ag-specific CD4+ T cells, given their expected low precursor frequency in vivo and their ability to be stained non-homogeneously with MHC class II tetramers, the present strategy may represent an interesting and powerful alternative for the monitoring of spontaneous or induced Ag-specific CD4+ T cells with already identified TCR usage. This latter limitation requires to have previously analyzed the TCR rearrangements (i.e. the CDR3 lengths) of such induced Ag-specific CD4+ T cells, which then enables their sensitive immunomonitoring using the MHC class II multimer and TCR immunoscope combined approach focusing on the most relevant T cell clonotypes.

In conclusion, we report herein that the combination of MHC class II multimer bead sorting and TCR immunoscope analysis greatly increases the sensitivity of detection of rare Ag-specific CD4+ T cells. Such a strategy may be particularly useful to tightly follow induced tumor Ag-specific CD4+ T cells during the immunomonitoring of patients under anti-tumor vaccination protocols. More generally, MHC class II bead-sorted Ag-specific CD4+ T cells could also be analyzed in more detail for the genes they express using real-time quantitative RT-PCR, and may be used to initiate various further in vitro experiments.

3 Materials and methods

3.1 Production of MHC class II tetramers

The two expression vectors used for the production of HLA-DRA1*0101 and HLA-DRB1*0401 in insect cells were constructed as recently described by Fourneau et al. 37. Briefly, leucine zippers chimeric MHC class II containing DR α and DR β chain constructs were first generated in the dual promoter baculovirus transfer vector pAcUW51 (PharMingen, San Jose, CA), before subcloning into the appropriately modified copper-inducible Drosophila expression vector pRmHa-3 (kindly provided by Dr. L. Goldstein, University of Arizona, AZ), to enable inducible expression of DR4 heterodimers in D. melanogaster Schneider (S2) cells.

S2 cells cotransfected with the pRmHa-3-DRA1*0101 and DRB1*0401 vectors were grown in spinner bottles up to around 5 millions cells/ml and induced with 0.5 mM CuSO4 during 5 days to allow the production of DR4 molecules. Culture supernatants were concentrated tenfold by ultrafiltration on 30-kDa membranes to eliminate unpaired chains, and soluble DR4 heterodimers were purified by immunoaffinity as previously described 9, 17, using the L243 Ab (kindly provided by Dr. N. Mooney, INSERM U396, Paris, France) cross-linked to protein A Sepharose (Amersham Biosciences, Orsay, France). DR4 was then buffer-exchanged to 10 mM Tris/10 mM NaCl, pH 8.0, on 10-kDa ultrafiltration filters, before biotinylation with the BirA enzyme (Avidity, Denver, CO) for 3 h at 30°C following manufacturer's recommendations. After excess biotin removal and buffer exchange to 10 mM Tris/50 mM NaCl, pH 6.0, as above, biotinylated molecules were stored at –80°C. Final products were analyzed by gel filtration (Superdex 75, Amersham Biosciences) and Western blot on a native 12% polyacrylamide gel, using rabbit anti-human DRα and DRβ antisera kindly provided by H. L. Ploegh (Harvard Medical School, Boston, MA).

Peptide loading was performed according to Novak et al. 9 in 100 mM sodium phosphate pH 5.9, containing 2.5 mg/ml n-octyl-β-D-glucopyranoside (Sigma, St Quentin Fallavier, France) for 72 h at 37°C. DR4-peptide aliquots were finally stored at –80°C. Peptides corresponding to the epitopes 306–318 of HA (PKYVKQNTLKLAT), 119–130 of NY-ESO-1 (PGVLLKEFTVSG), 119–143 of NY-ESO-1 (PGVLLKEFTVSGNILTIRLTAADHR), 51–73 of MelanA/MART1 (RNGYRALMDKSLHVGTQCALTRR), and 171–185 of MAGE3 (PIGHLYIFATCLGLS) were purchased from Neosystem, Strasbourg, France.

Tetramerization of peptide-loaded DR4 molecules was performed at room temperature by addition of ultravidin-PE (Leinco Technologies, St. Louis, MO) at a 4:1-molar ratio, in five equal aliquots added at 5-min intervals.

3.2 Conformational tests of DR4 tetramers

The LCL 676 was coated via its Fc receptor either with conformational anti-DR αβ chain L243 mAb or with an isotype control (mouse IgG2a; PharMingen, Le Pont de Claix, France) on ice for 20 min. The cells were then stained with 20 μg/ml of DR4-HA-PE tetramer for 1 h at 37°C, washed and incubated with anti-CD19-allophycocyanin (PharMingen) on ice for 20 min. The cells were analyzed on a FACSCalibur flow cytometer using CellQuest software (Becton Dickinson, Le Pont de Claix, France).

