Soluble HLA/peptide monomers cross-linked with co-stimulatory antibodies onto a streptavidin core molecule efficiently stimulate antigen-specific T cell responses

Abstract

Soluble MHC–peptide complexes, commonly referred to as tetramers, have been shown to induce strong cross-linking of TCR and CD8, resulting in a vigorous activation followed by a rapid non-apoptotic CD8⁺ T cell death. This has limited tetramer use for antigen-specific T cells isolation and cloning, as sorted tetramer positive cells were shown to possess compromised functional integrity. Here we show that the cross-linking of a secondary co-stimulatory signal into oligomeric MHC:peptide complexes prevents such cell death, and in contrast strongly stimulates antigen-specific T cell responses. Such soluble antigen-presenting complexes (sAPCs) containing MHC:peptide complexes linked to either anti-CD27 or anti-CD28 antibodies were capable of priming and expanding HLA-A*0201 restricted CMV specific T cells and also of generating functional HLA-A*0301 restricted BCR/ABL-specific T cell responses. These sAPCs constitute an encouraging alternative method for generating antigen-specific T cells that could be applied to a variety of antigens.

Introduction

The current standard protocol available for the priming and/or expansion of low frequency and often low avidity tumour-specific T cells utilises monocyte-derived DCs pulsed with protein or peptides derived from the antigen [4, 13, 19, 24, 29]. Antigen-specific T cells can then be cloned and expanded in a cocktail of cytokines designed to maximise their activation and maturation into memory and effector phenotypes [27]. Unfortunately this technique has many drawbacks. The generation of DCs from individual patients is not only laborious and time-consuming but due to the low frequency of DC precursors in the peripheral blood, it also requires a large volume of donated blood which often limits the number of stimulations. The variability in both the quantity and the quality of DCs generated from patients also renders the reproducibility of the technique very difficult. Additionally the culture conditions are less than ideal, often not meeting the GMP requirement for clinical applications.

To attempt to generate a more defined and reproducible system for priming and expanding T cells, soluble MHC–peptide complexes were assessed for their potential immunomodulatory capacities, since it has been demonstrated that the engagement of trimeric or tetrameric HLA/peptide complexes was sufficient for triggering and transducing T cell receptor signals [5]. Moreover, the infusion of MHC/peptide dimers or tetramers has been demonstrated in a number of animal models to successfully suppress or activate antigen-specific T cell responses [11, 21, 25]. However the preservation of growth capacity and functional integrity of antigen-specific T cells isolated and FACs sorted with tetramers is known to be difficult. More recently, it has been shown that the exposure of activated CTLs with soluble MHC/peptide complexes induced strong activation of the CD8-associated Lck kinases and provoked rapid CTL death that was characteristic of programmed necrosis [8]. This was only observed with tetramers that contained short linkers as compared to MHC/peptide complexes containing long linkers (pentamers and octamers). As only tetramers containing short linkers were capable of inducing strong activation-dependent calcium mobilisation and CTL death, it was suggested that the key determinant for such tetramer-induced CTL death was the distance between individual MHC/peptide units, which largely determined their ability to activate CTLs [8]. Such strong TCR-CD8 cross-linking and subsequent activation may be useful for the generation of antigen-specific T cell responses if provided with appropriate secondary costimulatory signals.

Alternative methods for the rapid generation and expansion of high numbers of antigen-specific T cells have been developed with the new generation of “ready-to-use” artificial APCs (aAPCs). HLA/peptide complexes, along with costimulatory antibodies or recombinant proteins including B7.1 (CD80) or anti-CD28 antibody and ICAM-1 (CD54), have been immobilised onto magnetic beads or streptavidin-coated microbeads (Fig. 1a–c; [22, 26, 28, 32, 36]). These acellular artificial antigen-presenting cells were shown to be capable of rapidly generating higher yields of functional CMV, melanoma and mHAg specific CD8⁺ T cells than yields which could be achieved using conventional autologous DCs as APCs.

Fig. 1.

Examples of acellular aAPC systems are schematised. a Acellular aAPCs were developed by coating streptavidin-labelled microbeads with biotinylated HLA/peptide monomers along with biotinylated anti-CD28 co-stimulatory antibody [22, 32, 36]. b In some cases, the recombinant co-stimulatory molecules, such as B7.1 and ICAM-1 were non-covalently immobilised onto the beads [28]. c HLA/peptide molecules have also been conjugated to an immunoglobulin heavy chain, producing a dimeric HLA/Immunoglobulin fusion protein. These fusion proteins were then immobilised onto beads along with anti-CD28 co-stimulatory antibody [26]. d In this study, soluble antigen-presenting complexes were developed by cross-linking biotinylated HLA/peptide complexes with biotinylated co-stimulatory antibody onto a streptavidin core molecule

Here, we report a novel artificial system for stimulating antigen-specific T cell responses using soluble antigen-presenting complexes (sAPCs), which consisted of cross-linked biotinylated HLA/peptide complexes with a biotinylated co-stimulatory antibody bound onto a streptavidin core molecule (Fig. 1d). The feasibility of these sAPCs to expand antigen-specific CD8⁺ T cells in a memory setting or to prime from a naïve individual, was first assessed in a viral model, using a HLA-A*0201 restricted CMV immuno-dominant peptide (pp65, NLVPMVATV) [35, 37]. After validating the experimental protocol conditions for the generation of CMV-specific T cells with this model, these sAPCs were tested for their capacity in generating anti-tumour CTLs in the context of HLA-A*0301 restricted BCR/ABL (b3a2) described tumour-specific antigen (KQSSKALQR, [3, 6, 9]). Our data demonstrate that these sAPCs can expand CMV-specific T cells, and generate functional BCR/ABL-specific T cell responses from healthy donors, and therefore represent an encouraging alternative method for generating functional antigen-specific T cells. In addition, these sAPCs could be used to purify tetramer positive cells without affecting their functional integrity.

Materials and methods

Donor samples

Peripheral blood samples were obtained from HLA-A*0201, CMV-seropositive and -seronegative and HLA-A*0301 healthy volunteers with informed consent. Cells were separated using Ficoll–Hypaque density gradient centrifugation (Cedarlane^® Laboratories Ltd, Canada) and cryopreserved in RPMI 1640 Complete Medium (1 U/ml penicillin, 1 μg/ml streptomycin, BioWhittaker) supplemented with 20% heat-inactivated foetal calf serum (FCS, BioWhittaker) and 10% dimethyl sulfoxide (DMSO) according to standard protocols. Before use, cells were thawed, washed and suspended in RPMI and 10% pooled AB serum (BioWhittaker).

Synthetic peptides

In this study, the HLA-A*0201 associated cytomegalovirus peptide NLVPMVATV [35],and the HLA-A*0301 associated BCR/ABL peptide KQSSKALQR [3] were synthesised on an ABI synthesiser using Fmoc chemistry and purified by high-performance liquid chromatography (HPLC) by Alta Bioscience, the University of Birmingham, UK. Their purity of more than 90% was confirmed by mass spectrometry. The peptides were dissolved in DMSO at 10 mg/ml.

HLA/peptide complex synthesis

The HLA-A*0201/NLVPMVATV and the HLA-A*0301/KQSSKALQR tetramers were produced as described elsewhere [1]. Briefly, the extracellular portion of the HLA coding cDNA was amplified by polymerase chain reaction (PCR) and cloned into the pET-3d vector (Novagen), modified to contain a C-terminal peptide tag substrate for Bir A-dependent biotinylation. The HLA heavy chains were expressed in BL21(DE3)pLysS E. coli as insoluble inclusion bodies. The β2-microglobulin was produced in a similar fashion. Both molecules were solubilised in 8 M urea and added with the appropriate peptide into a dilution refolding buffer [400 mM arginine (Sigma), 100 mM Tris pH 8, 5 mM reduced glutathione (Sigma), 0.5 mM oxidised glutathione (Sigma), 2 mM EDTA (GibcoBRL)]. The refolded molecules were then purified by Fast Protein Liquid Chromatography (FPLC) gel filtration on a Superdex 75 column (Pharmacia, UK). Biotinylation was performed overnight using bacterially expressed Bir A enzyme, with comparable efficiency to commercially available Bir A (Avidity, Denver, CO). Monomeric biotinylated complexes were further purified by gel filtration followed by anion exchange chromatography. Tetrameric molecules were then formed by the addition of phycoerythrin (PE)-labelled streptavidin at a 4:1 molar ratio.

Soluble artificial antigen-presenting complex generation

Soluble artificial antigen-presenting complexes (sAPCs) were generated by cross-linking biotinylated HLA/peptide monomers with a biotinylated co-stimulatory antibody. Goat-anti-human biotinylated CD27 (clone 57703), CD28 (clone 37407) and CD40L (clone 40804) antibodies (R&D, Minneapolis, USA) were resuspended in the same buffer as the HLA/peptide monomers (20 mM Tris–Hcl pH 8, 150 mM NaCl) and added to the biotinylated HLA/peptide monomers at a theoretical 1:3 molar ratio. 0.8 μg of costimulatory antibody was mixed with 1 μg of HLA/peptide monomer at 4°C with shaking for 1 h. The sAPCs were formed by the addition of PE-labelled streptavidin at a 4:1 molar ratio. The final concentration of these sAPCs was 0.53 μg of HLA/peptide and 0.43 μg of co-stimulatory antibody per μl of complex.