3.3 Peptide binding assays

Peptide binding assays were performed as previously described 19. HLA-DR4 molecules were purified from the HLA-homozygous EBV LCL BOLETH by affinity chromatography using L243 mAb coupled to protein A Sepharose (Amersham Biosciences). The biotinylated peptide HA p306–318 (PKYVKQNTLKLAT) was used as a tracer in the binding assays. Recombinant HLA-DR4 or LCL purified HLA-DR4 molecules were respectively diluted in 100 mM phosphate pH 6.0, 0.25% n-octyl-β-D-glucopyranoside buffer, or in 10 mM phosphate, 10 mM citrate, 150 mM NaCl, 1 mM n-dodecyl-β-D-maltoside. They were incubated with 30 nM biotinylated HA peptide and serial dilutions of competitor peptides, in 96-well polypropylene plates (Nunc, Roskilde, Denmark) at 37°C for 24 h. After neutralization of the pH, samples were applied to 96-well maxisorp ELISA plates (Nunc) previously coated with 10 μg/ml L243 mAb and saturated with 100 mM Tris pH 7.5/0.3% BSA buffer. After a 2-h incubation and washing, bound biotinylated peptide was detected by incubating streptavidin-alkaline phosphatase conjugate (Amersham Biosciences), and 4-methylumbelliferyl phosphate substrate (Sigma). Emitted fluorescence was measured at 450 nm upon excitation at 365 nm. Maximal binding was determined by incubating the biotinylated peptide with the MHC class II molecule in the absence of competitor.

3.4 HA-specific CD4+ T cell clones

The HA-specific HA1.7 CD4+ T cell clone 38 was kindly provided by J. Lamb (Imperial College of Science, London, UK). This clone recognizes the HA p306–318 peptide presented by DR4 or DR1 molecules 39 and bears a Vα1.2/Vβ3.1-Jβ1.2 TCR 33. The LR51 HA p306–318-specific CD4+ T cell clone was kindly provided by G. Lombardi (University of Edinburgh Medical School, Edinburgh, UK). Both clones were rested for 7–11 days after stimulation prior to tetramer experiments.

3.5 NY-ESO-1-specific CD4+ T cell lines

Specific NY-ESO-1 T cell lines were generated from PBMC of a DRB1*0401 healthy donor stimulated with 10 μg/ml of NY-ESO-1 p119–130 peptide at 105 cells per well in a 96-well U-bottom plate in complete RPMI medium. IL-2 at 5 IU/ml was added at days 5 and 8. A second stimulation was performed on day 12 with irradiated T2/DR4 cells loaded for 2 h with NY-ESO-1 peptide (105 cells per well) in complete medium containing 5 IU/ml of IL-2, and was repeated every 10 days. The specificity of the generated T cell lines was assessed by a proliferation assay using loaded or unloaded T2 cells as stimulators.

3.6 Methyl-β-cyclodextrine experiments

HA1.7 T cells were treated with various concentrations of βMCD (Sigma), ranging from 0 to 20 mM, in RPMI medium at 37°C for 30 min. After washing, cells were stained with DR4-HA-PE tetramer at 37°C for 1 h, then with anti-CD4-FITC Ab (Caltag, Burlingame, CA) for 20 min at 4°C. Cells were finally washed and immediately analyzed on a FACSCalibur flow cytometer.

3.7 Intracellular IFN-γ production

HA1.7 T cells were stained with 20 μg/ml of DR4-HA-PE tetramers for 1 h at 37°C. After washing, they were incubated or not at 37°C with PMA and ionomycin (respectively 20 ng/ml and 1 μM) in presence of brefeldin A (10 μg/ml). After 2 h, cells were washed and stained with anti-CD4-allophycocyanin Ab (PharMingen). For intracellular staining, Cytofix/Cytoperm was used according to the manufacturer's recommendation (PharMingen) with anti-IFN-γ Ab (R&D Systems, Lille, France) in parallel with its isotype control (mouse IgG2b) for 30 min on ice. Cells were washed and immediately analyzed on a FACSCalibur flow cytometer. IFN-γ production was analyzed after gating on the tetramer-positive population.

3.8 HA1.7 T cell clone dilution experiments and combination of DR4 multimer bead sorting with TCR immunoscope analysis

PBMC were obtained from HLA-DR4 healthy donors and were used to dilute HA1.7 T cells at appropriate concentrations. For the experiment testing the combination of DR4 multimer sorting with TCR immunoscope analysis, specific dilutions were generated by adding 0, 148, 740, 3,700, 18,500 or 92,500 HA1.7 T cells to 37 millions PBMC, giving frequencies of HA1.7 T cells ranging from 0 up to 0.25×10–2 among total PBMC. These dilutions were used to determine the threshold of detection of Ag-specific T cells using TCR immunoscope analysis on DR4-HA multimer bead-sorted or unsorted T cells. Multimer bead sorting was performed as described by Bodinier et al. 32. Briefly, 6.7×105 avidin-coated beads (Dynal, Compiègne, France) were incubated for 60 min with 1.5 μg of biotinylated DR4-HA monomers, before addition to the cell samples in 600 μl of 0.1% BSA-PBS buffer. Cells were rotated for 4 h at room temperature, magnetically sorted, and washed extensively. After total RNA extraction and RT, TCR BV repertoire analysis was performed by quantitative immunoscope technology as previously described 29 using the Immunoscope software 40.


This work was supported by the Ligue Contre le Cancer (Comité de Paris), Association pour la Recherche Contre le Cancer (ARC), INSERM and the Pasteur Institute in Paris. M. V. was supported by a poste d'accueil INSERM and M.-S. C. by a poste vert INSERM. We are indebted to Dr. Véronique Baron and Annick Lim for helpful support in dilution and immunoscope analysis experiments, to Joëlle Tréton for providing us with blood samples from healthy donors, to Nicolas Fazilleau and Dr. Philippe Bousso for helpful discussions, and to Dr. Larry Goldstein (pRmHa-3), Dr. Nuala Mooney (L243), Dr. Hidde Ploegh (DRα and DRβ antisera), Dr. Jonathan Lamb (HA1.7) and Dr. Giovanna Lombardi (LR51) for kindly providing us with reagents.


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