ELISA for testing correct HLA class I/co-stimulatory molecules cross-linking

The presence of both co-stimulatory antibody and correctly refolded HLA class I molecules in the sAPCs was verified in a modified Enzyme Linked Immuno-Sorbent Assay (ELISA). Maxisorb immuno-plates (Nunc^® Nalge Nunc International, Roskilde, Denmark) were coated for 3 h at 37°C with anti-goat IgG antibody (Sigma, 1:3000 dilution in PBS). 5 μg of sAPC protein sample or controls including no antibody, HLA/peptide tetramer alone and soluble co-stimulatory antibody were aliquoted and incubated for 1 h at room temperature (RT). The plates were blocked with goat serum (Sigma, 1:100 dilution in PBS) for an additional 2 h at RT. Plates were incubated with rabbit anti-human β2m antibody (DAKO, UK; 1:5,000) for 20 min at RT. Then 100 μl of alkaline phosphatase goat-anti rabbit IgG (Sigma, 1:5,000) was added to each well and incubated for 20 min at RT. Finally, the alkaline phosphatase substrate was added (Sigma Fast™ p-Nitrophenyl phosphate, Sigma) and the plates were left at RT for 10–30 min. The optical density values were measured at 405 nm in an ELISA plate reader (Titertek Multiskan^® MCC/340, Labsystems, Finland).

Generation of monocyte-derived dendritic cells (mDCs)

PBMC (10 × 10⁶) were plated in 12-well plates for 6 h in complete X-Vivo 10 medium at 37°C. The non-adherent cells were removed and cryopreserved using standard protocol. The adherent cells were incubated in 2 ml of complete X-Vivo 10 medium (BioWhittaker) supplemented with 200 ng/ml GMCSF (R&D) and 100 ng/ml IL-4 (R&D) at 37°C. On days 2 and 4, 2 ml of medium supplemented with 400 ng/ml GMCSF and 200 ng/ml IL-4 was added. On day 6, DCs were matured with TNFα (10 ng/ml, R&D), poly I:C (12.5 μg/ml, Sigma) and sCD40L (1 μg/ml, PeproTech Inc, Peterborough, UK). DCs were pulsed with 20 μg/ml of peptide on day 7 and their maturation was confirmed by cell surface up-regulation of co-stimulatory molecules CD80, CD83 and CD86.

Stimulation of antigen-specific T cells with sAPCs

PBMCs were plated in 24-well plates at a density of 3 × 10⁶ cells/well in X-Vivo 10 medium supplemented with 10% pooled human AB serum and 10 ng/ml of IL-7. Cells were stimulated with 1.5 μl of sAPC complexes or conventional HLA/peptide tetramers plus soluble co-stimulatory antibodies (0.8 μg HLA/peptide and 0.64 μg co-stimulatory antibody) every 5 days, for up to 3 weeks. In parallel, cells were stimulated with autologous irradiated monocyte-derived DCs (ratio of T cells:DC of 10:1) on day 1 and 10. A third stimulation was performed with autologous irradiated PBMCs (ratio of T cells:APC of 5:1) on day 17. Three days following stimulation, cells were washed from the remaining sAPCs and resuspended in complete X-Vivo 10 medium supplemented with IL-7 (10 ng/ml), IL-15 (10 ng/ml) and IL-2 (10 UI/ml).

CFSE proliferation assay

PBMCs were aliquoted at a density of 10 × 10⁶ cells/ml in RPMI 1640 and stained prior to the second and the fourth stimulation with 1 μM CFSE for 10 min at 37°C (CFSE, Molecular Probes, Eugene, Oregon, USA). Cells were recovered by two washes with pre-warmed RPMI 1640 supplemented with 1% FCS. CFSE-labelled lymphocytes were incubated with antigenic stimuli in accordance with the priming protocol. CFSE is an amine reactive fluorescein probe that diffuses passively into cells and couples with the amine group chains of intracellular and cell surface proteins. CFSE is equally distributed between two dividing cells, resulting in a halving of cellular fluorescence as cells proliferate. Cell proliferation was followed by flow cytometry as CFSE emits at 525 nm.

Flow cytometry/HLA tetramer analysis

A minimum of 1 × 10⁶ PBMCs were incubated for 30 min at 37°C with 2 μg of the relevant HLA/peptide tetramer in staining medium (PBS supplemented with 10% heat inactivated FCS and 0.1% sodium azide). Cells were washed and incubated for 15 min at 4°C in the dark with 5 μl CD3-FITC and CD8-PerCP (BD Pharmingen, San Diego, USA). For the assessment of Ag-specific T cells differentiation phenotype, activated T cells were stained with CD27-FITC and CD45R0-APC (BD Pharmingen). Cells were washed and fixed with 1% w/v of paraformaldehyde in PBS. Data were collected on a FACSCalibur flow cytometer (BD) within 48 h using CellQuest™ software version 3.3 (BD) and their subsequent analysis was performed using FlowJo software (Tree Star).

Cytotoxicity assay

Cytotoxic T lymphocyte activity was tested in triplicate in standard ⁵¹Cr release assays. Allogeneic CML target cells were labelled with 150 μCi of Na⁵¹₂CrO₄ and incubated for 2 h at 37°C. A total of 5,000 target cells per well were aliquoted in 96-well U bottom plates. CTLs and target cells were incubated in complete RPMI supplemented with 10% FCS at a ratio of 20:1 for 4 h at 37°C. Each target was plated in triplicate with medium alone (spontaneous chromium release) and with 5% Triton X-100 (BDH) in PBS (total chromium release). Chromium release was measured using a 1450 Microbeta Plus liquid scintillation counter (Wallac). The percentage of specific lysis was calculated using the formula: (experimental release – spontaneous release)/(total release – spontaneous release) × 100.

Statistical analysis

The statistical analyses of the data presented here were performed using Prism^® 4 Software (GraphPad, San Diego, USA). P values ≤0.05 were considered statistically significant.

Results

The soluble antigen-presenting complexes (sAPCs) retain their HLA/peptide specificity

The soluble antigen-presenting complexes (sAPCs) contain both the MHC:peptide complexes capable of binding to cognate T cell receptors, and antibodies specific for T cell co-stimulatory receptors (Fig. 2). The ratio of HLA/peptide complexes and co-stimulatory antibodies required for cross-linking in these sAPCs was established on the basis of maintaining the TCR ligand specificity. For this, sAPCs for HLA-A*0201/CMV peptide and anti-CD28 antibody were constructed at the different ratios of 3:1 (HLA/peptide: co-stimulatory antibody), 2:2 and 1:3. PBMCs derived from HLA-A*0201 CMV-seropositive and -seronegative donors were stained with the different sAPCs, as well as with HLA-A2/CMV conventional tetramers (Fig. 3). The frequency of tetramer positive T cells detected with the sAPC at a 3:1 ratio was similar to the frequency detected with the conventional HLA/peptide tetramers. The decrease of HLA/peptide-co-stimulatory antibody ratio from 3:1 to 1:3 resulted in an increase in the frequency of CD8/sAPCs tetramer T positive cells detected from both the CMV-seropositive donor and the CMV-seronegative donor (from 0.26 to 5.04% and from 0.04 to 1.74%, Fig. 3a, b). In addition, sAPCs at a 3:1 ratio recognise and bind to the same CMV specific T cell population recognised by a conventional HLA-A2/CMV tetramer (Fig. 3c). Thus sAPCs constructed at a ratio of 3:1 retained their TCR ligand specificity and were subsequently used for the stimulation of antigen specific T cells.

Fig. 2.

The HLA/peptide-costimulatory antibody complexes (sAPCs) were tested for the presence of both refolded HLA class I molecules and costimulatory antibody (CD27, CD28, CD40^L and IgG isotype control) in a modified ELISA assay as described in “Materials and methods”. Serial dilutions (1 in 2) of sAPCs and negative controls, including HLA/peptide tetramers and soluble costimulatory antibody, were assessed in parallel. Both Fc regions of the costimulatory antibodies and HLA/peptide complexes were detected in all the sAPCs, above the background level of detection obtained with conventional HLA/peptide tetramers or soluble co-stimulatory antibodies

Fig. 3.

sAPCs were assessed for their TCR ligand specificity. An example is shown for HLA-A*0201/CMV monomers tetramerised with anti-CD28 antibodies at the ratio of 3:1 (three HLA/peptide complexes for one co-stimulatory antibody), 2:2 and 1:3. PBMCs derived from HLA-A*0201 CMV-seropositive (a) or CMV-seronegative (b) healthy donors were stained with 2 μg of HLA-A*0201/CMV tetramer (CMV tet) and each sAPC complexes. c PBMCs derived from a HLA-A*0201 CMV-seropositive patient with high frequency CMV specific T cells were double stained with CMV/CD28 PE sAPC (ratio 3:1) and CMV Tet-APC. The frequencies of tetramer positive cells were assessed by flow cytometry. Cells are gated on CD3⁺ T cells and the percentage of CD8⁺ tetramer⁺ cells are shown in each upper right gate

sAPCs efficiently expand CMV specific CD8⁺ T cells derived from HLA-A*0201 CMV-seropositive and -seronegative donors

PBMCs derived from HLA-A*0201 CMV-seropositive and -seronegative donors were stimulated with CMV-sAPCs providing both anti-CD27 and anti-CD28 co-stimulatory signals. Lymphocytes were re-stimulated with either sAPC-CD27/28 (mixture of equimolar amounts of sAPC-CD27 and sAPC-CD28 complexes) or with sAPC-CD27/28/40^L from the second and for the following stimulations. PBMCs were also stimulated with sAPCs constructed with an irrelevant IgG isotype antibody control (3:1 ratio) and with conventional HLA/peptide tetramers plus soluble co-stimulatory antibodies. Specific CMV T cell activation and expansion following the different types of stimulation was assessed by HLA/CMV tetramer staining three days after the fourth stimulation and a representative HLA/tetramer dot plot is shown in Fig. 4.

Fig. 4.

Representative HLA/tetramer staining of CMV-specific CD8⁺ T cells stimulated with sAPCs. PBMCs derived from an HLA-A*0201 CMV-seropositive donor (a) and from an HLA-A*0201 CMV-seronegative donor (b) were incubated without antigen (Medium) stimulated with HLA/CMV-IgG tetramer (CMV Tet/IgG), sAPC-CD27/28, sAPC-CD27/28.40^L or HLA/tetramer plus soluble co-stimulatory antibodies. Activated T cells were stained with HLA-A2/CMV Allophycocyanin Tetramer and analysed by flow cytometry. Cells are gated on CD3⁺ T cells and the percentage of CMV specific CD8⁺ T cells are shown in each upper right gate

The stimulation of PBMCs from HLA-A*0201 CMV-seropositive donors with the sAPC complexes induced an increase in the number of CMV specific T cells compared to the frequency detected in the negative “media alone” control (Fig. 4a). In addition, the priming of PBMCs derived from CMV-seronegative donors with sAPC-CD27/28 successfully induced the expansion of tetramer positive cells up to 0.68% (Fig. 4b). HLA/peptide tetramers and soluble costimulatory molecules were capable of inducing a greater expansion of CMV memory T cells, derived from a seropositive donor (Fig. 4a, tet + sol CD27/CD28). However, the cross-linking of both HLA/peptide complexes and co-stimulatory molecules on sAPCs was required for the efficient triggering of TCR activation and expansion of CMV-specific T cells (Fig. 4a).

The nature of the co-stimulatory molecules provided in sAPCs affects the kinetics of antigen-specific T cell responses

The highest percentages of tetramer positive T cells were detected after stimulation with sAPCs-CD27/CD28 (Fig. 4a, b). The addition of anti-CD40^L co-stimulatory signals from the second and for the following stimulations consistently resulted in a lower number of CMV-specific T cells detected in the cultures. In this sAPC system, a maximum of four ligands can be cross-linked onto the streptavidin core molecule. Thus, for comparative purposes between the different stimulation conditions, we chose to maintain the signal delivered through the TCR at a constant level. Accordingly, the addition of anti-CD40^L antibody in the stimulation resulted by necessity in the decrease of anti-CD27 and anti-CD28 co-stimulatory signals. The dilution of the early anti-CD27 and/or anti-CD28 co-stimulatory signals may have diminished the CMV-specific T cell activation threshold and subsequently their expansion. It is also possible that the delivery of the late CD40^L signal induced the differentiation of CMV specific T cells into a more mature, and less proliferative compartment. The proliferation and differentiation of CMV specific T cells under the different sAPC stimulation conditions were further characterised. PBMCs derived from a HLA-A*0201 CMV-seropositive donor were labelled with CFSE prior to the second antigen stimulation and analysed for the dilution of CFSE fluorescence upon each cell division (Fig. 5a). Three days after the second antigen stimulations, CMV specific T cells demonstrated very little or no proliferative response, as shown by the high level of CFSE fluorescence detected for each stimulation condition. Only the CMV specific T cells stimulated with PHA demonstrated one or two cellular divisions. Three days after the fourth stimulation, all CMV specific T cells from the positive control have divided and lost CFSE fluorescence (PHA, fourth stimulation). The majority of CMV-specific T cells stimulated with sAPC-CD27/28 demonstrated a complete loss of CFSE fluorescence compared to the negative control (Medium). A significant number of cells remained however in their fourth or fifth cellular divisions (Fig. 5a, sAPC-CD27/28). Following stimulation with sAPC-CD27/28/40^L, all the CMV-specific T cells demonstrate a complete loss of CFSE fluorescence (Fig. 5a, sAPC-CD40^L). Thus the stimulation of CMV-specific T cells with sAPC-CD27/28/40^L appears to induce a faster antigen specific proliferative response compared to the stimulation with sAPC-CD27/28. In addition, the stimulation of CMV-specific T cells with sAPC-CD27/28 induced the preferential differentiation and expansion of CMV-specific T cells into an effector phenotype (67.8% of CD27⁻ CD45RO⁺ cells, Fig. 5b). The addition of anti-CD40^L co-stimulatory antibodies from the second and onward stimulations induced the CMV-specific T cells to further differentiate into terminal effectors (Fig. 5b, sAPCs-CD27/28/40^L, 62% of CD27⁻ CD45RO⁻ cells). Similarly, stimulation with the sAPCs constructed with an irrelevant IgG isotype control induced the differentiation of the low percentage of CMV-specific T cells into terminal effectors (81.5%, CMVtet/IgG). In this case, however, the lack of co-stimulation signalling has probably triggered the apoptosis of these terminally differentiated antigen-specific T cells. Thus, the lower frequency of tetramer positive cells detected after stimulation with sAPC-CD27/28/40^L appears to result from the faster proliferation and differentiation of CMV-specific T cells into a more mature phenotype, and their subsequent activation-induced cell death triggered by CD40^L signalling.

Fig. 5.

The proliferation and differentiation kinetic of CMV-specific T cells stimulated with sAPCs is dependent on the costimulatory signal provided. a PBMCs derived from an HLA-A*0201 CMV-seropositive donor were labelled with CFSE prior to the second and to the fourth stimulation as described in material and method and shown in Fig. 4. A negative control (Medium alone) and positive control (polyclonal stimulus via PHA) were also included. Activated T cells were harvested three days after the second (two stimulations) and fourth stimulations (four stimulations), stained with HLA-A2/CMV Allophycocyanin tetramer and acquired by flow cytometry. Cells were gated on CD3⁺ CD8⁺ CMV tetramer⁺ cells, and analysed for the loss of CFSE fluorescence. b Activated T cells were harvested 3 days after the fourth stimulation and stained with HLA/CMV Allophycocyanin tetramer, CD27 and CD45RO cell surface markers. Lymphocytes were acquired by flow cytometry and gated on CD3⁺ CD8⁺ CMV tetramer⁺ cells. The frequencies of naïve (CD27⁺CD45RO⁻), memory (CD27⁺CD45RO⁺), effector (CD27⁻CD45RO⁺) and terminal effector (CD27⁻CD45RO⁻) CMV specific CD8⁺ T cells is shown for the different stimulation conditions

BCR/ABL-specific T cells can be generated with CML-sAPCs from healthy donors

The capacity of the sAPCs to prime antigen-specific T cell responses was also assessed in the context of the BCR/ABL tumour antigen. BCR/ABL-derived antigenic peptides have been demonstrated to be presented on the surface of HLA-A*0301 positive CML cells, however, the antigen appears to be poorly immunogenic [9, 31]. HLA/BCR-ABL peptide monomers were cross-linked with anti-CD27, anti-CD28 or anti-CD40^L co-stimulatory antibodies. PBMCs derived from five HLA-A*0301 healthy donors were stimulated with sAPCs, in combination with anti-CD27 or with both anti-CD27 and anti-CD28 co-stimulatory antibodies, respectively. Lymphocytes were re-stimulated following the same conditions, with or without the addition of anti-CD40^L co-stimulatory antibody from the second and subsequent stimulations. In addition, controls including stimulation with no antigen, HLA/tetramers-IgG, HLA/tetramers plus soluble costimulatory antibodies and peptide-pulsed DCs were performed in parallel. The BCR/ABL-specific T cell responses generated from these donors were assessed by HLA/tetramer staining. The stimulation of PBMCs-with sAPCs complexes successfully generated BCR/ABL-specific T cells from at least two donors. The frequencies of tetramer positive cells detected from these five HLA-A*0301 healthy donors after stimulation with sAPCs varied between 0.06 and 3.61% (Fig. 6a, b), with BCR/ABL-specific T cells successfully being generated from at least two donors. The variability in the number of tetramer positive T cells detected from the different donors may reflect the differences in the nature and frequency of antigen-specific T cells in each individual’s T cell repertoire. The frequency of BCR/ABL-specific T cells detected from the peripheral blood of healthy donors ranged from 0.01 to 0.12% (data not shown). No or few tetramer positive T cells were detected in the negative control (Medium) or after stimulations with either peptide-pulsed autologous DCs (DCs-peptide) or HLA/tetramers alone (CML Tet/IgG) (≤mean of 0.4%, Fig. 6a). In addition, no tetramer positive T cells were detected from the cultures stimulated with sAPC-CD27 using an irrelevant HLA/peptide tetramer. An example of such HLA/tetramer control staining is shown in Fig. 6b, right column. As no appropriate negative peptide control was available (HLA-A*0301 restricted tumour derived peptide described for CML), activated T cells were stained with the HLA-B*0801/GFKQSSKAL tetramers [3, 6].

Fig. 6.

BCR/ABL-specific T cells were generated from HLA-A*0301 healthy donors following stimulation with sAPCs. PBMCs derived from five HLA-A*0301 healthy donors were stimulated with sAPC-CD27, sAPC-CD27/28 or sAPC-CD27/28/40^L. In parallel, PBMCs were incubated without antigen (Medium), or stimulated with either autologous peptide pulsed-irradiated DCs (DCs-peptide), or with HLA/CML tetramers (CML tetramer) plus soluble co-stimulatory antibodies (Tet + sol). Activated T cells were harvested three days after the fourth stimulation and stained with HLA-A3/CML or HLA-B8/CML (control) Allophycocyanin Tetramers and analysed by flow cytometry. The frequency of tetramer positive cells is expressed as a percentage of CD3⁺CD8⁺ T cells and the mean frequency values are shown for each condition (a). b Representative HLA/tetramer dot plots of BCR/ABL-specific T cells

Similar to the CMV-specific T cell responses, the nature of the co-stimulatory signals provided in the sAPCs complexes appears to affect the levels of priming and/or expansion of BCR/ABL-specific T cells. The stimulation with sAPC-CD27 generated the highest percentages of tetramer positive cells from HLA-A*0301 healthy donors (mean values of 1.21% ± 2.4%, Fig. 6a). The early anti-CD27 co-stimulatory signal was found to be crucial for the successful expansion of BCR/ABL-specific T cells, as its dilution (sAPC-CD27/28, sAPC-CD27/28/40^L) reproducibly reduced the frequency of tetramer positive cells detected (mean values from 1.21 to 0.63% and 0.91%, Fig. 6). It is possible that the signalling via CD40L costimulatory molecule triggered Ag-specific T cells to differentiate into a more mature phenotype as observed in the context of CMV-specific T cell responses.

BCR/ABL-specific T cells generated using CML sAPCs showed specific cytotoxic activity against HLA-matched CML targets

The level of cytotoxic activity of the BCR/ABL-specific T cells generated from a healthy donor following stimulations with sAPCs is shown in Fig. 7. Only T cells stimulated with sAPC-CD27 and sAPC-CD27/28/40^L demonstrated specific cytotoxic activity against HLA-matched CML target cells above the controls (from 12.8 to 18.2% above HLA-unmatched or b2a2 CML target cells). These stimulation conditions had also been previously shown to expand the highest frequency of tetramer positive cells (from 3.61 to 4.28%, Fig. 6b). The BCR/ABL-specific T cells were successfully expanded following sAPC-CD27/28 stimulation (1.5% tetramer positive cells) did not recognise and kill CML target cells above the background. Thus, it appears that the co-stimulatory signals provided for the activation of antigen-specific T cells influence the specific cytotoxic function of these cells. None of the T cells stimulated with peptide-pulsed autologous DCs cells demonstrated specific cytotoxic activity above the background (HLA unmatched or b2a2 targets). This was expected, as the frequency of tetramer positive T cells detected from these cultures was low. This demonstrates the feasibility of generating functional BCR/ABL-specific T cells in the context of HLA-A*0301 from healthy donors.

Fig. 7.

BCR/ABL-specific CD8⁺ T cells generated with sAPCs demonstrated specific cytotoxic activity against HLA-matched CML cells. PBMCs derived from HLA-A*0301 healthy donor were stimulated with the various combinations of sAPCs and were assessed for their cytotoxic activity by Chromium release assay. Controls cells (Medium, DCs-peptide) were also included in the assay. Activated T cells were harvested three days after the fourth stimulation and incubated with HLA-matched b3a2⁺ or b2a2⁺ CML target cells and HLA-unmatched b3a2⁺ CML target cells. The specific cytotoxic activity was measured after 4 hours incubation of target cells with effector T cells at a ratio 1:20, respectively. The percentage of specific lysis was calculated using the following formula: 100 × (cpm experimental release − cpm spontaneous release)/(cpm maximal release − cpm spontaneous release) where cpm represents the count per minute. The error bars represent the standard deviation between triplicate samples

Discussion

We demonstrated the successful modulation of antigen specific CD8⁺ T cell responses using soluble antigen-presenting complexes (sAPCs), which consisted of cross-linking HLA/peptide monomers with co-stimulatory antibody onto a streptavidin core molecule. These sAPCs are devoid of natural antigen processing and presentation machinery and thus their application is limited to known HLA-restricted antigen-specific responses. Their simplicity is however very attractive as they are easy to generate, and the number as well as the nature of molecules presented to the T cells can be completely controlled. The binding of these sAPC complexes (ratio of MHC peptide: co-stimulatory antibody of 3:1) was specifically driven by the pMHC complex and not by the antibody binding to the accessory molecule, as a CMV-seropositive donor showed 0.26% of sPAC binding whereas a CMV-seronegative donor only showed 0.04% of sPAC binding (Fig. 3). The modulation of antigen-specific T cell responses was especially successful for the priming of CMV-specific T cells and for the expansion of low frequency BCR/ABL-specific T cells. In fact, HLA/peptide tetramer and soluble costimulatory molecules were capable of inducing a greater expansion of memory CMV-specific T cells. The amount of both HLA/peptide complexes and costimulatory molecules were identical for all the different stimulation conditions. It is possible that the stimulation with soluble costimulatory antibodies induced a polyclonal T cell activation, which may have enhanced the activation of the CMV-specific T cells. Another contribution factor may be that the presence of four HLA/peptide molecules increased the avidity of the T cell interaction with these tetramers, which in turn induced a stronger antigen-specific T cell activation. However the cross-linking of HLA/peptide and costimulatory molecules was mandatory for the successful expansion of primary responses in the context of both CMV and BCR/ABL naïve responses.

For both CMV and BCR/ABL responses, the nature, amount and timing of the co-stimulation(s) influenced the magnitude and the differentiation of the antigen-specific T cells generated. The stimulation of CMV-specific responses providing anti-CD27 antibody and/or anti-CD28 efficiently induced a significant expansion of antigen specific CD8⁺ T cells. The signalling via anti-CD27 co-stimulatory molecules was shown to be mandatory for the efficient priming of both CMV and BCR/ABL-specific T cell responses and confirms the major role of CD27 co-stimulation for the activation and proliferation of human naïve T cells, particularly with respect to tumour responses [10, 16, 20]. In our experiments, the co-stimulation of BCR/ABL responses with sAPC-CD28 antibody alone was not sufficient to generate detectable tetramer positive T cells (data not shown). However it has been previously demonstrated that high levels of CD28 signalling were required for the expansion of tumour specific T cells [18]. The dilution of the initial co-stimulatory signal by the addition of a second co-stimulatory signal was also demonstrated to affect the magnitude of both CMV and BCR/ABL-specific responses. Antigen stimulations with sAPC-CD27 alone induced the expansion of higher numbers of CMV or BCR/ABL-specific T cells than did stimulations with sAPC-CD27/28 or sAPC-CD27/40^L. The addition of CD40^L signal from the second stimulations was also shown in the context of CMV-specific T cell responses to induce a faster proliferation and differentiation of tetramer positive cells into terminal effector cells, and was believed to result in their activation-induced cell death. Additionally, anti-CD40^L signalling has also been shown to play a major role as a late activation signal and to bypass the need for helper T cells [2, 33]. Its signalling function has been shown in our experiments and by others to be crucial for the generation of tumour specific T cell responses [10, 12, 14].

In this study, the modulation of antigen-specific T cell responses was tested using anti-CD27, anti-CD28 and/or anti CD40^L co-stimulatory molecules. However, other co-stimulatory molecules may also serve to enhance these responses and need to be assessed in the future. For example the expression of the adhesion molecules LFA-3 (CD58) and/or ICAM-1 (CD54) in conjunction with CD80 (anti-CD28) on cellular or acellular artificial APCs has been demonstrated to significantly increase the expansion of functional antigen-specific T cells in the context of CMV, melanoma and mHAgs [15, 18, 28, 30]. These adhesion molecules were shown to induce cellular membrane rearrangement and to initiate the formation of the immunological synapse between the T cells and the APCs. In the context of the soluble antigen-presenting complexes assessed here, the signalling via these adhesion molecules may not be critical since there is no requirement for linking two cell surfaces together. Another co-stimulatory molecule expressed on activated T cells, the 4-1BB ligand, was shown to induce a strong proliferation and amplification of cytotoxic T cell functions [7, 34]. 4-1BB signalling was specifically demonstrated to protect T cell from activation-induced cell death and to maintain antigen-specific T cell survival and proliferation over 70 days of culture [17, 23]. Such signalling may be beneficial for the long-term survival of CMV memory specific T cells stimulated with sAPCs.

The use of HLA/peptide tetramers has been demonstrated in vivo in a mouse model [21]. The clinical application of such vaccination in humans remains however unclear. The presence of Streptavidin in these sAPCs complexes would be of primary concern as it has been shown that streptavidin (or avidin) is immunogenic. In addition, the Fc regions of the co-stimulatory antibodies cross-linked on these sAPCs could induce the undesirable effects of complement-dependent killing of the targeted cells. Nevertheless these sAPCs are proving to be efficient in priming and/or expanding a high number of antigen specific T cells ex vivo, which could be purified and safely re-infused into patients as a specific T cell adoptive immunotherapy.

The incubation of CTLs with tetramers containing short linkers (conventional tetramers) has been demonstrated to induce a vigorous and rapid CTL activation-induced death [8]. However the cross-linking of HLA/peptide monomers with co-stimulatory antibody (sAPCs) shows their ability to maintain their TCR ligand specificity and to efficiently modulate antigen specific CD8⁺ T cell responses in the context of CMV and to generate functional BCR/ABL-specific T cells from HLA-A*0301 healthy donors. These sAPCs constitute an encouraging alternative method for priming tumour-specific T cell responses and could be easily applied to a variety of HLA/peptide complexes. In addition, such sAPCs could be an ideal alternative reagent for the isolation of antigen-specific T cells by FACs, which would prevent any alterations of their growth capacity or functional integrity.

Abbreviations

CMV

Cytomegalovirus

CTL

Cytotoxic T lymphocyte

FCS

Foetal calf serum

HLA

Human leukocyte antigen

MHC

Major histocompatibility complex

sAPC

Soluble antigen-presenting complex